Contraryto previous research, some patients with atopy who undergo allogeneichematopoietic cell transplantation (AHCT) may be cured even if the donor isatopic, according to research published in Bone Marrow Transplantation.

Investigatorsaimed to determine whether atopic disease could be cured or transferred fromdonor to recipient, as there is evidence, though unconfirmed, that AHCT caneither transfer atopy from donor to recipient when the donor has the conditionand the recipient does not (D+R-), or cure atopy in a recipient with thecondition when the donor does not have it (D-R+).

Of the 54 participants including in the study, the median age at transplant was 46 years (range, 4-64 years), one-third of patients had acute myeloid leukemia, and 50% had a human leukocyte antigenmatched sibling. The median donor age at transplant was 34 years (range, 7-60 years).

Amongall AHCT procedures included in this study, 18 (33%) had an atopic donor andrecipient (D+R+), 13 (24%) had a nonatopic donor and recipient (D-R-), 11(20.4%) had an atopic donor and nonatopic recipient, and 12 (22.2%) had anonatopic donor and an atopic recipient.

At afollow-up of at least 2-years post-HCT, 7 of 12 (58%) D-R+ patients becamenonatopic, while only 1 of 11 (9%) D+R- patients became atopic. While 11 of 13(85%) D-R- patients remained nonatopic, 11 of 18 (61%) D+R+ patients became nonatopic,as well.

Inconclusion, cure of atopy with HCT occurs in about half patients, albeitprobably not due to the replacement of atopic with nonatopic immune system butpossibly due to an immune reset analogous to that observed in some autoimmunedisease patients after autologous HCT, the authors concluded. They added thatprospective trials are needed to confirm these findings.

Reference

Whiteside S, Chin A, Tripathi G, et al. Curtability and transferability of atopy with allogeneic hematopoietic cell transplantation [published online March 30, 2020]. Bone Marrow Transplant. doi: 10.1038/s41409-020-0876-7

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Atopy: Weighing Risk With the Possibility of Cure Using Hematopoietic Cell Transplantation - Hematology Advisor

Bone Marrow Stem Cells Comments Off on Atopy: Weighing Risk With the Possibility of Cure Using Hematopoietic Cell Transplantation – Hematology Advisor | April 10th, 2020

Abstract

Dyskeratosis congenita is a cancer-prone inherited bone marrow failure syndrome caused by telomere dysfunction. A mouse model recently suggested that p53 regulates telomere metabolism, but the clinical relevance of this finding remained uncertain. Here, a germline missense mutation of MDM4, a negative regulator of p53, was found in a family with features suggestive of dyskeratosis congenita, e.g., bone marrow hypocellularity, short telomeres, tongue squamous cell carcinoma, and acute myeloid leukemia. Using a mouse model, we show that this mutation (p.T454M) leads to increased p53 activity, decreased telomere length, and bone marrow failure. Variations in p53 activity markedly altered the phenotype of Mdm4 mutant mice, suggesting an explanation for the variable expressivity of disease symptoms in the family. Our data indicate that a germline activation of the p53 pathway may cause telomere dysfunction and point to polymorphisms affecting this pathway as potential genetic modifiers of telomere biology and bone marrow function.

TP53 is the gene most frequently mutated in human tumors (1), and germ lineinactivating p53 mutations cause the Li-Fraumeni syndrome of cancer predisposition (2). In addition, accelerated tumorigenesis has been associated with polymorphisms increasing the expression of MDM2 or MDM4, the essential p53 inhibitors (3, 4). Alterations of the p53/MDM2/MDM4 regulatory node are, thus, mainly known to promote cancer. Unexpectedly, however, we recently found that mice expressing p5331, a hyperactive mutant p53 lacking its C terminus, recapitulated the complete phenotype of patients with dyskeratosis congenita (DC) (5).

DC is a telomere biology disorder characterized by the mucocutaneous triad of abnormal skin pigmentation, nail dystrophy, and oral leukoplakia; patients are also at very high risk of bone marrow failure, pulmonary fibrosis, and cancer, especially head and neck squamous cell carcinoma (HNSCC) and acute myeloid leukemia (AML) (6). Patients with DC are known to exhibit disease diversity in terms of age of onset, symptoms, and severity due to the mode of inheritance and causative gene (7, 8). DC is caused by germline mutations in genes encoding key components of telomere biology: the telomerase holoenzyme (DKC1, TERC, TERT, NOP10, and NHP2), the shelterin telomere protection complex (ACD, TINF2, and POT1), telomere capping proteins (CTC1 and STN1), and other proteins interacting with these cellular processes (RTEL1, NAF1, WRAP53, and PARN) (6). Twenty to 30% of affected individuals remain unexplained at the molecular level.

Our finding that p5331/31 mice were remarkable models of DC was initially unexpected for two reasons. First, an increased p53 activity was not expected to cause telomere dysfunction, given the well-accepted notion that p53 acts as the guardian of the genome. However, p53 is now known to down-regulate the expression of many genes involved in genome maintenance (5, 9, 10), and this might actually contribute to its toolkit to prevent tumor formation (11). Second, telomere biology diseases are usually difficult to model in mice because of differences in telomere length and telomerase expression between mice and humans. Mice that lack telomerase exhibited short telomeres only after three or four generations (G3/G4) of intracrosses (12, 13). However, mice with a telomerase haploinsufficiency and a deficient shelterin complex exhibited telomere dysfunction and DC features in a single generation (G1) (14). Because DC features were observed in G1 p5331/31 mice, we supposed that p53 might exert pleiotropic effects on telomere maintenance. Consistent with this, we found that murine p53 down-regulates several genes implicated in telomere biology (5, 9). Because some of these genes were also down-regulated by p53 in human cells (5, 9), our data suggested that an activating p53 mutation might cause features of DC in humans. However, this conclusion remained speculative in the absence of any clinical evidence.

Here, we report the identification of a germline missense mutation in MDM4, encoding an essential and specific negative regulator of p53, in a family presenting some DC-like phenotypic traits. We used a mouse model to demonstrate that this mutation leads to p53 activation, short telomeres, and bone marrow failure. Together, our results provide compelling evidence that a germline mutation affecting a specific p53 regulator may cause DC-like features in both humans and mice.

Family NCI-226 first enrolled in the National Cancer Institute (NCI) inherited bone marrow failure syndrome (IBMFS) cohort in 2008 (Fig. 1A and table S1). At the time, the proband (226-1) was 17 years of age and had a history of neutropenia, bone marrow hypocellularity, vague gastrointestinal symptoms, and chronic pain. His mother (226-4) also had intermittent neutropenia and a hypocellular bone marrow. Notably, his maternal aunt (226-7) had a history of melanoma and died at age 52 because of AML. The maternal aunts daughter (probands cousin, 226-8) had HNSCC at age 27 years, intermittent neutropenia, and bone marrow hypocellularity, while her son (probands cousin, 226-9) was diagnosed with metastatic HNSCC at 42 years of age. The probands father (226-3) was healthy with the exception of hemochromatosis. An IBMFS was suspected on the basis of the family history of cancer and neutropenia. Chromosome breakage for Fanconi anemia was normal, while lymphocyte telomeres were between the 1st and 10th percentiles in the proband and maternal cousin (226-8) (Fig. 1, B and C). The proband was tested for mutations in known DC-causing genes, and a TERT variant (p.W203S) was identified. Unexpectedly, however, the variant was found to be inherited from his father. TERT p.W203S is not present in gnomAD, but it is predicted to be tolerated by MetaSVM (15).

(A) Pedigree of family NCI-226. Arrow indicates proband. Cancer histories include oral squamous cell carcinoma for 226-8 at age 27 years and for 226-9 at age 42 years, and melanoma at 51 years and AML at 52 years for 226-7 (see table S1 for further details). 226-5 had lung cancer at age 69 years. 226-6 had non-Hodgkin lymphoma at age 91 years. In addition, four siblings of 226-6 had cancer: one with breast, two with lung, and one with ovary or uterus (not specified). Sequencing of 226-5, 226-6, 226-7, and 226-9 was not possible because of lack of available DNA. (B and C) Lymphocyte telomere lengths (TL) of study participants. Total lymphocyte telomere lengths are shown and were measured by flow cytometry with in situ hybridization. (B) Graphical depiction of telomere length in relation to age. Four individuals had telomeres measured twice. Legend is in (C). Percentiles (%ile) are based on 400 healthy individuals (50). (C) Age at measurement(s) and telomere length in kilobases. (D) Sequence of the MDM4 RING domain (residues 436 to 490) with secondary structure residues indicated (black boxes). The P-loop motif is highlighted in gray, and the mutated residue in red. (E) The mutant RING domain retains ATP-binding capacity. Wild-type (WT) and mutant (TM) glutathione S-transferase (GST)RING proteins, or GST alone, were incubated with 10 nM ATP and 5 Ci ATP-32P for 10 min at room temperature, filtered through nitrocellulose, and counted by liquid scintillation CPM, counts per minute. Results from two independent experiments. (F) The mutant MDM4 RING domain has an altered capacity to dimerize with the MDM2 RING. Two-hybrid assays were carried out as described (47). -LW, minus leucine and tryptophan; -LWHA, minus leucine, tryptophan, histidine and adenine; OD, optical density. Growth on the -LWHA medium indicates protein interaction, readily observed between MDM2 (M2-BD) and WT MDM4 (M4-AD WT) but faintly visible between MDM2 and MDM4T454M (M4-AD TM). (G) Impact of the mutation in transfected human cells. U2OS cells were transfected with an empty vector (EV) or an expression plasmid encoding a Myc-tagged MDM4 (WT or T454M) protein and then treated or not with cycloheximide (CHX) to inhibit protein synthesis, and protein extracts were immunoblotted with antibodies against Myc, p21, or actin. Bands were normalized to actin, and a value of 1 was assigned to cells transfected with the WT MDM4 expression plasmid (for Myc) or with the empty vector (for p21).

Since the TERT variant did not track with disease inheritance, whole-exome sequencing (WES) was performed to search for a causal gene. The whole-exome data were filtered by maternal autosomal inheritance and revealed three genes with heterozygous missense mutations potentially deleterious according to bioinformatics predictions: MDM4, KRT76, and REM1 (table S2). Given the limited knowledge of the function of KRT76 and REM1, and our prior knowledge of a DC-like phenotype in p5331/31 mice, we chose to focus on the mutation affecting MDM4 because it encodes a major negative regulator of p53. Although the T454M mutation does not affect the p53 interaction domain of MDM4, it might affect p53 regulation because it affects the MDM4 RING domain: Residue 454 is both part of a P-loop motif thought to confer adenosine triphosphate (ATP)binding capacity (16) and part of a strand important for MDM2-MDM4 heterodimerization (Fig. 1D) (17). The mutant RING domain had fully retained its capacity to bind ATP specifically (Fig. 1E and fig. S1A) but exhibited an altered capacity to interact with the MDM2 RING domain in a yeast two-hybrid assay (Fig. 1F). We next used transfection experiments to evaluate the consequences of this mutation on the full-length protein in human cells. We transfected U2OS cellsknown to have a functional but attenuated p53 pathway due to MDM2 overexpression (18)with either an empty vector or an expression plasmid encoding a Myc-tagged MDM4WT or MDM4T454M protein. Compared with cells transfected with the empty vector, cells transfected with a MDM4WT or a MDM4T454M expression plasmid exhibited decreased p21 levels, indicating MDM4-mediated p53 inhibition in both cases (Fig. 1G). However, the decrease in p21 levels was less pronounced in cells expressing MDM4T454M than in cells expressing MDM4WT (Fig. 1G) despite similar transfection efficiencies (fig. S1B). The lower expression levels of the MDM4T454M protein likely contributed to its decreased capacity to inhibit p53 (Fig. 1G). In this experimental setting, the treatment with cycloheximide did not reveal any significant difference in stability between the mutant and wild-type (WT) MDM4 proteins (Fig. 1G and quantification in fig. S1C), raising the possibility that the observed lower MDM4T454M protein levels might result from differences in mRNA translation efficiency. Together, these preliminary results argued for an impact of the mutation on MDM4 function, leading to p53 activation.

The MDM4 RING domain is remarkably conserved throughout evolution, e.g., with 91% identity between the RING domains of human MDM4 and mouse Mdm4 (19). Thus, we decided to create a mouse model to precisely evaluate the physiological impact of the human mutation. We used homologous recombination in embryonic stem (ES) cells to target the p.T454M mutation at the Mdm4 locus (Fig. 2A). Targeted recombinants were identified by long-range polymerase chain reaction (PCR) (Fig. 2B), confirmed by DNA sequencing (Fig. 2C), and the structure of the recombinant allele was further analyzed by Southern blots with probes located 5 and 3 of the targeted mutation (Fig. 2D). Recombinant ES clones were then microinjected into blastocysts to generate chimeric mice, and chimeras were mated with PGK-Cre mice to excise the Neo gene. PCR was used to verify transmission through the germ line of the Mdm4T454M (noted below Mdm4TM) mutation and to genotype the mouse colony and mouse embryonic fibroblasts (MEFs) (Fig. 2E). We first isolated RNAs from Mdm4TM/TM MEFs and sequenced the entire Mdm4 coding sequence: The Mdm4TM sequence was identical to the WT Mdm4 sequence except for the introduced missense mutation (not shown). Furthermore, like its human counterpart, the Mdm4 gene encodes two major transcripts: Mdm4-FL, encoding the full-length oncoprotein that inhibits p53, and Mdm4-S, encoding a shorter, extremely unstable protein (20, 21). We observed, in unstressed cells as well as in cells treated with Nutlin [a molecule that activates p53 by preventing Mdm2-p53 interactions (22) without altering Mdm4-p53 interactions (23, 24)], that the Mdm4TM mutation affected neither Mdm4-FL nor Mdm4-S mRNA levels (Fig. 2F). In Western blots, however, Mdm4-FL was the only detectable isoform, and it was expressed at lower levels in the mutant MEFs (Fig. 2G).

(A) Targeting strategy. Homologous recombination in ES cells was used to target the T454M mutation at the Mdm4 locus. For the Mdm4 WT allele, exons 9 to 11 are shown [black boxes, coding sequences; white box, 3 untranslated region (3UTR)] and Bam HI (BH) restriction sites. Above, the targeting construct contains the following: (i) a 2.9-kb-long 5 homology region encompassing exon 10, intron 10, and exon 11 sequences upstream the mutation; (ii) the mutation (asterisk) within exon 11; (iii) a 2.6-kb-long fragment encompassing the 3 end of the gene and sequences immediately downstream; (iv) a neomycin selection gene (Neo) flanked by loxP sequences (gray arrowheads) and an additional BH site; (v) a 2.1-kb-long 3 homology region containing sequences downstream Mdm4; and (vi) the Diphtheria toxin a gene (DTA) for targeting enrichment. (B to D) screening of G418-resistant ES clones as described in (A), with asterisks (*) indicating positive recombinants: (B) PCR with primers a and b; (C) sequencing after PCR with primers c and d: the sequence for codons 452 to 456 demonstrates heterozygosity at codon 454; (D) Southern blot of Bam HIdigested DNA with the 5 (left) or 3 (right) probe. (E) Examples of fibroblast genotyping by PCR with primers e and f. (F) The Mdm4T454M mutation does not alter Mdm4 mRNA levels. Mdm4-FL (left) and Mdm4-S (right) mRNAs were extracted from WT and Mdm4TM/TM MEFs before or after treatment for 24 hours with 10 M Nutlin, quantified using real-time PCR, and normalized to control mRNAs, and then the value in Nutlin-treated WT MEFs was assigned a value of 1. Results from five independent experiments and >4 MEFs per genotype. ns, not significant in a Students t test. (G) Decreased Mdm4 protein levels in Mdm4TM/TM MEFs. Protein extracts, prepared from MEFs treated as in (F), were immunoblotted with antibodies against Mdm4 or actin. Bands were normalized to actin, and then the values in Nutlin-treated WT cells were assigned a value of 1. p53P/P Mdm4E6/E6 MEFs do not express a full-length Mdm4 protein (20): They were loaded to unambiguously identify the Mdm4(-FL) band in the other lanes.

Mdm4TM/TM MEFs contained higher mRNA levels for the p53 targets p21(Cdkn1a) and Mdm2, indicating increased p53 activity (Fig. 3A). Consistent with this, Mdm4TM/TM MEFs exhibited increased p21 and Mdm2 protein levels (Fig. 3B and fig. S2). Moreover, Mdm4TM/TM MEFs prematurely ceased to proliferate when submitted to a 3T3 protocol (Fig. 3C), which also suggests an increased p53 activity. The mean telomere length was decreased by 11% in Mdm4TM/TM MEFs, and a subset of very short telomeres was observed in these cells, hence demonstrating a direct link between the Mdm4TM mutation, p53 activation, and altered telomere biology (Fig. 3D). In p5331/31 MEFs, subtle but significant decreases in expression were previously observed for several genes involved in telomere biology, and in particular, small variations in Rtel1 gene expression were found to have marked effects on the survival of p5331/31 mice (5, 9). Similarly, Mdm4TM/TM MEFs exhibited subtle but significant decreases in expression for Rtel1 and several other genes contributing to telomere biology (Fig. 3E). We previously showed that p53 activation correlates with an increased binding of the E2F4 repressor at the Rtel1 promoter (9). Hence, the decreased Rtel1 mRNA levels in Mdm4TM/TM MEFs most likely resulted from increased p53 signaling. Consistent with this, a further increase in p53 activity, induced by Nutlin, led to further decreases in Rtel1 mRNA and protein levels, in both WT and Mdm4TM/TM cells (fig. S3A). Recently, in apparent contradiction with our finding that p53 activation can cause telomere shortening (5), p53 was proposed to prevent telomere DNA degradation by inducing subtelomeric transcripts, including telomere repeat-containing RNA (TERRA) (25, 26), which suggested a complex, possibly context-dependent impact of p53 on telomeres (27). This led us to compare TERRA transcripts in WT and Mdm4TM/TM cells. Consistent with an earlier report (26), p53 activation led to increased TERRA at the mouse Xq subtelomeric region in WT cells (fig. S3B). However, Mdm4TM/TM cells failed to induce TERRA in response to stress (fig. S3B). Together, our data suggest that the telomere shortening observed in Mdm4TM/TM cells results from a p53-dependent decrease in expression of several telomere-related genes and, notably, Rtel1, a gene mutated in several families with DC (6). In addition, although evidence that altered TERRA levels can cause DC is currently lacking, we cannot exclude that an altered regulation of TERRA expression might contribute to telomere defects in Mdm4TM/TM cells.

