New York, Dec 12 (IANS) Researchers have revealed the appealing array of fruit and candy flavours that entice millions of young people to take up vaping are cardiotoxic and disrupt the heart's normal electrical activity.

Mounting studies indicate that the nicotine and other chemicals delivered by vaping, while generally less toxic than conventional cigarettes, can damage the lungs and heart.

"But so far there has been no clear understanding about what happens when the vaporized flavouring molecules in flavoured vaping products, after being inhaled, enter the bloodstream and reach the heart," said study author Sami Noujaim from the University of South Florida in the US.

In the study, published in the American Journal of Physiology-Heart and Circulatory Physiology, the research team reported on a series of experiments assessing the toxicity of vape flavourings in cardiac cells and in young mice.

The flavoured electronic nicotine delivery systems widely popular among teens and young adults are not harm-free.

"Altogether, our findings in the cells and mice indicate that vaping does interfere with the normal functioning of the heart and can potentially lead to cardiac rhythm disturbances," Noujaim said.

In mouse cardiac muscle cells (HL-1 cells), the researchers tested the toxicity of three different popular flavours of e-liquid: fruit flavour, cinnamon, and vanilla custard.

All three were toxic to HL-1 cells exposed to e-vapour bubbled into the laboratory dish where the cells were cultured.

Cardiac cells derived from human pluripotent stem cells were exposed to three distinct e-vapours.

The first e-vapour containing the only solvent interfered with the electrical activity and beating rate of cardiac cells in the dish. A second e-vapour with nicotine added to the solvent increased the toxic effects on these cells.

The third e-vapour comprised of nicotine, solvent, and vanilla custard flavouring (the flavour previously identified as most toxic) augmented damage to the spontaneously beating cells even more.

"This experiment told us that the flavouring chemicals added to vaping devices can increase harm beyond what the nicotine alone can do," Noujaim said.

The findings showed that mice exposed to vaping were more prone to an abnormal and dangerous heart rhythm disturbance known as ventricular tachycardia compared to control mice.

"Our research matters because regulation of the vaping industry is a work in progress," Noujaim noted.

--IANS

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Flavours added to vaping devices can damage the heart: Study - Sify News

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Abstract

Enzyme replacement therapy, in which a functional copy of an enzyme is injected either systemically or directly into the brain of affected individuals, has proven to be an effective strategy for treating certain lysosomal storage diseases. The inefficient uptake of recombinant enzymes via the mannose-6-phosphate receptor, however, prohibits the broad utility of replacement therapy. Here, to improve the efficiency and efficacy of lysosomal enzyme uptake, we exploited the strategy used by diphtheria toxin to enter into the endolysosomal network of cells by creating a chimera between the receptor-binding fragment of diphtheria toxin and the lysosomal hydrolase TPP1. We show that chimeric TPP1 binds with high affinity to target cells and is efficiently delivered into lysosomes. Further, we show superior uptake of chimeric TPP1 over TPP1 alone in brain tissue following intracerebroventricular injection in mice lacking TPP1, demonstrating the potential of this strategy for enhancing lysosomal storage disease therapy.

Lysosomal storage diseases (LSDs) are a group of more than 70 inherited childhood diseases characterized by an accumulation of cellular metabolites arising from deficiencies in a specific protein, typically a lysosomal hydrolase. Although each individual disease is considered rare, LSDs have a combined incidence of between 1/5000 and 1/8000 live births, and together, they account for a substantial proportion of the neurodegenerative diseases in children (1). The particular age of onset for a given LSD varies depending on the affected protein and the percentage of enzymatic activity still present; however, in most cases, symptoms manifest early in life and progress insidiously, affecting multiple tissues and organs (2). In all but the mildest of cases, disease progression results in severe physical disability, possible intellectual disability, and a shortened life expectancy, with death occurring in late childhood or early adolescence.

As they are monogenic diseases, reintroducing a functional form of the defective enzyme into lysosomes is in principle a viable strategy for treating LSDs. Enzyme replacement therapy (ERT) is now approved for the treatment of seven LSDs, and clinical trials are ongoing for five others (3). However, delivering curative doses of recombinant lysosomal enzymes into lysosomes remains a major challenge in practice. ERT typically takes advantage of a specific N-glycan posttranslational modification, mannose-6-phosphorylation (M6P), which controls trafficking of endogenous lysosomal enzymes, as well as exogenous uptake of lysosomal enzymes from circulation by cells having the cation-independent M6P receptor (CIMPR) (4). Hence, a combination of factors including (i) the abundance of the M6P receptor in the liver, (ii) poor levels of CIMPR expression in several key target tissue types such as bone and skeletal muscle, (iii) incomplete and unpredictable M6P labeling of recombinant enzymes, and (iv) the highly variable affinity of recombinant lysosomal enzymes for CIMPR [viz., Kds (dissociation constants) ranging from low to mid micromolar (5, 6)] all contribute to diminishing the overall effectiveness of therapies using CIMPR for cell entry (3).

To improve the delivery of therapeutic lysosomal enzymes, we drew inspiration from bacterial toxins, which, as part of their mechanism, hijack specific host cellsurface receptors to gain entry into the endolysosomal pathway. While we and others have explored exploiting this pathway to deliver cargo into the cytosol (7, 8), here we asked whether this same approach could be used to enhance the delivery of lysosomal enzymes into lysosomes. We choose the diphtheria toxin (DT)diphtheria toxin receptor (DTR) system owing to the ubiquitous nature of the DTR, in particular its high expression levels on neurons.

Corynebacterium diphtheriae secretes DT exotoxin, which is spread to distant organs by the circulatory system, where it affects the lungs, heart, liver, kidneys, and the nervous system (9). It is estimated that 75% of individuals with acute disease also develop some form of peripheral or cranial neuropathy. This multiorgan targeting results from the fact that the DTR, heparin-binding EGF (epidermal growth factor)like growth factor (HBEGF), is ubiquitously expressed. The extent to which DT specifically targets difficult-to-access tissues such as muscle and bone, however, is not currently known.

DT is a three-domain protein that consists of an N-terminal ADP (adenosine diphosphate)ribosyl transferase enzyme (DTC), a central translocation domain (DTT), and a C-terminal receptorbinding domain (DTR). The latter is responsible for both binding cell surface HBEGF with high affinity [viz., Kd = 27 nM (10)] and triggering endocytosis into early endosomes (Fig. 1A). Within endosomes, DTT forms membrane-spanning pores that serve as conduits for DTC to enter the cytosol where it inactivates the host protein synthesis machinery. The remaining portions of the toxin remain in the endosomes and continue to lysosomes where they are degraded (11, 12). We hypothesized that the receptor-binding domain, lacking any means to escape endosomes, would proceed with any attached cargo to lysosomes and, thus, serve as a means to deliver cargo specifically into lysosomes following high-affinity binding to HBEGF.

(A) DT intoxication pathway (left), DT domain architecture, and LTM structure (right). (B and C) DTK51E/E148K, LTM, mCherry-LTM, and LTM-mCherry compete with wild-type DT for binding and inhibit its activity in a dose-dependent manner with IC50 (median inhibitory concentration) values of 46.9, 10.1, 52.7, and 76.1 nM, respectively (means SD; n = 3). (D and E) C-terminal and N-terminal fusions of LTM to mCherry were immunostained (red) and observed to colocalize with the lysosomal marker LAMP1 (39). (F) Fractional co-occurrence of the red channel with the green channel (Manders coefficient M2) were calculated for mCherry-LTM and LTM-mCherry and were found to be 0.61 0.10 and 0.52 0.11, respectively (means SD; n = 6).

In this study, we generated a series of chimeric proteins containing the DTR-binding domain, DTR, with the goal of demonstrating the feasibility of delivering therapeutic enzymes into lysosomes through the DT-HBEGF internalization pathway. We showed that DTR serves as a highly effective and versatile lysosome-targeting moiety (LTM). It can be placed at either the N or C terminus of the cargo, where it retains its high-affinity binding to HBEGF and the ability to promote trafficking into lysosomes both in vitro and in vivo. On the basis of its advantages, over M6P-mediated mechanisms, we further investigated the utility of LTM for the lysosomal delivery of human tripeptidyl peptidase-1 (TPP1) with the long-term goal of treating Batten disease.

To evaluate whether the DTR-binding fragment could function autonomously to traffic cargo into lysosomes, we first asked whether the isolated 17-kDa DTR fragment could be expressed independently from DT holotoxin and retain its affinity for HBEGF. We cloned, expressed, and purified the receptor-binding fragment and evaluated its ability to compete with full-length DT for the DTR, HBEGF. Before treating cells with a fixed dose of wild-type DT that completely inhibits protein synthesis, cells were incubated with a range of concentrations of LTM or a full-length, nontoxic mutant of DT (DTK51E/E148K). LTM-mediated inhibition of wild-type DT-mediated toxicity was equivalent to nontoxic DT (Fig. 1B), demonstrating that the receptor-binding fragment can be isolated from the holotoxin without affecting its ability to fold and bind cell surface HBEGF. Next, we evaluated whether LTM had a positional bias (i.e., was able to bind HBEGF with a fusion partner when positioned at either terminus). To this end, we generated N- and C-terminal fusions of LTM to the model fluorescent protein mCherry (i.e., mCherry-LTM and LTM-mCherry). To determine binding of each chimera to HBEGF, we quantified the ability of each chimera to compete with wild-type DT on cells in the intoxication assay. Both constructs competed with wild-type DT to the same extent as LTM alone and DTK51E/E148K (Fig. 1C), demonstrating that LTM is versatile and autonomously folds in different contexts.

To evaluate intracellular trafficking, HeLa cells were treated with either LTM-mCherry or mCherry-LTM and then fixed and stained 4 hours later with an antibody against the lysosomal marker LAMP1. In both cases, we observed significant uptake of the fusion protein (Fig. 1, D and E). We calculated Manders coefficients (M2) to quantify the extent to which signal in the red channel (LTM-mCherry and mCherry-LTM) was localizing with signal in the green channel (LAMP1). The fraction of red/green co-occurrence was calculated to be 0.61 for mCherry-LTM and 0.52 for LTM-mCherry, indicating trafficking to the lysosomal compartments of the cells and no significant difference (P = 0.196) between the two orientations of chimera (Fig. 1F). Together, these results confirm that the LTM is capable of binding HBEGF and trafficking associated cargo into cells and that the LTM can function in this manner at either terminus of a fusion construct.

With minimal positional bias observed in the mCherry fusion proteins, we next screened LTM fusions to TPP1 to identify a design that maximizes expression, stability, activity, and, ultimately, delivery. TPP1 is a 60-kDa lysosomal serine peptidase encoded by the CLN2 gene, implicated in neuronal ceroid lipofuscinosis type 2 or Batten disease. Loss of function results in the accumulation of lipofuscin, a proteinaceous, autofluorescent storage material (13). Exposure to the low-pH environment of the lysosome triggers autoproteolytic activation of TPP1 and release of a 20-kDa propeptide that occludes its active site. From a design perspective, we favored an orientation in which the LTM was N terminal to TPP1, as autoprocessing of TPP1 would result in the release of the upstream LTM-TPP1 propeptide, liberating active, mature TPP1 enzyme in the lysosome (Fig. 2A). Given the need for mammalian expression of lysosomal enzymes, we generated synthetic genetic fusions of the LTM to TPP1, in which we converted the codons from bacterially derived DT into the corresponding mammalian codons. Human embryonic kidney (HEK) 293F suspension cells stably expressing recombinant TPP1 (rTPP1) and TPP1 with an N-terminal LTM fusion (LTM-TPP1) were generated using the piggyBac transposon system (14). A C-terminal construct (TPP1-LTM) was also produced; however, expression of this chimera was poor in comparison with rTPP1 and LTM-TPP1 (~0.4 mg/liter, cf. 10 to 15 mg/liter).

(A) Design of LTM-TPP1 fusion protein and delivery schematic. (B) Enzyme kinetics of rTPP1 and LTM-TPP1 against the synthetic substrate AAF-AMC are indistinguishable. Michaelis-Menten plots were generated by varying [AAF-AMC] at a constant concentration of 10 nM enzyme (means SD; n = 3). Plots and kinetic parameters were calculated with GraphPad Prism 7.04. (C) Maturation of TPP1 is unaffected by the N-terminal fusion of LTM. (D) LTM-TPP1 inhibits wild-type DT activity in a dose-dependent manner (IC50 of 17.2 nM), while rTPP1 has no effect on protein synthesis inhibition by DT (means SD; n = 3). (E) LTM and DTR-TPP1 bind HBEGF with apparent Kds of 13.3 and 19.1 nM, respectively. (F) LTM-TPP1 (39) colocalizes with LAMP1 staining (red).

The activity of rTPP1 and LTM-TPP1 against the tripeptide substrate Ala-Ala-Phe-AMC (AAF-AMC) was assessed to determine any effects of the LTM on TPP1 activity. The enzyme activities of rTPP1 and LTM-TPP1 were determined to be equivalent, as evidenced through measurements of their catalytic efficiency (Fig. 2B), demonstrating that there is no inference by LTM on the peptidase activity of TPP1. Maturation of LTM-TPP1 through autocatalytic cleavage of the N-terminal propeptide was analyzed by SDSpolyacrylamide gel electrophoresis (PAGE) (Fig. 2C). Complete processing of the zymogen at pH 3.5 and 37C occurred between 5 and 10 min, which is consistent with what has been observed for the native recombinant enzyme (15).

The ability of LTM-TPP1 to compete with DT for binding to extracellular HBEGF was first assessed with the protein synthesis competition assay. Similar to LTM, mCherry-LTM, and LTM-mCherry, LTM-TPP1 prevents protein synthesis inhibition by 10 pM DT with an IC50 (median inhibitory concentration) of 17.2 nM (Fig. 2D). As expected, rTPP1 alone was unable to inhibit DT-mediated entry and cytotoxicity. To further characterize this interaction, we measured the interaction between LTM and LTM-TPP1 and recombinant HBEGF using surface plasmon resonance (SPR) binding analysis (Fig. 2E). By SPR, LTM and LTM-TPP1 were calculated to have apparent Kds of 13.3 and 19.1 nM, respectively, values closely corresponding to the IC50 values obtained from the competition experiments (10.1 and 17.2 nM, respectively). Consistent with these results, LTM-TPP1 colocalizes with LAMP1 by immunofluorescence (Fig. 2F).

To study uptake of chimeric fusion proteins in cell culture, we generated a cell line deficient in TPP1 activity. A CRISPR RNA (crRNA) was designed to target the signal peptide region of TPP1 in exon 2 of CLN2. Human HeLa Kyoto cells were reverse transfected with a Cas9 ribonucleoprotein complex and then seeded at low density into a 10-cm dish. Single cells were expanded to colonies, which were picked and screened for TPP1 activity. A single clone deficient in TPP1 activity was isolated and expanded, which was determined to have ~4% TPP1 activity relative to wild-type HeLa Kyoto cells plated at the same density (Fig. 3A). The small residual activity observed is likely the result of another cellular enzyme processing the AAFAMC (7-amido-4-methlycoumarin) substrate used in this assay, as there is no apparent TPP1 protein being produced (Fig. 3B). Sanger sequencing of the individual alleles confirmed complete disruption of the CLN2 gene (fig. S1). In total, three unique mutations were identified within exon 2 of CLN2: a single base insertion resulting in a frameshift mutation and two deletions of 24 and 33 base pairs (bp), respectively.

(A) CLN2 knockout cells exhibit ~4% TPP1 activity relative to wild-type HeLa Kyoto cells (means SD; n = 3). (B) Western blotting against TPP1 reveals no detectable protein in the knockout cells. (C) (Left) In vitro maturation of pro-rTPP1 and LTM-TPP1 (16 ng) was analyzed by Western blot. (Right) TPP1 present in wild-type (WT) and TPP1/ cells, and TPP1/ cells treated with 100 nM rTPP1 and LTM-TPP1. (D) Uptake of rTPP1 and LTM-TPP1 into HeLa Kyoto TPP1/ cells was monitored by TPP1 activity (means SD; n = 4). (E) TPP1 activity present in HeLa Kyoto TPP1/ cells following a single treatment with 50 nM LTM-TPP1 (means SD; n = 3).

Next, we compared the delivery and activation of rTPP1 and LTM-TPP1 into lysosomes by treating TPP1/ cells with a fixed concentration of the enzymes (100 nM) and by analyzing entry and processing by Western blot (Fig. 3C). In both cases, most enzymes were present in the mature form, indicating successful delivery to the lysosome; however, the uptake of LTM-TPP1 greatly exceeded the uptake of rTPP1. As both rTPP1 and LTM-TPP1 receive the same M6P posttranslational modifications promoting their uptake by CIMPR, differences in their respective uptake should be directly attributable to uptake by HBEGF. To quantify the difference in uptake and lysosomal delivery, cells were treated overnight with varying amounts of each enzyme, washed, lysed, and assayed for TPP1 activity. The activity assays were performed without a preactivation step, so signal represents protein that has been activated in the lysosome. For both constructs, we observed a dose-dependent increase in delivery of TPP1 to the lysosome (Fig. 3D). Delivery of LTM-TPP1 was significantly enhanced compared with TPP1 alone at all doses, further demonstrating that uptake by HBEGF is more efficient than that by CIMPR alone. TPP1 activity in cells treated with LTM-TPP1 was consistently ~10 greater than that of cells treated with rTPP1, with the relative difference increasing at the highest concentrations tested. This may speak to differences in abundance, replenishment, and/or recycling of HBEGF versus CIMPR, in addition to differences in receptor-ligand affinity. Uptake of LTM-TPP1 and rTPP1 into several other cell types yielded similar results (fig. S2). To assess the lifetime of the delivered enzyme, cells were treated with LTM-TPP1 (50 nM) and incubated overnight. Cells were washed and incubated with fresh media, and TPP1 activity was assayed over the course of several days. Cells treated with LTM-TPP1 still retained measurable TPP1 activity at 1 week after treatment (Fig. 3E).

While the DT competition experiment demonstrated that HBEGF is involved in the uptake of LTM-TPP1 but not rTPP1 (Fig. 2D), it does not account for the contribution of CIMPR to uptake. Endoglycosidase H (EndoH) cleaves between the core N-acetylglucosamine residues of high-mannose N-linked glycans, leaving behind only the asparagine-linked N-acetylglucosamine moiety. Both rTPP1 and LTM-TPP1 were treated with EndoH to remove any M6P moieties, and delivery into Hela TPP1/ was subsequently assessed. While rTPP1 uptake is completely abrogated by treatment with EndoH, LTM-TPP1 uptake is only partially decreased (Fig. 4), indicating that while HBEGF-mediated endocytosis is the principal means by which LTM-TPP1 is taken up into cells, uptake via CIMPR still occurs. The fact that CIMPR uptake is still possible in the LTM-TPP1 fusion means that the fusion is targeted to two receptors simultaneously, increasing its total uptake and, potentially, its biodistribution.

Uptake of LTM-TPP1 via the combination of HBEGF and CIMPR was shown to be 3 to 20 more efficient than CIMPR alone in cellulo (fig. S2). To interrogate this effect in vivo, TPP1-deficient mice (TPP1tm1pLob or TPP1/) were obtained as a gift from P. Lobel at Rutgers University. Targeted disruption of the CLN2 gene was achieved by insertion of a neo cassette into intron 11 in combination with a point mutation (R446H), rendering these mice TPP1 null by both Western blot and enzyme activity assay (16). Prior studies have demonstrated that direct administration of rTPP1 into the cerebrospinal fluid (CSF) via intracerebroventricular or intrathecal injection results in amelioration of disease phenotype (17) and even extension of life span in the disease mouse (18). To compare the uptake of LTM-TPP1 and rTPP1 in vivo, the enzymes were injected into the left ventricle of 6-week-old TPP1/ mice. Mice were euthanized 24 hours after injection, and brain homogenates of wild-type littermates, untreated, and treated mice were assayed for TPP1 activity (Fig. 5A). Assays were performed without preactivation, and therefore, the results report on enzyme that has been taken up into cells, trafficked to the lysosome, and processed to the mature form.

(A) Assay schematic. (B) TPP1 activity in brain homogenates of 6-week-old mice injected with two doses (5 and 25 g) of either rTPP1 or LTM-TPP1 (5 g, P = 0.01; 25 g, P = 0.002). (C) TPP1 activity in brain homogenates following a single 25-g dose of LTM-TPP1, 1, 7, and 14 days postinjection. Data are presented as box and whisker plots, with whiskers representing minimum and maximum values from n 4 mice per group. Statistical significance was calculated using paired t tests with GraphPad Prism 7.04.

While both enzymes resulted in a dose-dependent increase in TPP1 activity, low (5 g) and high (25 g) doses of rTPP1 resulted in only modest increases of activity, representing ~6 and ~26% of the wild-type levels of activity, respectively (Fig. 5B). At the same doses, LTM-TPP1 restored ~31 and ~103% of the wild-type activity. To assess the lifetime of enzyme in the brain, mice were injected intracerebroventricularly with 25 g of LTM-TPP1 and euthanized either 1 or 2 weeks postinjection. Remarkably, at 1 week postinjection, ~68% of TPP1 activity was retained (compared with 1 day postinjection), and after 2 weeks, activity was reduced to ~31% (Fig. 5C).

ERT is a lifesaving therapy that is a principal method of treatment in non-neurological LSDs. Uptake of M6P-labeled enzymes by CIMPR is relatively ineffective due to variable receptor affinity (5, 6), heterogeneous expression of the receptor, and incomplete labeling of recombinantly produced enzymes (19). Despite its inefficiencies and high cost (~200,000 USD per patient per year) (20), it remains the standard of care for several LSDs, as alternative treatment modalities (substrate reduction therapy, gene therapy, and hematopoietic stem cell transplantation) are not effective, not as well developed, or inherently riskier (2125). Improving the efficiency and distribution of recombinant enzyme uptake may help address some of the current shortcomings in traditional ERT.

Several strategies have been used to increase the extent of M6P labeling on recombinantly produced lysosomal enzymes: engineering mammalian and yeast cell lines to produce more specific/uniform N-glycan modification (19, 26, 27), chemical or enzymatic modification of N-glycans posttranslationally (28), and covalent coupling of M6P (29). M6P-independent uptake of a lysosomal hydrolase by CIMPR has been demonstrated for both -glucuronidase (28) and acid -glucosidase (30, 31). In the latter work, a peptide tag (GILT) targeting insulin-like growth factor II receptor (IGF2R) was fused to recombinant alpha glucosidase, which enabled receptor-mediated entry into cells. CIMPR is a ~300-kDa, 15-domain membrane protein with 3 M6P-binding domains and 1 IGF2R domain. By targeting the IGF2R domain with a high-affinity (low nanomolar) peptide rather than the low-affinity M6P-binding domain, the authors were able to demonstrate a >20-fold increase in the uptake of a GAA-peptide fusion protein in cell culture and a ~5-fold increase in the ability to clear built-up muscle glycogen in GAA-deficient mice.

In this study, we have demonstrated efficient uptake and lysosomal trafficking of a model lysosomal enzyme, TPP1, via a CIMPR-independent route, using the receptor-binding domain of a bacterial toxin. HBEGF is a member of the EGF family of growth factors, and DT is its only known ligand. Notably, it plays roles in cardiac development, wound healing, muscle contraction, and neurogenesis; however, it does not act as a receptor in any of these physiological processes (32). Intracellular intoxication by DT is the only known process in which HBEGF acts as a receptor, making it an excellent candidate receptor for ERT, as there is no natural ligand with which to compete. Upon binding, DT is internalized via clathrin-mediated endocytosis and then trafficked toward lysosomes for degradation (33, 34). Acidification of endosomal vesicles by vacuolar ATPases (adenosine triphosphatases) promotes insertion of DTT into the endosomal membrane and subsequent translocation of the catalytic DTC domain into the cytosol. In the absence of an escape mechanism, the majority of internalized LTM should be trafficked to the lysosome, as we have demonstrated with our chimera (Figs. 2F and 3C). Uptake of LTM-TPP1 in vitro is robustly relative to rTPP1 (Fig. 3D and fig. S2), and TPP1 activity is sustained in the lysosome for a substantial length of time (Fig. 3E). We have also demonstrated that the increase in uptake efficiency that we observed in cell culture persists in vivo. TPP1 activity in the brains of CLN2-null mice was significantly greater in animals treated with intracerebroventricularly injected LTM-TPP1, as compared with those treated with TPP1 at two different doses (Fig. 5B), and, remarkably, this activity persists with an apparent half-life of ~8 days (Fig. 5C).

An important consideration for further development of the LTM platform for clinical development is the potential immunogenicity of using a bacterial fragment in this context. Previously, we demonstrated that the receptor-binding fragment of DT could be replaced with a human scFv (single-chain fragment variable) targeting HBEGF (8). With our demonstration of the potential for targeting HBEGF for LSDs, future efforts will focus on increasing the affinity and specificity of these first-generation humanized LTMs to develop high-affinity chimeras with greatly reduced immunogenicity for further development.

While the ability of LTM-TPP1 to affect disease progression has yet to be determined, recent positive clinical trial results (35) and the subsequent approval of rTPP1 (cerliponase alfa) for treatment of neuronal ceroid lipofuscinosis 2 (NCL2) provide support for this approach. In that clinical trial, 300 mg of rTPP1 was administered by biweekly intracerebroventricular injection to 24 affected children, and this was able to prevent disease progression. While this dose is of the same order of magnitude as other approved ERTs (<1 to 40 mg/kg) (36, 37), it represents a substantial dose, especially considering that it was delivered to a single organ. Improving the efficiency of uptake by targeting an additional receptor as we have done here, is expected to greatly decrease the dose required to improve symptoms, while at the same time decreasing costs and the chances of dose-dependent side effects.

DTK51E/E148K, LTM, LTM-mCherry, mCherry-LTM, and HBEGF constructs were cloned using the In-Fusion HD cloning kit (Clontech) into the Champion pET SUMO expression system (Invitrogen). Recombinant proteins were expressed as 6His-SUMO fusion proteins in Escherichia coli BL21(DE3)pLysS cells. Cultures were grown at 37C until an OD600 (optical density at 600 nm) of 0.5, induced with 1 mM IPTG (isopropyl--d-thiogalactopyranoside) for 4 hours at 25C. Cell pellets harvested by centrifugation were resuspended in lysis buffer [20 mM tris (pH 8.0), 160 mM NaCl, 10 mM imidazole, lysozyme, benzonase, and protease inhibitor cocktail] and lysed by three passages through an EmulsiFlex C3 microfluidizer (Avestin). Following clarification by centrifugation at 18,000g for 20 min and syringe filtration (0.2 m), soluble lysate was loaded over a 5-ml His-trap FF column (GE Healthcare) using an AKTA FPLC. Bound protein was washed and eluted over an imidazole gradient (20 to 150 mM). Fractions were assessed for purity by SDS-PAGE, pooled, concentrated, and frozen on dry ice in 25% glycerol for storage at 80C.

TPP1 cDNA was obtained from the SPARC BioCentre (The Hospital for Sick Children) and cloned into the piggyBac plasmid pB-T-PAF (J.M.R., University of Toronto) using Not I and Asc I restriction sites to generate two expression constructs (pB-T-PAF-ProteinA-TEV-LTM-TPP1 and pB-T-PAF-ProteinA-TEV-TPP1). Stably transformed expression cell lines (HEK293F) were then generated using the piggyBac transposon system, as described (14). Protein expression was induced with doxycycline, and secreted fusion protein was separated from expression media using immunoglobulin G (IgG) Sepharose 6 fast flow resin (GE Healthcare) in a 10-ml Poly-Prep chromatography column (Bio-Rad). Resin was washed with 50 column volumes of wash buffer [10 mM tris (pH 7.5) and 150 mM NaCl] and then incubated overnight at 4C with TEV (Tobacco Etch Virus) protease to release the recombinant enzyme from the Protein A tag. Purified protein was then concentrated and frozen on dry ice in 50% glycerol for storage at 80C.

Cellular intoxication by DT was measured using a nanoluciferase reporter strain of Vero cells (Vero NlucP), as described previously (8). Briefly, Vero NlucP cells were treated with a fixed dose of DT at EC99 (10 pM) and a serial dilution of LTM, LTM-mCherry, mCherry-LTM, DTK51E/E148K, LTM-TPP1, or rTPP1 and incubated overnight (17 hours) at 37C. Cell media was then replaced with a 1:1 mixture of fresh media and Nano-Glo luciferase reagent (Promega), and luminescence was measured using a SpectraMax M5e (Molecular Devices). Results were analyzed with GraphPad Prism 7.04.

SPR analysis was performed on a Biacore X100 system (GE Healthcare) using a CM5 sensor chip. Recombinant HBEGF was immobilized to the chip using standard amine coupling at a concentration of 25 g/ml in 10 mM sodium acetate (pH 6.0) with a final response of 1000 to 2500 resonance units (RU). LTM and LTM-TPP1 were diluted in running buffer [200 mM NaCl, 0.02% Tween 20, and 20 mM tris (pH 7.5)] at concentrations of 6.25 to 100 nM and injected in the multicycle analysis mode with a contact time of 180 s and a dissociation time of 600 s. The chip was regenerated between cycles with 10 mM glycine (pH 1.8). Experiments were performed in duplicate using two different chips. Binding data were analyzed with Biacore X100 Evaluation Software version 2.0.2, with apparent dissociation constants calculated using the 1:1 steady-state affinity model.

HeLa cells were incubated with LTM-mCherry (0.5 M), mCherry-LTM (0.5 M), or LTM-TPP1 (2 M) for 2 hours. Cells were washed with ice-cold phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, and permeabilized with 0.5% Triton X-100. mCherry constructs were visualized with a rabbit polyclonal antibody against mCherry (Abcam, ab16745) and anti-rabbit Alexa Fluor 568 (Thermo Fisher Scientific). LAMP1 was stained with a mouse primary antibody (DSHB 1D4B) and anti-mouse Alexa Fluor 488 (Thermo Fisher Scientific).

Colocalization was quantified using the Volocity (PerkinElmer) software package to measure Manders coefficients of mCherry signal with LAMP1 signal. The minimal threshold for the 488- and 568-nm channels was adjusted to correct the background signal. The same threshold for both channels was used for all the cells examined.

CLN2/ fibroblast 19494 were incubated with LTM-TPP1 (2 M) for 2 hours. Cells were washed with ice-cold PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.5% Triton X-100. LTM-TPP1 was visualized with a mouse monoclonal against TPP1 (Abcam, ab54685) and anti-mouse Alexa Fluor 488 (Thermo Fisher Scientific). LAMP1 was stained with rabbit anti-LAMP1 and anti-rabbit Alexa Fluor 568 (Thermo Fisher Scientific).

TPP1 protease activity was measured using the synthetic substrate AAF-AMC using a protocol adapted from Vines and Warburton (38). Briefly, enzyme was preactivated in 25 l of activation buffer [50 mM NaOAc (pH 3.5) and 100 mM NaCl] for 1 hour at 37C. Assay buffer [50 mM NaOAc (pH 5.0) and 100 mM NaCl] and substrate (200 M AAF-AMC) were then added to a final volume of 100 l. Fluorescence (380 nm excitation/460 nm emission) arising from the release of AMC was monitored in real time using a SpectraMax M5e (Molecular Devices). TPP1 activity in cellulo was measured similarly, without the activation step. Cells in a 96-well plate were incubated with 25 l of 0.5% Triton X-100 in PBS, which was then transferred to a black 96-well plate containing 75 l of assay buffer with substrate in each well.

crRNA targeting the signal peptide sequence in exon 2 of CLN2 was designed using the Integrated DNA Technologies (www.idtdna.com) design tool. The gRNA:Cas9 ribonucleoprotein complex was assembled according to the manufacturers protocol (Integrated DNA Technologies) and reverse transfected using Lipofectamine RNAiMAX (Thermo Fisher Scientific) into HeLa Kyoto cells (40,000 cells in a 96-well plate). Following 48 hours of incubation, 5000 cells were seeded into a 10-cm dish. Clonal colonies were picked after 14 days and transferred to a 96-well plate. Clones were screened for successful CLN2 knockout by assaying TPP1 activity and confirmed by Sanger sequencing and Western blot against TPP1 antibody (Abcam, ab54385).

The pro-form of TPP1 was matured in vitro to the active form in 50 mM NaOAc (pH 3.5) and 100 mM NaCl for 1 to 30 min at 37C. The autoactivation reaction was halted by the addition of 2 Laemmli SDS sample buffer containing 10% 2-mercaptoethanol and boiled for 5 min. Pro and mature TPP1 were separated by SDS-PAGE and imaged on a ChemiDoc gel imaging system (Bio-Rad).

Proteins or cellular lysate were separated by 4 to 20% gradient SDS-PAGE before being transferred to a nitrocellulose membrane using the iBlot (Invitrogen) dry transfer system. Membranes were then blocked for 1 hour with a 5% milktris-buffered saline (TBS) solution and incubated overnight at room temperature with a 1:100 dilution of mouse monoclonal antibody against TPP1 (Abcam, ab54685) in 5% milk-TBS. Membranes were washed 3 5 min with 0.1% Tween 20 (Sigma-Aldrich) in TBS before a 1-hour incubation with a 1:5000 dilution of sheep anti-mouse IgG horseradish peroxidase secondary antibody (GE Healthcare) in 5% milk-TBS. Chemiluminescent signal was developed with Clarity Western ECL substrate (Bio-Rad) and visualized on a ChemiDoc gel imaging system (Bio-Rad).

rTTP1 and LTM-TPP1 were treated with EndoH (New England Biolabs) to remove N-glycan modifications. Enzymes were incubated at 1 mg/ml with 2500 U of EndoH for 48 hours at room temperature in 20 mM tris (pH 8.0) and 150 mM NaCl in a total reaction volume of 20 l. Cleavage of N-glycans was assessed by SDS-PAGE, and concentrations were normalized to native enzyme-specific activities.

Cryopreserved TPP1+/ embryos were obtained from P. Lobel at Rutgers University and rederived in a C57/BL6 background at The Centre for Phenogenomics in Toronto. Animal maintenance and all procedures were approved by The Centre for Phenogenomics Animal Care Committee and are in compliance with the CCAC (Canadian Council on Animal Care) guidelines and the OMAFRA (Ontario Ministry of Agriculture, Food, and Rural Affairs) Animals for Research Act.

TPP1/ mice (60 days old) were anesthetized with isoflurane (inhaled) and injected subcutaneously with sterile saline (1 ml) and meloxicam (2 mg/kg). Mice were secured to a stereotactic system, a small area of the head was shaved, and a single incision was made to expose the skull. A high-speed burr was used to drill a hole at stereotaxic coordinates: anteroposterior (A/P), 1.0 mm; mediolateral (M/L), 0.3 mm; and dorsoventral (D/V), 3.0 mm relative to the bregma, and a 33-gauge needle attached to a 10-l Hamilton syringe was used to perform the intracerebroventricular injection into the left ventricle. Animals received either 1 or 5 l of enzyme (5 g/l), injected at a constant rate. Isoflurane-anesthetized animals were euthanized by transcardial perfusion with PBS. Brains were harvested and frozen immediately, then thawed and homogenized in lysis buffer [500 mM NaCl, 0.5% Triton X-100, 0.1% SDS, and 50 mM Tris (pH 8.0)] using 5-mm stainless steel beads in TissueLyser II (Qiagen). In vitro TPP1 assay was performed, as described, minus the activation step.

Acknowledgments: We thank P. Lobel at Rutgers University for providing the TPP1-deficient mice. Funding: We are grateful to the Canadian Institutes of Health Research for funding. Author contributions: S.N.S.-M. devised and performed experiments and drafted the initial manuscript. G.L.B. provided materials and assisted in conceptualization and experimental design. X.Z., D.Z., and R.H. contributed to the experimental design and performed experiments. P.K.K. and B.A.M. contributed to the experimental design. J.M.R. contributed to the experimental design and revised the manuscript. R.A.M. assisted in conceptualization, contributed to the experimental design, and assisted in writing the manuscript. Competing interests: B.A.M. is a chief medical advisor at Taysha Gene Therapies. The authors declare that they have no other competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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Exploiting the diphtheria toxin internalization receptor enhances delivery of proteins to lysosomes for enzyme replacement therapy - Science Advances

Cardiac Stem Cells Comments Off on Exploiting the diphtheria toxin internalization receptor enhances delivery of proteins to lysosomes for enzyme replacement therapy – Science Advances | December 13th, 2020

Researchers with affiliations to Dalhousie University, Nova Scotia Health and the IWK Health Centre are the recipients of over $1.3 million in funding from Research Nova Scotia.

The funding has been provided by the New Health Investigator Grant, which supports new health researchers who are engaged in work that aligns with the provinces health research priorities. The grant aims to provide two years of support of up to $100,000 for researchers who are within the first five years of their academic appointment in Nova Scotia, or who are new to the field of health research.

There has never been a greater need to support new health researchers in Nova Scotia to help inform practice, policy and decision making, says Stefan Leslie, CEO of Research Nova Scotia in a news release. Were pleased to announce funding for these researchers and are confident their work will positively impact the health of Nova Scotians.

For the 2020-21 academic year, funding for this grant is provided by the Nova Scotia Department of Health and Wellness. It will support the establishment of independent programs of research and support and expand the research productivity necessary for obtaining long-term funding from national and external agencies and provide opportunities for early-career investigators to make significant contributions in their field.

Congratulations to all the recipients of funding from Research Nova Scotia, says Dr. Alice Aiken, vice president research and innovation at Dalhousie. With projects that span a wide range of topics, like diabetes, cancer, dementia care, and the COVID-19 pandemic, these researchers are improving health care and helping people in the Maritimes and beyond to be healthier.

Highlights of some of the funded projects:

Dr. Christine Cassidy, Faculty of Health

Designing an integrated pediatric inpatient-ambulatory care service delivery model

The health care system is facing challenges related to poor quality of care, rising health care costs, and outdated technology. Efforts are needed to redesign health services to improve outcomes for patients, health care providers, and the overall health system. One way to address these challenges is to integrate care across multiple health care providers and services. This means that care is coordinated to meet patient needs and preferences.

During the COVID-19 pandemic, the IWK Health Care Centre identified gaps in their current approach to delivering services to children, youth, and their families which includes the need to improve the integration of care across their outpatient and inpatient settings. Healthcare interventions are more effective when patients and care providers are included in the design process, and the integrated approach developed by Dr. Cassidy and her research team will help strengthen the delivery of care within the pediatric health system.

Dr. Parisa Ghanouni, Faculty of Health

Community-based services for individuals with developmental disabilities: Transition to adult care

Despite the great progress signaled by the United Nations Convention on the Rights of Persons with Disabilities, individuals with disabilities worldwide continue to confront barriers to equitable access to the health resources and social supports that enable their full participation in society. Gaps in access have improved for many, especially for children, but the transition to adulthood continues to represent a services cliff that people with disabilities confront in their late teens.

Through their research, Dr. Ghanouni and her team plan to uncover barriers and facilitators related to community-based healthcare services during the transition of adolescents with developmental disabilities to adulthood in rural areas, and co-develop a toolkit with stakeholders that outlines implementation strategies to promote successful transitions. This initiative will advance knowledge on services available that support the transition to adulthood in rural areas, highlight service gaps, point to important areas for investment, and contribute to academic, policy and community understandings and capacity around services for people with disabilities.

Dr. Brendan Leung, Faculty of Dentistry

Harnessing oral microbiota to prevent chemotherapy-induced oral mucositis: Functional screening using a bio-printed mammalian-microbe co-culture model

Chemotherapy induced oral mucositis (CIOM) is a painful and debilitating side effect of cancer treatment that affects 20-40% of cancer patients. Chemotherapy kills cancer cells, but it also affects fast growing normal cells in the body, especially those that line the mouth. When those are damaged, painful mouth ulcers form. These can affect patients ability to eat, drink, talk and even rest, and significantly reduce their quality of life. Currently there is no effective way to prevent CIOM from happening, and the only way to treat it is to provide supportive care such as numbing gels, ice chips and painkillers.

Research has found that the types of bacteria that normally live in the mouth change when someone develops CIOM. It is difficult to study cause and effect between bacteria and CIOM, partly because it is difficult to grow bacteria and human cells together in the lab in a controlled and repeatable way. Through his research, Dr. Leung will use a unique method to grow oral bacteria to investigate how microbes interact with oral cells during chemotherapy in order to identify microbial species that may offer protection against CIOM.

Dr. Elaine Moody, Faculty of Health

Primary healthcare for people with dementia: Exploring care provided by collaborative family practice teams in Nova Scotia

There is an increasing need to improve the health care of people with dementia in Nova Scotia. As the population ages, it will become even more important to provide good care to people with dementia to ensure they can live well in the community. In Nova Scotia, there has been a move to develop collaborative family practice teams, where physicians, nurse practitioners, family practice nurses and other healthcare providers work together to address the primary health care needs of individuals. Primary care providers in these teams require dementia-specific knowledge, skills, resources and supports to enable people with dementia and their caregivers to live well in the community.

Dr. Moody and her research team hope to better understand how collaborative family practice teams in Nova Scotia are addressing the needs of people living with dementia in the community, and to identify ways to improve their care. To achieve their goal, the researchers will gather the perspectives of people living with dementia and caregivers on how collaborative family practice teams provide care in order to identify gaps in current service provision and opportunities to improve care, with a particular focus on diversity and inclusion. Additionally, they will explore how care provided by collaborative family practice teams to people with dementia has been affected by the COVID-19 outbreak.

Other funded projects include:

Dr. Leah Cahill, Faculty of Medicine

Does a simple blood test predict who needs strict blood sugar control to prevent heart disease?

Dr. Sylvain Charlebois, Faculty of ManagementHome food gardening in response to the COVID-19 pandemic: Lessons for food security considerations

Dr. Ketul Chaudhary, Faculty of MedicineCardiac Vascular Stem Cells in Right Heart Failure

Dr. Jon Dorling, Faculty of MedicinePreterm Infant Gut microbiome associations with Environment and Outcomes in the NICU (PIGEON)

Dr. Denys Khaperskyy, Faculty of MedicineRole of stress granule formation in immune responses to respiratory viruses

Dr. Michael Kucharczyk, Faculty of MedicineCan Magnetic Resonance Imaging of the prostate combined with a Radiomics Evaluation determine the invasive capacity of a tumour (Can MRI-PREDICT)

Dr. Paula McLaughlin, Faculty of MedicineIdentifying, understanding, and mitigating gaps in dementia care

Dr. Sandra Meier, Faculty of MedicineAn app responding to behaviour of people to promote mental wellbeing in anxious youth

Dr. Deniz Top, Faculty of MedicineDifference in the regulation of behaviour genes as a proposed mechanism for mental illness

Dr. Igor Yakovenko, Faculty of ScienceScreening, self-management and referral to treatment for young cannabis users: Fulfilling an unmet need

For a complete list of recipients and projects, visit the Research Nova Scotia website.

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New health researchers at Dal, IWK and Nova Scotia Health receive funding from Research Nova Scotia - Dal News

Cardiac Stem Cells Comments Off on New health researchers at Dal, IWK and Nova Scotia Health receive funding from Research Nova Scotia – Dal News | December 11th, 2020

PHOENIX, Dec. 10, 2020 /PRNewswire/ --Creative Medical Technology Holdings trading under the ticker symbol CELZ announced today its patent filing based on data covering utilization of the Company's ImmCelz product at generating what is termed in the field of immunology as "infectious tolerance."

Using an animal model of rheumatoid arthritis, investigators demonstrated administration of ImmCel protected mice from immunologically mediated joint damage. Importantly, cells from treated mice were able to reverse disease when transferred to arthritic mice. Detailed scientific analysis revealed that ImmCelz administration caused generation of T regulatory cells and tolerogenic dendritic cells. Both of these cell types have previously been described to possess ability to suppress autoimmunity.

"In 2003, Dr. Weiping Min from the University of Western Ontario and myself published a paper describing the Tolerogenic Loop, in which we were able to perform fully mis-matched cardiac transplants without need for long term immune suppression1." Said Dr. Thomas Ichim, Chief Scientific Officer of the Company. "We are extremely enthusiastic to discover that ImmCelz, which is a personalized immunotherapy can induce similar biological processes and in this case suppress autoimmunity."

Creative Medical Technology Holdings possesses numerous issued patents in the area of cellular therapy including patent no. 10,842,815 covering use of T regulatory cells for spinal disc regeneration, patent no. 9,598,673 covering stem cell therapy for disc regeneration, patent no. 10,792,310 covering regeneration of ovaries using endothelial progenitor cells and mesenchymal stem cells, patent no. 8,372,797 covering use of stem cells for erectile dysfunction, and patent no. 7,569,385 licensed from the University of California covering a novel stem cell type.

"Given that our issued intellectual property covers multi-billion dollar markets, it is critical in our development plans to establish scientific mechanisms of action. By understanding how our products work at a cellular and molecular level, we feel we have an advantage when engaging Big Pharma in discussions for licensing/partnering interactions." Said Timothy Warbington, President and CEO of the Company.

The company intends to publish an update on the overall 2020 activities in the coming weeks.

About Creative Medical Technology Holdings

Creative Medical Technology Holdings, Inc. is a commercial stage biotechnology company specializing in stem cell technology in the fields of urology, neurology and orthopedics and trades on the OTC under the ticker symbol CELZ. For further information about the company, please visitwww.creativemedicaltechnology.com.

Forward Looking Statements

OTC Markets has not reviewed and does not accept responsibility for the adequacy or accuracy of this release. This news release may contain forward-looking statements including but not limited to comments regarding the timing and content of upcoming clinical trials and laboratory results, marketing efforts, funding, etc. Forward-looking statements address future events and conditions and, therefore, involve inherent risks and uncertainties. Actual results may differ materially from those currently anticipated in such statements. See the periodic and other reports filed by Creative Medical Technology Holdings, Inc. with the Securities and Exchange Commission and available on the Commission's website atwww.sec.gov.

Timothy Warbington, CEO[emailprotected] CreativeMedicalHealth.com

Creativemedicaltechnology.comwww.StemSpine.comwww.Caverstem.comwww.Femcelz.com

1 https://www.jimmunol.org/content/170/3/1304

SOURCE Creative Medical Technology Holdings, Inc.

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Creative Medical Technology Holdings files Patent on Induction of Infectious Tolerance by Ex Vivo Reprogrammed Immune Cells Utilizing ImmCelz Cellular...

Cardiac Stem Cells Comments Off on Creative Medical Technology Holdings files Patent on Induction of Infectious Tolerance by Ex Vivo Reprogrammed Immune Cells Utilizing ImmCelz Cellular… | December 10th, 2020

Less than two months after Peter Kolchinsky and Raj Shah announced a new $461 million fund, the partners at RA Capital Management appear to have made another investment.

RA is headlining a $45 million Series A round for the Oxford, UK-based biotech PepGen, which focuses on severe neuromuscular diseases like Duchenne muscular dystrophy. The company will use the funding to advance a slate of what theyre calling cell-penetrating peptides combined with some of their proprietary conjugates into the clinic.

We believe PepGens PPMOs have enormous potential for the treatment of severe neuromuscular and cardiac disorders, RA venture partner Ramin Farzaneh-Far told Endpoints News in an email. The financing reflects our confidence, and that of our syndicate partners, in the technology.

Oxford Sciences Innovation, PepGens seed investor, also participated in the round, as well as the University of Oxford and CureDuchenne Ventures. Wednesdays cash will also allow PepGen to build out a corporate team in the new Boston headquarters and expand the R&D hub in the UK, Farzaneh-Far said.

The move from RA comes shortly after Shah told Endpoints News in October that the cash for its Nexus I life sciences fund, roughly $300 million, was churned through at a relatively rapid pace. In just 15 months of investment, RA had spent about 80% of their fund, which prompted the Nexus II raise.

Though the new fund built off largely the first, the cash pools remain separate. Farzaneh-Far declined to comment to which Nexus fund Wednesdays investment belonged.

PepGen itself was spun out of Oxford in 2018 in order to further develop the peptides at the heart of its research. The biotech says that the cell-penetrating nature of the peptides, when conjugated with phosphorodiamidate morpholino oligomers or PPMOs, could allow for enhanced delivery of oligonucleotides to key tissues, while also improving safety compared to other medicines.

Specifically, PepGen is hoping to leapfrog the exon-skipping approaches already available in order to restore dystrophin expression in DMD patients, CEO and co-founder Caroline Godfrey said in a statement.

One of the areas where PepGen says its programs are beneficial is in the cardiovascular comorbidities that often accompany DMD. Because the peptides can penetrate cells, the company says its drug candidates strongly distribute to cardiac tissue.

With the recent approvals of treatments that generate small increases in dystrophin in skeletal muscle, patients may be ambulating and living longer, but this in turn is expected to shift the burden of morbidity and mortality towards an epidemic of heart disease, which is not adequately addressed by current DMD therapies, Farzaneh-Far said in an earlier statement.

This past summer, the FDA green-lit the third DMD drug when Japanese developer NS Pharma gained an accelerated approval for viltolarsen. That followed a wild back-and-forth between regulators and Sarepta, who originally rejected their DMD candidate in August 2019 but reversed course later that year.

The agency, however, still doesnt have full efficacy data on any of the three approved DMD drugs, as the OKs were all based on the same disease biomarker.

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RA Capital backs Oxford spinout PepGen with a $45M Series A, seeking to treat Duchenne and other similar diseases - Endpoints News

Cardiac Stem Cells Comments Off on RA Capital backs Oxford spinout PepGen with a $45M Series A, seeking to treat Duchenne and other similar diseases – Endpoints News | December 9th, 2020

CAMBRIDGE, Mass.--(BUSINESS WIRE)--bluebird bio, Inc. (Nasdaq: BLUE) announced that new data from Group C of its ongoing Phase 1/2 HGB-206 study of investigational LentiGlobin gene therapy (bb1111) for adult and adolescent patients with sickle cell disease (SCD) show a complete elimination of severe VOEs and VOEs between six and 24 months of follow-up. These data are being presented at the 62nd American Society of Hematology (ASH) Annual Meeting and Exposition, taking place virtually from December 5-8, 2020.

Now with more than two years of data, we continue to observe promising results in our studies of LentiGlobin for SCD that further illustrate its potential to eliminate the symptoms and devastating complications of sickle cell disease. Consistently achieving the complete resolution of severe vaso-occlusive events (VOEs) and VOEs between Month 6 and Month 24 follow-up is unprecedented other than with allogeneic stem cell transplantation. Importantly, our data show the potential for LentiGlobin for SCD to produce fundamentally disease-modifying effects with sustained pancellular distribution of gene therapy-derived anti-sickling HbAT87Q and improvement of key markers of hemolysis that approach normal levels, said David Davidson, M.D., chief medical officer, bluebird bio. In addition to these clinical outcomes, for the first time with a gene therapy we now have patient-reported outcomes through the validated PROMIS-57 tool, showing reduction in pain intensity at 12 months after treatment with LentiGlobin for SCD. These results provide insight into the potential real-life impact LentiGlobin for SCD may offer patients.

SCD is a serious, progressive and debilitating genetic disease. In the U.S., the median age of death for someone with sickle cell disease is 43 46 years. SCD is caused by a mutation in the -globin gene that leads to the production of abnormal sickle hemoglobin (HbS). HbS causes red blood cells to become sickled and fragile, resulting in chronic hemolytic anemia, vasculopathy and unpredictable, painful VOEs.

In the HGB-206 study of LentiGlobin for SCD, VOEs are defined as episodes of acute pain with no medically determined cause other than a vaso-occlusion, lasting more than two hours and severe enough to require care at a medical facility. This includes acute episodes of pain, acute chest syndrome (ACS), acute hepatic sequestration and acute splenic sequestration. A severe VOE requires a 24-hour hospital stay or emergency room visit or at least two visits to a hospital or emergency room over a 72-hour period, with both visits requiring intravenous treatment.

LentiGlobin for SCD was designed to add functional copies of a modified form of the -globin gene (A-T87Q-globin gene) into a patients own hematopoietic (blood) stem cells (HSCs). Once patients have the A-T87Q-globin gene, their red blood cells can produce anti-sickling hemoglobin (HbAT87Q) that decreases the proportion of HbS, with the goal of reducing sickled red blood cells, hemolysis and other complications.

As a hematologist, I regularly see the debilitating effects of pain events caused by sickle cell disease. Pain has an overwhelmingly negative impact on many facets of my patients lives and can lead to prolonged hospitalizations, said presenting study author Alexis A. Thompson, M.D., professor of pediatrics at Northwestern University Feinberg School of Medicine and head of hematology at Ann and Robert H. Lurie Childrens Hospital of Chicago. The results observed with LentiGlobin gene therapy for SCD include the complete elimination of severe vaso-occlusive pain episodes, which is certainly clinically meaningful, but also for the first time, we have documented patients reporting that they are experiencing improved quality of life. This degree of early clinical benefit is extraordinarily rewarding to observe as a provider."

As of the data cut-off date of August 20, 2020, a total of 44 patients have been treated with LentiGlobin for SCD in the HGB-205 (n=3) and HGB-206 (n=41) clinical studies. The HGB-206 total includes: Groups A (n=7), B (n=2) and C (n=32).

HGB-206: Group C Updated Efficacy Results

The 32 patients treated with LentiGlobin for SCD gene therapy in Group C of HGB-206 had up to 30.9 months of follow-up (median of 13.0; min-max: 1.1 30.9 months).