(A) Quantification of p21 and Mdm2 mRNAs extracted from WT, Mdm4+/TM, and Mdm4TM/TM MEFs, treated or not for 24 hours with 10 M Nutlin. mRNA levels were quantified using real-time PCR and normalized to control mRNAs, and then the value in Nutlin-treated WT MEFs was assigned a value of 1. Results from 10 independent experiments. (B) Protein extracts, prepared from p53/, WT, and Mdm4TM/TM MEFs treated as in (A), were immunoblotted with antibodies against Mdm2, Mdm4, p53, p21, or actin. Bands were normalized to actin, and then the values in Nutlin-treated WT MEFs were assigned a value of 1. (C) Proliferation of MEFs in a 3T3 protocol. Each point is the average value of three independent MEFs. (D) Decreased telomere length in Mdm4TM/TM MEFs, as measured by quantitative FISH with a telomeric probe. Results from two MEFs per genotype, and 68 to 75 metaphases per MEF [means + 95% confidence interval (CI) are shown in yellow]. a.u., arbitrary units. (E) Telomere-related genes down-regulated in Mdm4TM/TM MEFs. mRNAs were extracted from unstressed WT and Mdm4TM//TM MEFs, quantified using real-time PCR, and normalized to control mRNAs, and the value in WT MEFs was assigned a value of 1. Results from >3 independent experiments and two MEFs per genotype. In relevant panels: P = 0.08, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Students t (A, C at passage 7, and E) or Mann-Whitney (D) statistical tests.

Mdm4TM/TM mice were born in Mendelian proportions from Mdm4+/TM intercrosses (Fig. 4A) but were smaller than their littermates and died within 0 to 30 min after birth, with signs of severe respiratory distress (Fig. 4, B and C). Consistent with this, Mdm4TM/TM pups at postnatal day 0 (P0) appeared hypoxic (Fig. 4C), and their lungs were very small and dysfunctional (Fig. 4D). Thus, Mdm4TM/TM pups most likely died from neonatal respiratory failure. Tissues from Mdm4TM/TM pups exhibited increased p21 mRNA levels, suggesting an increase in p53 activity in these animals (fig. S4). We next used flowFISH (fluorescence in situ hybridization) with a telomere-specific probe to evaluate the impact of the mutation on telomere length in vivo. Lung cells from Mdm4TM/TM pups (and control G3 Terc/ mice) exhibited a 25% decrease in mean telomere length compared with cells from WT or Mdm4+/TM littermates, indicating altered telomere biology in G1 homozygous mutants (Fig. 4E). Notably, p53 loss or haploinsufficiency rescued the perinatal lethality of Mdm4TM/TM pups, illustrating that the premature death of Mdm4TM/TM mice likely resulted from increased p53 activity (Fig. 4F). However, p53/ and Mdm4TM/TM p53/ mice exhibited similar survival curves, with a fraction of the mice (respectively 4 of 12 and 1 of 6) succumbing to thymic lymphoma in less than 180 days. In contrast, after 180 days, all the p53+/ mice remained alive, whereas most Mdm4TM/TM p53+/ mice had died. Mdm4TM/TM p53+/ mice were smaller than their littermates (Fig. 4G) and exhibited hyperpigmentation of the footpads (Fig. 4H), and 120-day-old Mdm4TM/TM p53+/ mice exhibited abnormal hemograms (Fig. 4I). Furthermore, the Mdm4TM/TM p53+/ mice that died 60 to 160 days after birth exhibited bone marrow hypocellularity (Fig. 4J), indicating bone marrow failure as the likely cause for their premature death.

(A) Mendelian distribution of the offspring from 8 Mdm4+/TM intercrosses. (B) Mdm4TM/TM mice die at birth. Cohort sizes are in parentheses. (C) Mdm4TM/TM neonates are smaller than their littermates and appear hypoxic. (D) Lungs from Mdm4TM/TM P0 pups are hypoplastic and sink in phosphate-buffered saline owing to a lack of air inflation. (E) Flow-FISH analysis of P0 lung cells with a telomere-specific peptide nucleic acid (PNA) probe. Top: Representative results from a WT, a Mdm4+/TM, a Mdm4TM/TM, and a G3 Terc/ mouse are shown. Right: Green fluorescence (fluo.) with black histograms for cells without the probe (measuring cellular autofluorescence) and green histograms for cells with the probe. The shift in fluorescence intensity is smaller in Mdm4TM/TM and Terc/ cells (c or d < a or b), indicating reduced telomere length. Left: Propidium iodide (PI) fluorescence histograms are superposed for cells with or without the probe. Below: Statistical analysis of green fluorescence shifts (see Materials and Methods). Means + 95% CI are shown; data are from two to three mice and >3800 cells per genotype. (F) Impact of decreased p53 activity on Mdm4TM/TM animals. Cohort sizes are in parentheses. (G) Examples of littermates with indicated genotypes. (H) Hind legs of mice with indicated genotypes. (I) Mdm4TM/TM p53+/ mice exhibit abnormal hemograms. Counts for white blood cells (WBC), red blood cells (RBC), and platelets (PLT) for age-matched (120 days old) animals are shown. (J) Hematoxylin and eosin staining of sternum sections from WT and Mdm4TM/TM p53+/ mice. In relevant panels: ns, not significant; *P < 0.05, ***P < 0.001, and ****P < 0.0001 by Mantel-Cox (B and F), Students t (C, D, G, and I), or Mann-Whitney (E) statistical tests. Photo credits: E.T. and R.D., Institut Curie (C, G, and H); R.D., Institut Curie (D).

Although Mdm4TM/TM MEFs and mice were useful to demonstrate that the Mdm4T454M mutation leads to p53 activation and short telomeres, a detailed analysis of Mdm4+/TM mice appeared more relevant to model the NCI-226 family, in which all affected relatives were heterozygous carriers of the MDM4T454M mutation. Unlike Mdm4TM/TM mice, most Mdm4+/TM animals remained alive 6 months after birth and had no apparent phenotype, similarly to WT mice (Fig. 5A). This was consistent with our analyses in fibroblasts because Mdm4+/TM MEFs behaved like WT cells in a 3T3 proliferation assay (Fig. 3C). However, p53 target genes appeared to be transactivated slightly more efficiently in Mdm4+/TM than in WT cells (Fig. 3A), and 30% of Mdm4+/TM mice exhibited a slight hyperpigmentation of the footpads, suggesting a subtle increase in p53 activity (Fig. 5B). We reasoned that a further, subtle increase in p53 activity might affect the survival of Mdm4+/TM mice. We tested this hypothesis by mating Mdm4+/TM animals with p53+/31 mice. p53+/31 mice were previously found to exhibit a slight increase in p53 activity and to remain alive for over a year (5). Notably, unlike Mdm4+/TM or p53+/31 heterozygous mice, Mdm4+/TM p53+/31 compound heterozygotes died in less than 3 months (Fig. 5A) and exhibited many features associated with strong p53 activation. Mdm4+/TM p53+/31 mice exhibited intense skin hyperpigmentation (Fig. 5C), were much smaller than their littermates (Fig. 5D), and exhibited heart hypertrophy (Fig. 5E) and thymic hypoplasia (Fig. 5F) and the males had testicular hypoplasia (Fig. 5G). Bone marrow failure was the likely cause for the premature death of Mdm4+/TM p53+/31 mice, as indicated by abnormal hemograms of 18-day-old (P18) compound heterozygotes (Fig. 5H) and bone marrow hypocellularity in the sternum sections of moribund Mdm4+/TM p53+/31 animals (Fig. 5I). We next used flow-FISH to analyze telomere length in the bone marrow cells of P18 WT, Mdm4+/TM, p53+/31, and Mdm4+/TM p53+/31 mice. We found no significant difference between telomere lengths in cells from five WT and three Mdm4+/TM mice with normal skin pigmentation, whereas cells from two Mdm4+/TM mice with increased skin pigmentation (or from p53+/31 mice) exhibited marginal (5 to 7%) decreases in mean telomere length. Notably, in G1 Mdm4+/TM p53+/31 cells, the average telomere length was decreased by 34% (Fig. 5J). Together, these results demonstrate that Mdm4+/TM mice are hypersensitive to subtle increases in p53 activity. Consistent with this, Mdm4+/TM p53+/31 MEFs also exhibited increased p53 signaling and accelerated proliferation arrest in a 3T3 protocol (fig. S5). In sum, the comparison between Mdm4TM/TM and Mdm4TM/TM p53+/ mice, or between Mdm4+/TM and Mdm4+/TM p53+/31 animals, indicated that subtle variations in p53 signaling had marked effects on the phenotypic consequences of the Mdm4T454M mutation (table S3).

(A) Impact of increased p53 activity on Mdm4+/TM animals. Cohort sizes are in parentheses. (B) Footpads from Mdm4+/TM mice appear normal (top) or exhibit a subtle increase in pigmentation (bottom). (C) Mdm4+/TM p53+/31 mice exhibit strong skin hyperpigmentation. (D) Mdm4+/TM p53+/31 mice are smaller than age-matched WT mice. (E to G) Mdm4+/TM p53+/31 mice exhibit heart hypertrophy (E) as well as thymic (F) and testicular (G) hypoplasia. (H) Mdm4+/TM p53+/31 mice exhibit abnormal hemograms. Counts for white blood cells, red blood cells, and platelets for five age-matched (P18) animals per genotype are shown. (I) Hematoxylin and eosin staining of sternum sections from mice of the indicated genotypes. (J) Flow-FISH analysis of P18 bone marrow cells with a telomere-specific PNA probe. Top: Representative results for a WT, a Mdm4+/TM with normal skin pigmentation (nsp), a Mdm4+/TM with increased footpad skin pigmentation (isp), a p53+/31, and a Mdm4+/TM p53+/31 mouse are shown; black histograms, cells without the probe; green histograms, cells with the probe. The smallest shift in fluorescence intensity (e) was observed with Mdm4+/TM p53+/31 cells. Bottom: Statistical analysis of green fluorescence shifts. Means + 95% CI are shown; data are from >1500 cells per genotype. In relevant panels: ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Mantel-Cox (A), Students t (D and E to H), or Mann-Whitney (J) statistical tests. Photo credits: R.D. and P.L., Institut Curie (B); E.T. and R.D., Institut Curie (C and D).

The carriers of the MDM4T454M mutation exhibited considerable heterogeneity in their phenotypes (Fig. 1 and table S1). The data from our mouse model suggested that variations in p53 activity might account for the variable expressivity and penetrance of clinical features among the NCI-226 MDM4+/T454M relatives. Hence, we analyzed nine known common polymorphisms reported to affect p53 activity and tumorigenesis (four at the TP53 locus, two at the MDM2 locus, and three at the MDM4 locus) (3,4,2832). Among the four MDM4+/T454M relatives, the proband (NCI-226-1) is more difficult to interpret because the potential contribution of the TERT p.W203S variant to his phenotype cannot be ruled out (even though it appears unlikely according to in silico predictions). The MDM4 allele encoding the mutant protein (p.T454M) appears associated with the C allele of single-nucleotide polymorphism (SNP) rs4245739, the G allele of SNP rs11801299, and the G allele of SNP rs1380576 (Fig. 6A). These three MDM4 variant alleles are associated with increased p53 activity (4,32) and might, thus, synergize with the MDM4T454M mutation in this family.

(A) Genotyping of polymorphisms that may affect the p53 pathway. The SNPs rs1800371 and rs1042522 modify the p53 protein sequence (28,29), whereas rs17878362 and rs17880560 are singlets (A1) or doublets (A2) of G-rich sequences in noncoding regions of TP53 that affect p53 expression (30). SNPs rs117039649 and rs2279744, in the MDM2 promoter, affect MDM2 mRNA levels (3,31). Three SNPs are at the MDM4 locus: rs4245739 in the 3UTR region affects MDM4 protein levels (4), whereas rs11801299 and rs1380576 were associated with an increased risk of developing retinoblastoma (32), a cancer type with frequent MDM4 alterations (51). Polymorphisms that differ among family members are in bold, with the allele (or haplotype) associated with increased p53 activity in green (because it may synergize with the effects of the MDM4T454M mutation). Alleles (or haplotypes) for which there is evidence of decreased p53 activity, or for which the effect is uncertain, are highlighted in red or blue, respectively. Please note that the clinical effects of the TP53 rs1042522 SNP have recently been contested (33), so that all alleles for this SNP were labeled in blue. MAF, minor allele frequency reported for all gnomAD populations combined. https://gnomad.broadinstitute.org (52). (B) Comparative analysis of primary fibroblasts from family members 226-4 and 226-8. p21 and RTEL1 mRNAs, extracted from cells from relatives NCI 226-4 and NCI 226-8 or two unrelated patients with DC carrying a TINF2 or a TERT mutation, were quantified using real-time PCR, normalized to control mRNAs, and then expressed relative to the mean values in TINF2 and TERT mutant cells. ns, not significant, **P < 0.01 and ***P < 0.001 in a Students t test.

The probands affected cousin (226-8) exhibited a very early onset of disease, with lymphocyte telomere length within or below the first percentile of age-matched control participants and tongue squamous cell carcinoma at age 27 (Fig. 1 and table S1). The WT MDM4 allele of 226-8 carried the rs4245739 C, the rs11801299 G, and the rs1380576 G variants associated with increased p53 activity. This suggests a potential disease-modifying effect of these MDM4 SNPs. In contrast, the probands mother (226-4) was much less severely affected, with telomere length between the 10th and 50th percentiles (Fig. 1). Although we cannot rule out that disease anticipation might contribute to her milder phenotype, note that her WT MDM4 allele carried variants that might correlate with decreased p53 activity and could antagonize the MDM4T454M mutation (rs4245739 A, rs11801299 A, and rs1380576 C; Fig. 6A). Family members 226-4 and 226-8 shared the same genotypes for all the other tested variants, except for TP53 rs1042522, a SNP first reported to affect apoptotic or cell cycle arrest responses (28), but with a clinical effect that now appears controversial (33). The probands sister (226-2), with a B cell deficiency and telomere lengths around the 10th percentile, also appeared less affected than 226-8. All the tested variants at the MDM2 and MDM4 loci were identical between 226-2 and 226-8. However, unlike 226-8, 226-2 exhibited a TP53 allele with an A1A1 haplotype for variants rs17878362 and rs17880560 that might decrease p53 activity (30) and antagonize the effects of the MDM4T454M mutation (Fig. 6A).

We had primary fibroblasts available for two of these family members, 226-4 and 226-8, allowing us to directly assess the functional effect of the MDM4T454M variant in these cells. These fibroblasts were grown in parallel with primary fibroblasts from patients with DC carrying either a TINF2K280E mutation or a TERTP704S mutation, and mRNA levels for p21 and RTEL1 were quantified. In agreement with the notion that a MDM4T454M heterozygous mutation activates p53 signaling in NCI-226 family members, fibroblasts from both 226-4 and 226-8 exhibited increased p21 mRNA levels compared with TINF2 or TERT mutant cells (Fig. 6B). However, cells from 226-4 only exhibited a 2-fold increase in p21 levels, whereas a 12-fold increase was observed for cells from 226-8, consistent with the notion that SNPs affecting the p53 pathway might counteract (for 226-4) or strengthen (for 226-8) the effect of the MDM4T454M mutation. Furthermore, we previously showed that RTEL1 mRNA levels are down-regulated upon p53 activation in human cells (5). RTEL1 mRNA levels appeared normal in cells from 226-4 but were markedly decreased in cells from 226-8, raising the possibility that a threshold in p53 activation might be required to affect RTEL1 expression (Fig. 6B).

Although MDM4 is primarily known for its clinical relevance in cancer biology, our study shows that a germline missense MDM4 mutation may cause features suggestive of DC. In humans, the MDM4 (p.T454M) mutation was identified in this family with neutropenia, bone marrow hypocellularity, early-onset tongue SCC, AML, and telomeres between the 1st and 10th percentiles in the younger generation. In mice, the same Mdm4 mutation notably correlated with increased p53 activity, short telomeres, and bone marrow failure. In both human transfected cells and MEFs, the mutant protein was expressed at lower levels than its WT counterpart, likely contributing to increased p53 activity. Together, these results demonstrate the importance of the MDM4/p53 regulatory axis on telomere biology and DC-like features in both species. Notably, p5331/31 mice were previously found to phenocopy DC (5), but whether this finding was relevant to human disease had remained controversial. When a mutation in PARN was found to cause DC (34), it first appeared consistent with the p5331 mouse model because PARN, the polyadenylate-specific ribonuclease, had been proposed to regulate p53 mRNA stability (35). However, whether PARN regulates the stability of mRNAs is now contested (36). Rather, PARN would regulate the levels of over 200 microRNAs, of which only a few might repress p53 mRNA translation (37). Furthermore, PARN regulates TERC, the telomerase RNA component (38), and TERC overexpression increased telomere length in PARN-deficient cells (39). Thus, whether a germline mutation that specifically activates p53 can cause DC-like features remained to be demonstrated in humans, and our report provides compelling evidence for this, because unlike PARN, MDM4 is a very specific regulator of p53.

A germline antiterminating MDM2 mutation was recently identified in a patient with a Werner-like syndrome of premature aging. Although multiple mechanisms might contribute to the clinical features in that report, a premature cellular senescence resulting from p53 hyperactivation was proposed to play a major role in his segmental progeroid phenotype (40). In that regard, our finding that increased p53 activity correlates with short telomeres appears relevant because telomere attrition is a primary hallmark of aging, well known to trigger cellular senescence (41). Furthermore, germline TP53 frameshift mutations were recently reported in two patients diagnosed with pure red blood cell aplasia and hypogammaglobulinemia, resembling but not entirely consistent with Diamond Blackfan anemia (DBA) (42). In addition to the pure red cell aplasia diagnostic of DBA, those patients were found to exhibit relatively short telomeres (although not as short as telomeres from patients with DC), which may also seem consistent with our results. Our finding of an MDM4 missense mutation in a DC-like family, together with recent reports linking an antiterminating MDM2 mutation to a Werner-like phenotype and TP53 frameshift mutations to DBA-like features, indicates that the clinical impact of germline mutations affecting the p53/MDM2/MDM4 regulatory network is just emerging. An inherited hyperactivation of the p53 pathwayvia a germline TP53, MDM2, or MDM4 mutationmay thus cause either DBA, Werner-like, or DC-like features, but additional work will be required to determine whether mutations in any of these three genes can cause any of these three syndromes. Likewise, several mouse models have implicated p53 deregulation in features of other developmental syndromes including the CHARGE, Treacher-Collins, Waardenburg, or DiGeorge syndrome (43), and it will be important to know whether germline mutations in TP53, MDM2, or MDM4 may cause these additional syndromes in humans.