In patients with six or more months of follow-up whose hemoglobin fractions were available (n=22), median levels of gene therapy-derived anti-sickling hemoglobin, HbAT87Q, were maintained with HbAT87Q contributing at least 40% of total hemoglobin at Month 6. At last visit reported, total hemoglobin ranged from 9.6 15.1 g/dL and HbAT87Q levels ranged from 2.7 8.9 g/dL. At Month 6, the production of HbAT87Q was associated with a reduction in the proportion of HbS in total hemoglobin; median HbS was 50% and remained less than 60% at all follow-up timepoints. All patients in Group C were able to stop regular blood transfusions by three months post-treatment and remain off transfusions as of the data cut-off.

Nineteen patients treated in Group C had a history of severe VOEs, defined as at least four severe VOEs in the 24 months prior to informed consent (annualized rate of severe VOE min-max: 2.0 10.5 events) and at least six months follow-up after treatment with LentiGlobin for SCD. There have been no reports of severe VOEs in these Group C patients following treatment with LentiGlobin for SCD. In addition, all 19 patients had a complete resolution of VOEs after Month 6.

Hemolysis Markers

In SCD, red blood cells become sickled and fragile, rupturing more easily than healthy red blood cells. The breakdown of red blood cells, called hemolysis, occurs normally in the body. However, in sickle cell disease, hemolysis happens too quickly due to the fragility of the red blood cells, which results in hemolytic anemia.

Patients treated with LentiGlobin for SCD in Group C demonstrated near-normal levels in key markers of hemolysis, which are indicators of the health of red blood cells. Lab results assessing these indicators were available for the majority of the 25 patients with 6 months of follow-up.

The medians for reticulocyte counts (n=23), lactate dehydrogenase (LDH) levels (n=21) and total bilirubin (n=24) continued to improve compared to screening values and stabilized by Month 6. In patients with Month 24 data (n=7), these values approached the upper limit of normal by Month 24. These results continue to suggest that treatment with LentiGlobin for SCD may improve biological markers to near-normal levels for SCD.

Pancellularity

As previously reported, assays were developed by bluebird bio to enable the detection of HbAT87Q and HbS protein in individual red blood cells, as well as to assess if HbAT87Q was pancellular, or present throughout all of a patients red blood cells. In 25 patients with at least six months of follow-up, on average, more than 80% of red blood cells contained HbAT87Q, suggesting near-complete pancellularity of HbAT87Q distribution and with pancellularity further increasing over time.

HGB-206: Improvements in Health-Related Quality of Life

Health-related quality of life (HRQoL) findings in Group C patients treated with LentiGlobin for SCD in the HGB-206 study were generated using the Patient Reported Outcomes Measurement Information System 57 (PROMIS-57), a validated instrument in SCD.

Data assessing pain intensity experienced by nine Group C patients were analyzed according to baseline pain intensity scores relative to the general population normative value: 2.6 on a scale of 0-10, where 10 equals the most intense pain. Data were assessed at baseline, Month 6 and Month 12.

Of the five patients with baseline scores worse than the population normative value average, four demonstrated clinically meaningful reductions in pain intensity at Month 12; the group had a mean score of 6.0 at baseline and a mean score of 2.4 at Month 12. Of the four patients with better than or near population normative values at baseline, two reported improvement and two remained stable with a mean score of 2.3 at baseline and 0.8 at Month 12.

HGB-206: Group C Safety Results

As of August 20, 2020, the safety data from Group C patients in HGB-206 remain generally consistent with the known side effects of hematopoietic stem cell collection and myeloablative single-agent busulfan conditioning, as well as underlying SCD. One non-serious, Grade 2 adverse event (AE) of febrile neutropenia was considered related to LentiGlobin for SCD. There were no serious AEs related to LentiGlobin for SCD.

One patient with significant baseline SCD-related and cardiopulmonary disease died 20 months post-treatment; the treating physician and an independent monitoring committee agreed his death was unlikely related to LentiGlobin for SCD and that SCD-related cardiac and pulmonary disease contributed.

LentiGlobin for SCD Data at ASH

The presentation of HGB-206 Group C results and patient reported outcomes research are now available on demand on the ASH conference website:

About HGB-206

HGB-206 is an ongoing, Phase 1/2 open-label study designed to evaluate the efficacy and safety of LentiGlobin gene therapy for sickle cell disease (SCD) that includes three treatment cohorts: Groups A (n=7), B (n=2) and C (n=32). A refined manufacturing process designed to increase vector copy number (VCN) and further protocol refinements made to improve engraftment potential of gene-modified stem cells were used for Group C. Group C patients also received LentiGlobin for SCD made from HSCs collected from peripheral blood after mobilization with plerixafor, rather than via bone marrow harvest, which was used in Groups A and B of HGB-206.

About LentiGlobin for SCD (bb1111)

LentiGlobin gene therapy for sickle cell disease (bb1111) is an investigational treatment being studied as a potential treatment for SCD. bluebird bios clinical development program for LentiGlobin for SCD includes the completed Phase 1/2 HGB-205 study, the ongoing Phase 1/2 HGB-206 study, and the ongoing Phase 3 HGB-210 study.

The U.S. Food and Drug Administration granted orphan drug designation, fast track designation, regenerative medicine advanced therapy (RMAT) designation and rare pediatric disease designation for LentiGlobin for SCD.

LentiGlobin for SCD received orphan medicinal product designation from the European Commission for the treatment of SCD, and Priority Medicines (PRIME) eligibility by the European Medicines Agency (EMA) in September 2020.

bluebird bio is conducting a long-term safety and efficacy follow-up study (LTF-307) for people who have participated in bluebird bio-sponsored clinical studies of LentiGlobin for SCD. For more information visit: https://www.bluebirdbio.com/our-science/clinical-trials or clinicaltrials.gov and use identifier NCT04628585 for LTF-307.

LentiGlobin for SCD is investigational and has not been approved in any geography.

About bluebird bio, Inc.

bluebird bio is pioneering gene therapy with purpose. From our Cambridge, Mass., headquarters, were developing gene and cell therapies for severe genetic diseases and cancer, with the goal that people facing potentially fatal conditions with limited treatment options can live their lives fully. Beyond our labs, were working to positively disrupt the healthcare system to create access, transparency and education so that gene therapy can become available to all those who can benefit.

bluebird bio is a human company powered by human stories. Were putting our care and expertise to work across a spectrum of disorders: cerebral adrenoleukodystrophy, sickle cell disease, -thalassemia and multiple myeloma, using gene and cell therapy technologies including gene addition, and (megaTAL-enabled) gene editing.

bluebird bio has additional nests in Seattle, Wash.; Durham, N.C.; and Zug, Switzerland. For more information, visit bluebirdbio.com.

Follow bluebird bio on social media: @bluebirdbio, LinkedIn, Instagram and YouTube.

LentiGlobin and bluebird bio are trademarks of bluebird bio, Inc.

Forward-Looking Statements

This release contains forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995. Any forward-looking statements are based on managements current expectations of future events and are subject to a number of risks and uncertainties that could cause actual results to differ materially and adversely from those set forth in or implied by such forward-looking statements. These risks and uncertainties include, but are not limited to: regarding the potential for LentiGlobin for Sickle Cell Disease to treat SCD; the risk that the efficacy and safety results from our prior and ongoing clinical trials will not continue or be repeated in our ongoing or planned clinical trials; the risk that the current or planned clinical trials of our product candidates will be insufficient to support regulatory submissions or marketing approval in the United States and European Union; the risk that regulatory authorities will require additional information regarding our product candidates, resulting in delay to our anticipated timelines for regulatory submissions, including our applications for marketing approval; and the risk that any one or more of our product candidates, will not be successfully developed, approved or commercialized. For a discussion of other risks and uncertainties, and other important factors, any of which could cause our actual results to differ from those contained in the forward-looking statements, see the section entitled Risk Factors in our most recent Form 10-Q, as well as discussions of potential risks, uncertainties, and other important factors in our subsequent filings with the Securities and Exchange Commission. All information in this press release is as of the date of the release, and bluebird bio undertakes no duty to update this information unless required by law.

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Treatment with Investigational LentiGlobin Gene Therapy for Sickle Cell Disease (bb1111) Results in Complete Elimination of SCD-Related Severe...

Cardiac Stem Cells Comments Off on Treatment with Investigational LentiGlobin Gene Therapy for Sickle Cell Disease (bb1111) Results in Complete Elimination of SCD-Related Severe… | December 8th, 2020

It sounds too good to be true - and it is. But Jose Bianco Moreira and the CERG research group at the Norwegian University of Science and Technology (NTNU) are convinced that some of the positive health effects of physical exercise can be achieved using gene therapy and medication.

"We're not talking about healthy people and everyone who can exercise. They still have to train, of course," says Moreira. He and his colleagues at NTNU's Department of Circulation and Medical Imaging are studying the effect of exercise on our cells.

"But some people can't train, or only in a limited way. This could include individuals who've been in accidents, who are in wheelchairs, or who have diseases that prevent the possibility of physical expression. We want to create hope for these folks."

"A small group of healthy people out there also obtain very little effect from physical exercise - so-called low responders - and would benefit from a method that worked at the cellular level," says Moreira.

A lot of research confirms the health benefits of physical exercise, but we know far less about what happens in the cells that provides the positive effects.

"International research in this field is brand new. We've barely scratched the surface," says the researcher.

"We think increasing our knowledge about what happens at the cellular level will be important for discovering medications and treatments for heart disease. My group studies genes, proteins and mitochondria that produce energy and are key for chemical processes in the cells."

Moreira believes that gene therapy is the most effective method for reproducing the health benefits we normally get through physical exercise.

A medicine that uses gene therapy is already in use for spinal muscle atrophy, a serious disease that leads to muscle wasting. The drug uses a harmless virus to deliver a copy that replaces the damaged motor neuron network in patients.

This form of therapy can inhibit or enhance the expression of a gene. This is a very expensive medicine and has not been tried for heart disease, for example.

Moreira believes CRISPR will be the future go-to gene therapy method. He believes this method of editing the genes will revolutionize a lot of disease treatments.

"CRISPR is easier to use, faster and cheaper than today's gene therapy, which only attenuates or enhances the expression of a gene. CRISPR's potential is almost limitless. It can alter the gene itself. The parts of the gene that don't work properly are replaced with well-functioning parts."

Experiments on rats and mice have shown that the method works. Experiments have also been performed on human cells in the laboratory to confirm CRISPR's effectiveness, but it has not yet been tested on humans.

"CRISPR still has to be tested in large clinical studies. I'd be optimistic if I say gene editing will come into regular use in 10-15 years," says Moreira.

Moreira's research group has used CRISPR in its research, but the results are not yet ready for publication.

"We believe gene therapy is the most powerful method because patients don't have to take a pill every day. Usually, gene therapy changes the gene forever, perhaps with an injection or two. The challenge is to find the right gene that needs change, and an effective method to repair it," he says.

NTNU researchers are focusing on the heart. They have identified a protein that heart-diseased rats are deficit in, but which increases when the rats go through training.

"By increasing the amount of this protein through gene therapy, we've managed to strengthen the muscle cells and have replicated some of the positive effects of physical exercise," says Moreira.

Medications are another possible method of mimicking the effects of exercise. Some existing medicines might even be able to recreate some of the positive effect on the heart.

"The research now has powerful technology platforms to find possible other uses for medicines we already have. One problem, of course, is that medicine is chemistry that affects the whole body, not just the organ you want to help. Something that's good for the heart could be detrimental for the liver, for example. Compared to gene therapy, though, the potential for medications is much more limited," Moreira says.

When the research group at NTNU started their study, they had no idea which genes were affected by exercise. They performed experiments where rats with heart defects underwent training. Afterwards, the hearts were removed and examined. Then these hearts were compared with those from untrained rats with heart disease. Afterwards, the hearts of the trained and untrained rats with heart disease were compared to healthy rat hearts.

"We observed that genes were altered in the diseased hearts, but discovered that some of them were repaired in the rats that had trained. This way, we find genes that we can target. Through our measurements, we can find out exactly what training changes at the cellular level," says Moreira.

Reference: Moreira, J.B.N., Wohlwend, M. & Wislff, U. Exercise and cardiac health: physiological and molecular insights. Nat Metab. 2020;2,829839. doi:10.1038/s42255-020-0262-1

This article has been republished from the following materials. Note: material may have been edited for length and content. For further information, please contact the cited source.

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Could Gene Therapy Be Used To Mimic the Positive Effects of Exercise? - Technology Networks

Cardiac Stem Cells Comments Off on Could Gene Therapy Be Used To Mimic the Positive Effects of Exercise? – Technology Networks | December 8th, 2020

Editor's note: SpaceX has successfully launched the Dragon CRS-21 cargo mission for NASA and landed its Falcon 9 rocket. Read our launch wrap story here.

CAPE CANAVERAL, Fla. The next SpaceX resupply launch to the International Space Station, scheduled for Sunday (Dec. 6), will carry a host of science gear to the astronauts living and working on the orbiting laboratory.

The robotic flight, called CRS-21, marks the 21st mission for SpaceX under its commercial cargo resupply services contract with NASA. Launch is scheduled for 11:17 a.m. EST (1617 GMT) on Sunday from NASA's Kennedy Space Center in Florida, and you can watch the action live here at Space.com, courtesy of NASA. You can also watch directly via NASA TV or SpaceX.

SpaceX initially aimed to launch the CRS-21 cargo mission for NASA on Saturday (Dec. 5), but foul weather prompted a delay. "Due to poor weather in the recovery area for todays attempt, now targeting Sunday, December 6 at 11:17 a.m. EST for launch of CRS-21," SpaceX wrote in an update early Saturday morning. SpaceX plans to recover the mission's Falcon 9 booster for later reuse.

The upgraded Dragon cargo capsule that will launch atop a veteran SpaceX Falcon 9 rocket is filled with 6,400 lbs. (2,903 kilograms) of supplies and science investigations. The research gear will support a variety of experiments in the life sciences, regenerative medicine and many other fields.

Related: How SpaceX's Dragon space capsule works (infographic)

Saturday's flight will mark the first time SpaceXs upgraded Dragon spacecraft will carry cargo. (Up until now, the advanced Dragon variant has solely carried astronauts.) The vehicle is a modified version of the Crew Dragon spacecraft that lacks the systems necessary for human missions, such as seats, cockpit controls and a life-support system, as well as the SuperDraco thrusters that provide a special emergency escape system that's only used if a problem occurs during launch.

This new Dragon allows more science to ride skyward. Costello explained that the interior of Dragon can now support more powered payloads, which is a huge benefit for the life sciences as it allows for more cold storage and other types of investigations. It also allows for the crew to store some of the powered payloads onboard Dragon while the craft is on orbit.

Several of the payloads on Dragon feature a unique piece of hardware called a tissue chip. Human cells and tissue grow on the chip scaffold, creating a 3D structure in microgravity that researchers can observe to learn more about how fundamental processes work in space, including aging and bone and muscle loss.

One such investigation, run by the University of Florida, will study how muscles atrophy in space. Sixteen samples of skeletal muscle will be sent to the space station, where the bundles of muscle tissue will be observed in microgravity. Half of the muscle samples were donated by younger, active individuals while the other half are from older, more sedentary volunteers.

Half of the samples in each group will be subjected to electric stimuli to see how the muscles contract in the absence of gravity. Researchers will use this experiment as a starting point for future research that will eventually test therapies to see if muscle degradation can be prevented.

Another payload will look at brain organoids created using stem cell technology. This investigation seeks to understand how microgravity affects the survival and function of brain cells, which could lead to advances in treatments for autism and Alzheimers disease, researchers said.

"Space travel mimics the effects of aging we see on Earth, only in a much shorter time span, making it easier to examine the processes that are taking place," Bill McLamb, chief scientist at Kentucky-based company Space Tango, told Space.com. "Its hard to study human brains in space, which is why these types of experiments are so beneficial."

The investigation will take stem cells and convert them into brain cells that will form three-dimensional structures called brain organoids. Stored in a special container called a well, these types of mini organs are able to mimic both the cellular variety and the function of the developing human brain.

This type of research could help NASA and its partners prepare for crewed missions to distant destinations such as Mars, which will expose astronauts to the rigors of space for long stretches, and also help combat degenerative brain disease here on Earth, researchers said.

A team of researchers from Stanford University will be looking at how engineered heart tissue behaves in microgravity. The Cardinal Heart investigation will send tissue samples that consist of cardiomyocytes, endothelial cells and cardiac fibroblasts to study how changes in gravity affect the heart at the cellular level.

Researchers know that microgravity causes changes in the workload and shape of the human heart, but it's still unknown if these changes could become permanent if a person lived for long periods of time in space.

The project's tissue bundles will be affixed to tissue chips. The experiment's results could help identify new treatments and support development of screening measures to predict cardiovascular risk prior to spaceflight, team members said. Follow-on investigations will include therapies that could treat heart disease.

The HemoCue investigation will look at how white blood cells react in space. Here on Earth, doctors use the total number of white blood cells, as well as the various types observed, to diagnose illness. HemoCue will debut a new type of technology that will allow users to do white blood cell counts on orbit.

The goal is to test how well the device works in microgravity. If effective, it could be a valuable tool in an astronauts medical kit, researchers said.

Another payload called Micro-14 looks at how yeast, in particular Candida albicans, responds to the space environment. C. albicans is an opportunistic pathogen, capable of causing severe and even life-threatening illness in immunocompromised hosts. Micro-14 will evaluate how the yeast responds to microgravity, looking for changes at the cellular and molecular levels.

Since astronauts can become immunocompromised during spaceflight, researchers are especially interested in how best to predict the health risks from this organism. Previous research has shown that many microbes exhibit increased virulence in a microgravity environment, but more research is needed on this particular pathogen.

NASAs Jet Propulsion Laboratory in Southern California is spearheading a project that will take swab samples from various locations within the station to look at the relationship between bacteria and their metabolites (chemicals produced by bacterial growth). The project will help researchers better understand the distribution of microbes and metabolites within closed environments and how this distribution affects human health. The research could aid administrators of hospitals and nursing homes, where residents are often immunocompromised.

Related: SpaceX rocket launches for record 7th time, nails landing at sea

Sunday's launch marks the 101st flight overall for SpaceXs workhorse two-stage Falcon 9 rocket. The liftoff is expected to feature a veteran Falcon 9 first stage, designated B1058, that already has three flights under its belt. This frequent flyer previously launched SpaceX's Demo-2 mission, which sent two NASA astronauts to the space station this past summer, well as a communications satellite for the South Korean military and a batch of the companys own Starlink satellites.

Flying previously flown boosters has become commonplace for SpaceX, as the company continues to prove the Falcon 9's reliability. In fact, CRS-21 marks the 24th flight of 2020 for SpaceX, with the majority of those missions having flown on veteran rockets rather than brand-new ones.

To date, SpaceX has successfully landed its first-stage boosters 67 times. Now that the company has two fully operational drone-ship landing platforms "Of Course I Still Love You" and "Just Read the Instructions" in Florida, its able to launch (and land) more rockets. "Of Course I Still Love You" is already at the recovery zone waiting for its turn to catch B1058 when it returns to Earth shortly after liftoff.

Weather was a concern for SpaceX going into the weekend. Forecasts predicted iffy weather for a Saturday launch attempt, with the 45th Weather Squadron predicting a 50% chance of favorable conditions for liftoff. The primary concerns were thick clouds and cumulus clouds. The backup attempt on Sunday looks much better, with the forecast improving to 70% favorable on that day.

If all goes as planned, the Dragon will arrive at the station and dock at the Harmony modules space-facing port just over 24 hours after it blasts off.

Editor's note: This story was updated at 8:22 a.m. EST to include SpaceX's launch delay to Sunday, Dec. 6, due to bad weather.

Follow Amy Thompson on Twitter @astrogingersnap. Follow us on Twitter @Spacedotcom or Facebook.

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Tissue chips and organoids: SpaceX is launching lots of science to space for NASA on Sunday - Space.com

Cardiac Stem Cells Comments Off on Tissue chips and organoids: SpaceX is launching lots of science to space for NASA on Sunday – Space.com | December 6th, 2020

The Autologous Stem Cell Based Therapies Market was valued at US$ XX million in 2019 and is projected to reach US$ XX million by 2025, at a CAGR of XX percentage during the forecast period. In this study, 2019 has been considered as the base and 2020 to 2025 as the forecast period to estimate the market size for Autologous Stem Cell Based Therapies Market

Deep analysis about market status (2016-2019), competition pattern, advantages and disadvantages of products, industry development trends (2019-2025), regional industrial layout characteristics and macroeconomic policies, industrial policy has also been included. From raw materials to downstream buyers of this industry have been analysed scientifically. This report will help you to establish comprehensive overview of the Autologous Stem Cell Based Therapies Market

Get a Sample Copy of the Report at: https://i2iresearch.com/report/global-autologous-stem-cell-based-therapies-market-2020-market-size-share-growth-trends-forecast-2025/

The Autologous Stem Cell Based Therapies Market is analysed based on product types, major applications and key players

Key product type:Embryonic Stem CellResident Cardiac Stem CellsUmbilical Cord Blood Stem Cells

Key applications:Neurodegenerative DisordersAutoimmune DiseasesCardiovascular Diseases

Key players or companies covered are:RegeneusMesoblastPluristem Therapeutics IncU.S. STEM CELL, INC.Brainstorm Cell TherapeuticsTigenixMed cell Europe

The report provides analysis & data at a regional level (North America, Europe, Asia Pacific, Middle East & Africa , Rest of the world) & Country level (13 key countries The U.S, Canada, Germany, France, UK, Italy, China, Japan, India, Middle East, Africa, South America)

Inquire or share your questions, if any: https://i2iresearch.com/report/global-autologous-stem-cell-based-therapies-market-2020-market-size-share-growth-trends-forecast-2025/

Key questions answered in the report:1. What is the current size of the Autologous Stem Cell Based Therapies Market, at a global, regional & country level?2. How is the market segmented, who are the key end user segments?3. What are the key drivers, challenges & trends that is likely to impact businesses in the Autologous Stem Cell Based Therapies Market?4. What is the likely market forecast & how will be Autologous Stem Cell Based Therapies Market impacted?5. What is the competitive landscape, who are the key players?6. What are some of the recent M&A, PE / VC deals that have happened in the Autologous Stem Cell Based Therapies Market?

The report also analysis the impact of COVID 19 based on a scenario-based modelling. This provides a clear view of how has COVID impacted the growth cycle & when is the likely recovery of the industry is expected to pre-covid levels.

Contact us:i2iResearch info to intelligenceLocational Office: *India, *United State, *GermanyEmail: [emailprotected]Toll-free: +1-800-419-8865 | Phone: +91 98801 53667

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Autologous Stem Cell Based Therapies Market Research Report 2020: Market Competition Trend and Price by Manufacturers till 2026 - Factory Maintenance

Cardiac Stem Cells Comments Off on Autologous Stem Cell Based Therapies Market Research Report 2020: Market Competition Trend and Price by Manufacturers till 2026 – Factory Maintenance | December 6th, 2020

ESA astronaut Tim Peake during his 4 hour 43 minute spacewalk to replace a failed power regulator and install cabling on the ISS. Credit: ESA/NASA

Tim Peake is the first official British astronaut to walk in space. The former Army Air Corps officer has spent six months in space, after blasting off on a Russian Soyuz rocket to the International Space Station on December 15, 2016, but the spacewalk doubtless was his most gruelling test.

But what exactly did he go through, during his remarkable spell aboard the space station? Space travel leads to many changes in the human body, many of which have been investigated since Yuri Gargarin made the first manned spaceflight in 1961 and an extensive team provides guidance and preparation for astronauts before, during and after any spaceflight. But if youre planning an out-of-this-world trip, here are some of the things to expect.

The skeletal muscle system is the largest organ system of the human body. Hundreds of muscles are used for maintaining posture sitting, standing and performing a wide range of movements, with different loading conditions imposed by the forces of gravity on Earth.

Skeletal muscles have the ability to adapt to different purposes and the different loads placed on them, a quality known as plasticity. But like inactivity, space flight leads to loss of both skeletal muscle mass (atrophy) and strength.

During long spaceflights on the ISS, research found that 37 crew members experienced a decrease in mean isokinetic strength of between 8% and 17%. Men and women were similarly affected. In fact, this degradation occurs even when astronauts follow a strict exercise regime, meaning that it has profound implications for humans embarking on even longer journeys, such as to Mars. Data suggests that around 30% of muscle strength is lost after spending 110 to 237 days in microgravity.

Many parts of the cardiovascular system (including the heart) are influenced by gravity. On Earth, for example, the veins in our legs work against gravity to get blood back to the heart. Without gravity, however, the heart and blood vessels change and the longer the flight, the more severe the changes.

The size and shape of the heart, for example, changes with microgravity and the right and left ventricles decrease in mass. This may be because of a decrease in fluid volume (blood) and changes in myocardial mass. A human heart rate (number of beats per minute) is lower in space than on Earth, too. In fact, it has been found that the heart rate of individuals standing upright on the ISS is similar to their rate while lying down pre-flight on Earth. Blood pressure is also lower in space than on Earth.

The cardiac output of the heart the amount of blood pumped out of the heart each minute decreases in space, too. Without gravity, there is also a redistribution of the blood more blood stays in the legs and less blood is returned to the heart, which leads to less blood being pumped out of the heart. Muscle atrophy also contributes to reduced blood flow to the lower limbs.

This reduced blood flow to the muscles, combined with the loss of muscle mass, impacts aerobic capacity (below).

Aerobic capacity is a measure of aerobic fitness the maximum amount of oxygen that the body can use during exercise. This can be measured by VO2max and VO2peak tests. Changes to both the muscles and cardiovascular system caused by spaceflight contribute to reduced aerobic fitness.

After nine to 14 days of spaceflight, for example, research shows that aerobic capacity (VO2peak) is reduced by 20%-25%. But the trends are interesting. During longer spells in space say, five to six months after the initial reduction in aerobic capacity, the body appears to compensate and the numbers begin improving although they never return to pre-trip levels.

On Earth, the effects of gravity and mechanical loading are needed to maintain our bones. In space, this doesnt happen. Bone normally undergoes continual remodelling and two types of cells are involved: osteoblasts (these make and regulate the bone matrix) and osteoclasts (these absorb bone matrix). During spaceflight, however, the balance of these two processes is altered which leads to reduced bone mineral density. Research shows that a 3.5% loss of bone occurs after 16 to 28 weeks of spaceflight, 97% of which is in weight-bearing bones, such as the pelvis and legs.

The immune system, which protects the body against disease, is also affected. There are a number of variables which contribute to this, including radiation, microgravity, stress, isolation and alterations in the circadian rhythm, the 24-hour cycle of sleep and wakefulness that we follow on Earth. Also, while in space, astronauts will interact with microbes from themselves, other crew members, their food, their environment and these can alter their immune response, which may lead to challenging situations and increase the potential for infections among the crew as well as contamination of extraterrestrial sites.

This article is republished from The Conversation under a Creative Commons license.

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Five things that happen to your body in space - RocketSTEM

Cardiac Stem Cells Comments Off on Five things that happen to your body in space – RocketSTEM | December 6th, 2020

Hematologists and oncologists working in the community setting encounter multiple obstacles when prescribing chimeric antigen receptor (CAR) T-cell therapy to patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL). The challenges involve matters of processes, treatment cost, and access to treatment.

To further understand the issues and the solutions needed for physicians who treat relapsed/refractory DLBCL, researchers at Cardinal Health conducted 2 live survey sessions to collect information from clinicians. A total of 114 oncologists and hematologists from community practices and hospital settings participated in the survey. The population of hematologists/oncologists see roughly 20 patients per day, and the majority have been in practice for 11 to 20 years. Overall, 46% of the clinicians who attended the first live survey session, and 26% of those who attended the second reported that they had not referrer any patient for CAR T-cell therapy, and of those who did refer patients 32% and 22% of patients, respectively had not yet been infused with CAR T cells.1

The results of the survey revealed that while the use of CAR T-cell therapy increased in community practices over the past year, there remain issues with high cost and toxicity of treatment. It was also reported that the processing of insurance was a barrier to getting patients treated. These challenges continue to limit the number of clinicians who recommend CAR T-cell therapy to their patients.

In an interview withTargeted Oncology, Ajeet Gajra, MD, FACP, vice president, Cardinal Health, discussed the ongoing challenges community oncologists face with prescribing CAR T-cell therapy to patients with relapsed/refractory DLBCL.

TARGETED ONCOLOGY: Can you explain the overall prognosis for patients with DLBCL? What are outcomes generally like with existing standard of care therapy?

Gajra: The outlook for DLBCL improved with the advent of chemoimmunotherapy, better risk stratification, and improved supportive care. Recent studies demonstrate that despite aggressive biology, over 60% of patients with DLBCL treated with chemoimmunotherapy achieve long-term remissions and cures. However, the improvements reached a plateau in the past decade, especially for patients who relapse after initial chemoimmunotherapy. These patients typically have poor prognostic features as defined by the International Prognostic Index (IPI) with high likelihood of relapse and death. Patients with relapsed or refractory disease are typically treated with salvage immunochemotherapy such as rituximab, ifosfamide, carboplatin and etoposide (RICE) or rituximab, cisplatin high dose Ara-C and dexamethasone (RDHAP), and those with chemotherapy-sensitive disease receive autologous stem cell transplant (ASCT). Using this approach, complete response (CR) rates are 35% to 40%, and in a recent study the 3-year event-free survival (EFS) and overall survival (OS) were 31% and 50%, respectively. Outcomes with ASCT are much worse for patients with refractory DLBCL as demonstrated in the SCHOLAR trial wherein the objective response rate was 26% (CR rate, 7%) with a median OS of 6.3 months and only 20% of patients were alive at 2 years.

Thus, prior to 2017 when the first CAR T therapy was approved in DLBCL with progression after 2 prior lines of therapy, there had been a significant unmet need for patients with relapsed DLBCL. The approval of 2 CAR-T therapies, axicabtagene ciloleucel (axi-cel) in October of 2017 and tisagenlecleucel (Kymriah; tisa-cel) in May 2018, in the treatment of large-cell lymphoma (LBCL), has ushered in a new mode of treatment which offers the potential of long-term remission in what was essentially a fatal disease.

TARGETED ONCOLOGY: What has been your observation experience with using CAR T cell therapy in patients with DLBCL by US community oncologists?

Gajra: Axi-cel and tisa-cel are both CD19-directed, genetically modified autologous T cell immunotherapy agents. Since the process of obtaining CAR T therapy for an individual patient is quite complex, we sought to assess the uptake of these agents among United States community oncologists. We conducted a study of community oncologists at two time points to assess perceptions and use of approved CAR T therapies in relapsed DLBCL. At each time point over 50 distinct oncologists participated. At the early timepoint, 46% of participants indicated that they had not referred any patients for CAR T therapy but at the later timepoint, this number decreased to 29% suggesting increasing use over the course of the 10-month interval. Of those participants who had referred patients for CAR T therapy, 32% at the early timepoint reported that none of their patients had yet received the CAR T infusion but the percentage of non-receipt decreased to 22% at the later timepoint again suggesting improved uptake and utilization.

TARGETED ONCLOGY: How do patient characteristics factor into how oncologists select patients to administer CAR T cells to? What are the barriers to CAR-T use?

Gajra: CAR T therapies approved in DLBCL have limitations as defined by the FDA approval and are to be used in adult patients with relapsed or refractory large B-cell lymphoma, including DLBCL, after 2 or more lines of systemic therapy. Neither agent is approved for the use of CNS lymphoma. As with the pivotal trials for the 2 agents, patients must have good ECOG performance status, adequate organ function including marrow, hepatic, cardiac and renal function, no active infection and no CNS involvement. Both agents carry black box warnings for neurotoxicity and cytokine release syndrome (CRS) which can be potentially fatal. Thus, the patients selected need to have good physiologic reserve and be willing to accept risks associated with the therapies. With the approval of a new CD19-directed monoclonal antibody, tafasitamab, it is not clear if patients exposed to that agent can still benefit from CAR T therapies.

In addition to patient specific factors, CAR T therapy represents a complex manufacturing process that is unlike traditional drug therapy or stem cell transplant. After identification of a potential patient with relapsed LBCL who has received at least two prior systemic therapies, a benefits verification and referral to a designated CAR T-cell therapy center is required. If deemed appropriate by the CAR T center, the patient undergoes apheresis for T-cell collection. The cells are then transported to the manufacturers facility where they are isolated, activated and undergo gene transfer, creating the chimeric cells which go through a process of expansion to generate the numbers needed for therapeutic effect. This process takes from 10 days to a few weeks. The CAR T cells are then cryopreserved and transferred back to the CAR T facility and reinfused into the patient. Thus, it is critical to maintain vein to vein integrity. Thus, unlike traditional cytotoxic or monoclonal antibody products, these agents are patient specific, living cell products that have a complex process for their manufacture, storage and shipping, leading to high costs to the healthcare system and the patient.

Given this information, not surprisingly, the oncologists surveyed identified the high cost of therapy as a major barrier to uptake and utilization at both time points respectively. Over half the participants identified cumbersome logistics of administering therapy and following patients as another major barrier. Further exploration of logistical issues identified barriers encountered during the referral process could be attributed to the payer or the CAR T center.

The payer specific challenges identified include slow approval process by 27% of payers (and high rates of denials by in 13% of payers. The challenges specific to the CAR-T center include slow intake process by 23% of CAR T centers lack of a CAR T center in geographic vicinity in 13%. CAR T center choosing stem cell transplant rather than CAR T for the patient was also seen 10% of the time. Other commonly encountered clinical challenges reported by the participants included deterioration of the patient prior to CAR T administration, and the need to administer bridging chemotherapy while awaiting manufacture of CAR T therapy. The lack of communication from the CAR T center during the process was identified by a minority as an impediment to recommending CAR T therapies, including lack of instructions to the primary oncologist and the patient.

TARGETED ONCOLOGY: Can you discuss the toxicities observed with CAR T cell therapy in this patient population? Do you haveany insight into toxicities observed in the real-world setting?

Gajra: As stated, both approved products carry black box warnings for CRS and neurotoxicity, now called Immune Effector Cell Associated Neurologic Syndrome (ICANS). CRS is an acute systemic inflammatory syndrome characterized by fever, hypotension, tachycardia, hypoxia and multiple organ dysfunction. ICANS is a neuropsychiatric complex manifested by encephalopathy, headache, tremor, dizziness, aphasia, delirium, insomnia and anxiety. The treating team needs to maintain a high index of suspicion for these potentially life-threatening agents and patients need to have access to facilities with advanced critical care. Tumor debulking ahead of CAR T infusion and prophylactic use of tocilizumab may reduce the risk of CRS. Use of corticosteroids early can alleviate the severity and duration of ICANS.

The scientific team at Cardinal Health has studied the real-world adverse events (AEs) to CAR T agents in DLBCL.2 We analyzed the postmarketing case reports from the FDA, AEs reporting system involving axicel and tisa-cel for large B-cell lymphomas were analyzed. Of 804 AE cases identified 67% of axi-cel cases and 26% of tisa-cel cases reported neurological AEs. Compared with cases without neurological AEs, significant associations were observed between neurological AEs and use of axi-cel, age 65 years, CRS and the outcome of hospitalization. These findings and those of other investigators suggest that there may be differences in neurological toxicity based on the agent used.

TARGETED ONCOLOGY: Can you provide background on how this web-based survey can about at Cardinal Health Specialty Solutions? What is the overall goal with it?

Gajra: We are continuously engaged in research with healthcare providers, including medical oncologists/hematologists, to assess their perspectives on issues they face in their day-to-day practice, including the impact of new therapies on patient care. We share our research findings with healthcare stakeholders through peer-reviewed manuscripts and abstracts, as well as through our Oncology Insights report, which is published twice a year.

TARGETED ONCOLOGY:How can the information obtained from this survey impact practice? Where are you in the process of response collect and obtaining results?

Gajra: Our research on CAR-T therapy, collected via web-based and in-person surveys, has helped us identify the challenges to the use of these therapies encountered by community oncologists. Given that over 50% of cancer care is rendered in the community setting, it is important to identify these barriers with a goal of mitigating them and facilitating timely access to these potentially life-saving therapies for patients. With a new CAR-T approval in mantle cell lymphoma this year and other potential approvals in newer indications on the horizon, streamlining access to CAR-T therapies will continue to be a priority.

We have a follow-up to this paper that will be presented at ASH 2020 where additional research with community oncologists in early 2020 has revealed that the rate of non-receipt of CAR-T therapies in DLBCL is relatively constant at around 30%. In addition, we are exploring interest and uptake of CAR-T therapies in the outpatient setting as oncologists gain more confidence in preventing, minimizing and managing the toxicity of CAR-T therapies.

References:

1. Gajra A, Jeune-Smith Y, Yeh T, et al. Perceptions of community hematologists/oncologists on barriers to chimeric antigen receptor T-celltherapy for the treatment of diffuse large B-cell lymphoma. Immunotherapy. 202012(10);725-732. doi: 10.2217/imt-2020-0118

2. Gajra A, Zettler ME, Phillips EG Jr, Klink AJ, Jonathan K Kish, Fortier S, Mehta S, Feinberg BA. Neurological adverse events following CAR T-cell therapy: a real-world analysis. Immunotherapy. 2020 Oct;12(14):1077-1082. doi: 10.2217/imt-2020-0161

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Not All Patients With Relapsed DLBCL Referred for CAR T in Community Setting - Targeted Oncology

Cardiac Stem Cells Comments Off on Not All Patients With Relapsed DLBCL Referred for CAR T in Community Setting – Targeted Oncology | December 3rd, 2020

Global 3D Cardiac Mapping Systems Market: Overview

Cardiac mapping is a special type of technique which helps in gathering and displaying the information from cardiac electrograms. Such technique is mainly used in the diagnosis of heart rhythms. Therefore, cardiac mapping technique has gained immense popularity in case of arrhythmia. The cardiac mapping procedure involves the percutaneous insertion of catheter into the heart chamber and recording the cardiac electrograms sequentially. Such procedure helps in correlating the cardiac anatomy with the electrograms. The latest 3D cardiac mapping systems provide the three dimensional model of hearts chamber, which further helps in tracking the exact location of the catheter. Such advantages are majorly driving the global 3D cardiac mapping systems market.

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From the perspective of technology, the global 3D cardiac mapping systems market is segmented into basket catheter mapping, electroanatomical mapping, and real-time positional management (Cardiac pathways) EP system. Among these segments, electroanatomical mapping segment accounts for the maximum share in the global 3D cardiac mapping systems market. This mapping are extensively used in several healthcare industry due to its potential in increasing the safety, accuracy, and efficiency of catheter. A research report by TMR Research (TMR) thoroughly explains the new growth opportunities in the global 3D cardiac mapping systems market. Additionally, the report also provides a comprehensive analysis of the markets competitive landscape.

Global 3D Cardiac Mapping Systems Market: Notable Developments

Some of the recent developments are contouring the shape of the global 3D cardiac mapping systems market in a big way:

Key players operating in the global 3D cardiac mapping systems market include BioScience Webster, Boston Scientific Corporation, and Abbott.

Global 3D Cardiac Mapping Systems Market: Key Growth Drivers

Rising Number of Patients with Cardiac Disorders and Arrhythmia Fillips Market

The global 3D cardiac mapping systems market has grown steadily over the years, owing to the convenience it provides to the patients with heart problem. Growing number of people with cardiovascular diseases and rising cases of arrhythmia are the major factors fueling growth in the global 3D cardiac mapping systems market. Along with this, increasing pressure for reducing diagnosis errors and rapidly rising healthcare expenditure are also responsible for boosting the global 3D cardiac mapping systems market. However, above all such factors, the global 3D cardiac mapping systems market is majorly fueled by the accuracy and patient safety provided through real-time monitoring. Such 3D cardiac mapping systems are mainly designed to improve the resolution. This system also helps in gaining prompt of cardiac activation maps. All such advantages are also providing impetus to the growth of the global 3D cardiac mapping systems market.

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Furthermore, rising ageing population who are prone to heart-attack and several chronic heart disorders and increasing diagnosis rate of cardiac illness are the factors stoking demand in the global 3D cardiac mapping systems market. Moreover, this 3D cardiac mapping helps in reducing the diagnosis time. Such factor is also contributing to the growth of the global 3D cardiac mapping systems market.

Global 3D Cardiac Mapping Systems Market: Regional Outlook

On the regional front, North America is leading the global 3D cardiac mapping systems market as the region has seen rapid growth in healthcare industry. Along with this, increasing prevalence of heart attacks, rising healthcare expenditure, and burgeoning population is also responsible for fueling growth in the 3D cardiac mapping systems market in this region.

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3D Cardiac Mapping Systems Market Key Vendors, Analysis by Growth and Revolutionary Opportunities by 2028 - Murphy's Hockey Law

Cardiac Stem Cells Comments Off on 3D Cardiac Mapping Systems Market Key Vendors, Analysis by Growth and Revolutionary Opportunities by 2028 – Murphy’s Hockey Law | December 3rd, 2020

Hopes for new treatments for osteoporosis and cartilage degeneration have been raised after scientists identified a gene that plays a key role in the ageing of bone, tendon, ligament and cartilage.

The researchers hope that they can use their findings to slow down treat age-related diseases connected to the skeletal system by creating treatments that slow down the ageing process behind them.

The i newsletter latest news and analysis

Our findings are novel and significant in finding a critical answer to how skeletal tissues lose their capability to maintain their properties and functions when we age, said Wan-Ju Li, of the University of Wisconsin-Madison.

We can also develop new pharmacological therapies to treat age-associated diseases based on our findings [although] it will take a few years before we can see the application happens, he said.

The study is published in the journal Stem Cells. The journals editor-in-chief, Jan Nolta, of the University of California at Davis, said the discovery is a very important accomplishment.

Researchers said it is possible that the same mechanism that has been identified for the skeletal system may also be present in neural stem cells and cardic stem cells, where it may play a role in causing diseases associated with those areas of the body.

We dont know if the molecule and mechanism we have identified in the paper also play the same role in other stem cells, such as neural stem cells and cardiac stem cells, in causing Parkinsons disease and heart diseases, respectively, since we havent tested it with these cells, Dr Lin said.

But I am sure that other scientists in the fields of aging and brain and heart will follow our study to answer these questions in the future, Dr Lin said.

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Osteoporosis treatments could be on the way after scientists identify aging gene - iNews

Cardiac Stem Cells Comments Off on Osteoporosis treatments could be on the way after scientists identify aging gene – iNews | December 1st, 2020

Stem Cell Assay Market: Snapshot

Stem cell assay refers to the procedure of measuring the potency of antineoplastic drugs, on the basis of their capability of retarding the growth of human tumor cells. The assay consists of qualitative or quantitative analysis or testing of affected tissues andtumors, wherein their toxicity, impurity, and other aspects are studied.

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With the growing number of successfulstem cell therapytreatment cases, the global market for stem cell assays will gain substantial momentum. A number of research and development projects are lending a hand to the growth of the market. For instance, the University of Washingtons Institute for Stem Cell and Regenerative Medicine (ISCRM) has attempted to manipulate stem cells to heal eye, kidney, and heart injuries. A number of diseases such as Alzheimers, spinal cord injury, Parkinsons, diabetes, stroke, retinal disease, cancer, rheumatoid arthritis, and neurological diseases can be successfully treated via stem cell therapy. Therefore, stem cell assays will exhibit growing demand.

Another key development in the stem cell assay market is the development of innovative stem cell therapies. In April 2017, for instance, the first participant in an innovative clinical trial at the University of Wisconsin School of Medicine and Public Health was successfully treated with stem cell therapy. CardiAMP, the investigational therapy, has been designed to direct a large dose of the patients own bone-marrow cells to the point of cardiac injury, stimulating the natural healing response of the body.

Newer areas of application in medicine are being explored constantly. Consequently, stem cell assays are likely to play a key role in the formulation of treatments of a number of diseases.

Global Stem Cell Assay Market: Overview

The increasing investment in research and development of novel therapeutics owing to the rising incidence of chronic diseases has led to immense growth in the global stem cell assay market. In the next couple of years, the market is expected to spawn into a multi-billion dollar industry as healthcare sector and governments around the world increase their research spending.

The report analyzes the prevalent opportunities for the markets growth and those that companies should capitalize in the near future to strengthen their position in the market. It presents insights into the growth drivers and lists down the major restraints. Additionally, the report gauges the effect of Porters five forces on the overall stem cell assay market.

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Global Stem Cell Assay Market: Key Market Segments

For the purpose of the study, the report segments the global stem cell assay market based on various parameters. For instance, in terms of assay type, the market can be segmented into isolation and purification, viability, cell identification, differentiation, proliferation, apoptosis, and function. By kit, the market can be bifurcated into human embryonic stem cell kits and adult stem cell kits. Based on instruments, flow cytometer, cell imaging systems, automated cell counter, and micro electrode arrays could be the key market segments.

In terms of application, the market can be segmented into drug discovery and development, clinical research, and regenerative medicine and therapy. The growth witnessed across the aforementioned application segments will be influenced by the increasing incidence of chronic ailments which will translate into the rising demand for regenerative medicines. Finally, based on end users, research institutes and industry research constitute the key market segments.

The report includes a detailed assessment of the various factors influencing the markets expansion across its key segments. The ones holding the most lucrative prospects are analyzed, and the factors restraining its trajectory across key segments are also discussed at length.

Global Stem Cell Assay Market: Regional Analysis

Regionally, the market is expected to witness heightened demand in the developed countries across Europe and North America. The increasing incidence of chronic ailments and the subsequently expanding patient population are the chief drivers of the stem cell assay market in North America. Besides this, the market is also expected to witness lucrative opportunities in Asia Pacific and Rest of the World.

Global Stem Cell Assay Market: Vendor Landscape

A major inclusion in the report is the detailed assessment of the markets vendor landscape. For the purpose of the study the report therefore profiles some of the leading players having influence on the overall market dynamics. It also conducts SWOT analysis to study the strengths and weaknesses of the companies profiled and identify threats and opportunities that these enterprises are forecast to witness over the course of the reports forecast period.

Some of the most prominent enterprises operating in the global stem cell assay market are Bio-Rad Laboratories, Inc (U.S.), Thermo Fisher Scientific Inc. (U.S.), GE Healthcare (U.K.), Hemogenix Inc. (U.S.), Promega Corporation (U.S.), Bio-Techne Corporation (U.S.), Merck KGaA (Germany), STEMCELL Technologies Inc. (CA), Cell Biolabs, Inc. (U.S.), and Cellular Dynamics International, Inc. (U.S.).

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Stem Cell Assay Market In-Depth Analysis and Forecast 2017-2025 - Khabar South Asia

Cardiac Stem Cells Comments Off on Stem Cell Assay Market In-Depth Analysis and Forecast 2017-2025 – Khabar South Asia | December 1st, 2020

The researchers noted a weakening of possible repair mechanisms of blood vessels in patients who showed clinically significant vasoplegia

Vasoplegia, where vaso refers to blood vessels and plegia stands for paralysis, is a condition where the patient exhibits a low blood pressure, even in the presence of normal or increased output of blood from the heart. When this occurs as a complication of cardiopulmonary bypass surgery, there is a chance that it can lead to multiple organ failure and even death. Now, a diverse group of researchers including clinicians, computational biologists and biotechnologists have come together to study how this may be predicted early on based on clinical observations, so that effective treatment may be given.

Also Read | Mumbais first robot-assisted cardiac surgery

In a pilot study involving 19 patients who underwent elective cardiac surgery, the researchers measured the circulating counts of endothelial progenitor cells and hematopoietic stem cells at different points in time starting from when the patient was being anaesthetised to until 24 hours after the surgery. They find that in a statistically significant number of people in the group that showed clinically significant vasoplegia, there was a blunting of the endothelial progenitor cell response. Also, in the group that did not show clinically significant vasoplegia, they observed that there was no such blunting.

We can say there appears to be a pattern, which is well worth exploring in a larger cohort of patients and further delineating this particular response as a biomarker in predicting a potentially devastating complication following cardiac surgeries, says Dr. Paul Ramesh Thangaraj, from the department of cardiothoracic surgery, Apollo Hospitals, Chennai, who is one of the PIs of the study. This research is published in the journal PLOS ONE.

Hematopoietic and endothelial progenitor cells play an important role in repair of damaged tissues and inner lining of the blood vessels called the endothelium, respectively. Usually, these cells reside in the bone marrow; however, in response to injury to a tissue or a blood vessel, they come out into the circulation from the bone marrow and home into the site of injury for tissue repair, says Madhulika Dixit from the Department of Biotechnology, Indian Institute of Technology Madras, in an email to The Hindu.

Prof Dixit describes using flow cytometry to measure the counts during the surgery and afterwards. The cells were identified by means of expression of specialised cell surface receptors. For this, at regular intervals the blood withdrawn from the patient was subjected to flow cytometry. We checked for time-dependent changes in circulating counts of progenitor cells during the course of cardiopulmonary bypass in patients.

Also Read | Minimally invasive cardiac surgery the best bet

One of the key challenges was to get significant patterns in this small dataset, according to Rahul Siddharthan, from The Institute of Mathematical Sciences, Chennai, who was one of the people involved in formal analysis. In this case, we have two data sets, with two-valued outcomes [non-vasoplegic or vasoplegic], and the goal is to see how other measured parameters can predict them, he says. There are very sophisticated machine-learning algorithms available these days for such tasks. In this case the most basic algorithm, logistic regression, is good enough, says Prof. Siddharthan.

As he explains, in both cases, the idea is to look at a single value (change in circulating progenitor cells at two timepoints) and in seeing its predictive power for the output. The trend is clear, that for non-vasoplegic patients, the level of circulating progenitor cells increases, while for vasoplegic patients, it stays flat or decreases. There are exceptions but the finding is statistically significant even on this small study, says Prof. Siddharthan.

With a larger study, Dr.. Paul Ramesh envisages even developing a risk score for predicting vasoplegia as a complication following surgery.

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Pilot study finds potential signal indicative of loss of tone in blood vessels after cardiac surgery - The Hindu

Cardiac Stem Cells Comments Off on Pilot study finds potential signal indicative of loss of tone in blood vessels after cardiac surgery – The Hindu | November 30th, 2020

Myocardial Infarction Drug used to treat Heart Attack. Medicines and chemical substances that can cause myocardial infarction. Treatment ranges from lifestyle changes and cardiac rehabilitation to medication, stents, and bypass surgery.

Myocardial Infarction Drug Market is anticipated to grow at a CAGR of +6% during the forecast period 2020-2028.

A Global Myocardial Infarction Drug Market analysis and forecast is released based on a wide study of the market. Statistics about the approaching market trends as well as the current scenario of the market is a vital implement for existence and development in the constantly developing industry.

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Due to the pandemic, we have included a special section on the Impact of COVID 19 on the Myocardial Infarction Drug Market which would mention How the Covid-19 is affecting the Myocardial Infarction Drug Industry, Market Trends and Potential Opportunities in the COVID-19 Landscape, Covid-19 Impact on Key Regions and Proposal for Myocardial Infarction Drug Players to Combat Covid-19 Impact.

The Top Key Players of the global Myocardial Infarction Drug Market:

BioCardia, Inc., Laboratoires Pierre Fabre SA, Human Stem Cells Institute, CSL Limited, Capricor Therapeutics, Inc., Hemostemix Ltd, Compugen Ltd., Celyad SA, FibroGen, Inc., Lees Pharmaceutical Holdings Limited, Juventas Therapeutics, Inc., Cynata Therapeutics Limited, CellProthera, Biscayne Pharmaceuticals, Inc., HUYA Bioscience International, LLC, LegoChem Biosciences, Inc, Immune Pharmaceuticals Inc.

Segmentation by Product type:

Segmentation by Application:

Market Segmentation by Region:

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The Global Myocardial Infarction Drug Market has demonstrated an increasing need to alter the policies that are being currently used by the players so as to exhibit commercial capacities of the manufacturers, distributors, and vendors. This helps the key players in developing a firm strategy that is flexible enough to keep up with future events in the market space.

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Global Myocardial Infarction Drug Market to have sustainable growth over the forecast period 2020-2028| Leading Players BioCardia, Inc., Laboratoires...

Cardiac Stem Cells Comments Off on Global Myocardial Infarction Drug Market to have sustainable growth over the forecast period 2020-2028| Leading Players BioCardia, Inc., Laboratoires… | November 30th, 2020

New York, Nov. 25, 2020 (GLOBE NEWSWIRE) -- Reportlinker.com announces the release of the report "Global Induced Pluripotent Stem Cell (iPSC) Industry" - https://www.reportlinker.com/p05798831/?utm_source=GNW 4 billion by the year 2027, trailing a post COVID-19 CAGR of 6.6%, over the analysis period 2020 through 2027. Stem cells are undifferentiated cells that hold the capability to divide, and differentiate into specialized cells in the body. Stem cells act as repair system and replenish adult tissues, maintaining the turnover of regenerative organs such as the blood and skin. In organs, such as the bone marrow, stem cells frequently form replacement cells to repair the worn out tissue. These cells can respond to signals from the body and transverse a particular developmental pathway to differentiate into one specific cell type. Due to their regenerative properties, stem cells are being researched for therapeutic applications in diabetes, cardiovascular disease, neurodegenerative disease, cancer, autoimmune diseases, spinal cord defects, among others. Stem Cell research is an exciting field where continuous discoveries are being made on new sources of stem cells and new methods of their acquisition and harvesting. Of late, adult stem cells have garnered a lions share of the stem cell space, purely based on the fact that they require less expensive clinical trials, need to comply with fewer regulatory norms and ethical issues compared to other stem cell variants such as embryonic stem cells.