Heterozygous Mdm4+/TM mice appeared normal but were hypersensitive to variations in p53 activity, and, perhaps most notably, Mdm4+/TM p53+/31 compound heterozygous mice rapidly died from bone marrow failure. Thus, the p5331 mutation acted as a strong genetic modifier of the Mdm4TM mutation. It is tempting to speculate that similarly, among the NCI-226 family members heterozygous for the MDM4T454M allele, differences in the severity of phenotypic traits (e.g., lymphocyte telomere length and bone marrow cellularity) may result, in part, from modifiers affecting the p53 pathway and synergize or antagonize with the effects of the MDM4T454M mutation. To search for potentially relevant modifiers, we looked at nine polymorphisms at the TP53, MDM2, and MDM4 loci that were previously reported to affect p53 activity. Notably, we found that the family member most severely affected (226-8, the probands cousin) carried a TP53 haplotype, as well as SNPs on the WT MDM4 allele, that might synergize with the effects of the MDM4T454M mutation. Conversely, a TP53 haplotype for the probands sister (226-2), or SNPs at the WT MDM4 locus for the probands mother (226-4), might antagonize the impact of MDM4T454M allele. Consistent with this, primary fibroblasts from 226-4 and 226-8 exhibited increased p53 activity, but p53 activation was much stronger in cells from 226-8. Our data, thus, appear consistent with the existence of genetic modifiers at the TP53 and MDM4 loci that may affect DC-like phenotypic traits among family members carrying the MDM4 (p.T454M) mutation. However, this remains speculative given the small number of individuals that could be analyzed. Furthermore, nonexonic variants affecting other genes might also contribute to DC-like traits (44). Last, the TP53 and MDM4 polymorphisms considered here were previously evaluated for their potential impact on tumorigenic processes, rather than DC-like traits such as telomere length or bone marrow hypocellularity. Our data suggest that polymorphisms at the TP53 and MDM4 (and possibly MDM2) loci should be evaluated for their potential impact on bone marrow function and telomere biology.

The individuals in this study are participants in an Institutional Review Boardapproved longitudinal cohort study at the NCI entitled Etiologic Investigation of Cancer Susceptibility in Inherited Bone Marrow Failure Syndromes (www.marrowfailure.cancer.gov, ClinicalTrials.gov NCT00027274) (7). Patients and their family members enrolled in 2008 and completed detailed family history and medical history questionnaires. Detailed medical record review and thorough clinical evaluations of the proband, his sister, parents, and maternal cousin were conducted at the National Institutes of Health (NIH) Clinical Center. Telomere length was measured by flow cytometry with in situ hybridization (flow-FISH) (45) in leukocytes of all patients and family members reported. DNA was extracted from whole blood using standard methods. DNA was not available from 226-7 or 226-9 (Fig. 1). Given the time frame of participant enrollment, Sanger sequencing of DKC1, TINF2, TERT, TERC, and WRAP53 was performed first, followed by exome sequencing.

WES of blood-derived DNA for family NCI-226 was performed at the NCIs Cancer Genomics Research Laboratory as previously described (46). Exome enrichment was performed with NimbleGens SeqCap EZ Human Exome Library v3.0 + UTR (Roche NimbleGen Inc., Madison, WI, USA), targeting 96 Mb of exonic sequence and the flanking untranslated regions (UTRs) on an Illumina HiSeq. Annotation of each exome variant locus was performed using a custom software pipeline. WES variants of interest were identified if they met the following criteria: heterozygous in the proband, his mother, and maternal cousin; nonsynonymous; had a minor allele frequency <0.1% in the Exome Aggregation Consortium databases; and occurred <5 times in our in house database of 4091 individuals. Variants of interest were validated to rule out false-positive findings using an Ion 316 chip on the Ion PGM Sequencer (Life Technologies, Carlsbad, CA, USA).

Primers flanking the MDM4 RING domain were used to amplify RING sequences, and PCR products were cloned (or cloned and mutagenized) in the pGST-parallel2 plasmid. Glutathione S-transferase (GST) fusion proteins were expressed in BL21 (DE3) cells. After induction for 16 hours at 20C with 0.2 mM IPTG (isopropyl--d-thiogalactopyranoside), soluble proteins were extracted by sonication in lysis buffer [50 mM tris (pH 7.0), 300 mM LiSO4, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 0.2% NP-40, complete Protease inhibitors (Roche) 1]. The soluble protein fraction was incubated with Glutathione Sepharose beads (Pharmacia) at 4C for 2 hours, and the bound proteins were washed with 50 mM tris (pH 7.0), 300 mM LiSO4, and 1 mM DTT and then eluted with an elution buffer [50 mM tris-HCl (pH 7.5), 300 mM NaCl, 1 mM DTT, and 15 mM glutathione]. WT and mutant GST-RING proteins (0, 1, 2, 4, or 8 g) or GST alone (0 or 8 g) was incubated with 10 nM ATP and 5 Ci ATP-32P for 10 min at room temperature, filtered through nitrocellulose, and counted by liquid scintillation. Alternatively, 7 g of either WT or mutant GST-RING proteins was incubated with 5 Ci ATP-32P for 10 min at room temperature and increasing amounts (0, 0.02, 2, 20, and 200 M) of ATP or guanosine triphosphate (GTP), filtered through nitrocellulose, and counted by liquid scintillation.

The yeast two-hybrid assays were performed as described (47). Briefly, MDM4 and MDM2 RING open reading frames were cloned in plasmids derived from the two-hybrid vectors pGADT7 (Gal4-activating domain) and pGBKT7 (Gal4-binding domain) creating N-terminal fusions and transformed in yeast haploid strains Y187 and AH109 (Clontech). Interactions were scored, after mating and diploid selection on dropout medium without leucine and tryptophan, as growth on dropout medium without leucine, tryptophan, histidine, and adenine.

U2OS cells (106) were transfected by using Lipofectamine 2000 (Invitrogen) with pCDNA3.1 (6 g), or 5 106 cells were transfected with 30 g of pCDNA3.1-MycTag-MDM4WT or pCDNA3.1-MycTag-MDM4TM. Twenty-four hours after transfection, cells were treated with cycloheximide (50 g/ml; Sigma-Aldrich, C4859), then scratched in phosphate-buffered saline (PBS) after 2, 4, or 8 hours, pelleted, and snap frozen in liquid nitrogen before protein or RNA extraction with standard protocols.

The targeting vector was generated by recombineering from the RP23-365M5 BAC (bacterial artificial chromosome) clone (CHORI BACPAC Resources) containing mouse Mdm4 and downstream sequences of C57Bl6/J origin. A loxP-flanked neomycin cassette (Neo) and a diphtheria toxin gene (DTA) were inserted downstream of the Mdm4 gene, respectively, for positive and negative selections, and a single-nucleotide mutation encoding the missense mutation T454M (TM) was targeted in the exon 11 of Mdm4. The targeting construct was fully sequenced before use.

CK-35 ES cells were electroporated with the targeting construct linearized with Not I. Recombinant clones were identified by long-range PCR, confirmed by Southern blot, PCR, and DNA sequencing (primer sequences in table S4). Two independent recombinant clones were injected into blastocysts to generate chimeras, and germline transmission was verified by genotyping their offspring. Reverse transcription PCR (RT-PCR) of RNAs from Mdm4TM/TM MEFs showed that the mutant complementary DNA (cDNA) differed from an Mdm4 WT sequence only by the engineered missense mutation. The genotyping of p53+/, p53+/31, and G3 Terc/ mice was performed as previously described (5, 12). All experiments were performed according to Institutional Animal Care and Use Committee regulations.

MEFs isolated from 13.5-day embryos were cultured in a 5% CO2 and 3% O2 incubator, in Dulbeccos modified Eagles medium GlutaMAX (Gibco), with 15% fetal bovine serum (Biowest), 100 M 2-mercaptoethanol (Millipore), 0.01 mM Non-Essential Amino Acids, and penicillin/streptavidin (Gibco) for five or fewer passages, except for 3T3 experiments, performed in a 5% CO2 incubator for seven passages. Cells were treated for 24 hours with 10 M Nutlin 3a (Sigma-Aldrich) (22) or 15 M cisplatin (Sigma-Aldrich). Primary human fibroblasts at low passage (p.2 for TINF2K280E, p.3 for NCI-226-4 and NCI-226-8, and p.4 for TERTP704S) were thawed and cultured in fibroblast basal medium (Lonza) with 20% fetal calf serum, l-glutamin, 10 mM Hepes, penicillin/streptavidin, and gentamicin before quantitative PCR (qPCR) analysis.

Total RNA, extracted using NucleoSpin RNA II (Macherey-Nagel), was reverse transcribed using SuperScript IV (Invitrogen), with, for TERRA quantification, a (CCCTAA)4 oligo as described (48). Real-time qPCRs were performed with primer sequences as described (5, 9, 48) on a QuantStudio using Power SYBR Green (Applied Biosystems).

Protein detection by immunoblotting was performed using antibodies against Mdm2 (4B2), Mdm4 (M0445; Sigma-Aldrich), p53 (AF1355, R&D Systems), actin (A2066; Sigma-Aldrich), p21 (F5; Santa Cruz Biotechnology), Myc-Tag (SAB2702192; Sigma-Aldrich), and Rtel1 (from J.-A.L.-V.). Chemiluminescence revelation was achieved with SuperSignal West Dura (Perbio). Bands of interest were quantified by using ImageJ and normalized with actin.

Cells were treated with colcemide (0.5 g/ml) for 1.5 hours, submitted to hypotonic shock, fixed in an (3:1) ethanol/acetic acid solution, and dropped onto glass slides. Quantitative FISH was then carried out as described (5) with a TelC-Cy3 peptide nucleic acid (PNA) probe (Panagene). Images were acquired using a Zeiss Axioplan 2, and telomeric signals were quantified with iVision (Chromaphor).

Flow-FISH with mouse cells was performed as described (45). For each animal, either the lungs were collected or the bone marrow from two tibias and two femurs was collected and red blood cells were lysed; then, 2 106 cells were fixed in 500 l of PNA hybridization buffer [70% deionized formamide, 20 mM tris (pH 7.4), and 0.1% Blocking reagent; Roche] and stored at 20C. Either nothing (control) or 5 l of probe stock solution was added to cells [probe stock solution: 10 M TelC-FAM PNA probe (Panagene), 70% formamide, and 20 mM tris (pH 7.4)], and samples were denatured for 10 min at 80C before hybridization for 2 hours at room temperature. After three washes, cells were resuspended in PBS 1, 0.1% bovine serum albumin, ribonuclease A (1000 U/ml), and propidium iodide (12.5 g/ml) and analyzed with an LSR II fluorescence-activated cell sorter. WT and G3 Terc/ mice were included in all flow-FISH experiments, respectively, as controls of normal and short telomeres. For fluorescence shift analyses, the green histograms (corresponding to cells with the telomeric probe) were sliced into 18 windows of equal width and numbered 0 to 17 according to their distance from the median value in cells without the probe, and the number of cells in each window was quantified with ImageJ. The data from two to five mice per genotype were typically used to calculate mean telomere lengths, expressed relative to the mean in WT cells.

Organs were fixed in formol 4% for 24 hours and then ethanol 70% and embedded in paraffin wax. Serial sections were stained with hematoxylin and eosin using standard procedures (49). For hemograms, 100 l of blood from each animal was recovered retro-orbitally in a 10-l citrate-concentrated solution (S5770; Sigma-Aldrich) and analyzed using an MS9 machine (Melet Schloesing Laboratoires).

DNA extracted from Epstein-Barr virustransformed lymphocytes of NCI-226 family members was amplified with primers flanking nucleotide polymorphisms of interest (primer sequences in table S5), and then PCR products were analyzed by Sanger DNA sequencing.

Analyses with Students t, Mann-Whitney, or Mantel-Cox statistical tests were performed by using GraphPad Prism, and values of P < 0.05 were considered significant.

This is an open-access article distributed under the terms of the Creative Commons Attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Acknowledgments: We are grateful to the family for valuable contributions to this study. We thank I. Grandjean, C. Caspersen, A. Fosse, and M. Garcia from the Animal Facility, C. Alberti and C. Roulle from the Transgenesis Platform, M. Richardson and A. Nicolas from the Pathology Service, and Z. Maciorowski from the Cell-Sorting Facility of the Institut Curie. We thank A. Chor for help with qPCRs, A. Pyanitskaya, C. Adam, V. Borde, M. Schertzer, and M. Perderiset for plasmids and technical advices, and A. Fajac for comments on the manuscript. F.T. would like to acknowledge the talent, kindness, and loyal support of I. Simeonova and S.J., two exceptional PhD students whose pioneering work led to this study. Funding: The Genetics of Tumor Suppression laboratory received funding from the Ligue Nationale contre le Cancer (Labellisation 2014-2018 and Comit Ile-de-France), the Fondation ARC and the Gefluc. PhD students were supported by fellowships from the Ministre de lEnseignement Suprieur et de la Recherche (to S.J., E.T., and R.D.), the Ligue Nationale contre le Cancer (to S.J.), and the Fondation pour la Recherche Mdicale (to E.T.). The work of S.A.S., N.G., and B.P.A. was supported by the intramural research program of the Division of Cancer Epidemiology and Genetics, NCI, and the NIH Clinical Center. Author contributions: V.L. created the Mdm4T454M mouse model, genotyped mouse cohorts, and performed transfections, yeast two-hybrid assays, protein purifications, and molecular cloning. E.T., R.D., and V.L. managed mouse colonies. E.T., R.D., and P.L. performed mouse anatomopathology. I.D., E.T., R.D., F.T., and J.-A.L.-V. determined mouse telomere lengths. V.L. and S.J. genotyped human polymorphisms and analyzed human fibroblasts. E.T. and R.D. genotyped MEFs and performed 3T3 assays. V.L., R.D., and E.T. performed Western blots. E.T., R.D., V.L., S.J., and P.L. performed qPCRs. B.B. and V.L. performed ATP-binding assays. B.P.A. supervised the NCI IBMFS study. N.G. and S.A.S. evaluated study participants. S.A.S. analyzed the exome sequencing data. F.T. and S.A.S. supervised the project and wrote the manuscript. Competing interests: The authors declare that they have no 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. The human samples can be provided by S.A.S. pending scientific review and a completed material transfer agreement. Requests for human cells should be submitted to S.A.S.

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Germline mutation of MDM4, a major p53 regulator, in a familial syndrome of defective telomere maintenance - Science Advances

Bone Marrow Stem Cells Comments Off on Germline mutation of MDM4, a major p53 regulator, in a familial syndrome of defective telomere maintenance – Science Advances | April 10th, 2020

Advance Market Analyticsreleased the research report ofGlobal Stem CellsMarket, offers a detailed overview of the factors influencing the global business scope.Global Stem Cells Market research report shows the latest market insights with upcoming trends and breakdown of the products and services.The report provides key statistics on the market status, size, share, growth factors of the Global Stem Cells.This Report covers the emerging players data, including: competitive situation, sales, revenue and global market.

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The stem cell is used for treating chronic diseases such as cardiovascular disorders, cancer, diabetes, and others. Growing research and development in stem cell isolation techniques propelling market growth. For instance, a surgeon from Turkey developed a method for obtaining stem cells from the human body without enzymes which are generally used for the isolation of stem cells. Further, growing healthcare infrastructure in the developing economies and government spending on the life science research and development expected to drive the demand for stem cell market over the forecasted period.

The Global Stem Cellsis segmented by following Product Types:

Type (Adult Stem Cells (Neuronal, Hematopoietic, Mesenchymal, Umbilical Cord, Others), Human Embryonic Stem Cells (hESC), Induced Pluripotent Stem Cells, Very Small Embryonic-Like Stem Cells), Application (Regenerative Medicine (Neurology, Orthopedics, Oncology, Hematology, Cardiovascular and Myocardial Infraction, Injuries, Diabetes, Liver Disorder, Incontinence, Others), Drug Discovery and Development), Technology (Cell Acquisition (Bone Marrow Harvest, Umbilical Blood Cord, Apheresis), Cell Production (Therapeutic Cloning, In-vitro Fertilization, Cell Culture, Isolation), Cryopreservation, Expansion and Sub-Culture), Therapy (Autologous, Allogeneic)

Region Included are: North America, Europe, Asia Pacific, Oceania, South America, Middle East & Africa

Country Level Break-Up: United States, Canada, Mexico, Brazil, Argentina, Colombia, Chile, South Africa, Nigeria, Tunisia, Morocco, Germany, United Kingdom (UK), the Netherlands, Spain, Italy, Belgium, Austria, Turkey, Russia, France, Poland, Israel, United Arab Emirates, Qatar, Saudi Arabia, China, Japan, Taiwan, South Korea, Singapore, India, Australia and New Zealand etc.Enquire for customization in Report @:https://www.advancemarketanalytics.com/enquiry-before-buy/72815-global-stem-cells-market-1

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Chapter 5: Displaying the by Type, End User and Region 2013-2018

Chapter 6: Evaluating the leading manufacturers of the Global Stem Cells market which consists of its Competitive Landscape, Peer Group Analysis, BCG Matrix & Company Profile

Chapter 7: To evaluate the market by segments, by countries and by manufacturers with revenue share and sales by key countries in these various regions.

Chapter 8 & 9: Displaying the Appendix, Methodology and Data Source

Finally, Global Stem Cells Market is a valuable source of guidance for individuals and companies.

Data Sources & Methodology

The primary sources involves the industry experts from the Global Stem Cells Market including the management organizations, processing organizations, analytics service providers of the industrys value chain. All primary sources were interviewed to gather and authenticate qualitative & quantitative information and determine the future prospects.

In the extensive primary research process undertaken for this study, the primary sources Postal Surveys, telephone, Online & Face-to-Face Survey were considered to obtain and verify both qualitative and quantitative aspects of this research study. When it comes to secondary sources Companys Annual reports, press Releases, Websites, Investor Presentation, Conference Call transcripts, Webinar, Journals, Regulators, National Customs and Industry Associations were given primary weight-age.

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Stem Cells Market Expected to Boost the Global Industry Growth in the Near Future - Germany English News

Bone Marrow Stem Cells Comments Off on Stem Cells Market Expected to Boost the Global Industry Growth in the Near Future – Germany English News | April 10th, 2020

When Jeff Bezos, the Amazon kingpin, debuted his new muscular physique at the Sun Valley Conference in 2017, he almost broke the internet. His Vin Dieselesque guns launched countless memes about how the dweebs dweeb had transformed himself into a jacked-up specimen worthy of an action franchise.

In interviews Bezos credits his diet (which includes roast iguana and octopus for breakfast), his unwavering commitment to working out, and eight hours of sleep. But not everyone is buying it.

Clean livingthats the catchphrase, isnt it? quips Patricia Wexler, the ne plus ultra of Manhattan dermatologists. Very few admit to doing any procedures.

Not a chance its just diet and exercise, says Roberta Del Campo, a dermatologist based in Miami, the countrys plastic surgery capital. Behind the scenes these people are getting all sorts of injectables and body sculpting treatments, such as Emsculpt and Trusculpt Flex, which have surged in popularity, especially among men, in the last couple of years.

Drew AngererGetty Images

Other experts suspect that captains of industry such as Bezos, who is 56, are going to even greater lengths to project vigor for both boards and broads. The tech titans are all looking much better than they used to, says Jessie Cheung, a Chicago-based cosmetic dermatologist whose holistic approach often involves testosterone and growth hormone substitutes, especially for men of a certain age who are lacking in muscle and look frail.

Access to bio-hacking tools such as stem cells and hormones is allowing men to look, perform, and think better. Its worth noting that Bezos, along with fellow billionaire Peter Thiel, invested in Unity Biotechnology, a company researching drugs and treatments to keep aging at bay. Im pretty sure hes gotten a taste of some good stuff, Cheung says.

Welcome to the new male vanity, in which even Silicon Valley bigwigs considerably younger than Bezos are resorting to newfangled procedures to avoid aging out of the workforce. The stakes have never been higher. American men underwent 1.1 million noninvasive cosmetic procedures in 2018a 72 percent increase since 2000, a trend that shows no signs of abating. In its forecast for 2020, the American Society for Aesthetic Plastic Surgery predicts the continued rise of the Daddy-Do-Over, the male equivalent of the Mommy Makeover, as men look to boost their confidence and improve their physical appearance.

Its a lesson in maintenance the men in the presidential race would do well to learn. In the not so distant past politicians could dismiss reporters questions about whether theyd had a face-lift, as Arnold Schwarzenegger did during his 2003 run for governor of California, when he joked that they must be confusing him with Cher. Now pols and pundits of every party are being grilled as mercilessly about their appearance as about their Medicare plans.

"Unfortunately for Biden, you can see the work thats been done," says one NYC dermatologist.

Joe Bidens forehead and Donald Trumps hair flap and skin color are dissected with the rigor of Kremlinologists (some of them actually are Kremlinologists, in Trumps case). And with good reason: If Hillary Clintons wrinkles, Elizabeth Warrens glasses, and Amy Klobuchars eyebrows are fair game, why not the nipped and tucked peacocks strutting around on Capitol Hill?

Denials about the scars on the side of Bidens face are, according to the experts, malarkey. Unfortunately for Biden, who has obviously had hair transplants and Botox, among other things, you can see the work thats been done, says Wexler. Nobody should be talking about work. When you have work done, the last thing you want is for people to notice it.

The queen of Fraxels laser focus on male primping is not partisan. Mr. Trump has definitely had workand not great work, at that, she adds. Give him his crumb, though: He wasnt bad looking when he was younger and in better shape.

Trumps penchant for cosmetic adjustments has been an open if much denied secret since at least 1991, when Ivana Trump disclosed his scalp reduction surgery and chin and waist liposuction in their divorce papers. In February the world was served a fresh reminder, when the president was photographed, in an image that quickly went viral, stepping out of Marine One with a windswept rug and a fake tan for the history books.

At tony dermatologist practices from coast to coast, man-tans like Trumps and obvious old-school work like the kind favored by Vladimir Putin is frowned uponif anyone can move any facial muscles at all. Instead, next-gen lasers such as NeoSkin by Aerolase, IBeam, and Nd:YAG are used to eliminate redness and discoloration.

Instead of surgical face-lifts, which, to be fair, remain popular in certain parts of the country (I definitely see them more on the West Coast, Wexler says, where its been around longer and is more accepted), men of means are turning to noninvasive procedures, most notably Ultherapy, a relatively painless FDA-cleared ultrasound treatment that requires no downtime.

Edward George/Alamy Stock Photo

For the ultimate injection of masculine vigor, though, Cheung works with membersand not necessarily of Congress. We make penises bigger and better, she says. Self-confidence for men is tied up with their penises and how well they work. We give them their swagger back.

Men looking for an extra glide in their stride are considering the augmented Priapus Shot, or P-shot, Cheung says, a treatment thats the male equivalent of the O-shot. She is also increasingly recommending a machine called Emsella, better known as the Orgasm Throne, which generates approximately 11,000 Kegel contractions in 30 minutes (it was originally developed for female incontinence). It really gives you an invigorating kick in the pants, Cheung says.

If the recent past is anything to go by, theres no guarantee that the candidates who end up squaring off in November will provide anything resembling accurate medical recordswhich is a shame, as they would make interesting reading. Like Bezos and less heralded moguls across the country, they are unlikely to reveal any touch-ups to anyone but their best pals.

Men will come in and ask for something their friend has had done, Wexler says. But you wont hear anyone on Jimmy Fallon saying, Im so tired: I was at the dermatologist all day.

This story appears in the May 2020 issue of Town & Country.

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Jeff Bezos and the New Face of Male Vanity - TownandCountrymag.com

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Scientists at Lund University in Sweden showed long ago they could reprogram human cells into nerve cells and implant them into the brains of rats after a stroke. But would the cells form the vital connections needed to restore mobility and sensations like touch?

Now, they have early evidence that the answer to that question isyes. The Lund team turned skin cells into nerve cells, transplanted them into the brains of the rodent stroke models and observed them for six months. The new cells repaired the damage caused by strokes in the animals, the researchers reported in the journal PNAS.

The Lund University team transplanted the reprogrammed skin cells into the rats cerebral cortices, the region of the brain thats most commonly damaged by stroke. Then they used electron microscopy and other technologies to track the cells. That allowed them to see that the cells were making the connections needed to repair damaged nerve circuits.

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This virtual event will bring together industry experts to discuss the increasing pace of pharmaceutical innovation, the need to maintain data quality and integrity as new technologies are implemented and understand regulatory challenges to ensure compliance.

We have been able to see that the fibers from the transplanted cells have grown to the other side of the brain, the side where we did not transplant any cells, and created connections, said co-author Zaal Kokaia, professor of neurology at Lund, in a statement.

RELATED: Restoring neurons to preserve memory after heart attack or stroke

Cell therapy has been proposed for treating stroke damage in the past, but efforts to make it a reality have hit some roadblocks. A stem cell therapy being developed by British biotech ReNeuron failed to hit its primary trial endpoint of improving arm and leg movements. ReNeuron has since turned in better results from a trial of its cell therapy for improving vision in patients with retinitis pigmentosa.

Meanwhile, academic researchers are testing a variety of other therapies aimed at repairing stroke damage. Last year, for example, Stanford researchers showed that blocking a particular microRNA prompted star-shaped brain cells called astrocytes to become neurons, which helped restore memory in rats.

The Lund team is now planning additional animal trials to study how their transplanted cells affect memory and other intellectual functions, they said. They will also watch the rats closely to make sure they arent experiencing side effects, and theyll study the impact of the transplants on regions of the brain.

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Cell therapy restores mobility and sensations in rodent models of stroke - FierceBiotech

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Newswise (New York, NY April 9, 2020) Mount Sinai Health System is the first in the country to use an innovative allogeneic stem cell therapy in COVID-19 patients and will play a central role in developing and conducting a rigorous clinical trial for patients with severe acute respiratory distress syndrome, the breathing illness that afflicts people who have severe cases of COVID-19.

The therapy, known as remestemcel-L, has previously been tested in bone marrow transplant patients, who can experience an overactive immune response similar to that seen in severe cases of COVID-19.

Mount Sinai began administering the therapy, known as remestemcel-L, to patients in late March under the Food and Drug Administrations compassionate use program, which allows patients with an immediately life-threatening condition to gain access to an investigational therapy. Ten patients with moderate to severe cases of COVID-19-related acute respiratory distress syndrome (ARDS), most of whom were on ventilators, were given the therapy and doctors saw encouraging results.

We are encouraged by what we have seen so far and look forward to participating in the randomized controlled trial starting soon that would better indicate whether this is an effective therapy for patients in severe respiratory distress from COVID-19, said Keren Osman, MD, Medical Director of the Cellular Therapy Service in the Bone Marrow and Stem Cell Transplantation Program at The Tisch Cancer Institute at Mount Sinai and Associate Professor of Medicine (Hematology and Medical Oncology) at the Icahn School of Medicine at Mount Sinai. Dr. Osman oversaw the treatment of the first Mount Sinai patients with this innovative therapy.

Under the leadership of Annetine Gelijns, PhD, Alan Moskowitz, MD, and Emilia Bagiella, PhD, of Mount Sinais Institute of Transformative Clinical Trials, Mount Sinai will serve as the clinical and data coordinating center for a randomized clinical trial evaluating the therapeutic benefit and safety of this stem cell therapy in 240 patients with COVID-related ARDS in the United States and Canada. The trial will be conducted as a public-private partnership between the Cardiothoracic Surgical Trials Network, which was established as a flexible clinical trials platform by the National Heart, Lung, and Blood Institute, and Mesoblast, the manufacturer of the cells.

The coronavirus pandemic has caused exponential increases of people suffering with acute respiratory distress syndrome, requiring intubation and mechanical ventilation with many dying, said Dr. Gelijns, who is also the Edmond A. Guggenheim Professor of Health Policy at the Icahn School of Medicine at Mount Sinai. We have designed a clinical trial that will expeditiously determine whether the stem cell therapy will offer a life-saving therapy for a group of patients with a dismal prognosis.

We are interested to study the potential of this anti-inflammatory cell therapy to make an impact on the high mortality of lung complications in COVID-19 patients, said CSTN Chairman A. Marc Gillinov, MD. This randomized controlled trial is in line with our mandate to rigorously evaluate novel therapies for public health imperatives.

The therapy consists of mesenchymal stem cells. These cells are found in bone marrow and serve many functions including aiding tissue repair and suppressing inflammation. The therapy was previously tested in a phase 3 trial in children who had an often-fatal inflammatory condition called graft-versus-host disease (GVHD) that can occur after bone marrow transplants.

The inflammation that occurs in GVHD is the result of a cytokine storm, which activates immune cells that attack healthy tissue. A similar cytokine storm that causes damage to the lungs and other organs appears to be taking place in COVID-19 patients who develop acute respiratory distress syndrome, said John Levine, MD, Professor of Medicine (Hematology and Medical Oncology), and Pediatrics, at the Icahn School of Medicine at Mount Sinai, who helped implement the compassionate use of the drug at Mount Sinai.

These stem cells have shown excellent response rates in severe graft-versus-host disease in children, said Dr. Levine, who is also the co-director of the Mount Sinai Acute GVHD International Consortium (MAGIC). Mesenchymal stem cells have a natural property that dampens excessive immune responses.

Several people were instrumental in quickly and efficiently working through the complex application process for each patient to gain compassionate use of the therapy. Three key players involved were Stacey-Ann Brown, MD, MPH, Assistant Professor of Medicine (Pulmonary, Critical Care and Sleep Medicine) at the Icahn School of Medicine at Mount Sinai; Tiffany Drummond, Assistant Director of Regulatory Affairs at The Tisch Cancer Institute at Mount Sinai; and Camelia Iancu-Rubin, PhD, Director of the Cellular Therapy Laboratory at the Icahn School of Medicine at Mount Sinai.

About the Mount Sinai Health System

The Mount Sinai Health System is New York City's largest academic medical system, encompassing eight hospitals, a leading medical school, and a vast network of ambulatory practices throughout the greater New York region. Mount Sinai is a national and international source of unrivaled education, translational research and discovery, and collaborative clinical leadership ensuring that we deliver the highest quality carefrom prevention to treatment of the most serious and complex human diseases. The Health System includes more than 7,200 physicians and features a robust and continually expanding network of multispecialty services, including more than 400 ambulatory practice locations throughout the five boroughs of New York City, Westchester, and Long Island. The Mount Sinai Hospital is ranked No. 14 onU.S. News & World Report's"Honor Roll" of the Top 20 Best Hospitals in the country and the Icahn School of Medicine as one of the Top 20 Best Medical Schools in country. Mount Sinai Health System hospitals are consistently ranked regionally by specialty and our physicians in the top 1% of all physicians nationally byU.S. News & World Report.

For more information, visithttps://www.mountsinai.orgor find Mount Sinai onFacebook,TwitterandYouTube.

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Mount Sinai Leading the Way in Innovative Stem Cell Therapy for COVID-19 Patients - Newswise

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ANKARA

Amid the closure of Turkish airspace due to the coronavirus outbreak, it opened it up to an Italian jet to save the life of a 2-year-old Italian child.

On March 14, Dr. Massimo Cardillo, the head of Italys National Transplant Center, sent an email asking Turkish authorities for help with the ailing toddler, according to Turkish healthcare sources.

The mail said that after a comprehensive search of international donor banks, the compatible donor for the toddler awaiting a stem cell transplant was found in Turkey, added the sources, who asked not to be named due to restrictions on speaking to the media.

Although by that time Turkey had closed its airspace due to the COVID-19 threat, Turkeys Health Ministry and Foreign Ministry made a special exception in this case to save the toddlers life.

The compatible donor was found and provided by Turkey's Stem Cell Coordination Center.

On March 31, a jet took off from Rome and was allowed to land at Istanbul Airport.

The stem cells were then delivered to the Italian team by Turkish doctors in an isolated room at the airport.

The Italian team took the cells to Rome without incident and delivered them to the hospital for transplantation to the toddler.

Nicoletta Sacchi, director of the Italian Bone Marrow Donor Registry, said they will never forget Turkeys help during this difficult period.

"I extend my thanks to the donor, the main hero of the event. Were grateful to both Turkey and the donor," he said.

Due to the coronavirus pandemic, which has killed tens of thousands of people worldwide since emerging last December, many countries, including Turkey, have closed their airspace to both international and domestic flights.

*Writing by Fahri Aksut

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Turkey opens airspace to save Italian toddler's life - Anadolu Ajans

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Achieved Primary Investigational New Drug (IND) Approval in the U.S.; Positioned to Commence Patient Enrollment

Maintained Clinical Progress in Israel and Nearing Completion of Phase 1/2 Trial

Strategic Commercial Agreement with Canndoc Anticipated to Generate Significant Revenue; Closing of Merger Transaction Progressing as Planned

TEL AVIV, Israel, April 3, 2020 /PRNewswire/ -- Cellect Biotechnology Ltd. (Nasdaq: APOP), a developer of a novel stem cell production technology, today announced operating and financial results for the fourth quarter and full year ended December 31, 2019.

"We achieved a number of strategic priorities in 2019, including the IND approval to commence our first-ever trial in the U.S.," commented Dr. Shai Yarkoni, Chief Executive Officer."We plan to begin enrolling patients for this trial and completing the trial in Israel when the COVID-19 pandemic is mitigated. While these near-term events are value-enhancers, I believe that our recently announced prospective partnership with Canndoc could be a game-changer for Cellect and change our growth trajectory. It has the potential to significantly enhance our short and long term business prospects and shareholder value. As a player in the fast-growing pain management market, we would anticipate significant revenue opportunities already this year."

Recent Strategic Development

As previously announced, on March 4, 2020, the Company entered into a commercial binding Letter Of Intent (LOI) with Canndoc Ltd, a leading pharma grade medical cannabis pioneer and a wholly owned subsidiary of publicly-traded Intercure Ltd. (TASE: INCR),to acquire from Canndoc all rights to the use and sell Canndoc products for the reduction of opioid usage, including accumulated data, as well as on-going and pipeline of clinical trials. This commercial arrangement is subject to negotiation and approval by each company's board of directors and definitive agreements.

Additionally, the two companies signed a non-binding LOI for a full merger. Under preliminary details, Cellect will acquire from Intercure all of Canndoc outstanding shares, in exchange for additional Cellect ADRs to be in total ~95% (~93% on a fully diluted basis) of the merged company. The proposed merger is subject to independent valuation of both companies, fairness opinion by a third party, negotiation of a definitive agreement, approval of the agreement by the Company's Board of Directors and shareholders, internal approvals by Canndoc and Intercure, and customary closing conditions, including the approval of the IMCA (Israeli Medical Cannabis Agency). Upon the closing of the merger, Cellect and Canndoc will aim to fulfill all of the requirements to ensure the Company's ADRs and warrants continue trading on the Nasdaq Stock Market (Nasdaq) and, for this purpose, Intercure would commit to invest a cash sum of at least $3.0 million in any public offering that is undertaken by the Company, at a price of not less than $4.50 per ADR.

Based on the progress to date, the Company continues to expect the commercial and merger transactions will close in the second quarter of 2020.

Additional Operating Highlights:

Clinical Progress Update:

Due to the ongoing COVID-19 pandemic, the Company is experiencing clinical disruption such as:

The Company continues to take all the necessary precautions advised by global health officials to ensure the health and safety of its employees and partners. The Company is unaware of any impact on employees from pandemic related exposure or illness and is continuing to perform in-house research, including in the opioid/pain management area.

Fourth Quarter and Full Year 2019 Financial Results:

Balance Sheet Highlights:

For the convenience of the reader, the amounts have been translated from NIS into U.S. dollars, at the representative rate of exchange on December 31, 2019 (U.S. $1 = NIS 3.456).

About Cellect Biotechnology Ltd.

Cellect Biotechnology (NASDAQ: APOP) has developed a breakthrough technology, for the selection of stem cells from any given tissue, that aims to improve a variety of stem cell-based therapies.

The Company's technology is expected to provide researchers, clinical community and pharma companies with the tools to rapidly isolate stem cells in quantity and quality allowing stem cell-based treatments and procedures in a wide variety of applications in regenerative medicine. The Company's current clinical trial is aimed at bone marrow transplantations in cancer treatment.

Story continues

Forward Looking Statements

This press release contains forward-looking statements about the Company's expectations, beliefs and intentions. Forward-looking statements can be identified by the use of forward-looking words such as "believe", "expect", "intend", "plan", "may", "should", "could", "might", "seek", "target", "will", "project", "forecast", "continue" or "anticipate" or their negatives or variations of these words or other comparable words or by the fact that these statements do not relate strictly to historical matters. For example, forward-looking statements are used in this press release when we discuss Cellect's intent regarding the future potential of Cellect's technology. These forward-looking statements and their implications are based on the current expectations of the management of the Company only and are subject to a number of factors and uncertainties that could cause actual results to differ materially from those described in the forward-looking statements. In addition, historical results or conclusions from scientific research and clinical studies do not guarantee that future results would suggest similar conclusions or that historical results referred to herein would be interpreted similarly in light of additional research or otherwise. The following factors, among others, could cause actual results to differ materially from those described in the forward-looking statements: the Company's history of losses and needs for additional capital to fund its operations and its inability to obtain additional capital on acceptable terms, or at all; the Company's ability to continue as a going concern; uncertainties of cash flows and inability to meet working capital needs; the Company's ability to obtain regulatory approvals; the Company's ability to obtain favorable pre-clinical and clinical trial results; the Company's technology may not be validated and its methods may not be accepted by the scientific community; difficulties enrolling patients in the Company's clinical trials; the ability to timely source adequate supply of FasL; risks resulting from unforeseen side effects; the Company's ability to establish and maintain strategic partnerships and other corporate collaborations; the scope of protection the Company is able to establish and maintain for intellectual property rights and its ability to operate its business without infringing the intellectual property rights of others; competitive companies, technologies and the Company's industry; unforeseen scientific difficulties may develop with the Company's technology; and the Company's ability to retain or attract key employees whose knowledge is essential to the development of its products. Any forward-looking statement in this press release speaks only as of the date of this press release. The Company undertakes no obligation to publicly update or review any forward-looking statement, whether as a result of new information, future developments or otherwise, except as may be required by any applicable securities laws. More detailed information about the risks and uncertainties affecting the Company is contained under the heading "Risk Factors" in Cellect Biotechnology Ltd.'s Annual Report on Form 20-F for the fiscal year ended December 31, 2019 filed with the U.S. Securities and Exchange Commission, or SEC, which is available on the SEC's website, http://www.sec.gov, and in the Company's periodic filings with the SEC.