Researchers around the world have been focusing research activities to develop adult stem cell therapies in order to combat a variety of diseases ranging from diabetes to heart disease. Factually, adult stem cells are the only stem cells that have been approved for use in transplants for the treatment of diseases such as cancer. Interestingly, with drug development based on embryonic stem cells being challenged amid growing debate over ethics and regulation of this research, iPSCS offers an alternate step forward in the commercialization of stem cell therapies and regenerative medicine. Embryonic stem cell research continues to remain embroiled in ethical, religious, and political controversies across various countries around the world. Induced Pluripotent Stem Cells (iPSs), which are reprogrammed to mimic embryonic stem cell-like state allowing expression of genes and human cells needed for therapeutic purposes, offers an attractive alternate way forwarding in furthering the goals of stem cell research. Pioneered in 2006 and developed in the following year, these cells are created by conversion of somatic cells into PSCs by introducing certain genes including Myc, Klf4, Oct3/4 and Sox2.

Pluripotent stem cells hold tremendous potential in the regenerative medicine arena. Based on their ability to proliferate indefinitely and develop into desirable cell type such as heart, liver, neuronal and pancreatic cells, iPSCs offer a source of new cells that can replace lost or damaged cells. For instance, iPSCs can be developed into beta islet cells, blood cells or neuronal cells for the treatment of diabetes, leukemia and neurological disorders, respectively. Parkinsons, Alzheimers & spinal cord injuries are key neurologic diseases expected to benefit from iPS research. Dramatic rise in cancer cases worldwide and the need for novel anti-cancer therapies will emerge as a key driver for the growth of iPSCs. Interest in cancer research soars high on new hopes of direct reprogramming of cancer cells with enforced expression of pluripotency factors and the resulting dedifferentiation of transformed cancer cells. The ongoing pandemic is also opening up new opportunities for Human induced pluripotent stem cells (hiPSCs) by offering a reliable model for researchers involved in studying how coronavirus indirectly or directly affects different cells in the human body. Made from a small sample of blood or skin cells, hiPSCs are robust stem cells that can be developed into any cell type and then infected with the coronavirus in order to analyse the disease prognosis and the resulting effects. By deploying hiPSCs, researchers have identified that stem cell-derived cardiomyocytes (heart muscle cells) and blood vessels remain directly exposed to COVID-19 infection. Scientists identified that a significant portion of stem cell-derived cardiomyocytes ceased beating and expired within 3 days after being infected by coronavirus. Researchers can leverage the infected cardiomyocytes to screen for potential drug candidates that can restore their function and improve their survival; and also for identifying new antiviral drugs that potentially curtail coronavirus replication in the heart, reduce cardiac injury and curb the disease prognosis. Researchers can also utilize the infected cardiomyocytes to analyze COVID-induced myocarditis through addition of immune cells to their lab experiments.

Competitors identified in this market include, among others,

Read the full report: https://www.reportlinker.com/p05798831/?utm_source=GNW

I. INTRODUCTION, METHODOLOGY & REPORT SCOPE I-1

II. EXECUTIVE SUMMARY II-1

1. MARKET OVERVIEW II-1 Impact of Covid-19 and a Looming Global Recession II-1 Induced Pluripotent Stem Cells (iPSCs) Market Gains from Increasing Use in Research for COVID-19 II-1 Studies Employing iPSCs in COVID-19 Research II-2 Stem Cells, Application Areas, and the Different Types: A Prelude II-3 Applications of Stem Cells II-4 Types of Stem Cells II-4 Induced Pluripotent Stem Cell (iPSC): An Introduction II-5 Production of iPSCs II-6 First & Second Generation Mouse iPSCs II-6 Human iPSCs II-7 Key Properties of iPSCs II-7 Transcription Factors Involved in Generation of iPSCs II-7 Noteworthy Research & Application Areas for iPSCs II-8 Induced Pluripotent Stem Cell ((iPSC) Market: Growth Prospects and Outlook II-9 Drug Development Application to Witness Considerable Growth II-11 Technical Breakthroughs, Advances & Clinical Trials to Spur Growth of iPSC Market II-11 North America Dominates Global iPSC Market II-12 Competition II-12 Recent Market Activity II-13 Select Innovation/Advancement II-16

2. FOCUS ON SELECT PLAYERS II-17 Axol Bioscience Ltd. (UK) II-17 Cynata Therapeutics Limited (Australia) II-17 Evotec SE (Germany) II-17 Fate Therapeutics, Inc. (USA) II-17 FUJIFILM Cellular Dynamics, Inc. (USA) II-18 Ncardia (Belgium) II-18 Pluricell Biotech (Brazil) II-18 REPROCELL USA, Inc. (USA) II-18 Sumitomo Dainippon Pharma Co., Ltd. (Japan) II-19 Takara Bio, Inc. (Japan) II-19 Thermo Fisher Scientific, Inc. (USA) II-20 ViaCyte, Inc. (USA) II-20

3. MARKET TRENDS & DRIVERS II-21 Effective Research Programs Hold Key in Roll Out of Advanced iPSC Treatments II-21 Induced Pluripotent Stem Cells: A Giant Leap in the Therapeutic Applications II-21 Research Trends in Induced Pluripotent Stem Cell Space II-22 Exhibit 1: Worldwide Publication of hESC and hiPSC Research Papers for the Period 2008-2010, 2011-2013 and 2014-2016 II-22 Exhibit 2: Number of Original Research Papers on hESC and iPSC Published Worldwide (2014-2016) II-23 Concerns Related to Embryonic Stem Cells Shift the Focus onto iPSCs II-23 Regenerative Medicine: A Promising Application of iPSCs II-24 Induced Pluripotent: A Potential Competitor to hESCs? II-25 Exhibit 3: Global Regenerative Medicine Market Size in US$ Billion for 2019, 2021, 2023 and 2025 II-27 Exhibit 4: Global Stem Cell & Regenerative Medicine Market by Product (in %) for the Year 2019 II-27 Exhibit 5: Global Regenerative Medicines Market by Category: Breakdown (in %) for Biomaterials, Stem Cell Therapies and Tissue Engineering for 2019 II-28 Pluripotent Stem Cells Hold Significance for Cardiovascular Regenerative Medicine II-28 Exhibit 6: Leading Causes of Mortality Worldwide: Number of Deaths in Millions & % Share of Deaths by Cause for 2017 II-30 Leading Causes of Mortality for Low-Income and High-Income Countries II-30 Growing Importance of iPSCs in Personalized Drug Discovery II-31 Persistent Advancements in Genetics Space and Subsequent Growth in Precision Medicine Augur Well for iPSCs Market II-33 Exhibit 7: Global Precision Medicine Market (In US$ Billion) for the Years 2018, 2021 & 2024 II-34 Increasing Prevalence of Chronic Disorders Supports Growth of iPSCs Market II-34 Exhibit 8: Worldwide Cancer Incidence: Number of New Cancer Cases Diagnosed for 2012, 2018 & 2040 II-35 Exhibit 9: Number of New Cancer Cases Reported (in Thousands) by Cancer Type: 2018 II-36 Exhibit 10: Fatalities by Heart Conditions: Estimated Percentage Breakdown for Cardiovascular Disease, Ischemic Heart Disease, Stroke, and Others II-37 Exhibit 11: Rising Diabetes Prevalence Presents Opportunity for iPSCs Market: Number of Adults (20-79) with Diabetes (in Millions) by Region for 2017 and 2045 II-38 Aging Demographics Add to the Global Burden of Chronic Diseases, Presenting Opportunities for iPSCs Market II-38 Exhibit 12: Expanding Elderly Population Worldwide: Breakdown of Number of People Aged 65+ Years in Million by Geographic Region for the Years 2019 and 2030 II-39 Growth in Number of Genomics Projects Propels Market Growth II-39 Genomic Initiatives in Select Countries II-40 Exhibit 13: New Gene-Editing Tools Spur Interest and Investments in Genetics, Driving Lucrative Growth Opportunities for iPSCs: Total VC Funding (In US$ Million) in Genetics for the Years 2014, 2015, 2016, 2017 and 2018 II-41 Launch of Numerous iPSCs-Related Clinical Trials Set to Benefit Market Growth II-41 Exhibit 14: Number of Induced Pluripotent Stem Cells based Studies by Select Condition: As on Oct 31, 2020 II-43 iPSCs-based Clinical Trial for Heart Diseases II-43 Induced Pluripotent Stem Cells for Stroke Treatment II-44 ?Off-the-shelf? Stem Cell Treatment for Cancer Enters Clinical Trial II-44 iPSCs for Hematological Disorders II-44 Market Benefits from Growing Funding for iPSCs-Related R&D Initiatives II-44 Exhibit 15: Stem Cell Research Funding in the US (in US$ Million) for the Years 2016 through 2021 II-46 Human iPSC Banks: A Review of Emerging Opportunities and Drawbacks II-46 Human iPSC Banks Worldwide: An Overview II-48 Cell Sources and Reprogramming Methods Used by Select iPSC Banks II-49 Innovations, Research Studies & Advancements in iPSCs II-50 Key iPSC Research Breakthroughs for Regenerative Medicine II-50 Researchers Develop Novel Oncogene-Free and Virus-Free iPSC Production Method II-51 Scientists Study Concerns of Genetic Mutations in iPSCs II-52 iPSCs Hold Tremendous Potential in Transforming Research Efforts II-52 Researchers Highlight Potential Use of iPSCs for Developing Novel Cancer Vaccines II-54 Scientists Use Machine Learning to Improve Reliability of iPSC Self-Organization II-54 STEMCELL Technologies Unveils mTeSR? Plus II-55 Challenges and Risks Related to Pluripotent Stem Cells II-56 A Glance at Issues Related to Reprogramming of Adult Cells to iPSCs II-57 A Note on Legal, Social and Ethical Considerations with iPSCs II-58

4. GLOBAL MARKET PERSPECTIVE II-59 Table 1: World Current & Future Analysis for Induced Pluripotent Stem Cell (iPSC) by Geographic Region - USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World Markets - Independent Analysis of Annual Sales in US$ Thousand for Years 2020 through 2027 II-59

Table 2: World 7-Year Perspective for Induced Pluripotent Stem Cell (iPSC) by Geographic Region - Percentage Breakdown of Value Sales for USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World Markets for Years 2020 & 2027 II-60

Table 3: World Current & Future Analysis for Vascular Cells by Geographic Region - USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World Markets - Independent Analysis of Annual Sales in US$ Thousand for Years 2020 through 2027 II-61

Table 4: World 7-Year Perspective for Vascular Cells by Geographic Region - Percentage Breakdown of Value Sales for USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World for Years 2020 & 2027 II-62

Table 5: World Current & Future Analysis for Cardiac Cells by Geographic Region - USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World Markets - Independent Analysis of Annual Sales in US$ Thousand for Years 2020 through 2027 II-63

Table 6: World 7-Year Perspective for Cardiac Cells by Geographic Region - Percentage Breakdown of Value Sales for USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World for Years 2020 & 2027 II-64

Table 7: World Current & Future Analysis for Neuronal Cells by Geographic Region - USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World Markets - Independent Analysis of Annual Sales in US$ Thousand for Years 2020 through 2027 II-65

Table 8: World 7-Year Perspective for Neuronal Cells by Geographic Region - Percentage Breakdown of Value Sales for USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World for Years 2020 & 2027 II-66

Table 9: World Current & Future Analysis for Liver Cells by Geographic Region - USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World Markets - Independent Analysis of Annual Sales in US$ Thousand for Years 2020 through 2027 II-67

Table 10: World 7-Year Perspective for Liver Cells by Geographic Region - Percentage Breakdown of Value Sales for USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World for Years 2020 & 2027 II-68

Table 11: World Current & Future Analysis for Immune Cells by Geographic Region - USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World Markets - Independent Analysis of Annual Sales in US$ Thousand for Years 2020 through 2027 II-69

Table 12: World 7-Year Perspective for Immune Cells by Geographic Region - Percentage Breakdown of Value Sales for USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World for Years 2020 & 2027 II-70

Table 13: World Current & Future Analysis for Other Cell Types by Geographic Region - USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World Markets - Independent Analysis of Annual Sales in US$ Thousand for Years 2020 through 2027 II-71

Table 14: World 7-Year Perspective for Other Cell Types by Geographic Region - Percentage Breakdown of Value Sales for USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World for Years 2020 & 2027 II-72

Table 15: World Current & Future Analysis for Cellular Reprogramming by Geographic Region - USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World Markets - Independent Analysis of Annual Sales in US$ Thousand for Years 2020 through 2027 II-73

Table 16: World 7-Year Perspective for Cellular Reprogramming by Geographic Region - Percentage Breakdown of Value Sales for USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World for Years 2020 & 2027 II-74

Table 17: World Current & Future Analysis for Cell Culture by Geographic Region - USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World Markets - Independent Analysis of Annual Sales in US$ Thousand for Years 2020 through 2027 II-75

Table 18: World 7-Year Perspective for Cell Culture by Geographic Region - Percentage Breakdown of Value Sales for USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World for Years 2020 & 2027 II-76

Table 19: World Current & Future Analysis for Cell Differentiation by Geographic Region - USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World Markets - Independent Analysis of Annual Sales in US$ Thousand for Years 2020 through 2027 II-77

Table 20: World 7-Year Perspective for Cell Differentiation by Geographic Region - Percentage Breakdown of Value Sales for USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World for Years 2020 & 2027 II-78

Table 21: World Current & Future Analysis for Cell Analysis by Geographic Region - USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World Markets - Independent Analysis of Annual Sales in US$ Thousand for Years 2020 through 2027 II-79

Table 22: World 7-Year Perspective for Cell Analysis by Geographic Region - Percentage Breakdown of Value Sales for USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World for Years 2020 & 2027 II-80

Table 23: World Current & Future Analysis for Cellular Engineering by Geographic Region - USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World Markets - Independent Analysis of Annual Sales in US$ Thousand for Years 2020 through 2027 II-81

Table 24: World 7-Year Perspective for Cellular Engineering by Geographic Region - Percentage Breakdown of Value Sales for USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World for Years 2020 & 2027 II-82

Table 25: World Current & Future Analysis for Other Research Methods by Geographic Region - USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World Markets - Independent Analysis of Annual Sales in US$ Thousand for Years 2020 through 2027 II-83

Table 26: World 7-Year Perspective for Other Research Methods by Geographic Region - Percentage Breakdown of Value Sales for USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World for Years 2020 & 2027 II-84

Table 27: World Current & Future Analysis for Drug Development & Toxicology Testing by Geographic Region - USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World Markets - Independent Analysis of Annual Sales in US$ Thousand for Years 2020 through 2027 II-85

Table 28: World 7-Year Perspective for Drug Development & Toxicology Testing by Geographic Region - Percentage Breakdown of Value Sales for USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World for Years 2020 & 2027 II-86

Table 29: World Current & Future Analysis for Academic Research by Geographic Region - USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World Markets - Independent Analysis of Annual Sales in US$ Thousand for Years 2020 through 2027 II-87

Table 30: World 7-Year Perspective for Academic Research by Geographic Region - Percentage Breakdown of Value Sales for USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World for Years 2020 & 2027 II-88

Table 31: World Current & Future Analysis for Regenerative Medicine by Geographic Region - USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World Markets - Independent Analysis of Annual Sales in US$ Thousand for Years 2020 through 2027 II-89

Table 32: World 7-Year Perspective for Regenerative Medicine by Geographic Region - Percentage Breakdown of Value Sales for USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World for Years 2020 & 2027 II-90

Table 33: World Current & Future Analysis for Other Applications by Geographic Region - USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World Markets - Independent Analysis of Annual Sales in US$ Thousand for Years 2020 through 2027 II-91

Table 34: World 7-Year Perspective for Other Applications by Geographic Region - Percentage Breakdown of Value Sales for USA, Canada, Japan, China, Europe, Asia-Pacific and Rest of World for Years 2020 & 2027 II-92

III. MARKET ANALYSIS III-1

GEOGRAPHIC MARKET ANALYSIS III-1

UNITED STATES III-1 Table 35: USA Current & Future Analysis for Induced Pluripotent Stem Cell (iPSC) by Cell Type - Vascular Cells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cells and Other Cell Types - Independent Analysis of Annual Sales in US$ Thousand for the Years 2020 through 2027 III-1

Table 36: USA 7-Year Perspective for Induced Pluripotent Stem Cell (iPSC) by Cell Type - Percentage Breakdown of Value Sales for Vascular Cells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cells and Other Cell Types for the Years 2020 & 2027 III-2

Table 37: USA Current & Future Analysis for Induced Pluripotent Stem Cell (iPSC) by Research Method - Cellular Reprogramming, Cell Culture, Cell Differentiation, Cell Analysis, Cellular Engineering and Other Research Methods - Independent Analysis of Annual Sales in US$ Thousand for the Years 2020 through 2027 III-3

Table 38: USA 7-Year Perspective for Induced Pluripotent Stem Cell (iPSC) by Research Method - Percentage Breakdown of Value Sales for Cellular Reprogramming, Cell Culture, Cell Differentiation, Cell Analysis, Cellular Engineering and Other Research Methods for the Years 2020 & 2027 III-4

Table 39: USA Current & Future Analysis for Induced Pluripotent Stem Cell (iPSC) by Application - Drug Development & Toxicology Testing, Academic Research, Regenerative Medicine and Other Applications - Independent Analysis of Annual Sales in US$ Thousand for the Years 2020 through 2027 III-5

Table 40: USA 7-Year Perspective for Induced Pluripotent Stem Cell (iPSC) by Application - Percentage Breakdown of Value Sales for Drug Development & Toxicology Testing, Academic Research, Regenerative Medicine and Other Applications for the Years 2020 & 2027 III-6

CANADA III-7 Table 41: Canada Current & Future Analysis for Induced Pluripotent Stem Cell (iPSC) by Cell Type - Vascular Cells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cells and Other Cell Types - Independent Analysis of Annual Sales in US$ Thousand for the Years 2020 through 2027 III-7

Table 42: Canada 7-Year Perspective for Induced Pluripotent Stem Cell (iPSC) by Cell Type - Percentage Breakdown of Value Sales for Vascular Cells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cells and Other Cell Types for the Years 2020 & 2027 III-8

Table 43: Canada Current & Future Analysis for Induced Pluripotent Stem Cell (iPSC) by Research Method - Cellular Reprogramming, Cell Culture, Cell Differentiation, Cell Analysis, Cellular Engineering and Other Research Methods - Independent Analysis of Annual Sales in US$ Thousand for the Years 2020 through 2027 III-9

Table 44: Canada 7-Year Perspective for Induced Pluripotent Stem Cell (iPSC) by Research Method - Percentage Breakdown of Value Sales for Cellular Reprogramming, Cell Culture, Cell Differentiation, Cell Analysis, Cellular Engineering and Other Research Methods for the Years 2020 & 2027 III-10

Table 45: Canada Current & Future Analysis for Induced Pluripotent Stem Cell (iPSC) by Application - Drug Development & Toxicology Testing, Academic Research, Regenerative Medicine and Other Applications - Independent Analysis of Annual Sales in US$ Thousand for the Years 2020 through 2027 III-11

Table 46: Canada 7-Year Perspective for Induced Pluripotent Stem Cell (iPSC) by Application - Percentage Breakdown of Value Sales for Drug Development & Toxicology Testing, Academic Research, Regenerative Medicine and Other Applications for the Years 2020 & 2027 III-12

JAPAN III-13 Table 47: Japan Current & Future Analysis for Induced Pluripotent Stem Cell (iPSC) by Cell Type - Vascular Cells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cells and Other Cell Types - Independent Analysis of Annual Sales in US$ Thousand for the Years 2020 through 2027 III-13

Table 48: Japan 7-Year Perspective for Induced Pluripotent Stem Cell (iPSC) by Cell Type - Percentage Breakdown of Value Sales for Vascular Cells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cells and Other Cell Types for the Years 2020 & 2027 III-14

Table 49: Japan Current & Future Analysis for Induced Pluripotent Stem Cell (iPSC) by Research Method - Cellular Reprogramming, Cell Culture, Cell Differentiation, Cell Analysis, Cellular Engineering and Other Research Methods - Independent Analysis of Annual Sales in US$ Thousand for the Years 2020 through 2027 III-15

Table 50: Japan 7-Year Perspective for Induced Pluripotent Stem Cell (iPSC) by Research Method - Percentage Breakdown of Value Sales for Cellular Reprogramming, Cell Culture, Cell Differentiation, Cell Analysis, Cellular Engineering and Other Research Methods for the Years 2020 & 2027 III-16

Table 51: Japan Current & Future Analysis for Induced Pluripotent Stem Cell (iPSC) by Application - Drug Development & Toxicology Testing, Academic Research, Regenerative Medicine and Other Applications - Independent Analysis of Annual Sales in US$ Thousand for the Years 2020 through 2027 III-17

Table 52: Japan 7-Year Perspective for Induced Pluripotent Stem Cell (iPSC) by Application - Percentage Breakdown of Value Sales for Drug Development & Toxicology Testing, Academic Research, Regenerative Medicine and Other Applications for the Years 2020 & 2027 III-18

CHINA III-19 Table 53: China Current & Future Analysis for Induced Pluripotent Stem Cell (iPSC) by Cell Type - Vascular Cells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cells and Other Cell Types - Independent Analysis of Annual Sales in US$ Thousand for the Years 2020 through 2027 III-19

Table 54: China 7-Year Perspective for Induced Pluripotent Stem Cell (iPSC) by Cell Type - Percentage Breakdown of Value Sales for Vascular Cells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cells and Other Cell Types for the Years 2020 & 2027 III-20

Table 55: China Current & Future Analysis for Induced Pluripotent Stem Cell (iPSC) by Research Method - Cellular Reprogramming, Cell Culture, Cell Differentiation, Cell Analysis, Cellular Engineering and Other Research Methods - Independent Analysis of Annual Sales in US$ Thousand for the Years 2020 through 2027 III-21

Table 56: China 7-Year Perspective for Induced Pluripotent Stem Cell (iPSC) by Research Method - Percentage Breakdown of Value Sales for Cellular Reprogramming, Cell Culture, Cell Differentiation, Cell Analysis, Cellular Engineering and Other Research Methods for the Years 2020 & 2027 III-22

Table 57: China Current & Future Analysis for Induced Pluripotent Stem Cell (iPSC) by Application - Drug Development & Toxicology Testing, Academic Research, Regenerative Medicine and Other Applications - Independent Analysis of Annual Sales in US$ Thousand for the Years 2020 through 2027 III-23

Table 58: China 7-Year Perspective for Induced Pluripotent Stem Cell (iPSC) by Application - Percentage Breakdown of Value Sales for Drug Development & Toxicology Testing, Academic Research, Regenerative Medicine and Other Applications for the Years 2020 & 2027 III-24

EUROPE III-25 Table 59: Europe Current & Future Analysis for Induced Pluripotent Stem Cell (iPSC) by Geographic Region - France, Germany, Italy, UK and Rest of Europe Markets - Independent Analysis of Annual Sales in US$ Thousand for Years 2020 through 2027 III-25

Table 60: Europe 7-Year Perspective for Induced Pluripotent Stem Cell (iPSC) by Geographic Region - Percentage Breakdown of Value Sales for France, Germany, Italy, UK and Rest of Europe Markets for Years 2020 & 2027 III-26

Table 61: Europe Current & Future Analysis for Induced Pluripotent Stem Cell (iPSC) by Cell Type - Vascular Cells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cells and Other Cell Types - Independent Analysis of Annual Sales in US$ Thousand for the Years 2020 through 2027 III-27

Table 62: Europe 7-Year Perspective for Induced Pluripotent Stem Cell (iPSC) by Cell Type - Percentage Breakdown of Value Sales for Vascular Cells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cells and Other Cell Types for the Years 2020 & 2027 III-28

Table 63: Europe Current & Future Analysis for Induced Pluripotent Stem Cell (iPSC) by Research Method - Cellular Reprogramming, Cell Culture, Cell Differentiation, Cell Analysis, Cellular Engineering and Other Research Methods - Independent Analysis of Annual Sales in US$ Thousand for the Years 2020 through 2027 III-29

Table 64: Europe 7-Year Perspective for Induced Pluripotent Stem Cell (iPSC) by Research Method - Percentage Breakdown of Value Sales for Cellular Reprogramming, Cell Culture, Cell Differentiation, Cell Analysis, Cellular Engineering and Other Research Methods for the Years 2020 & 2027 III-30

Table 65: Europe Current & Future Analysis for Induced Pluripotent Stem Cell (iPSC) by Application - Drug Development & Toxicology Testing, Academic Research, Regenerative Medicine and Other Applications - Independent Analysis of Annual Sales in US$ Thousand for the Years 2020 through 2027 III-31

Table 66: Europe 7-Year Perspective for Induced Pluripotent Stem Cell (iPSC) by Application - Percentage Breakdown of Value Sales for Drug Development & Toxicology Testing, Academic Research, Regenerative Medicine and Other Applications for the Years 2020 & 2027 III-32

FRANCE III-33 Table 67: France Current & Future Analysis for Induced Pluripotent Stem Cell (iPSC) by Cell Type - Vascular Cells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cells and Other Cell Types - Independent Analysis of Annual Sales in US$ Thousand for the Years 2020 through 2027 III-33

Table 68: France 7-Year Perspective for Induced Pluripotent Stem Cell (iPSC) by Cell Type - Percentage Breakdown of Value Sales for Vascular Cells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cells and Other Cell Types for the Years 2020 & 2027 III-34

Table 69: France Current & Future Analysis for Induced Pluripotent Stem Cell (iPSC) by Research Method - Cellular Reprogramming, Cell Culture, Cell Differentiation, Cell Analysis, Cellular Engineering and Other Research Methods - Independent Analysis of Annual Sales in US$ Thousand for the Years 2020 through 2027 III-35

Table 70: France 7-Year Perspective for Induced Pluripotent Stem Cell (iPSC) by Research Method - Percentage Breakdown of Value Sales for Cellular Reprogramming, Cell Culture, Cell Differentiation, Cell Analysis, Cellular Engineering and Other Research Methods for the Years 2020 & 2027 III-36

Table 71: France Current & Future Analysis for Induced Pluripotent Stem Cell (iPSC) by Application - Drug Development & Toxicology Testing, Academic Research, Regenerative Medicine and Other Applications - Independent Analysis of Annual Sales in US$ Thousand for the Years 2020 through 2027 III-37

Table 72: France 7-Year Perspective for Induced Pluripotent Stem Cell (iPSC) by Application - Percentage Breakdown of Value Sales for Drug Development & Toxicology Testing, Academic Research, Regenerative Medicine and Other Applications for the Years 2020 & 2027 III-38

GERMANY III-39 Table 73: Germany Current & Future Analysis for Induced Pluripotent Stem Cell (iPSC) by Cell Type - Vascular Cells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cells and Other Cell Types - Independent Analysis of Annual Sales in US$ Thousand for the Years 2020 through 2027 III-39

Table 74: Germany 7-Year Perspective for Induced Pluripotent Stem Cell (iPSC) by Cell Type - Percentage Breakdown of Value Sales for Vascular Cells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cells and Other Cell Types for the Years 2020 & 2027 III-40

Table 75: Germany Current & Future Analysis for Induced Pluripotent Stem Cell (iPSC) by Research Method - Cellular Reprogramming, Cell Culture, Cell Differentiation, Cell Analysis, Cellular Engineering and Other Research Methods - Independent Analysis of Annual Sales in US$ Thousand for the Years 2020 through 2027 III-41

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Growing Value of Stem Cells in Medicine to Create a US$2,4 Billion Opportunity for Induced Pluripotent Stem Cell ((iPSC) - GlobeNewswire

Cardiac Stem Cells Comments Off on Growing Value of Stem Cells in Medicine to Create a US$2,4 Billion Opportunity for Induced Pluripotent Stem Cell ((iPSC) – GlobeNewswire | November 26th, 2020

INTRODUCTION

Tissue-resident macrophages regulate immunity and are pivotal for development, homeostasis, and repair (1). Major research efforts have uncovered roles for tissue-resident macrophages during infection, insult, and repair. However, in many cases, these studies disproportionally focus on certain organs in animals while disregarding tissue macrophages in other locations (2). Because function in macrophages is shaped by their tissue of residence and the local environment, specific phenotypes may not be universally applicable to all tissues (3). Notably, the bladder has generally been overlooked in macrophage studies; consequently, the function, origin, and renewal of bladder-resident macrophages in health and disease are poorly characterized or even completely unknown (4, 5).