Cellect Biotechnology Ltd

Consolidated Statement of Operation

Convenience

translation

Twelve months

ended

Twelve months ended

Three months ended

December 31,

December 31,

December 31,

2019

2019

2018

2019

2018

Unaudited

Audited

Audited

Unaudited

Unaudited

U.S. dollars

NIS

(In thousands, except share and pershare data)

Research and development expenses

3,508

12,122

13,513

2,571

4,040

General and administrative expenses

2,954

10,210

15,734

2,378

4,733

Operating loss

6,462

22,332

29,247

4,949

8,773

Financial expenses (income) due towarrants exercisable into ADS

(2,032)

(7,022)

(7,719)

998

(4,784)

Other financial expenses (income), net

433

1,498

(1,415)

129

(238)

Total comprehensive loss

4,863

16,808

20,113

6,076

3,751

Loss per share:

Basic and diluted loss per share

0.023

0.079

0.155

0.027

0.029

Weighted average number of sharesoutstanding used to compute basic anddiluted loss per share

212,642,505

212,6432,505

129,426,091

224,087,799

130,274,953

Cellect Biotechnology Ltd

Consolidated Balance Sheet Data

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Cellect Biotechnology Reports Fourth Quarter and Full Year 2019 Results - Yahoo Finance

Bone Marrow Stem Cells Comments Off on Cellect Biotechnology Reports Fourth Quarter and Full Year 2019 Results – Yahoo Finance | April 10th, 2020

Tyto Cares kit includes a connected otoscope among other things

Numerous startups offering telehealth or remote monitoring solutions closed funding rounds this week, despite slowing activity due to the Covid-19 pandemic. One of them is Tyto Care, a startup with a platform for at-home medical exams. It actually includes a kit with several tools that can allow physicians to remotely listen to a patients heart, measure their temperature, and image their throat and ears. Several hospitals in Israel, including Sheba Medical Center, deployed its technology to care for patients remotely.

On the biotech side, there were some notable rounds, too, including $70 million for Aspen Neuroscience, which is developing a new treatment for Parkinsons disease. The company was founded by Scripps Research Professor Emeritus Jeanne Loring, who developed a way to turn pluripotent skin cells derived from skin cells or other adult cells into neurons that produce dopamine.

Read more about the companies that recently raised funding:

Tyto Care

Funding amount: $50 million

Headquarters: New York, Israel

Tyto Care, a company that lets people conduct at-home medical exams, already saw rising demand before the Covid-19 pandemic. The company said it saw threefold growth in sales last year and has continued to see its users increase during the pandemic. Its at-home telehealth kit includes a handheld device with attachments that allow physicians to remotely listen to the heart and lungs, measure temperature, and look at the throat and ears during an exam.

The company closed an oversubscribed $50 million round, co-led by Insight Partners, Olive Tree Ventures and Qualcomm Ventures. Tyto Care plans to use the additional funds to further expand its footprint in the U.S., Europe and Asia, and add new features to its platform, such as home diagnostics.

Aspen Neuroscience

Funding amount: $70 million

Headquarters: San Diego, California

Aspen Neuroscience is developing a treatment for Parkinsons disease using a patients own cells. The company uses induced pluripotent stem cells to make dopamine-producing neurons, which are affected by the disease.

The company closed a $70 million series A round, led by New York-based healthcare investor OrbiMed, with participation from ARCH Venture Partners, Frazier Healthcare Partners, Domain Associates, Section 32 and Sam Altman.

We are impressed by the progress Aspen has made to date against its goals to develop innovative therapies to treat Parkinson disease and encouraged by the broader investment communitys support of the company, OrbiMed Managing Partner Jonathan Silverstein said in a news release.

The company plans to use the capital to fund the development of its lead candidate, including completing studies needed to submit an investigational new drug application to the FDA, and recruiting for clinical trials.

Tango Therapeutics

Funding amount: $60 million

Headquarters: Cambridge, Massachusetts

Tango Therapeutics, a biotechnology company focusing on developing cancer therapies, closed a $60 million series B round. The company is working on developing treatments to counteract the loss of tumor suppressor genes, reverse cancer cells ability to evade the immune system, and identify new combinations that are more effective than single-agent therapies. The oversubscribed financing was led by Boxer Capital, with additional new investors in Cormorant Asset Management and Casdin Capital.

SonderMind

Funding amount: $27 million

Headquarters: Denver, Colorado

SonderMind, a startup that matches users with in-network therapists, raised $27 million in funding. The series B round was led by prominent VC General Catalyst and F-Prime Capital. Existing investors include the Kickstart Seed Fund, Di?ko Ventures and Jonathan Bush.

The company has a large network of behavioral providers in Colorado, and is expanding in Texas and Arizona. It plans to use the proceeds of the funding round to expand its partnership with payors, employers and health systems.

SilverCloud

Funding amount: $16 million

Headquarters: Boston, Massachusetts

SilverCloud has seen an uptick in users tapping into its mental health programs for depression, anxiety and other conditions. The company raised a $30 million series B round, led by MemorialCare Innovation Fund, the VC arm of MemorialCare Health System. Other participating investors included LRVHealth, OSF Ventures and UnityPoint Health Ventures.

So far, the company had drummed up partnerships with more than 300 companies. Notably, it was also one of the products selected for Express Scripts first digital health formulary. SilverCloud said it would use the additional funds to expand access to mental health support services for healthcare professionals, as well as their families and their patients.

CyberMDX

Funding: $20 million

Headquarters: New York

Healthcare security startup CyberMDX closed a $20 million funding round. Sham, a French risk management and insurance provider, led the funding round, with participation from Pitango Venture Capital and Oure Ventures.

CyberMDX monitors a providers network for threats to its IT systems, connected medical devices, and other IoT devices. The company said it will use the $20 million to expand its platform to new markets.

Photo credit: Tyto Care

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Funding roundup: At-home medical exams and a Parkinson's treatment - MedCity News

Skin Stem Cells Comments Off on Funding roundup: At-home medical exams and a Parkinson’s treatment – MedCity News | April 10th, 2020

We can summon our cars with our smart phones, have a drone bring our wildest wishes to our door, and were just an Alexa and Roomba partnership away from having our own The Jetsonsstyle domestic assistant at our beck and call. In the world of aesthetics, futuristic procedures we never knew we needed are here now, too. These are todays top tweaks that prove the future is actually now.

Future FatMoving fat from one part of the body to another is a procedure that has been around since the late 1800s, and because fat transfers have been a reliable source of volume for faces, butts and breasts, their popularity continues to rise. Now, you dont even need your own fat to get a volume boost. Renuva is an alternative way to do fat transfers without liposuction, says Vero Beach, FL plastic surgeon Alan Durkin, MD. It can fill in scars and dimples, and plump hollow cheeks and hands. Instead of creating collagen, it induces natural human fat. When injected, Renuva acts as a scaffold that allows the body to stimulate its own fat cells to grow and divide creating organic fat. So, where does this fat come from? Dr. Durkin says its donated human tissue that is screened extensively and processed for quality and safety. It arrives in dehydrated form and we rehydrate it with saline before injecting it.

Glow GettersThink of microdroplets of filler as the Tiny House Nation of injectable rejuvenation. Although microdroplets have not yet been approved by the FDA in the United States, Juvderm Volite was created for this specific application and is being used extensively and successfully in Europe, says Bloomfield Hills, MI dermatologist Linda C. Honet, MD. Restylane Skinboosters Vital and Vital Lite are also used with the microinjection technique in Canada and marketed with a special microinjection syringe that delivers tiny amounts0.01 milliliters of fillerin a serial injection fashion. The main benefit of this approach? A consistent, superficial glow. We have found that when the hyaluronic acidbased filler is deposited in one area, deeper into the dermis, we still see the plumping and hydration in all areas of the skin,adds Beverly Hills, CA dermatologist Ava Shamban, MD.

A few years ago, under-eye carboxytherapy injection videos were going viral, as the insertion of carbon dioxide under the skin causes skin to inflate like a balloon. That visual hasnt stopped doctors from utilizing carboxytherapy to boost skin rejuvenation. The intent of carboxy injections is to increase oxygen in the skin by increasing capillary blood flow to eliminate carbon dioxide, says San Antonio dermatologist Vivian Bucay, MD. Now, a CO2 Lift mask gives similar benefits without the intense skin expansion. The mask is made of two gels that we mix together and apply on the skin, she adds. Although there are no formal studies to show carboxytherapy speeds recovery compared to other topicals, Dr. Bucay uses it after laser treatments, Ultherapy, microneedling and chemical peels to reduce healing time.

Miracle GrowWhen we think of getting something lasered, we tend to think of the skin- resurfacing treatments that obliterate layers of dead skin to reveal baby-fresh skin. But some doctors, like New York dermatologist Doris Day, MD, are harnessing laser energy to help hair grow in places where it hasnt for years. I use the Fotona laserit employs photobiomodulation, a form of gentle deep heatto stimulate the stem cells of dormant hair follicles and encourage regrowth. The laser energy penetrates the tissue, where it interacts with chromophores and induces a complex set of reactions that increases circulation, reduces inflammation and helps restore normal cellular function. Currently, there are no clinical studies to prove the efficacy of this hair growth treatment, but there are controlled studies being planned. Dr. Day has seen results with some patients as part of a long-term plan that also includes Nutrafol, DuoZyme supplements and quercetin, as well as topical treatments and sometimes platelet-rich plasma therapy.

Body MovinBelly buttons get an automatic upgrade during a tummy tuck or Mommy Makeover, but stand-alone umbilicoplasty is trending as patients continue to find small areas of their bodies to tinker with and perfect. And, its not just about turning an outie into an innie. Most of the belly button surgeries performed by Raleigh, NC plastic surgeon Michael Law, MD are on those who have had a tummy tuck with another doctor and are left with visible scarring, or their belly button has an odd, or operated-on look.

For tummies in need of extra tightening, theres a nonsurgical option being explored that involves the same polydioxanone (PDO) threads used in thread lifts for the face. Abdominal thread lifts are essentially retention sutures, which are placed into the lower, mid or upper abdomen to lift tissue, explains Spokane, WA dermatologist Wm. Philip Werschler, MD. Ideal candidates are those who arent surgical candidates, those who dont want surgery, or those whose concerns are less than what a typical tummy tuck would correct. The in-office procedure takes about one hour and is performed under local anesthetic. There are no current studies to show the efficacy or benefits of thread lifts in this area, but Dr. Werschler says he continues to see good results.

To slenderize the legs, calf reduction is actually a thing. Excess fat on the calves may result in the appearance of cankles being a bit shorter can also make the calves appear thicker, says Los Angeles plastic surgeon Peter Lee, MD. We can perform liposuction in order to trim them down to the patients goal size. Excision techniques may also be needed for the removal of excess tissue. To make calves look smaller without surgery, New York dermatologist Tatiana Khrom, MD uses Botox Cosmetic to reshape: We target the back of the lower leg, the gastrocnemius muscles, to slim the calves and help patients fit into their favorite boots or feel more confident in their shorts, with results lasting up to six months.

Important IntelAll the doctors included in this story mentioned how important creative, off-label use is to the medical communityand strongly stressed seeing a board-certified doctor, practicing within scope, who has vast experience and knowledge on the treatments in question. Off-label use can be safe when done by an experienced doctor who specializes in that off-label treatment; that doctor may also produce research showing efficacy of the off-label use, has been trained on the off-label use, or performs it regularly. Of course, all cosmetic treatments can have a potential risk whether on-or off-label and this is why its important to see a properly board-certified doctor.

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Cosmetic Treatments The New New Wave: Trending Treatments You've Never Heard of Until Now Apr - NewBeauty Magazine

Skin Stem Cells Comments Off on Cosmetic Treatments The New New Wave: Trending Treatments You’ve Never Heard of Until Now Apr – NewBeauty Magazine | April 10th, 2020

Bizizz Market Research has recently published a research report, 3D Bioprinting Market By Component (3D Bioprinters (Magnetic 3d bioprinting, Laser-assisted bioprinting, Inkjet 3d bioprinting, Micro extrusion Bioprinters, and Other) Bioprinters Bio inks (Natural bio inks, Synthetic bio inks, and Hybrid bio inks)), Material (Hydrogels, Extracellular Matrices, Living Cells, and Other Biomaterials), Application (Research Applications (Drug Research, Regenerative Medicine, 3d Cell Culture) Clinical Applications (Skin, Bone & Cartilage, Blood Vessels, and Others), End User (Hospitals, Research Organizations and Academic Institutes Biopharmaceutical, and Companies), and Region-Global Industry Trends, Estimation & Forecast, 2019 2027. As per the report,Global 3D Bioprinting Marketwas valued at US$ 623 Mn in 2018 and it is anticipated to reach at market value of US$ 1.4 Bn by 2027, witnessing a CAGR of 18.6 % during the forecast period. Key drivers of the market are increasing prevalence of chronic disorders like kidney and heart failures, growing elderly populace, and the insufficient number of organ donors. However, dearth of skilled professionals may hinder the growth of the market during the forecast period.

3D bioprinting technology has witnessed accelerated adoption in the healthcare industry. Bioprinting has emerged as a promising technological know-how for the fabrication of synthetic tissues and organs, which can revolutionize the analysis and cure of more than a few scientific conditions. Bioprinting businesses around the world are constantly innovating in regenerative medicine, tissue engineering, drug therapies, and stem cell therapy, which is gaining attention from healthcare authorities and pharmaceutical agencies to envision a future with higher patient care, custom-made medical treatment, and an alternative to organ transplantation.

Over the previous few years, essential technological advancements in the 3D bioprinting space have taken place for numerous scientific applications, inclusive of skin tissue development, most cancers therapeutics, bone and cartilage development, and liver modeling. Advanced technologies grant players with a competitive area and thereby help in strengthening their function and share in the market. For instance, in 2018, Poietis (France) launched the 3D bioprinted pores and skin model, Poieskin. The total human pores and skin model is made by using the bioprinting of essential human collagen and fibroblast for the dermal layer and major human keratinocytes for the epidermal layer.

The 3D Bioprinting market is anticipated to register a CAGR of over 18.6% during the forecast period.

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By Material, the living cells segment held a prominent share in Global3D BioprintingMarket in 2018.

On the basis of material, the 3D bioprinting market is segmented into hydrogels, extracellular matrices, living cells, and other biomaterials. In 2018, the living cells segment accounted for the biggest market share particularly due to the developing R&D in the fields of regenerative medicine and stem cell research, and increasing public and personal investments to help research

By Application, Skin Printing Segment of Global 3D Bioprinting Market Is Anticipated To Witness the Fastest CAGR during the Forecast Period

The clinical applications market is similarly segmented into skin printing, bone & cartilage printing, blood vessel printing, and other scientific applications. Among these, the pores and skin printing purposes segment is estimated to develop at the best CAGR of 19.8% in the course of the forecast period. This can be attributed to the technological developments and new product launches in this utility segment, and the growing wide variety of aesthetic and reconstruction surgeries across the globe.

North America is anticipated to dominate the Global 3D Bioprinting Market during the Forecast Period

Growing target populace base is in all likelihood to be the crucial cause boosting the regions 3D bioprinting market growth. The existence of well-established corporations and subtle healthcare set-up in consort with high income tiers in the location are also anticipated through the market development. Moreover, huge research and improvement activities carried out inside the place are said to make contributions to market expansion. Additionally, the accessibility of 3D printed drugs that can be tailor-made in accordance with the age and body weight of a person is supporting to boost up the market evolution.

Global 3D Bioprinting market was highly consolidated with key players accounting for significant share in 2018. Prominent players operating in the Global 3D Bioprinting Market are: Solidscape, Inc. (acquired by Prodways Group), TeVido BioDevices, LLC, 3Dynamics Systems Ltd., Bio3D Technologies Pte. Ltd., Aspect Biosystems Ltd., Stratasys Ltd., Luxexcel Group B.V., Materialise N.V., Cyfuse Biomedical K.K., Voxeljet A.G., Envision TEC, and Organovo Holding, Inc., among others.

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Global 3D Bioprinting Market is anticipated to reach at market value of US$ 1.4 Bn by 2027 - Galus Australis

Skin Stem Cells Comments Off on Global 3D Bioprinting Market is anticipated to reach at market value of US$ 1.4 Bn by 2027 – Galus Australis | April 10th, 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 and tumors, wherein their toxicity, impurity, and other aspects are studied.

With the growing number of successful stem cell therapy treatment 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.

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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.

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.

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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 Highlights On Future Development 2025 - Science In Me

Spinal Cord Stem Cells Comments Off on Stem Cell Assay Market Highlights On Future Development 2025 – Science In Me | April 8th, 2020

/ AChR subunit KO zebrafish lines

We generated a subunit gene KO zebrafish (KO) using CRISPR-Cas9 (Fig. 1A) and an subunit gene KO zebrafish (KO) using transcription activatorlike effector nucleases (TALEN) (Fig. 1A). The KO zebrafish did not show obvious phenotypes during development and matured in a fashion indistinguishable from wild-type (WT) siblings (fig. S1). In contrast, KO fish generally failed to form swim bladders, and most of them died prematurely within 2 weeks after fertilization. However, a fraction of KO fish (approximately 25%) survived to adulthood. A double KO (DKO) line was generated by crossing KO and K lines. DKO larvae also failed to form swim bladders (Fig. 1B) and died within 2 weeks after fertilization.

(A) Schematic diagram of targeted genes. Arrowheads indicate targeted regions of genome editing. Each box and line indicates an exon and an intron, respectively. Alignment of genomic DNA sequences of WT and KO lines showed a 7base pair (bp) insertion in the AChR subunit gene chrng and a 1-bp insertion in the AChR subunit gene chrne. (B) Photograph showing WT and / DKO larva at 6 dpf. Notice the lack of swim bladder (arrowheads) in DKO. Scale bar, 1 mm. (C) Trunk regions of a WT larva (6 dpf) and a DKO larva (6 dpf) were stained with -BTX conjugated with Alexa Fluor 488 (green). In WT, AChRs were distributed in myoseptal regions (arrows) and in punctae in middle regions (arrowhead). DKO had -BTX signals only in myoseptal regions. Scale bars, 100 m.