Tissue-resident macrophages in adult organisms originate from embryonic progenitors, adult bone marrow (BM), or a mixture of both (612). During development, hematopoiesis begins in the yolk sac, giving rise to erythrocytes and macrophages directly and to erythro-myeloid progenitors (EMPs) (6, 13, 14). As hematopoiesis declines in the yolk sac, an intraembryonic wave of definitive hematopoiesis begins in the aorta-gonad-mesonephro, generating hematopoietic stem cells (HSCs). EMPs and then HSCs colonize the fetal liver to give rise to fetal liver monocytes, macrophages, and other immune cells, whereas only HSCs migrate to the BM to establish hematopoiesis in postnatal animals (15). Embryo-derived macrophages can either self-maintain and persist into adulthood or undergo replacement by circulating monocytes at tissue-specific rates. For example, a majority of macrophages in the gut are continuously replenished by BM-derived cells, whereas brain macrophages, or microglia, are long-lived yolk sacderived cells that are not replaced in steady-state conditions (8, 14, 16, 17). In certain conditions, origin influences macrophage behavior; for example, following myocardial infarction, embryonic-derived cardiac macrophages promote tissue repair, whereas BM-derived macrophages induce inflammation (18). However, macrophage functions are also imprinted by their microenvironment (19, 20). In the small intestine, macrophages in the muscle express higher levels of tissue-protective genes, such as Retnla, Mrc1, and Cd163 compared to lamina propria macrophages, although both originate from adult BM (21).

While the origin and maintenance of bladder-resident macrophages are currently unknown, these macrophages do play a role in response to urinary tract infection (UTI), which affects up to 50% of all women at some point in their lifetimes (5, 22). The immune response to uropathogenic Escherichia coli (UPEC) infection in the bladder is characterized by robust cytokine expression leading to rapid infiltration of large numbers of neutrophils and classical Ly6C+ monocytes (2328). Although essential to bacterial clearance, neutrophil and monocyte infiltration likely also induce collateral tissue damage. Targeted depletion of one of these two cell types is associated with reduced bacterial burden after primary infection in mice, whereas elimination of both cell types together leads to unchecked bacteria growth (23, 25, 26). Tissue-resident macrophages also take up a large number of bacteria during UTI; however, depletion of resident macrophages just before infection does not change bacterial clearance in a first or primary UTI (23). The absence of macrophages in the early stages of a primary UTI significantly improves bacterial clearance during a second, or challenge, infection (23). Exactly how the elimination of resident macrophages improves the response to a challenge infection is unclear, particularly as tissue-associated macrophages return to homeostatic numbers in the time interval between the two infections. Of note, improved bacterial clearance is lost in macrophage-depleted mice that are also depleted of CD4+ and CD8+ T cells, suggesting that macrophages modulate T cell activation or limit differentiation of memory T cells, as observed in other tissues (2933). For example, ablation of embryonic-derived alveolar macrophages results in increased numbers of CD8+ resident memory T cells following influenza infection in mice (31). In the gut, monocyte-derived macrophages support the differentiation of CD8+ tissue-resident memory T cells by production of interferon- (IFN-) and interleukin-12 (IL-12) during Yersinia infection (32). The opposing roles of macrophages in modulating T cell responses in the lung and gut support the idea that tissue type and/or ontogeny determines how macrophages may influence adaptive immunity (13).

To understand the role of bladder-resident macrophages, we investigated the origin, localization, and function of these cells during infection. We identified two subpopulations of resident macrophages in nave mouse bladders with distinctive cell surface proteins, spatial distribution, and gene expression profiles. We found that bladder macrophage subsets were long-lived cells, slowly replaced by BM-derived monocytes over the lifetime of the mouse. During UTI, the macrophage subsets differed in their capacity to take up bacteria and survive infection; however, both subsets were replaced by BM-derived cells following resolution of infection. Thus, after a first infection, macrophage subsets had divergent transcriptional profiles compared to their nave counterparts, shaping the response to subsequent UTI.

We reported that macrophage depletion before a first UTI improves bacterial clearance during challenge infection (23). Thus, we initiated a follow-up study to investigate the role of bladder-resident macrophages during UTI. Using the macrophage-associated cell surface proteins CD64 and F4/80 (34, 35), we identified a clear CD64 and F4/80 double-positive resident macrophage population in nave bladders from 7- to 8-week-old female CX3CR1GFP/+ mice. This transgenic mouse is widely used to distinguish macrophage populations in other tissues as the chemokine receptor CX3CR1 is expressed by monocytes and macrophages at some point in their development (36). In most tissues, resident macrophages are either GFP+ as they express CX3CR1 or GFP because they no longer express CX3CR1 (10). Therefore, we were surprised to observe heterogeneity in green fluorescent protein (GFP) expression levels, revealing potentially two subpopulations (Maclo and Machi) of CD64+ F4/80+ macrophages in the bladder (Fig. 1A). Although the differences were small in magnitude, the Machi-expressing population was present in statistically significantly greater numbers and proportions compared to the Maclo population (Fig. 1A). As CX3CR1 deficiency results in decreased macrophage numbers and frequency in the intestine and brain, and the transgenic CX3CR1GFP/+ mouse we used is hemizygous for this receptor (3638), we investigated whether our putative bladder-resident macrophage subsets were similarly present in wild-type C57BL/6 mice. Using the same gating strategy and an anti-CX3CR1 antibody, we clearly identified that CX3CR1 expression levels distinguished two distinct macrophage populations in 7- to 8-week-old nave female wild-type mice (Fig. 1B). Notably, wild-type mice had similar numbers and proportions of each macrophage subset (Fig. 1B).

(A to C) Bladders from 7-week-old female CX3CR1GFP/+ and C57BL6/J mice were analyzed by flow cytometry. (A and B) Dot plots depict the gating strategy for macrophages subsets and graphs show the total cell number (log scale, left) and proportion (right) of bladder macrophage subset, derived from cytometric analysis in (A) CX3CR1GFP/+ and (B) C57BL6/J mice. (C) Histograms show the relative expression of CX3CR1, TIM4, and LYVE1 on macrophage subsets in C57BL6/J mice, Maclo is green and Machi is orange. (See fig. S1 for data on expression of additional proteins). (D) Representative confocal images of bladders from C57BL6/J mice at 20 and 40. Merged images and single channels with the target of interest are shown. DAPI, 4,6-diamidino-2-phenylindole. (E) Graphs show the proportion of each macrophage subset in the lamina propria and muscle of nave C57BL6/J mice. Data are pooled from three experiments, n = 3 to 6 mice per experiment. Each dot represents one mouse; lines are medians. Significance was determined using the nonparametric Mann-Whitney test to compare macrophage subset numbers (A and B) and the nonparametric Wilcoxon matched-pairs signed-rank test to compare the macrophage subset percentages (A, B, and E). All P values are shown; statistically significant P values (<0.05) are in red.

Next, we assessed the surface expression level of proteins known to define macrophage subsets in other tissues (39). We observed that the efferocytic receptor TIM4 and hyaluronan receptor LYVE1 were expressed by the Maclo population, whereas the Machi population was TIM4 and LYVE1 (Fig. 1C). Macrophage-associated proteins, such as CD64, F4/80, CD11b, CD11c, and MHC II, were differentially expressed between the subsets (fig. S1A), supporting the notion that these are distinct populations. A recent publication described several organs as having two distinct macrophage subsets, differentiated by their expression of LYVE1, CX3CR1, and, in particular, MHC II (39). To determine whether bladder macrophage subsets represented these two cell types, we used a similar gating strategy (fig. S1B); however, we observed that MHC II CD64+F4/80+ cells made up a very minor proportion (<2%) of bladder-resident macrophages (fig. SC). Last, to determine whether additional heterogeneity existed within the CD64+ F4/80+ bladder-resident macrophage population, we used the dimension reduction analyses tSNE and UMAP to visualize our data. In our analyses of the nave CD45+ cell population, a large CD64+ cluster contained two putative subsets that corresponded to traditionally gated Maclo and Machi populations and included the tiny proportion of MHC II macrophages (fig. S1D). tSNE (t-distributed stochastic neighbor embedding) and, more particularly, UMAP (uniform manifold approximation and projection) analysis of CD64+ F4/80+ macrophages revealed two groups, with differential expression of CX3CR1, F4/80, CD64, LYVE1, and TIM4, reflecting the data shown in the traditionally gated histograms (fig. S1, D and E). Thus, we concluded that two subsets of macrophages reside in nave mouse bladders with differential surface protein expression.

To determine the spatial orientation of the subsets, we stained nave female C57BL/6 bladders with antibodies to F4/80 and LYVE1 and phalloidin to demarcate the muscle layer from the lamina propria (Fig. 1D). We quantified the number of each subset in these two anatomical locations, observing a higher percentage of the LYVE1+ Maclo macrophage subset in the muscle compared to the LYVE1 Machi macrophage subset (Fig. 1E). Macrophages in the lamina propria were predominantly of the Machi phenotype (Fig. 1E). Thus, the phenotypic differences we observed in bladder-resident macrophage subsets extended to differential tissue localization. Given their spatial organization, we renamed the Maclo subset MacM for muscle and the Machi subset MacL for lamina propria. Together, these results reveal that two phenotypically distinct macrophage subsets reside in different regions of the nave bladder.

We next investigated whether macrophage heterogeneity in adult mouse bladders arose due to distinct developmental origins of the subsets. We analyzed bladders from newborn C57BL/6 pups by confocal imaging and by flow cytometry from CX3CR1-GFPexpressing E16.5 (embryonic day 16.5) embryos and newborn mice. We observed that, in E16.5 and newborn animals, a single CX3CR1hi macrophage population was present in the muscle and lamina propria of the bladder. By flow cytometry, these cells were uniformly positive for CD64 and negative for MHC II as expected for fetal macrophages (40) and stained positively for LYVE1 in confocal images of newborn mouse bladder, supporting that diversification of bladder macrophage subsets occurs after birth (Fig. 2A).

(A) Merged confocal and single channel images from a C57BL/6 newborn mouse bladder. Left image is enlarged at the right. Gating strategy in Cdh5-CreERT2Rosa26tdTomato CX3CR1GFP newborn mice and E16.5 embryos; histograms show CX3CR1 and MHC II expression. (B to E) Reporter recombination in microglia, monocytes, bladder macrophages, and MacM and MacL subsets in Cdh5-CreERT2Rosa26tdTomato mice: (B) E16.5 embryos, newborns 4-hydroxytamoxifen (4OHT)-treated at E7.5, (C) adults 4OHT-treated at E7.5, (D) E16.5 embryos, newborns 4OHT-treated at E10.5, (E) adults 4OHT-treated at E10.5. (F) Percentage of YFP+ cells in microglia, monocytes, MacM, and MacL macrophages in adult Flt3CreRosa26YFP mice. (G to I) Adult shield-irradiated C57BL/6 CD45.2 mice reconstituted with CCR2+/+ CD45.1 BM and C57BL/6 CD45.1 mice reconstituted with CCR2/ CD45.2 BM. Percentage of donor cells (G) in monocytes or (H) bladder-resident macrophages in mice transplanted with CCR2+/+ or CCR2/ BM at 3 and 6 months after transplantation. (See fig. S2 for data on blood leukocyte chimerism). (I) Bladder-resident macrophage replacement rate. Data pooled from two to three experiments, n = 2 to 6 mice per experiment. Each point represents one mouse; lines are medians. Significance determined using the Mann-Whitney test comparing (B to F) macrophages or subsets to monocytes or (G and H) CCR2+/+ to CCR2/ recipients, P values were corrected for multiple testing using the false discovery rate (FDR) method. All P values are shown; statistically significant P values (<0.05) are in red.

We hypothesized that, in adult mice, macrophage subsets arise following differentiation of cells seeded from embryonic progenitors or that one subset is derived from embryonic macrophages, whereas the second subset arises from BM-derived monocytes (41). To test these hypotheses, we used the Cdh5-CreERT2 Rosa26tdTomato transgenic mouse, in which the contribution of distinct hematopoietic progenitor waves to immune cell populations can be followed temporally, such that treatment of pregnant mice with 4-hydroxytamoxifen (4OHT) at E7.5 labels yolk sac progenitors and their progeny and treatment at E10.5 labels HSC that will settle in the BM (adult-type HSCs) and their cellular output (42). After treatment with 4OHT at E7.5, in which microglia were labeled as expected (8, 14), we found a significantly higher proportion of labeled bladder macrophages compared to monocytes in E16.5 embryos and newborn mice (Fig. 2B). Labeled bladder macrophage subsets were nearly absent, similar to monocytes, in adult (8- to 11-week-old) mice (Fig. 2C). These data support the fact that yolk sacderived bladder macrophages are diluted after birth in the adult and suggest that the subsets are composed of HSC-derived macrophages. Low levels of E10.5-labeled macrophages were detected in embryonic bladders (Fig. 2D), and their frequency increased in newborn and adult mice, although to a lesser degree than monocytes, supporting the idea that bladder macrophage subsets arise, at least in part, from adult-type HSCs (Fig. 2, D and E). Of note, both subsets found in the adult bladder showed similar frequencies of E10.5 labeling (Fig. 2E). Together, these results demonstrate that adult bladder macrophages are partially HSC-derived and the macrophage subsets cannot be distinguished from each other by their ontogeny.

To confirm that HSC-derived progenitors contribute to the bladder-resident macrophage pool, we analyzed bladders from adult Flt3Cre Rosa26YFP mice. In this transgenic mouse, expression of the tyrosine kinase receptor Flt3 in multipotent progenitors leads to expression of yellow fluorescent protein (YFP) in the progeny of these cells, such as monocytes, whereas microglia, arising from yolk sac progenitors, are essentially YFP (43). Recombination rates driven by Flt3 are very low during embryonic development, but blood monocyte labeling reaches 80 to 90% in adult mice (7). Therefore, if tissue-resident macrophages arise from postnatal BM-derived monocytes, labeling in adult mice should be similar to blood monocytes, whereas the presence of Flt3 tissue macrophages would indicate that they originated from either embryonic HSCs or adult Flt3-independent progenitors. We observed that, in 2- to 4-month-old and 22- to 24-month-old mice, ~50% of each macrophage subset was YFP+, which was significantly lower compared to circulating monocytes (Fig. 2F). This observation and those from the Cdh5-CreERT2 mice together support the fact that, in addition to adult HSCs, adult bladder macrophage subsets are derived from embryonic progenitors that may include fetal HSCs, and/or later yolk sac progenitors, but with no contribution from early yolk sac progenitors. In addition, the lack of equilibration of YFP labeling in the bladder with blood monocytes at 22 to 24 months suggests that tissue macrophages are not rapidly replaced over the lifetime of the mouse by BM-derived cells in the context of homeostasis.

To determine the replacement rate of bladder-resident macrophages by BM-derived cells in the adult mouse, we evaluated shielded irradiated mice, in which adult animals are irradiated with a lead cover over the bladder to protect this organ from radiation-induced immune cell death and nonhomeostatic immune cell infiltration. Animals were transplanted with congenic BM from wild-type or CCR2/ mice. Monocytes depend on CCR2 receptor signaling to exit the BM into circulation (44). At 3 and 6 months, we observed that a median of 27.7% (3 months) and 27.6% (6 months) of circulating Ly6C+ monocytes were of donor origin in mice reconstituted with wild-type BM, which is well in-line with published studies using this approach (45, 46), whereas only 6.1% (3 months) and 6.5% (6 months) of Ly6C+ monocytes were of donor origin in wild-type mice receiving CCR2/ BM (Fig. 2G). B and natural killer (NK) cells were replenished to a greater extent in mice reconstituted with CCR2/ BM compared to mice reconstituted with CCR2+/+ BM, which could be due to different engraftment efficiencies between CD45.1 and CD45.2 BM (fig. S2) (47, 48). In mice reconstituted with wild-type BM, 4.7% of MacM and 4.5% MacL were of donor origin at 3 months after engraftment. At 6 months after irradiation, 7% of MacM and 8.5% of MacL macrophages were of donor origin (Fig. 2H). Chimerism in bladder macrophage subsets was markedly reduced in CCR2/ BM recipients, suggesting that monocytes slowly replace bladder macrophage subsets in a CCR2-dependent manner (Fig. 2H). By dividing the median macrophage subset chimerism (7 or 8.5%) by the median circulating Ly6C+ monocyte chimerism at 6 months in mice receiving wild-type BM (27.6%), we determined that 25.3% of MacM and 30.8% MacL were replaced by BM-derived monocytes within 6 months (Fig. 2I).

Together, these results reveal that the establishment of distinct bladder-resident macrophage subsets occurs postnatally. Yolk sac macrophages initially seed the fetal bladder but are replaced by fetal HSC-derived macrophages. In adult mice, bladder macrophage subsets are partially maintained through a slow replacement by BM-derived monocytes, although a substantial number of fetally derived cells remain. The incomplete macrophage labeling we observed in our experiments supports the idea that a progenitor source, which cannot be labeled in either model, contributes to resident bladder macrophages. Currently, there is no fate-mapping model to discriminate or follow progeny specifically from late yolk sac EMPs or early fetally restricted HSC, as hematopoietic waves overlap in development. We can conclude that MacM and MacL macrophages do not differ in their developmental origin or rate of replacement by monocytes, supporting the view that one or more unique niches in adult tissue may be responsible for macrophage specialization into phenotypical and functionally distinct macrophage subsets.

Although bladder-resident macrophage subsets had similar ontogeny, their distinct spatial localization and surface protein expression suggested that they have different functions. To test this hypothesis, we first analyzed gene expression profiles of nave adult female MacM and MacL macrophages using bulk RNA sequencing (RNA-seq) (fig. S3A, gating strategy). To formally demonstrate that our cells of interest are macrophages, we aligned the transcriptomes of the bladder macrophage subsets with the macrophage core signature list published by the Immunological Genome Consortium and the bladder macrophage core list from the mouse cell atlas single-cell database (35, 49). The MacM and MacL subsets expressed 80% of the genes from the Immunological Genome Consortium macrophage core signature list and more than 95% of the genes in the bladder macrophage core list (fig. S3B), supporting the idea that our cells of interest are fully differentiated tissue-resident macrophages.

We observed that 1475 genes were differentially expressed between nave MacM and MacL macrophages, in which 899 genes were positively regulated and 576 genes were negatively regulated in the MacL subset relative to MacM macrophages (Fig. 3A). In the top 20 differentially expressed genes (DEG), MacM macrophages expressed higher levels of Tfrc, Ms4a8a, Serpinb6a, CCL24, Scl40a1, Clec10a, and Retnla, all of which are associated with an alternatively activated macrophage phenotype (5053); genes involved in iron metabolism, such as Tfrc, Steap4, and Slc40a1 (54); and genes from the complement cascade, including C4b and Cfp (Fig. 3B). In the same 20 most DEG, MacL macrophages expressed greater levels of Cx3cr1, Cd72, Itgb5, Axl, and Itgav, which are associated with phagocytosis, antigen presentation, and immune response activation (Fig. 3B) (5557). MacL macrophages also expressed inflammatory genes, such as Cxcl16, a chemoattractant for T and NKT cells (58, 59), and Lpcat2 and Pdgfb, which are involved in the metabolism of inflammatory lipid mediators (Fig. 3B) (60, 61). Using gene set enrichment analysis of the DEG to detect pathways up-regulated in the macrophage subsets, we observed that the MacM subset expressed genes linked to pathways such as endocytosis, mineral absorption, lysosome, and phagosome (Fig. 3C). Within the phagosome and endocytosis pathways, genes critical for bacterial sensing and alternative activation such as Tlr4, Mrc1 (encoding for CD206), Cd209, and Egfr (6264) were increased in the MacM subset. In the mineral absorption pathway, genes controlling iron metabolism that also enhance bacterial killing such as Hmox1 and Hmox2 were up-regulated in MacM macrophages (Fig. 3D) (65). In the MacL subset, genes linked to diverse inflammatory pathways, including Toll-like receptor signaling, apoptosis, antigen processing and presentation, and chemokine signaling, were present, as were many infectious and inflammatory diseaserelated pathways (Fig. 3E). Within these pathways, the MacL subset expressed genes related to bacterial sensing, such as Tlr1, Tlr2, and Cd14; initiation of inflammation, such as Il1b, Tnf, Ccl3, Ccl4, Cxcl10, Cxcl16, and Nfkb1; and apoptotic cell death, such as Mapk8, Pmaip1, Bcla1d, Cflar, Bcl2l11, and Birc2 (Fig. 3F).

MacM and MacL macrophages were sorted from 7- to 8-week-old female nave adult C57BL/6 mouse bladders and analyzed by RNA-seq (fig. S3, gating strategy). (A) Heatmaps show the gene expression profile of the 1475 differentially expressed genes and (B) the 20 most differentially expressed genes between the MacM and MacL subsets. (C to F) Using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of significantly up-regulated genes, the following are depicted: (C) pathways enriched in MacM macrophages, (D) up-regulated genes associated with selected pathways in MacM macrophages, (E) pathways enriched in MacL macrophages, and (F) up-regulated genes associated with selected pathways in MacL macrophages. In (C) and (E), the size of the nodes reflects the statistical significance of the term. (Q < 0.05; terms > 3 genes; % genes/term > 3; 0.4).

These findings suggest that MacM macrophages are more anti-inflammatory with increased endocytic activity, which is a common feature of highly phagocytic resident macrophages (66), and as such may play a prominent role in bacterial uptake or killing during infection. MacL, on the other hand, may play a greater role in antigen presentation and initiation or maintenance of inflammation.

As we observed enrichment of genes belonging to endocytosis, lysosome, and phagosome pathways in the MacM subset, we reasoned that the macrophage subsets differentially take up bacteria during infection. To test our hypothesis, we used a well-described mouse model of UTI, in which we transurethrally infect adult female mice via catheterization with 107 colony-forming units (CFU) of UPEC strain UTI89-RFP, which expresses a red fluorescent protein (RFP) (23). At 24 hours post-infection (PI), we investigated bacterial uptake by macrophage subsets (Fig. 4, A and B). Despite that MacM macrophages are farther from the infected urothelium than MacL macrophages, we observed that 20% of MacM and only 10% of MacL subsets contained bacteria at 24 hours PI, providing functional evidence to support the transcriptional data that MacM macrophages have a superior phagocytic capacity compared to MacL macrophages (Fig. 4B). Supporting this conclusion, we found that when we exposed sorted MacM and MacL macrophages to live UPEC in vitro, a greater proportion of MacM macrophages internalized bacteria after 2 hours compared to MacL macrophages (fig. S4A). In addition, despite very low levels of infection overall (~1% of macrophages), more UPEC could be found in MacM macrophages compared to MacL macrophages at 4 hours PI in vivo (fig. S4B). Taking the total population of UPEC-containing macrophages at 24 hours PI, we observed that ~80% of these cells were MacM macrophages, whereas the MacL subset comprised only 20% of this population, which was unusual given that MacM and MacL exist in the bladder in a 1:1 ratio (Fig. 4B and fig. S4C, gating strategy).

(A to H) Female C57BL6/J mice were infected with UTI89-RFP and bladders were analyzed by flow cytometry at (A to D) 24 hours or (E to H) 4 hours PI. (A) Gating strategy, resident macrophage subsets, and cells containing bacteria. (B) Percentage of infected macrophage subsets and UPEC distribution (fig. S4B, gating strategy). (C) IL-4R gMFI (geometric mean fluorescence intensity) in nave mice and 24 hours PI. (D) Total number and frequency of bladder macrophage subsets. (E) Gating strategy. (F) Total number and frequency of bladder macrophage subsets. Percentage of (G) macrophage subsets labeled with a live/dead marker (fig. S4D, gating strategy) and (H) dying macrophages containing UPEC. (I) MacM and MacL macrophage quantification in nave mice and 4 hours PI. (B to D and F to H) Data pooled from three experiments, n = 3 to 6 mice per experiment. (I) Data are pooled from two experiments, n = 2 to 3 mice per experiment. Each dot represents one mouse; lines are medians. In (D) and (F), Mann-Whitney test was used to compare the numbers and the nonparametric Wilcoxon matched-pairs signed-rank test was used to compare the percentages of each macrophage subset. (B and C and G to I) Mann-Whitney test. P values were corrected for multiple testing using the FDR method. All P values are shown; statistically significant P values (<0.05) are in red.

Given the predominance of genes associated with alternatively activated macrophages in the top 20 DEGs of MacM macrophages (Fig. 3B), we measured polarization of the macrophage subsets in nave and infected bladders by analyzing the expression of IL-4R by flow cytometry (Fig. 4C). IL-4R is the receptor of IL-4 and IL-13, two cytokines that drive alternative activation in macrophages (67). Both subsets had increased expression of IL-4R at 24 hours PI compared to their nave counterparts; however, MacM macrophages had consistently higher expression levels of IL-4R compared to MacL macrophages in nave and infected tissue (Fig. 4C).

In the course of our studies, we observed that the total number and proportion of MacL macrophages were significantly lower than those of MacM macrophages at 24 hours PI, whereas, in nave mice, both the number and proportion of the macrophage subsets were equivalent (Figs. 4D and 1B). To rule out the contribution of differentiated monocyte-derived cells to the macrophage pool, we assessed total macrophage cell numbers in the bladder at 4 hours PI, when there is minimal monocyte infiltration (Fig. 4E) (23). Macrophage subset numbers and proportions were significantly different at 4 hours PI (Fig. 4F). As the total numbers of each subset were not increased over nave levels (Fig. 1B), we hypothesized that macrophages die during infection, particularly as apoptosis pathways were more highly expressed in MacL macrophages (Fig. 3, C and D). Using a cell viability dye, which labels dying/dead cells, we found that a significantly higher proportion of MacL macrophages were dying compared to MacM macrophages at 4 hours PI (Fig. 4G and fig. S4D, gating strategy). As UPEC strains can induce macrophage death in vitro (68, 69), we asked whether macrophage cell death was induced by UPEC in vivo. We observed that only 20% of dying or dead cells in each subset were infected (Fig. 4H), suggesting that macrophage death was not primarily driven by UPEC uptake. To determine whether macrophage cell death was confined to a distinct location, we quantified macrophage subset numbers in the muscle and lamina propria. We observed that, at 4 hours PI, only MacL macrophages located in the lamina propria were reduced in numbers compared to nave mice (Fig. 4I). Given that, in the first hours after infection, the urothelium exfoliates massively (70), these results suggest that macrophage death, specifically in the lamina propria, may be due to the loss of a survival factor in this niche. To test whether alteration of the niche induced macrophage death, we chemically induced global urothelial exfoliation by intravesical instillation of protamine sulfate (71, 72). We observed that at 5 hours after treatment, the total numbers of both MacM and MacL subsets were reduced compared to macrophage subsets in nave mice (fig. S4E), suggesting that alterations in bladder urothelium are sufficient to reduce resident macrophage numbers in the bladder, although protamine sulfate may also directly induce macrophage death. Thus, we functionally validated the divergent gene expression observed between macrophage subsets, in which MacM macrophages are more phagocytic and MacL macrophages are more prone to die, supporting the idea that gene expression differences translate to divergent roles for the subsets in response to UTI.

As we observed macrophages dying during infection, we investigated the change in macrophage numbers over time as animals resolved their infection. Both populations significantly decreased at 24 hours PI, then subsequently increased nearly 10-fold at 7 days PI, and returned to numbers just above homeostatic levels at 4 weeks PI (Fig. 5A). With the dynamic increase of macrophage numbers over the course of UTI, we hypothesized that infiltrating monocytes replace resident macrophage subsets during infection, as we previously reported that infiltrating monocytes differentiate to cells resembling macrophages at 48 hours PI (23). To test this hypothesis, we used the CCR2CreERT2 Rosa26tdTomato mouse, in which administration of 4OHT induces recombination in CCR2-expressing cells, such as circulating Ly6C+ monocytes, leading to irreversible labeling of these cells in vivo (73). Blood monocytes and bladder-resident macrophages are not Tomato+ in untreated mice (fig. S5). We administered 4OHT to nave mice and, then, 24 hours later, infected half of the treated mice with 107 CFU of UTI89. At this time point, 24 hours after 4OHT treatment, we analyzed the labeling efficiency in circulating classical Ly6C+ monocytes, finding that approximately 80% of Ly6C+ monocytes were labeled in both nave and infected mice (Fig. 5B). After 6 weeks, when animals had resolved their infection, there were no labeled circulating Ly6C+ monocytes in nave or post-infected mice (Fig. 5B). When we analyzed the bladders of nave mice 6 weeks after the 4OHT pulse, only 2.9% of MacM and 2.1% of MacL macrophage subsets were labeled, supporting our earlier conclusion that monocytes contribute to bladder macrophage subsets at a very slow rate in the steady state (Fig. 5C). At 6 weeks PI, the total numbers of macrophage subsets finally returned to homeostatic levels (Fig. 5D), but PI MacM and MacL macrophages had two to three times more Tomato+ cells (median, MacM 8.4%, MacL 4.4%) than their nave counterparts. These data support the fact that, after monocytes infiltrate the bladder during infection, they remain in the tissue following resolution, integrating themselves into the resident macrophage pool, and thus contribute to the return of macrophage subsets to homeostatic levels.

(A) Total number of MacM (green) and MacL (orange) in nave and 1-, 7-, or 28-day PI mice. (B and C) CCR2CreERT2Rosa26tdTomato mice were pulsed with 4OHT. Twenty-four hours later, half were infected with UTI89-RFP. Percentage of Tomato+ (B) Ly6C+ monocytes 24 hours and 6 weeks after 4OHT-pulse or (C) bladder macrophage subsets 6 weeks after 4OHT-pulse. (D) Total number of macrophage subsets in nave and 6-week PI bladders. (E) Replicate-adjusted principal component analysis of all genes from nave and post-infected bladder macrophage subsets. Differentially expressed genes between nave and 6-week PI (F) MacM (513 genes) and (G) MacL (617 genes) macrophages. KEGG pathway analysis of significantly up-regulated genes, enriched in 6-week PI (H) MacM and (I) MacL macrophages. Up-regulated genes from selected pathways in 6-week PI (J) MacM and (K) MacL macrophages. (A, C, and D) Mann-Whitney test comparing infection to nave. P values were corrected for multiple testing using the FDR method. Higher left-shifted P values refer to MacM and lower right-shifted P values refer to MacL. (H and I) Node size reflects statistical significance of the term (Q < 0.05; terms > 3 genes; %genes/term > 3; 0.4). All P values are shown; statistically significant P values (<0.05) are in red.

As monocytes generally have different origins and developmental programs compared to tissue-resident macrophages, we used RNA-seq to determine whether the macrophage pool in post-infected bladders was different from nave tissue-resident cells. Using principal component analysis (PCA), we compared bladder macrophage subsets from 6-week post-infected mice to their nave counterparts. We found that macrophages clustered more closely together by subset, rather than by infection status, or, in other words, nave and post-infected MacL macrophages clustered more closely to each other than either sample clustered to nave or post-infected MacM macrophages (Fig. 5E). Five hundred thirteen genes (247 genes down-regulated and 266 genes up-regulated) were different between nave and post-infected MacM macrophages (Fig. 5F). Six hundred seventeen genes (401 genes down-regulated and 216 genes up-regulated) were differentially expressed between the nave and post-infected MacL subset (Fig. 5G). Applying gene set enrichment analysis to up-regulated genes in the post-infected macrophage subsets, we detected common pathways between the subsets including enrichment of genes linked to pathways such as antigen presentation; cell adhesion molecules; TH1, TH2, and TH17 cell differentiation; and chemokine signaling pathway (Fig. 5, H and I). Although the enriched genes were not identical within each subset for these pathways, some common up-regulated genes included those encoding for histocompatibility class 2 molecules, such as H2-Ab1, H2-Eb1, H2-DMb1, Ciita, and the Stat1 transcription factor (Fig. 5, I and J). As differentiation of monocytes into macrophages includes up-regulation of cell adhesion and antigen presentation molecules (74), including in the bladder (23), these data further support the idea that monocytes specifically contribute to the PI bladder-resident macrophage pool.

These results show that, in the context of UTI, dying macrophages are replaced by monocyte-derived cells. Tissue-resident macrophage subsets maintain their separate identities distinct from each other after infection, although each subset also takes on a different transcriptional profile compared to their nave counterparts, with up-regulated expression of genes related to adaptive immune responses.

We previously reported that macrophage depletion 24 hours before a primary UTI does not affect bacterial clearance (23). Given that post-infected macrophage subsets up-regulated pathways different from those associated with the transcriptomes of nave bladder macrophage subsets, and that these pathways were linked to inflammatory diseases and the adaptive immune response, we hypothesized that one or both macrophage subsets would mediate improved bacterial clearance to a challenge infection. To test this hypothesis, we infected mice with 107 CFU of kanamycin-resistant UTI89-RFP. Four weeks later, when the infection was resolved, mice were challenged with 107 CFU of the isogenic ampicillin-resistant UPEC strain, UTI89-GFP, and bacterial burden was measured at 24 hours PI. To test the contribution of the macrophage subsets to the response to challenge infection, we used different concentrations of anti-CSF1R depleting antibody to differently target the two macrophage subsets directly before challenge infection (Fig. 6A, experimental scheme). Using 500 g of anti-CSF1R antibody, we depleted 50% of MacM and 80% of MacL macrophages, whereas depletion following treatment with 800 g of anti-CSF1R antibody reduced MacM macrophages by 80% and the MacL subset by more than 90% (Fig. 6B and fig. S6A). Twenty-four hours after anti-CSF1R antibody treatment, the number of circulating neutrophils, eosinophils, NK, B, or T cells was not different from mock-treated mice at either concentration (fig. S6B). Classical Ly6C+ monocytes were modestly reduced in mice treated with 800 g of anti-CSF1R antibody but were unchanged in mice receiving 500 g of depleting antibody. Antibody treatment did not change circulating nonclassical monocyte numbers (fig. S6B). After challenge infection, the bacterial burden was not different in mice treated with 500 g of anti-CSF1R compared to mock-treated mice (Fig. 6C). By contrast, mice depleted with 800 g of anti-CSF1R had reduced bacterial burdens, indicative of a stronger response after challenge compared to nondepleted mice (Fig. 6D).

(A) Experimental scheme. (B) Efficacy of macrophage subset depletion in nave C57BL/6 mice treated with 500 or 800 g of anti-CSF1R antibody. (C and D) Bacterial burden per bladder 24 hours after challenge in female C57BL/6 mice infected with UTI89-RFP according to (A) and treated with phosphate-buffered saline (PBS) (mock) or (C) 500 g or (D) 800 g of anti-CSF1R antibody 72 hours before being challenged with the isogenic UTI89-GFP strain. (E to G) Mice were infected according to (A) and treated with 800 g of anti-CSF1R antibody 72 hours before challenge infection with 107 CFU of the isogenic UTI89-GFP strain. Graphs depict the (E) total number of the indicated cell type, (F) the percentage of the indicated cell type that was infected, and (G) the total number of the indicated cell type that contained UPEC at 24 hours after challenge in mice treated with PBS or 800 g of anti-CSF1R antibody. Data are pooled from three experiments, n = 3 to 6 mice per experiment. Each dot represents one mouse; lines are medians. (C to G) Mann-Whitney test, P values were corrected for multiple testing using the FDR method. All P values are shown; statistically significant P values (<0.05) are in red.

Neutrophils take up a majority of UPEC at early time points during UTI (23). Therefore, we hypothesized that the improved bacterial clearance in macrophage-depleted mice may be due to increased infiltration of inflammatory cells, such as neutrophils. At 24 hours after challenge infection, we observed that, while the numbers of resident macrophage subsets, MHCII+ monocytes, and MHCII monocytes in macrophage-depleted mice were reduced compared to mock-treated mice, as expected, the numbers of infiltrating neutrophils were unchanged by antibody treatment (Fig. 6E and fig. S6C, gating strategy). Fewer eosinophils infiltrated the tissue in macrophage-depleted mice, although the impact of this is unclear as their role in infection is unknown (Fig. 6E). Given that neutrophil infiltration was unchanged and that monocytes, which also take up a large number of bacteria during infection, were reduced in number, we considered that improved bacterial clearance in macrophage-depleted mice may be due to increased bacterial uptake on a per-cell basis during challenge infection. However, bacterial uptake was not different between depleted and mock-treated mice in neutrophils, MHCII+ and MHCII monocytes, or either macrophage subset (Fig. 6F). The lower numbers of the MacM subset in macrophage-depleted mice translated to lower numbers of infected MacM macrophages (Fig. 6, E and G, respectively). However, we observed no differences in the numbers of infected MacL macrophages, neutrophils, and MHCII+ or MHCII monocytes in macrophage-depleted mice compared to nondepleted animals (Fig. 6G). Together, these results support the notion that MacM macrophages negatively affect bacterial clearance in a challenge infection, but not at the level of direct bacterial uptake or myeloid cell infiltration.

As infiltration of inflammatory cells or the number of infected cells during challenge infection was not changed in macrophage-depleted mice, we questioned whether another host mechanism was involved in bacterial clearance. Exfoliation of infected urothelial cells is a host mechanism to eliminate bacteria (70, 75). We hypothesized that macrophage-depleted mice have increased urothelial exfoliation during challenge infection, leading to reduced bacterial numbers. We quantified the mean fluorescence intensity of uroplakins, proteins expressed by terminally differentiated urothelial cells (76), from bladders of post-challenged mice, depleted of macrophages or not (Fig. 7A). We did not detect a significant difference in urothelial exfoliation between mock-treated animals and mice depleted of macrophage before challenge infection, supporting that urothelial exfoliation is not the underlying mechanism behind improved bacterial clearance in macrophage-depleted mice (Fig. 7B). Infiltration of inflammatory cells is associated with bladder tissue damage and increased bacterial burden (26). As we observed fewer monocytes and eosinophils in macrophage-depleted mice during challenge infection, we investigated whether reduced cell infiltration was associated with less tissue damage. We assessed edema formation by quantifying the area of the lamina propria in post-challenged bladders, depleted of macrophages or not (Fig. 7A). We did not detect a difference in edema formation between nondepleted mice and mice depleted of macrophage before challenge infection (Fig. 7C).

Female C57BL/6 mice were infected according to the scheme shown in Fig. 6A and treated with 800 g of anti-CSF1R antibody 72 hours before challenge infection with 107 CFU of UTI89. (A) Representative confocal images of bladders from mice treated with PBS or 800 g of anti-CSF1R antibody 24 hours after challenge. Uroplakin, green; phalloidin, turquoise; DAPI, blue. (B) The graph shows the mean fluorescence intensity of uroplakin expression, quantified from imaging, at 24 hours after challenge. (C) The graph shows the area of the lamina propria, quantified from imaging, at 24 hours after challenge. (D to F) Graphs depict the (D and E) total number of the indicated cell type or (F) the total number of the indicated cell type expressing IFN- at 24 hours after challenge infection. Data are pooled from two experiments, n = 4 to 6 mice per experiment. Each dot represents one mouse; lines are medians. In (B) to (F), significance was determined using the nonparametric Mann-Whitney test and P values were corrected for multiple testing using the FDR method. All calculated/corrected P values are shown and P values meeting the criteria for statistical significance (P < 0.05) are depicted in red.

As we observed fewer eosinophils in macrophage-depleted mice during challenge infection, and our previous work demonstrated that type 2 immune responserelated cytokines are expressed early in UTI (24), we assessed the polarity of the T cell response to challenge infection (fig. S7, gating strategy). Macrophage depletion did not alter the infiltration of T regulatory cells or TH2 or TH17 T helper subsets (Fig. 7D). However, macrophage depletion did correlate with an increase in the numbers of TH1 T cells, NKT cells, NK cells, and type 1 innate lymphoid cells (ILC1s) (Fig. 7E). In macrophage-depleted mice, TH1 T cells, NKT cells, and NK cells had higher IFN- production compared to mock-treated mice (Fig. 7F), suggesting that, in the absence of post-infected macrophages, a more pro-inflammatory, bactericidal response to challenge infection arises in the bladder.

Despite numerous studies of macrophage ontogeny and function in many organs, the developmental origin and role of bladder macrophages are largely unknown. Here, we investigated this poorly understood compartment in homeostasis and a highly inflammatory infectious disease, UTI. A single macrophage population of yolk sac and HSC origin seeds the developing bladder; however, the yolk sac macrophage pool is ultimately replaced at some point after birth. After birth, two subsets, MacM and MacL, arise in the tissue, localizing to the muscle and the lamina propria, respectively. These subsets share similar developmental origin, in that they are primarily HSC-derived and, in adulthood, display a very slow turnover by Ly6C+ monocytes in the steady state. Their distinct transcriptomics support the idea that they play different roles in the bladder, at least in the context of infection. The MacM subset is poised to take up bacteria or potentially infected dying host cells, while polarizing toward a more alternatively activated profile during UTI. MacL macrophages express a profile with greater potential for the induction of inflammation and, whether due to direct consequences of this inflammation or potentially due to loss of the urothelium, undergo pronounced cell death during UTI.

In adult animals, steady-state tissue-resident macrophages are a mix of embryonic and adult monocyte-derived macrophages, with the exception of brain microglia (8, 14). The contributions from embryonic macrophages and circulating adult monocytes to the adult bladder macrophage compartment are similar to that of the lung and kidney (7, 11, 77). Although two macrophage subsets reside in the adult bladder, only a single LYVE1+CX3CR1+ macrophage population was identified in embryonic and newborn bladders. As the bladder is fully formed in newborn mice (78), it is unlikely that macrophage subsets arise to meet the needs of a new structure, as is the case for peritubular macrophages in the testis (41). Rather, although all structures are present, embryonic or prenatal bladder tissue demands are likely distinct from postnatal tissue remodeling in very young mice. For example, in the first weeks after birth, bladder macrophages may support urothelial cells undergoing increased proliferation to establish the three layers of urothelium in adult bladders (79). As these adult tissue niches become fully mature, they may provide different growth or survival factors, driving functional macrophage specialization in discrete locations in the tissue.

In the lung, spleen, BM, and liver, a subpopulation of pro-resolving macrophages are present that phagocytize blood-borne cellular material to maintain tissue homeostasis (66). These macrophages express Mrc1 (encoding for CD206), CD163, and Timd4 (encoding TIM4) (66). MacM macrophages likely represent this subpopulation in the bladder, as they expressed higher levels of genes associated with a pro-resolving phenotype, including the efferocytic receptor TIM4, CD206, and CD163. It is also possible that, similar to muscularis macrophages in the gut, MacM macrophages interact with neurons to control muscle contraction in the bladder and limit neuronal damage during infection (80, 81). By contrast, up-regulated pathways in the MacL subset, in combination with their localization under the urothelium, suggest that, similar to intestinal macrophages, they may regulate T cell responses to bladder microbiota or support urothelial cell integrity (82, 83).

Although it was somewhat unexpected, given that the MacM macrophage subset is located farther from the lumen and urothelium, where infection takes place, we favor the conclusion that MacM macrophages contain more bacteria because they are programmed to do so. This conclusion is supported by the higher expression of genes associated with complement, endocytosis, and phagosome pathways in the MacM subset. It is possible, although challenging to empirically demonstrate, that the MacM subset recognizes dying neutrophils, or even dying MacL macrophages, that have phagocytosed bacteria. We may also consider that, between the subsets, the rate at which bacteria are killed is different, UPEC may survive better in MacM macrophages, MacL macrophages may die after bacterial uptake, the near-luminal location of MacL macrophages may result in their disproportionate sloughing, or even that MacL macrophages break down phagocytosed content better. Additional genetic and knockout models would be needed to address these possibilities.

Significant numbers of MacL macrophages died in the first hours following infection, reflecting their enriched apoptosis pathway. The reduced numbers of both macrophage subsets in protamine sulfate-treated mice suggest that alterations in the urothelium may affect macrophage survival, although we cannot rule out the fact that protamine sulfate directly kills macrophages. Exfoliation induced by protamine sulfate is not comparable to infection, as protamine sulfate induces a rapid, large increase in trans-urothelial conductance (71), suggesting that it induces major disruptions in the urothelium. Protamine sulfate can also suppress cytokine activity and the inflammatory response in the bladder compared to UPEC infection (84). This severe disruption of the urothelium may lead to inadequate supplies of oxygen, nutrients, or survival factors, all of which would be detrimental to macrophage survival. It is less likely that bacteria induce macrophage death as only a small, and importantly equivalent, proportion of both subsets were infected. Instead, MacL macrophage death may be an important step to initiate immune responses to UTI. In the liver, Kupffer cell death by necroptosis during Listeria monocytogenes infection induces recruitment of monocytes, which, in turn, phagocytose bacteria (85). Here, macrophage depletion before challenge infection resulted in decreased infiltration of monocytes, likely due to diminished numbers of these cells in circulation, and fewer eosinophils; however, bacterial burden was also decreased. This suggests that macrophage-mediated immune cell recruitment is not their primary function in the bladder. Infiltration of inflammatory cells is not the only way macrophage cell death regulates infection, however. For instance, pyroptotic macrophages can entrap live bacteria and facilitate their elimination by neutrophils in vivo (86). As MacM macrophages express genes regulating iron metabolism, limiting iron to UPEC would also be a plausible mechanism to control bacterial growth (87).

In the steady state, tissue-resident macrophages can self-maintain locally by proliferation, with minimal input of circulating monocytes (9, 88). By contrast, under inflammatory conditions, resident macrophages are often replaced by monocyte-derived macrophages (85, 8890). Monocytes will differentiate into self-renewing functional macrophages if the endogenous tissue-resident macrophages are depleted or are absent (91, 92). Our results show that UPEC infection induces sufficient inflammation to foster infiltration and differentiation of newly recruited monocytes. It is likely, even, that greater macrophage replacement occurs than we actually measured, as we used a single 4OHT pulse in CCR2CreERT2 Rosa26tdTomato mice 24 hours before infection; however, these cells infiltrate infected bladders over several days. These experiments do not rule out a role for local proliferation in the bladder during UTI, but experiments to test this must be able to distinguish infiltrated monocytes that have already differentiated into tissue macrophages from bona fide tissue-resident macrophages when assessing proliferating cells. These data do support, however, the fact that infiltrating monocytes remain in the tissue, integrated into the resident macrophage pool, after tissue resolution.

Recruited monocyte-derived macrophages can behave differently than resident macrophages when activated, such as in the lung. Gamma herpes virus induces alveolar macrophage replacement by regulatory monocytes expressing higher levels of Sca-1 and MHC II (93). These post-infected mice have reduced perivascular and peribronchial inflammation and inflammatory cytokines, and fewer eosinophils compared to mock-infected mice when exposed to house dust mite to induce allergic asthma (93). Alveolar macrophages of mice infected with influenza virus are replaced by pro-inflammatory monocyte-derived macrophages. At 30 days PI, influenza-infected mice have more alveolar macrophages and increased production of IL-6 when challenged with S. pneumoniae compared to mock-infected mice, leading to fewer deaths (90). Although mechanisms regulating the phenotype of monocyte-derived macrophages are not known, the time of residency in the tissue and the nature of subsequent insults likely influence these cells. The longer that recruited macrophages reside in tissue, the more similar they become to tissue-resident macrophages and no longer provide enhanced protection to subsequent tissue injury (89, 90). In contrast to these studies in the lung, we found that elimination of macrophages, including those recruited during primary infection, led to improved bacterial clearance during secondary challenge, although it is not clear what the long-term consequences on bladder homeostasis might be when a more inflammatory type 1 immune response arises during infection.

Overall, our results demonstrate that two unique subsets of macrophages reside in the bladder. During UTI, these cells respond differently, and a proportion of the population dies. Thus, a first UPEC infection induces replacement of resident macrophage subsets by monocyte-derived cells. When sufficient numbers of MacM macrophages, composed of resident and replaced cells, are depleted, improved bacterial clearance follows, suggesting a major role of this subset in directing the immune response to challenge infection. While these findings greatly improve our understanding of this important immune cell type, much remains to be uncovered, such as the signals and niches that contribute to the establishment of two subsets of bladder-resident macrophages, their roles in the establishment and maintenance of homeostasis, and whether parallel populations and functions exist in human bladder tissue.

This study was conducted using a preclinical mouse model and transgenic mouse strains in controlled laboratory experiments to investigate the origin, maintenance, and function of bladder-resident macrophages in homeostasis and bacterial infection. At the onset of this study, our objective was to understand how bladder-resident macrophages negatively affect the development of adaptive immunity to UTI. Having found two resident macrophage subsets in the course of this work, our objectives were to determine whether these subsets have similar origins and homeostatic maintenance and whether they play divergent roles in response to primary or challenge infection. Mice were assigned to groups upon random partition into cages. In all experiments, a minimum of 2 and a maximum of 10 mice (and more typically 3 to 6 mice per experiment) made up an experimental group and all experiments were repeated two to three times. Sample size was based on our previous work and was not changed in the course of the study. In some cases, n was limited by the number of developing embryos available from timed pregnancies. Data collection is detailed below. Data from all repetitions were pooled before any statistical analysis. As determined a priori, all animals with abnormal kidneys (atrophied, enlarged, and white in color) at the time of sacrifice were excluded from all analyses, as we have observed that abnormal kidneys negatively affect resolution of infection. End points were determined before the start of experiments and researchers were not blinded to experimental groups.

All animals used in this study had free access to standard laboratory chow and water at all times. We used female C57BL/6J mice 7 to 8 weeks old from Charles River, France. Female CX3CR1GFP/+ mice 7 to 8 weeks old were bred in-house. CX3CR1GFP/GFP mice, used to maintain our hemizygous colony, were a gift from F. Chretien (Institut Pasteur). Cdh5-CreERT2 Rosa26tdTomato mice were crossed to CX3CR1GFP mice, producing Cdh5-CreERT2.Rosa26tdTomato.CX3CR1GFP mice at Centre dImmunologie de Marseille-Luminy. In Cdh5-CreERT2.Rosa26tdTomato.CX3CR1GFP mice, cells expressing the CX3CR1 receptor are constitutively GFP+, and treatment with 4OHT conditionally labels hemogenically active endothelial cells (42). We used female and male Cdh5-CreERT2.Rosa26tdTomato.CX3CR1GFP mice 8 to 11 weeks old, at E16.5, and newborns. Flt3Cre.Rosa26YFP mice were a gift from E.G.P. (Institut Pasteur). CCR2/ mice were a gift from M. Lecuit (Institut Pasteur). CCR2creERT2BB mice were a gift from B. Becher (University of Zurich) via S. Amigorena (Institut Curie). CCR2creERT2BB male mice were crossed to Rosa26tdTomato females to obtain CCR2creERT2BB-tdTomato mice at Institut Pasteur. We used female CCR2creERT2BB-tdTomato mice 7 to 8 weeks old. Additional details of the mouse strains used, including JAX and MGI numbers, are listed in table S1. Mice were anesthetized by injection of ketamine (100 mg/kg) and xylazine (5 mg/kg) and euthanized by carbon dioxide inhalation. Experiments were conducted at Institut Pasteur in accordance with approval of protocol number 2016-0010 and dha190501 by the Comit dthique en exprimentation animale Paris Centre et Sud (the ethics committee for animal experimentation), in application of the European Directive 2010/63 EU. Experiments with Cdh5-CreERT2 mice were performed in the laboratory of M. Bajenoff, Centre dImmunologie de Marseille-Luminy, in accordance with national and regional guidelines under protocol number 5-01022012 following review and approval by the local animal ethics committee in Marseille, France.