We histologically analyzed the expression of AChRs in the trunk region of 6 days post-fertilization (dpf) larvae by using -bungarotoxin (-BTX) conjugated with Alexa Fluor 488, a toxin that specifically binds to the assembled AChR (Fig. 1C). AChR clusters in DKOs were observed only in boundary regions between body segments (Fig. 1C), where slow muscles form NMJs (16). We initially expected that AChRs in fast muscles of DKO larvae would convert to the slow muscletype AChRs, comprising only , , and subunits. This conversion of subunit composition would not cause a change in AChR distribution visualized by -BTX, because both types of AChRs bind to -BTX. However, -BTX signals were absent in fast muscles, which suggested that fast muscles could not express AChRs composed of , , and subunits.

To correlate the AChR expression pattern observed by the -BTX staining with the synaptic function, we analyzed synaptic activities of fast and slow muscles in the DKO line at 6 dpf. We recorded spontaneous synaptic currents from muscle cells using the whole-cell patch clamp technique (Fig. 2, A to C). Traces show miniature endplate currents (mEPCs) from muscles of WT or DKO larvae (Fig. 2A). Slow muscles in the DKO line exhibited mEPCs. The frequency (14.5 3.1 Hz in WT, 15.5 3.2 Hz in DKO) and the amplitude of slow muscle mEPCs (260.0 74.1 pA in WT, 491.7 105.2 pA in DKO) showed no differences between WT and DKO lines (Fig. 2, B and C). However, fast muscles in DKO failed to produce mEPCs. To confirm that the lack of mEPCs is caused by the absence of functional receptors, we recorded currents in muscles generated by puff application of ACh (Fig. 2D). While fast muscles in WT larvae showed ACh-induced currents (756.4 138.6 pA), those in DKO larvae failed to show any response (0 0 pA). These results, in conjunction with the -BTX staining (Fig. 1C), showed that fast muscles of DKO larvae do not express any AChRs and receive no synaptic input.

(A) mEPC traces from fast or slow muscles of WT and DKO larvae (6 dpf) by whole-cell patch-clamp recordings. Fast muscle cells in DKO failed to exhibit mEPCs. (B and C) Frequencies (B) and amplitudes (C) of mEPCs were plotted for each muscle (n = 8 cells). (D) Representative traces of voltage-clamped slow and fast muscles in DKO larvae in response to the application of 30 M ACh. Calibration: 1 s, 500 pA. Amplitudes of ACh-induced currents in slow (n = 7 cells) and fast muscles (n = 7 cells) are shown. Each dot represents a muscle cell. (E) Construct used for Ca2+ imaging. Top: The GCaMP7a coding sequence was fused to the promoter region of the -actin promoter pact. Bottom: Schematic illustration showing the experimental procedure. The gene construct was injected into eggs of DKO at the one cell stage. Ca2+ response was analyzed at 6 dpf. Representative traces showing the increase of F/F in a fast muscle (black line) and a slow muscle (red line) during spontaneous contractions. (F) Overexpression of the subunit fused with an EGFP (-EGFP) in WT (3 dpf). Top panels: -EGFPs were expressed under the control of a slow musclespecific promoter, psmyhc. EGFP signals (green), expressed in the superficial slow muscles, filled the cytoplasm and did not colocalize with -BTX (magenta) signals. Bottom panels: -EGFPs were expressed under the regulation of pact. In deeper layer fast muscles, the clusters of EGFP and -BTX colocalized (arrowheads). Scale bars, 50 m.

We performed in vivo Ca2+ imaging in the DKO larvae at 6 dpf to further support the result of synaptic current recordings. We designed a gene construct in which a pan-muscle promoter, -actin promoter, drives the expression of a Ca2+ indicator, GCaMP7a (17), and injected the construct into fertilized eggs (Fig. 2E). In DKOs, we recorded Ca2+ response associated with spontaneous locomotion activities, induced by the application of N-methyl-d-aspartate (50 M) (18). The results showed that slow muscle cells exhibited Ca2+ transients, while fast muscle cells did not generate any Ca2+ response.

Considering that fast muscles do not allow composition of , , and subunits, we next examined whether slow muscles conversely allow incorporation of subunits in the AChR pentamer, by overexpressing the subunit in slow muscles. We designed a gene construct that expressed an subunit fused with enhanced green fluorescent protein (-EGFP) under the regulation of a slow musclespecific promoter, psmyhc (19). We injected the construct into fertilized WT eggs and observed the expression of EGFP at 3 to 4 dpf. EGFP signals typically filled the cytoplasm of the slow muscle cells and never colocalized with -BTX signals (Fig. 2F). In a control experiment, in which -EGFP was driven by the pan-muscle promoter (-actin promoter), the -EGFP signals made clusters in fast muscles, colocalizing with -BTX signals in deeper layers of the trunk region where fast muscles form NMJs. Together, fast muscles and slow muscles express specific types of AChR, and the alternate composition of subunits is prohibited.

To examine how silencing of synapses in fast muscles affect locomotion, we next analyzed swimming of WT and DKO larvae at 6 dpf. We induced escape responses by gentle tactile stimuli. Locomotion was recorded with a high-speed camera, and we measured angles between head and tail trajectories throughout each escape response (Fig. 3A and movie S1). WT fish turned their heads 120 to 140 in the initial stage of escape. The typical startle response of teleosts generally begins with a large turn of the head (termed C-bend), followed by a robust forward propulsion as described in previous studies (20).

(A) Escape behaviors in WT and DKO lines at 6 dpf in response to tactile stimuli. Images of representative larva on the left show superimposed frames of the complete escape response (the duration of movement is indicated in the top right corner). Scale bars, 2 mm. Kinematics for representative traces of 10 larvae are shown for the initial 50 ms of the response. Middle panels represent averaged traces. In the right panels, each trace represents a different larva. Body angles are shown in degrees, with 0 indicating a straight body, and positive and negative values indicating body bends in opposite directions. Scale bars, 10 ms. (B to D) Maximum turn angles, time to reach the maximum angle, and post-startle swimming speed were calculated for each group of fish (6 dpf). In DKO, the turn angle and the swimming speed were notably reduced, and it took longer to reach maximum angles (n = 10 fish). (E and F) Analyses of spontaneous locomotion. Images of representative larva (left) for WT or DKO showed superimposed frames of spontaneous swim bouts (the duration of movement indicated in the bottom right corner). Swimming speed was calculated for WT (n = 5 fish) and DKO (n = 5 fish), which showed no significant difference. Scale bars, 2 mm.

The initial turns of the DKO larvae were in sharp contrast to WT. Averaged maximum head turn angles in DKOs were markedly smaller compared to WT larvae (116.0 5.8 in WT, 20.2 4.0 in DKO; P < 0.001) (Fig. 3B), and time to reach the maximum angle was increased (8.7 0.2 ms in WT, 15.8 0.8 ms in DKO; P < 0.001) (Fig. 3C). In addition to the absence of C-bends, the post-startle swimming speed of the DKO line was also notably slower (84.9 8.1 mm/s in WT, 12.8 1.3 mm/s in DKO; P < 0.001) (Fig. 3D).

In addition to the escape response, we also analyzed spontaneous locomotion, which corresponds to the slow swim described by Budick and OMalley (21) or scoot reported by Burgess and Granato (22) (Fig. 3, E and F). Significant difference in swimming speed was not observed between WT and DKO (16.1 1.60 mm/s in WT, 13.2 0.9 mm/s in DKO; P = 0.20) (Fig. 3F). Thus, the contribution of fast muscles in spontaneous swimming is relatively small. These results strongly suggest that fast muscles in larval zebrafish play a key role in executing quick escape responses including the C-bend and fast forward propulsion behaviors, which corroborate earlier studies (23).

DKO fish die prematurely and do not develop into adults. However, KOs that reached the adult stage are expected to lack both and subunits, because subunit expression terminates early in development.

To dismiss the possibility of compensatory up-regulation of the subunit in adult KOs, we analyzed the expression of subunit mRNA with digital droplet polymerase chain reaction (ddPCR). Subunit mRNA was not detected in adult KOs, which were 3 to 5 months old (Fig. 4A). Interestingly, subunit mRNA was strongly up-regulated in larval KOs (Fig. 4B), which may account for functional escape response behavior at 6 dpf (fig. S1). Thus, our findings suggest that compensation by the subunit expression occurs only in larval KOs and not in adults.

(A) Quantification of or subunit mRNA in adult muscles. Subunit was not detected in WT. or subunit mRNA was not detected in KO (n = 6 fish in WT, n = 5 fish in KO). Sample numbers are shown in parentheses. (B) mRNA expression of subunit in 1-dpf larvae. Subunit was highly up-regulated in the KO (n = 5 fish) compared to WT (n = 5 fish). Sample numbers are shown in parentheses. (C) Schematic illustration of a transverse section of the trunk region. The area shown in micropictograms is indicated with a box. The distribution of AChRs in adults, WT or KO, was visualized by -BTX conjugated with Alexa Fluor 488 (green). Broken lines indicate the boundary of fast muscle area (arrowheads). Fast muscles in the KO fish lack -BTX signals. (D) Sections of adult fast muscles of WT and KO, stained with the fast musclespecific F310 antibody. Fast muscles in KO fish did not display atrophy. In the right panel, diameters of fast muscles in WT and KO were calculated (87 fibers, n = 3 fish). There was no significant difference. Scale bars, 100 m.

The expression of AChR in adult KO fish, visualized by -BTX, was consistent with the lack of compensation (Fig. 4C). Transverse sections of the trunk region were labeled with -BTX. Slow, intermediate, and fast muscles are spatially segregated (11). Slow muscles are located closest to the surface. WT fish displayed universally distributed, positive -BTX signals. In sharp contrast, -BTX signals in the KO fish were detected only in shallow, lateral regions, and fast muscles of the adult KO lacked AChR expression.

In spite of the absence of -BTXpositive signals, fast muscle fibers in KO fish unexpectedly lacked signs of prominent atrophy (24). A fast musclespecific F310 antibody used via immunohistochemistry allowed the visualization and diameter measurements of fast muscle fibers. Statistical analysis revealed no difference between KO and WT fiber size (58.7 0.5 m in WT, 58.3 0.7 m in KO; P = 0.945) (Fig. 4D).

We observed escape responses induced by objects dropping on water and subsequently analyzed C-bend angles and the swimming speed during escape (Fig. 5A) (25). We compared the maximum C-bend angles between the focal genetic lines. Similar to WT larvae (Fig. 3), WT adults start the escape response with the initial extreme head turn. Unexpectedly, we found that KO adult fish also display robust C-bends (Fig. 5, A and B). Although smaller in amplitude (103.0 7.5 in WT, 53.4 2.5 in KO), their time course did not exhibit any delay compared to WT. This is in sharp contrast to the complete loss of C-bend behavior observed in larval DKOs (Fig. 3). The duration of first turn also showed no significant difference between WTs and KOs (38.9 3.8 ms in WT, 46.6 4.9 ms in KO).

(A) Escape behaviors in WT and KO adults (3 to 4 months old). The startle response was induced by dropping objects on water. Images of representative fish to the left show superimposed frames of the complete escape response (the duration of movement is indicated in the bottom right corner). Kinematics for representative traces from 10 or 9 fish are shown for the initial 50 ms of response. Middle panels represent averaged traces. In right panels, each trace represents a different fish. Body angles are shown in degrees, with 0 indicating a straight body. Positive and negative values indicate body bends in opposite directions. (B) First turn angles were calculated for each group of fish (n = 10 fish in WT, n = 9 fish in KO). Turn angles were reduced in the KO fish. Sample numbers are shown in parentheses. (C) Post-startle swimming speed and total distance traveled were calculated for the first 120 ms. There was no significant difference between WT (n = 10 fish) and KO (n = 9 fish) adults.

Furthermore, the forward propulsion during escape of the KO adult zebrafish was almost intact. When the distance traveled was plotted against the time after stimulation, the curves for WT and KO nearly overlapped (Fig. 5C). The swimming speed (31.7 1.3 cm/s in WT, 25.5 3.0 cm/s in KO; P = 0.08) and total distance traveled (4.0 0.2 cm in WT, 3.2 0.4 cm in KO; P = 0.08) were similar between WT and KO adults.

Suspecting that compensation of locomotion occurred at the level of neural projection, we examined the projections of motor neurons by retrograde labeling using a fluorescent tracer, dextran conjugated with Alexa Fluor 488 (Fig. 6, A to C). We injected the tracer into muscles of WT and K fish following a method described in a previous report (26). Spinal motor neurons in adult zebrafish are classified on the basis of morphological features. Dorsomedial motor neurons with larger cell somas, which are called primary motor neurons (pMNs), specifically innervate fast muscles. Ventrolateral motor neurons with smaller somas, called secondary motor neurons (sMNs), are grouped in distinct populations depending on the innervation target: fast, intermediate, and slow muscles (2729). We analyzed the location of motor neuron somas in the spinal cord (Fig. 6B) by measuring the distance from the center of spinal cord to cell somas. In WT adults, fast muscles were innervated mainly by dorsomedial motor neurons (located close to the center), and slow muscles were innervated by ventrolateral motor neurons (Fig. 6, A and B).

(A) Schematic illustration of a transverse section of the trunk region showing the sites of dye injections. Right panels showing cell bodies of labeled motor neurons (arrowheads) in spinal cords. Broken lines indicating outlines of spinal cords. Scale bars, 50 m. (B) A graph showing the distance from the center of the spinal cord to cell bodies of motor neurons. In WT, motor neurons located close to the center innervate fast muscles, and ventrolateral motor neurons innervate slow muscles. In KO, slow muscles were innervated by motor neurons located close to the center. Numbers of labeled cells are shown in parentheses. (C) Graph showing the size of cell somas of motor neurons. In WT, large motor neurons innervate fast muscles, and smaller neurons innervate slow muscles. In KO, slow muscles were innervated by large motor neurons. (D) Schematic illustration of a transverse section of the trunk region showing the locations of the DiI crystal insertion. The right panel displays cell body of labeled pMN (arrowhead) in the spinal cord. The broken line indicates the outline of the spinal cord. Scale bar, 50 m. (E) Presynaptic structures were visualized by SV2A antibody. Broken lines indicate the boundary of slow muscle area (left side). Note the reduced signal in the fast muscles of the KO fish. Scale bars, 100 m. (F and G) Fast musclespecific myosins labeled by F310 antibody in WT (F) and KO (G). In (G), the boxed area is enlarged in the right panel. Broken lines indicate the boundary of slow muscle area (left side). Arrowheads indicate muscle cells with F310 signals in the slow muscle region. While a small number of slow muscle cells in WT sometimes showed immunoreactivity, the cell number was markedly increased in KO. Scale bars, 100 m. (H and I) Glycolytic muscle fibers were visualized by GPD staining in WT (H) and KO (I). Black broken lines indicate the boundary between slow and intermediate muscles, and the red broken line indicates the boundary between intermediate and fast muscles. Fast, intermediate, and slow muscle areas are labeled with F, I, and S, respectively. Note that the intermediate muscle region in KO is hard to distinguish from the fast muscle region, blurring the boundary (I). Arrowheads in the right panel indicate muscle cells with GPD signals in the slow muscle region. Scale bars, 100 m. (J) Schematic illustration showing the rerouted innervation of pMNs. In KO adults, synaptic silencing of fast muscles led to the innervation of fast musclespecific pMNs on slow muscle. This reinnervation caused conversion of slow to fast muscles. The projections of sMNs that innervate fast muscles may not change.

Both the location and the size of motor neuron somas suggested that slow muscles in KO adults were innervated by large motor neurons, which innervate only fast muscles in WT adults (Fig. 6C). Ventrolateral neurons did not seem to innervate slow muscles in KOs, as they were absent in retrograde labeling (Fig. 6, B and C). When we injected the tracer into fast muscles of KO adults, pMNs were not labeled (fig. S2). Motor neurons labeled in these preparations were presumably fast sMNs (26).

To rule out the possibility that pMN axons are inadvertently damaged by dye injections into slow muscles of KO adults, we used another method of retrograde labeling using a lipophilic tracer DiI (or DiIC18), which has a minimal possibility of causing pressure injection damage (30). After gently placing crystals of DiI onto slow muscles of KO adults, we found that pMNs were labeled in spinal cords of KO adults (Fig. 6D). We also analyzed the presynaptic input in muscles of WT and KO adults using SV2A antibody to visualize presynaptic proteins (Fig. 6E). The results showed that positive signals within fast muscles were reduced in KO compared to WT adults. Thus, fewer motor neurons innervated fast muscles in KO fish.

The muscle cell type is determined by the motor neuron input (31). Suspecting the signals from pMNs may convert the properties of slow muscles into those of fast muscles in adult KO fish, we examined the characteristics of slow muscle fibers. To do so, we analyzed the F310 antibody immunohistochemistry in adult KO fish, which labels fast musclespecific myosin (Fig. 6, F and G) (19). We also examined the -glycerophosphate dehydrogenase (-GPD) activity, which is a well-established method to visualize glycolytic muscles, i.e., fast muscles (Fig. 6, H and I) (32). Some tissue located in slow muscle regions stained positive for F310 (n = 3 fish; Fig. 6G) and -GPD signals (n = 3 fish; Fig. 6I), suggesting that some slow muscles expressed the fast muscletype isoform of myosin light chain and obtained glycolytic ability. Intermediate muscle fibers in KO also showed higher glycolytic ability compared to WT (Fig. 6, H and I). Thus, a subpopulation of slow and intermediate muscles was converted to fast muscles, presumably due to the innervation of fast muscle motor neurons (31).

In summary, the absence of AChRs in developing KOs is presumed to drive motor neuron axon innervation of fast muscles to instead reroute to slow muscles. These rewired pMNs presumably predominate over original axons in slow muscles, as a result of synaptic competition, and convert some slow and intermediate muscles to fast muscles (Fig. 6J).

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Synaptic silencing of fast muscle is compensated by rewired innervation of slow muscle - Science Advances

Spinal Cord Stem Cells Comments Off on Synaptic silencing of fast muscle is compensated by rewired innervation of slow muscle – Science Advances | April 8th, 2020

PERTH, Australia Australian stem cell therapy company Mesoblast Ltd. announced that the FDA gave it the green light to test its allogeneic mesenchymal stem cell (MSC) product candidate remestemcel-L in patients with acute respiratory distress syndrome (ARDS) caused by coronavirus (COVID-19).

Were going to be evaluating whether an injection of our cells intravenously can tone down the immune system just enough so it gets rid of the virus but doesnt destroy your lungs at the same time, Mesoblast CEO Silviu Itescu told BioWorld.

What people are dying of is acute respiratory distress syndrome, which is the bodys immune response to the virus in the lungs, and the immune system goes haywire, and in its battle with the virus it overreacts and causes severe damage to the lungs, Itescu said.

The FDA clearance provides a pathway in the United States for use of remestemcel-L in patients with COVID-19 ARDS, where the prognosis is very dismal, under both expanded access compassionate use and in a planned randomized controlled trial.

The company is in active discussions with various governments, regulatory authorities, medical institutions and pharmaceutical companies.