Antibodies, reagents, and software used in this study are listed in tables S2, S3, and S4, respectively.

Samples were acquired on a BD LSRFortessa using DIVA software (v8.0.1), and data were analyzed by FlowJo (Treestar) software, including the plugins for downsampling, tSNE, and UMAP (version 10.0). The analysis of bladder and blood was performed as described previously (23). Briefly, bladders were dissected and digested in buffer containing Liberase (0.34 U/ml) in phosphate-buffered saline (PBS) at 37C for 1 hour with manual agitation every 15 min. Digestion was stopped by adding PBS supplemented with 2% fetal bovine serum (FBS) and 0.2 M EDTA [fluorescence-activated cell sorting (FACS) buffer]. Fc receptors in single-cell suspensions were blocked with anti-mouse CD16/CD32 and stained with antibodies. Total cell counts were determined by addition of AccuCheck counting beads to a known volume of sample after staining, just before cytometer acquisition. To determine cell populations in the circulation, whole blood was incubated with BD PharmLyse and stained with antibodies (table S2). Total cell counts were determined by the addition of AccuCheck counting beads to 10 l of whole blood in 1-step Fix/Lyse Solution.

For intracellular staining, single-cell suspensions were resuspended in 1 ml of Golgi stop protein transport inhibitor, diluted (1:1500) in RPMI with 10% FBS, 1% sodium pyruvate, 1 Hepes, 1 nonessential amino acid, 1% penicillin-streptomycin, phorbol 12-myristate 13-acetate (50 ng/ml), and ionomycin (1 g/ml), and incubated for 4 hours at 37C. Samples were washed once with FACS buffer, and Fc receptors blocked with anti-mouse CD16/CD32. Samples were stained with antibodies listed in table S2 against surface markers and fixed and permeabilized with 1 fixation and permeabilization buffer and incubated at 4C for 40 to 50 min protected from light. After incubation, samples were washed two times with 1 permeabilization and wash buffer from the transcription factor buffer kit and stained with antibodies against IFN- and the transcriptional factors RORT, GATA3, T-bet, and FoxP3 (table S2), diluted in 1 permeabilization and wash buffer at 4C for 40 to 50 min protected from light. Last, samples were washed two times with 1 permeabilization and wash buffer and resuspended in FACS buffer. Total cell counts were determined by addition of counting beads to a known volume of sample after staining, just before cytometer acquisition.

Whole bladders were fixed with 4% paraformaldehyde (PFA) in PBS for 1 hour and subsequently washed with PBS. Samples were then dehydrated in 30% sucrose in PBS for 24 hours. Samples were cut transversally and embedded in optimal cutting temperature compound, frozen, and sectioned at 30 m. Sections were blocked for 1 hour with blocking buffer [3% bovine serum albumin (BSA) + 0.1% Triton X-100 + donkey serum (1:20) in PBS] and washed three times. Immunostaining was performed using F4/80, LYVE1 antibodies, or polyclonal asymmetrical unit membrane antibodies, recognizing uroplakins [gift from X.-R. Wu, NYU School of Medicine, (76)] (1:200) in staining buffer (0.5% BSA + 0.1% Triton X-100 in PBS) overnight. Sections were washed and stained with phalloidin (1:350) and secondary antibodies (1:2000) in staining buffer for 4 hours. Last, sections were washed and stained with 4,6-diamidino-2-phenylindole. Confocal images were acquired on a Leica SP8 confocal microscope. Final image processing was done using Fiji (version 2.0.0-rc-69/1.52p) and Icy software (v1.8.6.0).

Fate mapping of Cdh5-CreERT2 mice was performed as described previously (42). Briefly, for reporter recombination in offspring, a single dose of 4OHT supplemented with progesterone (1.2 mg of 4OHT and 0.6 mg of progesterone) was delivered by intraperitoneal injection to pregnant females at E7.5 or E10.5. Progesterone was used to counteract adverse effects of 4OHT on pregnancies. To fate map cells in CCR2creERT2BB-tdTomato mice, a single dose (37.5 g/g) of 4OHT injection was delivered intraperitoneally.

For shielded irradiation, 7- to 8-week-old wild-type female CD45.1 or CD45.2 C57BL6/J mice were anesthetized and dressed in a lab-made lead diaper, which selectively exposed their tail, legs, torso, and head to irradiation, but protected the lower abdomen, including the bladder. Mice were irradiated with 9 gray from an Xstrahl x-ray generator (250 kV, 12 mA) and reconstituted with ~3 107 to 4 107 BM cells isolated from congenic (CD45.1) wild-type mice or CD45.2 CCR2/ mice.

Samples were obtained from the whole bladders of nave and 6-week post-infected female C57BL/6J mice. Using FACS, four separate sorts were performed to generate biological replicates, and each sort was a pool of 10 mouse bladders. Macrophage subsets were FACS-purified into 350 l of RLT Plus buffer from the RNeasy Micro Kit plus (1:100) -mercaptoethanol. Total RNA was extracted using the RNeasy Micro Kit following the manufacturers instructions. Directional libraries were prepared using the Smarter Stranded Total RNA-Seq kit Pico Input Mammalian following the manufacturers instructions. The quality of libraries was assessed with the DNA-1000 kit on a 2100 Bioanalyzer, and quantification was performed with Quant-It assays on a Qubit 3.0 fluorometer. Clusters were generated for the resulting libraries with Illumina HiSeq SR Cluster Kit v4 reagents. Sequencing was performed with the Illumina HiSeq 2500 system and HiSeq SBS kit v4 reagents. Runs were carried out over 65 cycles, including seven indexing cycles, to obtain 65-bp single-end reads. Sequencing data were processed with Illumina Pipeline software (Casava version 1.9). Reads were cleaned of adapter sequences and low-quality sequences using cutadapt version 1.11. Only sequences of at least 25 nucleotides in length were considered for further analysis, and the five first bases were trimmed following the library manufacturers instructions. STAR version 2.5.0a (94), with default parameters, was used for alignment on the reference genome (Mus musculus GRCm38_87 from Ensembl version 87). Genes were counted using featureCounts version 1.4.6-p3 (95) from Subreads package (parameters: -t exon -g gene_id -s 1). Count data were analyzed using R version 3.4.3 and the Bioconductor package DESeq2 version 1.18.1 (96). The normalization and dispersion estimation were performed with DESeq2 using the default parameters, and statistical tests for differential expression were performed applying the independent filtering algorithm. A generalized linear model was set to test for the differential expression among the four biological conditions. For each pairwise comparison, raw P values were adjusted for multiple testing according to the Benjamini and Hochberg procedure and genes with an adjusted P value lower than 0.05 were considered differentially expressed. Count data were transformed using variance stabilizing transformation to perform samples clustering and PCA plot. The PCA was performed on the variance-stabilized transformed count matrix that was adjusted for the batch/replicate effect using the limma R package version 3.44.3.

To perform pathway analysis, gene lists of DEGs were imported in the Cytoscape software (version 3.7.2), and analyses were performed using the ClueGO application with the Kyoto Encyclopedia of Genes and Genomes as the database. Significant pathways were selected using the threshold criteria Q < 0.05; terms > 3 genes; % genes/term > 3; 0.4.

We used the human UPEC cystitis isolate UTI89 engineered to express the fluorescent proteins RFP or GFP and antibiotic-resistant cassettes to either kanamycin (UPEC-RFP) or ampicillin (UPEC-GFP) to infect animals for flow cytometric and bacterial burden analyses (23). We used the nonfluorescent parental strain UTI89 for confocal imaging experiments and flow cytometric experiments with CCR2CreERT2 Rosa26tdTomato mice (97). To allow expression of type 1 pili, necessary for infection (98), bacteria cultures were grown statically in Luria-Bertani broth medium for 18 hours at 37C in the presence of antibiotics [kanamycin (50 g/ml) or ampicillin (100 g/ml)]. Primary and challenge UTI were induced in mice as previously described (23, 99). For challenge infection, urine was collected twice a week, for 4 weeks, to follow the presence of bacteria in the urine. Once there were no UTI89-RFP bacteria in the urine, mice were challenged with UTI89-GFP bacteria and euthanized 24 hours after challenge infection. To calculate CFU, bladders were aseptically removed and homogenized in 1 ml of PBS. Serial dilutions were plated on LB agar plates with antibiotics, as required. For in vitro infections, macrophage subsets were sorted from a pool of 10 bladders of nave female C57BL/6J 7- to 8-week-old mice using FACS and 2 103 cells were incubated with 2 104 CFU of UPEC-RFP for 2 hours at 37C. Cells were acquired on a BD LSRFortessa using DIVA software (v8.0.1) and data were analyzed by FlowJo (Treestar) software (version 10.0).

Seven- to 8-week-old wild-type female C57BL6/J mice were anesthetized and instilled intravesically with 50 l of protamine sulfate (50 mg/ml) diluted in PBS and euthanized 5 hours after instillation for analysis.

To produce anti-CSF1R antibody, the hybridoma cell line AFS98 (gift from M. Merad at Icahn School of Medicine at Mount Sinai) (100) was cultured in disposable reactor cell culture flasks for 14 days, and antibodies were purified with disposable PD10 desalting columns. To deplete macrophages, wild-type C57BL/6 mice received intravenous injection of anti-CSF1R antibody (2 mg/ml) diluted in PBS. Animals received two or three intravenous injections, on consecutive days, of anti-CSF1R antibody or PBS. To deplete macrophages with a final concentration of 500 g of anti-CSF1R, we administered 250 g per mouse on day 1 and 250 g per mouse on day 2. To deplete macrophages with a final concentration of 800 g of anti-CSF1R, we administered 400 g per mouse on day 1, 200 g per mouse on day 2, and 200 g per mouse on day 3 to minimize the impact on circulating monocytes.

To quantify macrophage subsets in bladder tissue, six to seven images were randomly acquired of each of the areas of the muscle and lamina propria per mouse in wild-type C57BL/6 female mice with 40 magnification in an SP8 Leica microscope. Maximum intensity Z-projections were performed, and macrophage subsets were counted using Icy software (v1.8.6.0). To quantify urothelial exfoliation and tissue edema, images from whole bladder cross sections were acquired using 20 magnification in an SP8 Leica microscope. Maximum intensity Z-projections were performed, the urothelium was delimited, and mean fluorescence intensity of uroplakin staining was measured using Fiji (v1.51j) software. To quantify tissue edema, the lamina propria was delimited and the area was measured using Fiji software (v1.51j).

Statistical analysis was performed in GraphPad Prism 8 (GraphPad, USA) for Mac OS X applying the nonparametric Wilcoxon test for paired data or the nonparametric Mann-Whitney test for unpaired data in the case of two group comparisons. In the case that more than two groups were being compared or to correct for comparisons made within an entire analysis or experiment, calculated P values were corrected for multiple testing with the false discovery rate (FDR) method (https://jboussier.shinyapps.io/MultipleTesting/) to determine the FDR-adjusted P value. All calculated P values are shown in the figures, and those that met the criteria for statistical significance (P < 0.05) are denoted with red text.

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In the United States alone, one person dies every 36 seconds from cardiovascular disease. Globally, it is also the leading cause of death, claiming over 17 million lives each year. In cases of severe illness, heart transplants have shown great promise in increasing the life expectancy of patients with heart disease. About 75% of heart transplant recipients survive for 5 more years and about 56% survive for 10 more years. However, the average wait times for heart transplants are long, often exceeding 6 months, and some patients simply cannot afford to wait that long.

Therefore, scientists tend to refer to other modes of treatment which rely on managing chronic symptoms, such as hypertension (high blood pressure), diabetes mellitus, obesity, and high cholesterol. This approach, however, does not address the root cause of the problem, which is impaired heart functioning. Since heart cells do not have a mechanism to replace damaged tissue, scientists have become increasingly excited about the possibility of repairing or replacing damaged heart tissue using stem cells (unique cells that have the ability to divide for an extended period of time and differentiate into specialized cells, such as cardiac cells or nerve cells).

Regenerative medicine has been a topic of excitement among researchers for decades. In 1999, Anthony Atala, director of the Wake Forest Institute for Regenerative Medicine, was the first to implant lab-grown organs into several patients between 4 and 19 years old. In his method, he obtained bladder cells from the children and coaxed those cells into dividing on a scaffold (a structure that mimics the normal organ). The engineered bladders functioned normally and no ill effects were reported. Pretty much I was able to live a normal life after, said Luke, one of Atalas patients.

More recently, Yoshiki Sawa, a professor of cardiovascular surgery at the University of Osakas medical school, and his team of Japanese researchers successfully transplanted lab-grown cardiac muscles into a human patient. The researchers first extracted adult stem cells from the patients blood or skin and genetically reprogrammed them into induced pluripotent stem (iPS) cells. They were then coaxed into 0.1-millimeter-thick sheets of cardiac tissue and grafted onto the diseased human hearts. According to Sawa, the cells do not seem to integrate into the heart tissue but rather release growth factors (proteins) that help regenerate blood vessels in the damaged muscle tissue and improve cardiac function. The team has conducted an operation on a patient in January 2020, marking the worlds first transplant of cardiac muscle cells.

The United States is also home to major breakthroughs in regenerative medicine. For decades, scientists have utilized embryonic stem cells to engineer heart muscle cells that are able to maintain synchronous breathing in a dish for hours. Despite this major feat, the creation of a working heart called for a more sophisticated technique. Doris Taylor, director of regenerative medicine research at the Texas Heart Institute (THI), has grown in her lab over 100 ghost hearts using protein scaffolds. She creates these scaffolds by first obtaining an animal heart and then decellularizing it by pumping a detergent through its blood vessels to strip away lipids, DNA, soluble proteins, sugars and almost all the other cellular material from the heart, leaving only a pale mesh of collagen, laminins, and the extracellular matrix. This heart does not necessarily have to be a human heart. She often finds pig hearts to be promising tissue because of their considerable safety and unlimited supply. She then recellularizes the heart by injecting it with millions of stem cells and attaching it to artificial lungs and a blood pump. Although her technique has only been used so far for growing animal hearts, she believes that it will eventually be used to create human heart transplants, thus, revolutionizing cardiovascular surgery and putting an end to organ shortage and anti-rejection drugs.

These groundbreaking results in regenerative medicine altogether have taken years of painstaking research to achieve. Taylor believes that her research is exceptionally close to building a working, human-sized heart, and Sawa says that his technique of grafting healthy cardiac muscle sheets onto the patients diseased heart tissue has already helped one of his patients move out of intensive care in just a few days. As the researchers gain more knowledge and get closer to the solution, however, they encounter more challenging obstacles. Sawa, for instance, has found that grafted cells do not always beat in synchrony. Researchers are also split on how these grafts work. On the other hand, investigating the best way to deliver cells still remains a challenge in Taylors research.

Stem cell research in tissue engineering could save millions of lives around the world; therefore, Taylor believes that a coordinated approach among the researchers, clinicians, industry, regulatory bodies and, finally, society should be invigorated to catapult the field forward. For instance, the Twenty-first Century Cures Act can help advance her work by facilitating cooperation among experts and regulatory bodies, providing for accelerated approvals for therapeutic tools in regenerative medicine, and improving the regulation of biologics products. She also maintains that tissue engineering efforts remain poorly funded and believes that more resources must be allocated before her studies can come to life. There is a lot of dependence on societal benevolence, she said. In an interview with RedMedNet, she also said that intense collaboration on a national and an international level is crucial and should be a priority, even though it could be challenging due to scheduling issues and differences in time zones.

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Breakthroughs in Stem Cell Based Treatment of Heart Disease - The Connecticut College Voice

Cardiac Stem Cells Comments Off on Breakthroughs in Stem Cell Based Treatment of Heart Disease – The Connecticut College Voice | November 25th, 2020

This persuasive Exosome Therapeutic Market report makes to focus on the more important aspects of the market like what the market recent trends are. The company profiles of all the key players and brands that are dominating the Exosome Therapeutic Market have been taken into consideration here. The market analysis and competitor analysis helps the firm in determining the range in terms of sizes, colors, designs, and prices, etc within which its products are to be offered to the consumers. Important industry trends, market size, market share estimates are analyzed and mentioned in the Exosome Therapeutic Market business report.

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Market Analysis and Insights:Global Exosome Therapeutic Market

Exosome therapeutic market is expected to gain market growth in the forecast period of 2019 to 2026. Data Bridge Market Research analyses that the market is growing with a CAGR of 21.9% in the forecast period of 2019 to 2026 and expected to reach USD 31,691.52 million by 2026 from USD 6,500.00 million in 2018. Increasing prevalence of lyme disease, chronic inflammation, autoimmune disease and other chronic degenerative diseases are the factors for the market growth.

The major players covered in theExosome Therapeutic Marketreport areevox THERAPEUTICS, EXOCOBIO, Exopharm, AEGLE Therapeutics, United Therapeutics Corporation, Codiak BioSciences, Jazz Pharmaceuticals, Inc., Boehringer Ingelheim International GmbH, ReNeuron Group plc, Capricor Therapeutics, Avalon Globocare Corp., CREATIVE MEDICAL TECHNOLOGY HOLDINGS INC., Stem Cells Group among other players domestic and global.Exosome therapeutic market share data is available for Global, North America, Europe, Asia-Pacific, and Latin America separately. DBMR analysts understand competitive strengths and provide competitive analysis for each competitor separately.

Get Full TOC, Tables and Figures of Market Report @https://www.databridgemarketresearch.com/toc/?dbmr=global-exosome-therapeutic-market&rp

Exosomes are used to transfer RNA, DNA, and proteins to other cells in the body by making alteration in the function of the target cells. Increasing research activities in exosome therapeutic is augmenting the market growth as demand for exosome therapeutic has increased among healthcare professionals.

Increased number of exosome therapeutics as compared to the past few years will accelerate the market growth. Companies are receiving funding for exosome therapeutic research and clinical trials. For instance, In September 2018, EXOCOBIO has raised USD 27 million in its series B funding. The company has raised USD 46 million as series a funding in April 2017. The series B funding will help the company to set up GMP-compliant exosome industrial facilities to enhance production of exosomes to commercialize in cosmetics and pharmaceutical industry.

Increasing demand for anti-aging therapies will also drive the market. Unmet medical needs such as very few therapeutic are approved by the regulatory authority for the treatment in comparison to the demand in global exosome therapeutics market will hamper the market growth market. Availability of various exosome isolation and purification techniques is further creates new opportunities for exosome therapeutics as they will help company in isolation and purification of exosomes from dendritic cells, mesenchymal stem cells, blood, milk, body fluids, saliva, and urine and from others sources. Such policies support exosome therapeutic market growth in the forecast period to 2019-2026.

This exosome therapeutic market report provides details of market share, new developments, and product pipeline analysis, impact of domestic and localised market players, analyses opportunities in terms of emerging revenue pockets, changes in market regulations, product approvals, strategic decisions, product launches, geographic expansions, and technological innovations in the market. To understand the analysis and the market scenario contact us for anAnalyst Brief, our team will help you create a revenue impact solution to achieve your desired goal.

Global Exosome Therapeutic Market Scope and Market Size

Global exosome therapeutic market is segmented of the basis of type, source, therapy, transporting capacity, application, route of administration and end user. The growth among segments helps you analyse niche pockets of growth and strategies to approach the market and determine your core application areas and the difference in your target markets.

Based on type, the market is segmented into natural exosomes and hybrid exosomes. Natural exosomes are dominating in the market because natural exosomes are used in various biological and pathological processes as well as natural exosomes has many advantages such as good biocompatibility and reduced clearance rate compare than hybrid exosomes.

Exosome is an extracellular vesicle which is released from cells, particularly from stem cells. Exosome functions as vehicle for particular proteins and genetic information and other cells. Exosome plays a vital role in the rejuvenation and communication of all the cells in our body while not themselves being cells at all. Research has projected that communication between cells is significant in maintenance of healthy cellular terrain. Chronic disease, age, genetic disorders and environmental factors can affect stem cells communication with other cells and can lead to distribution in the healing process. The growth of the global exosome therapeutic market reflects global and country-wide increase in prevalence of autoimmune disease, chronic inflammation, Lyme disease and chronic degenerative diseases, along with increasing demand for anti-aging therapies. Additionally major factors expected to contribute in growth of the global exosome therapeutic market in future are emerging therapeutic value of exosome, availability of various exosome isolation and purification techniques, technological advancements in exosome and rising healthcare infrastructure.

Rising demand of exosome therapeutic across the globe as exosome therapeutic is expected to be one of the most prominent therapies for autoimmune disease, chronic inflammation, Lyme disease and chronic degenerative diseases treatment, according to clinical researches exosomes help to processes regulation within the body during treatment of autoimmune disease, chronic inflammation, Lyme disease and chronic degenerative diseases. This factor has increased the research activities in exosome therapeutic development around the world for exosome therapeutic. Hence, this factor is leading the clinician and researches to shift towards exosome therapeutic. In the current scenario the exosome therapeutic are highly used in treatment of autoimmune disease, chronic inflammation, Lyme disease and chronic degenerative diseases and as anti-aging therapy as it Exosomes has proliferation of fibroblast cells which is significant in maintenance of skin elasticity and strength.

Based on source, the market is segmented into dendritic cells, mesenchymal stem cells, blood, milk, body fluids, saliva, urine and others. Mesenchymal stem cells are dominating in the market because mesenchymal stem cells (MSCs) are self-renewable, multipotent, easily manageable and customarily stretchy in vitro with exceptional genomic stability. Mesenchymal stem cells have a high capacity for genetic manipulation in vitro and also have good potential to produce. It is widely used in treatment of inflammatory and degenerative disease offspring cells encompassing the transgene after transplantation.

Based on therapy, the market is segmented into immunotherapy, gene therapy and chemotherapy. Chemotherapy is dominating in the market because chemotherapy is basically used in treatment of cancer which is major public health issues. The multidrug resistance (MDR) proteins and various tumors associated exosomes such as miRNA and IncRNA are include in in chemotherapy associated resistance.

Based on transporting capacity, the market is segmented into bio macromolecules and small molecules. Bio macromolecules are dominating in the market because bio macromolecules transmit particular biomolecular information and are basically investigated for their delicate properties such as biomarker source and delivery system.

Based on application, the market is segmented into oncology, neurology, metabolic disorders, cardiac disorders, blood disorders, inflammatory disorders, gynecology disorders, organ transplantation and others. Oncology segment is dominating in the market due to rising incidence of various cancers such as lung cancer, breast cancer, leukemia, skin cancer, lymphoma. As per the National Cancer Institute, in 2018 around 1,735,350 new cases of cancer was diagnosed in the U.S. As per the American Cancer Society Inc in 2019 approximately 268,600 new cases of breast cancer diagnosed in the U.S.

Based on route of administration, the market is segmented into oral and parenteral. Parenteral route is dominating in the market because it provides low drug concentration, free from first fast metabolism, low toxicity as compared to oral route as well as it is suitable in unconscious patients, complicated to swallow drug etc.

The exosome therapeutic market, by end user, is segmented into hospitals, diagnostic centers and research & academic institutes. Hospitals are dominating in the market because hospitals provide better treatment facilities and skilled staff as well as treatment available at affordable cost in government hospitals.

Exosome therapeutic Market Country Level Analysis

The global exosome therapeutic market is analysed and market size information is provided by country by type, source, therapy, transporting capacity, application, route of administration and end user as referenced above.

The countries covered in the exosome therapeutic market report are U.S. and Mexico in North America, Turkey in Europe, South Korea, Australia, Hong Kong in the Asia-Pacific, Argentina, Colombia, Peru, Chile, Ecuador, Venezuela, Panama, Dominican Republic, El Salvador, Paraguay, Costa Rica, Puerto Rico, Nicaragua, Uruguay as part of Latin America.

Country Level Analysis, By Type

North America dominates the exosome therapeutic market as the U.S. is leader in exosome therapeutic manufacturing as well as research activities required for exosome therapeutics. At present time Stem Cells Group holding shares around 60.00%. In addition global exosomes therapeutics manufacturers like EXOCOBIO, evox THERAPEUTICS and others are intensifying their efforts in China. The Europe region is expected to grow with the highest growth rate in the forecast period of 2019 to 2026 because of increasing research activities in exosome therapeutic by population.

The country section of the report also provides individual market impacting factors and changes in regulation in the market domestically that impacts the current and future trends of the market. Data points such as new sales, replacement sales, country demographics, regulatory acts and import-export tariffs are some of the major pointers used to forecast the market scenario for individual countries. Also, presence and availability of global brands and their challenges faced due to large or scarce competition from local and domestic brands, impact of sales channels are considered while providing forecast analysis of the country data.

Huge Investment by Automakers for Exosome Therapeutics and New Technology Penetration

Global exosome therapeutic market also provides you with detailed market analysis for every country growth in pharma industry with exosome therapeutic sales, impact of technological development in exosome therapeutic and changes in regulatory scenarios with their support for the exosome therapeutic market. The data is available for historic period 2010 to 2017.

Competitive Landscape and Exosome Therapeutic Market Share Analysis

Global exosome therapeutic market competitive landscape provides details by competitor. Details included are company overview, company financials, revenue generated, market potential, investment in research and development, new market initiatives, global presence, production sites and facilities, company strengths and weaknesses, product launch, product trials pipelines, concept cars, product approvals, patents, product width and breadth, application dominance, technology lifeline curve. The above data points provided are only related to the companys focus related to global exosome therapeutic market.

Many joint ventures and developments are also initiated by the companies worldwide which are also accelerating the global exosome therapeutic market.

For instance,

Partnership, joint ventures and other strategies enhances the company market share with increased coverage and presence. It also provides the benefit for organisation to improve their offering for exosome therapeutics through expanded model range.

Customization Available:Global Exosome Therapeutic Market

Data Bridge Market Researchis a leader in advanced formative research. We take pride in servicing our existing and new customers with data and analysis that match and suits their goal. The report can be customised to include price trend analysis of target brands understanding the market for additional countries (ask for the list of countries), clinical trial results data, literature review, refurbished market and product base analysis. Market analysis of target competitors can be analysed from technology-based analysis to market portfolio strategies. We can add as many competitors that you require data about in the format and data style you are looking for. Our team of analysts can also provide you data in crude raw excel files pivot tables (Factbook) or can assist you in creating presentations from the data sets available in the report.

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Exosome Therapeutic Market to Record Escalated Growth in Revenue During the Forecast Period || Major Gaints Jazz Pharmaceuticals, Inc., Boehringer...

Cardiac Stem Cells Comments Off on Exosome Therapeutic Market to Record Escalated Growth in Revenue During the Forecast Period || Major Gaints Jazz Pharmaceuticals, Inc., Boehringer… | November 23rd, 2020