Recently published results from an investigator-initiated clinical study conducted in China reported that allogeneic MSCs cured or significantly improved functional outcomes in all seven treated patients. A post-hoc analysis of a randomized, placebo-controlled study in 60 patients with chronic obstructive pulmonary disease demonstrated that remestemcel-L significantly improved respiratory function in patients with the same elevated inflammatory biomarkers that are also observed in patients with COVID-19 ARDS.

Remestemcel-L is being developed for various inflammatory conditions and is believed to counteract the inflammatory processes implicated in those diseases by down-regulating the production of pro-inflammatory cytokines, increasing production of anti-inflammatory cytokines, and enabling recruitment of naturally occurring anti-inflammatory cells to involved tissues.

The safety and therapeutic effects of remestemcel-L intravenous infusions have been evaluated in more than 1,100 patients in various clinical trials.

The stem cell therapy was successful in a phase III trial for steroid-refractory acute graft-vs.-host disease (aGVHD) in children, a potentially fatal inflammatory condition due to a similar cytokine storm process as is seen in COVID-19 ARDS.

Cynata in preclinical ARDs studies

Fellow Aussie regenerative medicine company Cynata Therapeutics Ltd. is studying the utility of its Cymerus MSCs as a treatment for ARDS associated with COVID-19 with the Critical Care Research Group at Prince Charles Hospital in Brisbane, Australia.

Acute respiratory distress syndrome is a huge problem worldwide and is prevalent aside from COVID-19, but suddenly it is on the front page because people are dying of this. The data behooves us to see if MSC treatment can rescue people from this, Cynata CEO Ross Macdonald told BioWorld.

The Critical Care Research Group has long seen the need to improve interventions in patients who have ARDS, and they have an interest in MSCs and came to us, he said.

ARDS is an inflammatory process leading to the build-up of fluid in the lungs and respiratory failure. It can occur due to infection, trauma and inhalation of noxious substances. ARDS often affects previously healthy individuals and accounts for roughly 10% of all ICU admissions, with almost 25% of patients requiring mechanical ventilation. Survivors of ARDS are often left with severe long-term illness and disability.

The study will investigate Cynatas Cymerus MSCs as a treatment for ARDS, in combination with extracorporeal membrane oxygenation (ECMO). ECMO circulates blood through an artificial lung, oxygenating the blood before putting it back into the bloodstream of a patient. ECMO has emerged as a treatment adjunct to support the vital organs in patients with severe ARDS, which can provide short- to medium-term mechanical pulmonary support.

MSC therapy could be used as a possible treatment for ARDS due to the ability of MSCs to reduce inflammation, enhance clearance of pathogens and stimulate tissue repair.

The study will first seek to determine if Cymerus MSC treatment improves oxygenation in sheep with ARDS supported by ECMO, and to evaluate the effects on lung mechanics, blood flow, inflammation and lung injury, as well as safety.

If the study is successful, the data would support progression to a clinical trial of Cymerus MSCs in humans with ARDS undergoing ECMO support.

The study is being funded by the Queensland State Government, the National Health and Medical Research Council (NHMRC), the Intensive Care Society UK, and the Prince Charles Hospital Foundation.

If the FDA or TGA wants us to step in, were all ears. Our product is manufactured in the United States, and supply is not an issue. In theory, were ready to go, Macdonald said.

He was quick to point out that what differentiates Cynatas stem cell product from competitors is that its MSCs are derived from induced pluripotent stem cells (iPSCs), and most stem cell companies rely on multiple donors to donate either bone marrow or adipose tissue as their primary tissue sources. From those sources they derive a small number of MSCs, which represent the starting material of their manufacturing process.

Cynatas Cymerus MSC therapy comes from a single donor and can be produced in limitless quantities, giving it the potential to create a new standard, Macdonald said. The platform technology is based on versatile stem cells known as mesenchymoangioblasts (MCAs), which are a precursor of mesenchymal stem cells.

That process allows the company to make MSCs derived from iPSCs in large amounts without losing their potency, and that forms the basis for the companys platform technology, which it calls Cymerus.

Cynata is gearing up for three phase II trials with its Cymerus MSCs in graft-vs.-host disease (GVHD), critical limb ischemia and osteoarthritis.

Mesoblasts remestemcel-L is being studied in clinical trials across several inflammatory conditions, including in elderly patients with lung disease and adults and children with steroid-refractory aGVHD, heart failure and chronic low back pain due to intervertebral disc degeneration.

The FDA recently accepted Mesoblasts BLA for priority review for remestemcel-L for children with aGVHD. It has a PDUFA date of Sept. 30 for the product branded as Ryoncil.

Mesoblast shares (ASX:MSB) were up nearly 34% to AU$1.78 from AU$1.32 per share by market close April 6.

Cynatas shares (ASX:CYP) were trading at AU86 cents on April 7.

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MSC-based therapies from Mesoblast, Cynata advance to tackle COVID-19 ARDS - BioWorld Online

Bone Marrow Stem Cells Comments Off on MSC-based therapies from Mesoblast, Cynata advance to tackle COVID-19 ARDS – BioWorld Online | April 8th, 2020

The COVID-19 pandemic has created a unique challenge for patients with myelodysplastic syndromes and acute myeloid leukemia, creating many questions that experts tried to answer in a recent webinar from The Aplastic Anemia and MDS International Foundation.

The Aplastic Anemia and MDS International Foundation (AAMDSIF) recently hosted a webinar to address the questions of this patient population by connecting them with Dr. Gail J. Roboz, professor of medicine and director of the clinical and translational leukemia program at the Weill Medical College of Cornell University in the New York Presbyterian Hospital in New York City.

Over the course of the webinar, Dr. Roboz, also a member of the AAMDSIF Medical Advisory Board, answered questions from the audience about the various ways that COVID-19 is impacting patients, from treatment delays to transplant and beyond.

Audience: If a patient with MDS were to be diagnosed with COVID-19, what are the most important things that they should let their medical team know, as they may not be familiar with MDS?Roboz: I think that it's important that if you're being seen in a facility where they don't know you very well or they don't know much about MDS, you can tell them that MDS is a is a bone marrow failure problem. You can tell them about your own blood counts, do I usually run low neutrophils or low hemoglobin or low platelets or all three, so you can tell them about what your specific experience is. But with respect to therapy, it is not completely clear that the underlying diagnosis, in this case MDS, is going to change what they do.

I think one of the questions is going to be about potential interactions with any medications that you're taking for MDS. And that's, of course, something that would be discussed if you're hospitalized.

If you are not getting hospitalized and you are patient with MDS, I certainly think it's reasonable to have, if at all possible, a daily or every other day telemedicine visit, either by video or by phone or by email, or however you're communicating with your doctor as a check in to just see how you're doing, see how your symptoms are evolving.

Should I continue with routine blood tests under the conditions or should I hunker down and not leave the house? If the previous blood test that the patient has had is looking absolutely perfect, and if there is a track record over a period of time that we know that this patient is tolerating the drug well and hasn't had any issues, I would be willing to consider skipping a routine blood test.

That said, I think it's really important to discuss this individually with the physician. First of all, depending on where you are, I'm hearing that in some parts of the country, you can actually drive to the doctor's office and they have a check in system that's allowing you to check in from your car, so that you could actually get into the office, get a lab check and get out without seeing really anybody.

Is there a change in patient protocol for when patient should be concerned about a fever?That is a really important question, especially for neutropenic patients. I think that if you are neutropenic and running a fever, neutropenic fevers do have to be evaluated, especially in hematologic malignancy patients.

If you don't feel too bad, and you're not having shaking chills and you think you can get your doctor's office on the phone quickly, it's not unreasonable to try that. That said, most of the time, it's really tough to get seen urgently in an office at this point. Again, it depends on where you are.

If you're going into the ER, you have to be very specific with them and say, hey, listen, I have leukemia, or I have MDS. This is my doctor. I'm neutropenic. I'm coming in with neutropenic fever, and they will evaluate you simultaneously for all of the routine things for neutropenic fever, as well as for coronavirus.

What are the recommendations regarding patients moving forward with transplant?I think that the issue is that the intensive care units in many areas, and the infectious disease doctors and many of the pulmonary specialists and other supportive specialties that are so critical to get patients safely through transplant, are very occupied at the moment.

But we want to make sure that when you come in for a procedure with curative intent, that all of the backup that we need to get you through the procedure safely is 100% available. So, it is definitely the case that patients are being delayed in their transplant. However, there are situations in which people might proceed. And I think again, this has to be a very individual discussion with the physician.

In the New York area, we are anticipating a surge in mid-April. So we definitely have been making decisions for our transplant patients that we don't want to bring you in here literally at the moment when they're predicting that things are going to get much worse, because maybe things will be better at the end of the month or at the beginning of the next month. And then we can hopefully start breathing a sigh of relief and bring you in much more safely.

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COVID-19 Tips for Patients with Myelodysplastic Syndromes and Acute Myeloid Leukemia - Curetoday.com

Bone Marrow Stem Cells Comments Off on COVID-19 Tips for Patients with Myelodysplastic Syndromes and Acute Myeloid Leukemia – Curetoday.com | April 8th, 2020

Since the coronavirus outbreak hit Italy, Francesca Masi has felt her life has been hanging by a thread. She was diagnosed with myelofibrosis, a rare bone marrow cancer, in 2016 and was due to have a transplant this month, but now fears it will be postponed as the country deals with the pandemic.

Access for thousands of cancer patients in need of chemotherapy, scans, transplants and surgery has become difficult, if not impossible, in Italys Covid-19 emergency.

Across the country, dozens of specialist cancer wards and hospitals have been transformed to treat coronavirus, while others have closed after medical staff and patients were infected. There are now fewer beds in intensive care units for cancer patients.

At the beginning of my diagnosis I underwent other treatments with success, but since my conditions worsened, transplant became my only option. So to be stuck in this situation make me living in a constant state of anxiety, says Masi, who lives and works in Pontedera, in the province of Pisa. She is 46 and the mother of a 10-year-old boy. I now run the risk of dying, which isnt fair, because my doctors had finally located two foreign donors whose marrow was 100% compatible with mine. International flight restrictions to halt the spread ofcoronavirus mean marrow from overseas donors risks not arriving in Italy.

Research led by Codice Viola, a charity that supports pancreatic cancer patients, and seen exclusively by the Guardian, found that of 500 mostly breast or pancreatic patients appointments for chemotherapy or radiotherapy were postponed for 24% (11% with no arranged date), while 64% of surgical procedures were postponed indefinitely. More than half have had follow-up appointments rescheduled.

Dozens of patients and doctors who spoke to the Guardian fear that the restrictive measures to contain the virus are limiting access to proper medical care for cancer patients, who also represent 17% of Italian coronavirus fatalities, according to a recent study.

Francesca Pesce, 54, a professional translator and member of Codice Viola, has been living with metastatic pancreatic cancer for almost three years. This week she will leave Rome for a follow-up in Milan, one of the cities worst affected by the virus.

At least I have this option, which others dont, she said. On the one hand, cancer patients are afraid of contracting the virus in hospitals, so they forgo their treatments; on the other, hospitals have been forced to cancel their appointments as many oncologists and anaesthetists have been moved to other wards to assist in the Covid-19 emergency.

Paolo Ascierto, an oncologist at Naples Pascale hospital now treating coronavirus patients, said converting cancer wards to Covid-19 units could be risky. I understand the state of emergency, but we mustnt forget that cancer patients require dedicated and specialised treatments. There are special conditions, like patients in follow-up, that can be managed safely using online consultations to monitor the patients progress.

But there are other conditions, such as metastatic patients, that must be prioritised, because a lack of constant attention can mean the difference between life and death.

In Ortona, Abruzzo, protests erupted following the announcement by local authorities that the only hospital in the region specialised in womens cancer treatments was to be entirely converted to treat Covid-19 patients.

Where cancer wards are functioning, certain diagnostic procedures, such as endescopies, may be limited, and a decrease in blood donations is limiting surgical options. At the moment, only urgent surgical procedures are being performed, says Pesce. And even urgent procedures are now stymied, because of a shortage of blood due to the coronavirus emergency.

The closure of air routes to and from Italy has also made it virtually impossible for thousands of cancer patients to access treatment in other European hospitals.

Alessandra Capone, 47, a dancer, feminist and human rights activist, has been living with breast cancer for 10 years. In 2015 it spread to her liver and lymph nodes and last year she began a series of treatments at University Hospital in Frankfurt with just 5% of her liver cancer-free. She is now facing enormous difficulties travelling to Germany.

I contacted the Ministry of Foreign Affairs and International Cooperation but the line is always busy and I couldnt speak to anybody. Then I contacted the Italian consulate in Frankfurt. They told me I need a number of certifications for travelling to another country, even for health reasons. Not to mention that in Germany very few hotels are accepting reservations, especially from Italians, during this emergency. Its very stressful.

For the first time, however, the Italian government last week authorised an air force flight to Turkey to secure haemopoietic stem cells from a donor for a two-year-old boy whose condition had worsened.

Travel is also limited within Italy, where patients from Sicily, Puglia and Calabria often undergo treatments and operations in richer Veneto and Lombardy, which are among the worst affected regions in the country for Covid-19.

Grazia De Michele, 39, a blogger and researcher in the history of medicine, lives in Foggia, in Puglia, and has been living with breast cancer since 2010. A few months ago, her mother was diagnosed with pancreatic cancer. My mother was supposed to have a CT scan in March to see if the chemo shes on is working, but the scan was postponed, she says. I was supposed to undergo an oophorectomy, and my operation was postponed as well.

You have to imagine what its like for cancer patients, says Capone. Many live in a constant state of anxiety, with the fear of dying. The situation caused by the coronavirus emergency has put them under enormous psychological and physical distress. You see, you can protect yourself from coronavirus by staying at home, but its not the same with cancer. Cancer doesnt follow the diktat of quarantines or decrees. It keeps going, in war and in peace.

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Life 'hanging by a thread' for Italian cancer patients in coronavirus crisis - The Guardian

Bone Marrow Stem Cells Comments Off on Life ‘hanging by a thread’ for Italian cancer patients in coronavirus crisis – The Guardian | April 8th, 2020

Stempeutics Research, a group company of Manipal Education and Medical Group (MEMG), announced today that it has partnered with Global Consortium of cell therapy companies seeking European Commission Funding to Fight Against Corona! (FAC!). Under this partnership, Stempeutics will export its stem cell product Stempeucel (subject to regulatory approvals) for treating critically ill COVID-19 patients with lung disease. First the product will be clinically tested and upon successful outcomes, it intends to export the product on a regular basis. In this connection it is signing up an alliance with Educell Ltd, Slovenia.

Currently, no specific drugs or vaccines are available to cure the patients with COVID-19 infection. Mortality in COVID-19 infected patients with the inflammatory lung condition ARDS (Acute Respiratory Distress Syndrome)is reported to approach 50%, and is associated with older age, co-morbidities such as diabetes, cardiovascular disease, COPD (chronic obstructive pulmonary disease), higher disease severity, and elevated markers of inflammation. Current therapeutic interventions (with the exception of ventilators/respirators which are in very short supply) do not appear to be improving in-hospital survival. Hence, there is a large unmet need for a safe and effective treatment for COVID-19 infected patients, especially in severe cases. A promising new therapy for the ARDS, the terminal stage of COVID-19, using MSCs can quickly (2-4 days) reduce inflammation of the lung tissue, and allow patients to more quickly come off of the ventilatory support and hopefully fully recover with less significant lung damage.

Stempeucel is an allogeneic, off the shelf, pooled mesenchymal stromal cells having anti- inflammatory and immune-modulatory properties which prevents the over activation of the immune system. Stempeucel product exhibits a wide range of potent therapeutic properties. The product exhibits potent immunomodulatory and anti-inflammatory properties which could help in reducing the inflammation caused due to the cytokine storm elicited by the bodys immune cells in response to SARS-CoV-2 (COVID-19) related infection in the lungs. Also, the growth factor, Angiopoietin-1 (Ang-1) is effective in reducing alveolar epithelium permeability in the lung. Hence it is envisaged, Stempeucel will reduce the fatal symptoms of COVID 19 induced pneumonia and its progression to ARDS.

Commenting on this initiative, Dr. Miomir Knezevic, Leader of the Global Consortium and Founder of Educell said, We are happy to partner with Stempeutics since its product Stempeucel is already designated as an ATMP1 in Europe and also Stempeucel technology has been patented in many countries in Europe. Stempeutics manufacturing process is scalable and the product is affordable which are key to meet the demands of COVID-19 patients

Mr. BN Manohar, CEO of Stempeutics said, From the clinical data using Stempeucel in different clinical trials in other indications it may be postulated that Stempeucel has the potential capability for treating COVID-19 infection. Together with the safety profile observed from DCGI approved clinical trials involving more than 350 patients injected with Stempeucel by different routes of injection, this therapy may help in mitigating the lung tissue damaging effects of COVID-19 infection.

Dr. Stephen Minger, Scientific Advisor for the Global Consortium and ex Global Director of R&D, Cell Technologies GE Healthcare added The most severely affected CV-2 infected patients will often go on to develop ARDS which necessitates assisted ventilation to preserve breathing and lung function. Moreover, many ARDS patients will also experience an acute but severe life-threatening inflammatory response (cytokine storm) which can result in long-term damage to lung tissue and lung function. Treating ARDS patients with allogeneic expanded bone marrow derived MSCs could alleviate and ameliorate lung inflammation and compromised lung function and significantly reduce the time required for patients to be ventilated.

Dr. Raviraja N S, Sr. Director Business Development and Innovation, Stempeutics, said, Given the severe shortage of ventilators in the world, and the high mortality rate of patients who develop ARDS (approx. 50%), the clinical use of MSCs in COVID-19 ADRS patients could drastically impact on the healthcare burden currently occurring due to very large patient numbers, limited equipment and overworked medical personnel.

Mr. B N Manohar MD & CEO, Stempeutics Research

Manohar is the MD & CEO of Stempeutics Research a leading stem cell research and product development company in India. He earned his B.E. degree in Electronics & Communication from REC Trichy in 1977. Post that he did M.E. in Computer Science from College of Engineering, Guindy. Manohar has transformed Stempeutics a life science start-up from R&D to Commercialization stage with Global recognition. Stempeutics has developed an innovative drug called Stempeucel for addressing major unmet medical needs in India and Globally.

This drug developed by an Indian company has received many Global Recognitions. Fourteen countries including US & Japan has granted patent for the novelty and inventiveness of the drug. Europe has recognized this drug by granting Advanced Therapy Medicinal Product classification and Orphan Drug Designation. Recently it became the FIRST stem cell product to be approved by DCGI for conditional marketing for treating patients suffering from life threatening disease call Buergers Disease. Stempeutics has put India on the World map of Regenerative Medicine. Under Manohars leadership Stempeutics has been recognized as Indias hottest start-ups by Business Today in 2008 and Karnataka Government bestowed Emerging Company of the Year award in 2011 and 2013. In 2017 Manohar was awarded Biotechnologist of the Year award by Wockhardt Foundation, India. He raised US$ 10M in 2009 by establishing business alliance with major pharma company Cipla. Recently Stempeutics has tied up with Alkem Labs for Osteoarthritis indication. Prior to Joining Manipal Group, Manohar has had 12 years successful stints at Wipro GE Medical Systems. At GE Medical he has handled multiple senior assignments including Vice President Customer Service where he received GE Asia Service Award for highest revenue growth in 1998. Currently Manohar serves in the Boards of Stempeutics and MentisSoft.

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India Based Stem Cell Research Firm To Test Its Stem Cell Product For Acute Respiratory Disease Syndrome (ARDS) COVID-19 - IndianWeb2.com

Bone Marrow Stem Cells Comments Off on India Based Stem Cell Research Firm To Test Its Stem Cell Product For Acute Respiratory Disease Syndrome (ARDS) COVID-19 – IndianWeb2.com | April 8th, 2020

THERES a 70 per cent chance youre going to get a mum without MS for the first time.

Those were the words Amanda Weyman-Jones told her daughter before they hopped on a plane, in a last ditch-attempt for Amanda to take her future back.

In January, Amanda and daughter Chloe travelled halfway across the globe to Moscow, Russia for a life changing and experimental treatment in hopes it would effectively stop her three decade battle with multiple sclerosis in its tracks.

The trip was made possible with the support from the Roma community who banded together to help Amanda raise $80,000 to pay for the treatment only available in the Russian capital.

And according to Amanda, she has already seen a massive improvement with her condition.

Im walking and I would say I have improved 70 per cent already, and its only expected to get better as time goes on, the 58-year-old mother of six said.

They say that the treatment gives you an 80 per cent (chance) of curing your MS and at the moment, I feel like Im in that 80 per cent Im feeling really good about my chances.

Amanda underwent an experimental procedure called Autologous haematopoietic stem cell transplant (AHSCT) treatment, which rebuilds the patients immune system.

Seven weeks on and Amanda says she feels like a new person, and has been walking around the football field everyday, which she states is a miracle as she couldnt even walk to the field before the treatment.

Ever since returning to Roma from Russia in February, all she has wanted to do is shout from the rooftops that others living with MS can also have their lives changed.

I heard about a man on a property in Blackall with MS and hes young so I want him to know he doesnt have to have this disease, you can get better, Mrs Weyman-Jones said.

This treatment gives you that infinity with people. Its life saving stuff.

Amandas brother Hayward and sister Diana were both diagnosed with MS too; Hayward died last year, and Diana is now in a wheelchair.

All too familiar with the devastating effects of MS, Amanda is determined to not become a burden on her loved ones.

Amanda who has Primary Progressive MS was given an Expanded Disability Status Scale (EDSS) score of 4.5 which notes a limited walking mobility to approximately 300m without aide prior to treatment. With no action taken, she would probably have continued to progress until she was wheelchair bound. The EDSS is scored zero to 10, with 10 marking a person has died from MS.

Now, with more improvements expected to continue in the next six to 12 months, Amanda is hopeful her quality of life will improve and once her immunity has built up, to continue working at the family-owned-and-run Overlander Motel.

I will be forever grateful to Dr Frederinco, the brilliant medical team in Russia, Roma, its local businesses and the wider community for blessing me with a new life, Amanda said.

Through the generosity and support by all, I have realised how lucky I am to be surrounded by such a caring community.

Amanda said she knew that while there is a long road ahead of her, every passing day she is more feeling more hopeful.

I was told that recovery can be like a rollercoaster, so I will accept the bad days and make sure I remember the good, she said.

My walking is slowly getting safer and less hazardous to myself . and to all other pedestrians. Every morning I wake up, knowing that every days a better day.

I am a new person, it is just a miracle.

Stats about MS

With MS Queensland aware of nearly 4000 people living with Multiple Sclerosis in Queensland and over 25,600 people in Australia living with the neurological condition.

Most people with MS in Australia experience their first symptoms between 20 and 40 years of age, with about three quarters of people living with MS, female.

MS is not considered a classic genetic disease in that there is not one single gene that causes the condition. Rather, there are more than 200 different known genetic factors which contribute to the risk of developing MS. It has been estimated that genes may account for around half of the risk for MS, and those with a family history of MS are at greater risk than the general population. Even so, the majority of people with a family member with MS will not develop the disease so genes on their own are not enough.

MS is caused by a complex interaction between a persons genetics and environment factors.

Autologous haematopoietic stem cell transplant (AHSCT) is an immunosuppressive chemotherapy treatment combined with reinfusion of blood stem cells to help rebuild the immune system.

AHSCT has been used for decades for the treatment of blood cancers. However in the past ten or so years a number of international observational studies of several hundred patients have been published with some patients being followed for five to eight years.

The treatment consisted of four days of stimulation before the stem cells were collected and then Amanda was pumped full of high dose chemotherapy.

Amanda then had a rest day, and on January 29, her harvested stem cells were returned to her MS ravaged body, signalling the rebirth of her immune system with no memory of MS.

After that she was given daily steroid infusions and was put into isolation for six nights before one final dose of chemotherapy.

Response from MS Queensland about the treatment

CEO of MS Queensland Zane Ali said MS Queensland and MS Research Australia are continuing to support Australian research in the use of AHSCT to treat multiple sclerosis.

Rigorous evidence for the efficacy and safety of AHSCT in relation to other MS therapies, and the most appropriate circumstances for its use, is required for Australian hospitals and clinicians to provide this intervention with equity and with greater confidence in the potential outcomes, he said.

Australian hospitals and doctors are likely to recommend AHSCT as a possible treatment only if the other approved MS therapies are not working for an individual with MS or cannot be used in an individual for other reasons.

Despite Amandas MRI revealing her Central Nervous System was so progressed (with 35 lesions or more on the spine), she met the criteria for the treatment because of her mobility.

You have to be at a very healthy besides having MS, patients are tested from head to toe when they first arrive in hospital to ensure that they dont have any cancers or illnesses that could effect the viability of the treatment, said daughter Chloe.

The doctor was surprised after he saw how mobile mum still was considering the damage that he saw in her brain.

People arent accepted all of the time, some are told before they go and some are only told after all of the testing is completed in Moscow, then they are then sent home. International studies also suggest AHSCT does not halt or reverse progressive forms of the disease, and is therefore unlikely that

AHSCT would be recommended as a treatment for patients with secondary progressive or primary progressive MS.

Currently the treatment is provided in Australia through two observational clinical trials, at St Vincents, Sydney and Austin Health, Melbourne and by a small number of other centres on a case-by-case basis.

These centres have strict eligibility requirements that have been set by the hospital ethics committees and may only apply to limited numbers of patients with MS, Mr Ali said.

It is for this reason patients need to be referred to these centres by a neurologist, who can provide a detailed clinical history and MRI findings, Mr Ali said.

Mr Ali said that data from the large European Bone Marrow Transplant Registry suggests that in approximately 55 per cent of people with MS, treated with a range of different chemotherapy regimens, at three years of follow-up, inflammatory disease is halted with no evidence during the follow-up period of relapses, active brain lesions or disability progression.

After five years approximately 45 per cent of people remain progression free.

This case series included patients with both relapsing remitting and progressive disease, he said.

Other smaller studies have shown similar results, with remission of disease seen in these studies in at least 63 per cent of patients followed for a minimum of three years.

Most studies also show that the risk of disease activity returning gradually increases over longer periods of follow-up.

Mr Ali said in some but not all, of the people with MS who respond to AHSCT, some reversal of disability has been noted in some studies.

Other patients may continue to experience disease activity and disability progression (worsening) despite treatment with AHSCT, he said.

In people with progressive forms of MS or relapsing remitting MS of longer duration, the benefits of the procedure have been much less clear and accumulation of disability usually continues.

Amanda said they chose Moscow for the treatment because despite other countries offering the experimental procedure, after thorough research they found Russia had the highest rate of treatment success.

They accept advanced progressive MS where most of the other clinics offering it only treat relapsing remitting MS, she said.

I was still on my feet (only just) but people went over in wheelchairs, walkers, walking sticks and many were older than me.

For Chloe, who is training to become a nurse and who spent every spare moment researching AHSCT treatment said while they mostly compared the options available in Mexico and Russia, Russia stood out to them because it was cheaper and they had more experience.

One of the major influencing factors was that in Mexico patients stayed in a complex with they carer and in Russia patients stayed in a hospital, so we felt more reassured knowing that mum would have 24 hour care provided to her at the touch of a buzzer, she said.

Great lengths of a loving daughter

Although Amanda has lived with MS for 34 years, she feels like she is one of the lucky ones.

None of this would have been possible without Chloe. Her drive and determination surprises me every day, she said.

Researching, booking, fundraising and organising the whole trip, proves to me that I am the luckiest mum on earth.

Chloe was the driving force behind the push for treatment and the GoFundMe campaign which raised over half the $80,000 goal was with her mum every step of the journey.

I have found spending a month in Russia very interesting, the first couple of weeks for easy, but after that I just wanted to come home to Australia, the 19-year-old said.

Every day I would go and visit Mum in the morning and stay there with her until dark and then head on back to the hotel, I basically just did that every day.

I made some great friends with some other patients carers and so often we would catch up at the end of the day to recuperate and support each other.

Although the month spent away from her loved ones began to take its toll, Chloe has high hopes for the future.

It was very draining being over there, I felt like I wasnt doing much but I was just always so tired, she said.

Its amazing to see how quickly mum is healing after the treatment, but it will take some time to see what the true outcome for her is going to be.

In the end, we dont know what the future holds for mum and her MS, we are just thinking positively and hope that we see improvements over the next 12 months.

We feel very lucky that we had the opportunity to go over and are now advocating for other people to have the treatment as well.

Chloe and Amanda Weyman-Jones sightseeing in Moscow before the treatment began.

During the treatment.

Amanda Weyman-Jones with Greta and Theresa who were also going through the treatment.

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Roma woman tells of her 'life-changing' MS treatment - Observer

Bone Marrow Stem Cells Comments Off on Roma woman tells of her ‘life-changing’ MS treatment – Observer | April 8th, 2020

Autologous Stem Cell and Non-Stem Cell Based Therapies Market research report covers the existing situation and the development predictions of the industry for 2020. This report has prepared mainly on the basis of a common market assessment with input from industry experts. This estimated report consists of all have observed element about marketplace evaluation, increase Demand and forecast analysis in all over the world. This record gives a few edged examine and solution within the complicated international of polymer-based totally thermal interface materials market.

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U.S. STEM CELL, INC., Brainstorm Cell Therapeutics, Cytori, Dendreon Corporation, Fibrocell, Lion Biotechnologies, Caladrius Biosciences, Opexa Therapeutics, Orgenesis, Regenexx, Genzyme, Antria, Regeneus, Mesoblast, Pluristem Therapeutics Inc, Tigenix, Med cell Europe, Holostem, Miltenyi Biotec.

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USAEuropeJapanChinaIndiaSoutheast Asia

Scope of the Report:

This study focuses on the global market for Autologous Stem Cell and Non-Stem Cell Based Therapies especially in Europe, North America and Asia-Pacific, the Middle East and Africa, and South America. The report defines the market based on regions, size, manufacturers and applications.

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Market Segment On The Basis Of Product Type Includes:-

Embryonic Stem CellResident Cardiac Stem CellsAdult Bone MarrowDerived Stem CellsUmbilical Cord Blood Stem Cells

Applications Mentioned In This Report:-

Neurodegenerative DisordersAutoimmune DiseasesCancer and TumorsCardiovascular Diseases

The report then estimates 2020 market development trends of Autologous Stem Cell and Non-Stem Cell Based Therapies market. Outline of upstream raw materials, downstream trade and prevailing market dynamics is also carried out. In the end, the report makes some important proposals for a new project of Autologous Stem Cell and Non-Stem Cell Based Therapies market before evaluating its feasibility.

This report presents an extensive analysis of the current Autologous Stem Cell and Non-Stem Cell Based Therapies trends and emerging estimations & dynamics of the global Autologous Stem Cell and Non-Stem Cell Based Therapies industry. Likewise, explains the comprehensive analysis of factors that drive and restrict the growth of the Autologous Stem Cell and Non-Stem Cell Based Therapies market. Further covers a detailed analysis of the Autologous Stem Cell and Non-Stem Cell Based Therapies industry based on type and application help in understanding the Autologous Stem Cell and Non-Stem Cell Based Therapies trending products across geographies. Then highlights the potency of buyers and suppliers to understand the Autologous Stem Cell and Non-Stem Cell Based Therapies market potency. Finally, an extensive analysis of the Autologous Stem Cell and Non-Stem Cell Based Therapies market is conducted by key product positioning and monitoring of top players within the Autologous Stem Cell and Non-Stem Cell Based Therapies market framework.

Table of Contents:

1 Industry Overview of Autologous Stem Cell and Non-Stem Cell Based Therapies.2 Global Autologous Stem Cell and Non-Stem Cell Based Therapies Competition Analysis by Players.3 Company (Top Players) Profiles.4 Global Autologous Stem Cell and Non-Stem Cell Based Therapies Market Size by Type and Application (2020-2025).5 United States Autologous Stem Cell and Non-Stem Cell Based Therapies Development Status and Outlook.6 EU Ophthalmology DiagnosticsDevelopment Status and Outlook.7 Japan Autologous Stem Cell and Non-Stem Cell Based Therapies Development Status and Outlook.8 China Autologous Stem Cell and Non-Stem Cell Based Therapies Development Status and Outlook.9 India Autologous Stem Cell and Non-Stem Cell Based Therapies Development Status and Outlook.10 Southeast Asia Autologous Stem Cell and Non-Stem Cell Based Therapies Development Status and Outlook.11 Market Forecast by Regions, Type, and Application (2020-2025).12 Autologous Stem Cell and Non-Stem Cell Based Therapies Market Dynamics.13 Market Effect Factors Analysis.14 Research Finding/Conclusion.15 Appendix.

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Originally posted here:
Global Autologous Stem Cell and Non-Stem Cell Based Therapies Market [News 2020] Intelligence and Future Prospects 2025 - Fashion Trends News

Cardiac Stem Cells Comments Off on Global Autologous Stem Cell and Non-Stem Cell Based Therapies Market [News 2020] Intelligence and Future Prospects 2025 – Fashion Trends News | April 8th, 2020

CAMBRIDGE, England--(BUSINESS WIRE)--Metrion Biosciences Limited (Metrion), the specialist ion channel CRO and drug discovery company, today announced it has contributed to two new peer-reviewed papers under the U.S. Food and Drug Administrations (FDA) CiPA (Comprehensive in vitro Proarrhythmia Assay) initiative. The papers, in Nature Scientific Reports1 and Toxicology and Applied Pharmacology2, focus on application of improved cardiac safety testing protocols and recommendations for best practice for the drug discovery industry.

The CiPA Initiative (www.cipaproject.org), which began in July 2013 following a workshop at the US FDA, has the objective to revise and enhance the regulatory framework assessing cardiac safety of new chemical entities. Under current guidelines, new therapeutics undergo initial assessment of proarrhythmic risk by measuring activity against the hERG cardiac ion channel, before progressing to studies in preclinical animal models and ultimately, a Thorough QT interval study in the clinic. The CiPA initiative aims to extend the use of advances in early electrophysiology-based cardiac ion channel screening, in silico predictive modelling, and human induced pluripotent stem cell derived cardiomyocytes to improve the accuracy and reduce the cost of predicting the cardiac liability of new drug candidates. Metrions research forms part of the first stage of the proposed harmonisation work, to provide clarity on how to standardise cardiac ion channel assays to ensure they deliver consistent data for in silico models of clinical cardiac arrythmia risk.

The first paper1, published in Nature Scientific Reports on 27th March 2020 by an international group of authors drawn from 20 different commercial and academic laboratories, including Metrion Biosciences, was coordinated by the Health and Environmental Sciences Institute (HESI). It reviews data from a multi-year, multi-site collaboration across industry, academia and the FDA regulatory agency to optimize experimental protocols and reduce experimental variability and bias. The goal of the study was to guide the development of best practices for the use of automated patch clamp technologies in early cardiac safety screening. High quality in vitro cardiac ion channel data is required for accurate and reliable characterisation of the risk of delayed repolarisation and proarrhythmia in the human heart and to guide subsequent clinical studies and regulatory submissions.

The second paper2, to be published formally in Toxicology and Applied Pharmacology paper on 1st May 2020 but currently available online, uses automated patch clamp data from the CiPA consortium to address the lack of statistical quantification of variability, which hinders the use of primary hERG potency data to predict cardiac arrhythmia. The consortium establishes a more systematic approach to estimate hERG block potency and safety margins.

Dr Marc Rogers, CSO, Metrion Biosciences, said: The Metrion team has been a participant in the international CiPA Initiative since inception and we are now pleased to be able to announce the publication of our data from this global collaborative scientific effort. We believe these projects will make a significant contribution to the eventual revision of cardiac safety testing guidelines by the FDA and other international regulatory agencies. They also contribute to deepening our knowledge of the underlying causes of proarrhythmia, which will help prevent early attrition of potentially promising drugs.

Contributing organisations to the Nature Scientific Reports CiPA study include: Charles River Laboratories; Bayer AG; Sophion Bioscience A/S; Nanion Technologies; GlaxoSmithKline PLC; Pfizer; Sanofi R&D; Astra Zeneca; BSYS GmbH; Bristol-Myers Squibb Company; Eurofins Discovery; Merck; Metrion Biosciences Ltd.; Natural and Medical Science Institute at the University of Tbingen; Northwestern Feinberg School of Medicine, Chicago; Roche Innovation Center Basel; Novoheart; Health and Environmental Sciences Institute, Washington, DC; AbbVie.

Contributing organisations to the Toxicology and Applied Pharmacology hERG study include: Center for Drug Evaluation and Research, Food and Drug Administration; Eli Lilly and Company; AstraZeneca; CiPA LAB; NMI-TT GmbH; Sophion Bioscience A/S; B'SYS GmbH; The Ion Channel Company; F. Hoffmann-La Roche AG; Eurofins Discovery; Bristol-Myers Squibb; Merck & Co., Inc; Metrion Biosciences Ltd.; Nanion Technologies; Charles River Laboratories; Bayer AG; University of Nottingham; Universit de Lille.

For more information on Metrions fully integrated Cardiac Safety Screening / CiPA Screening service, please visit: https://www.metrionbiosciences.com/services/cardiac-safety-screening/

Merion Biosciences comprehensive cardiac safety testing White Paper The changing landscape of cardiac safety will also be available on the Companys website from 13th April 2020.

Link:
Metrion Biosciences and International Scientific Consortium Publish Data and New Recommendations for in Vitro Risk Assessment of the Cardiac Safety of...

Cardiac Stem Cells Comments Off on Metrion Biosciences and International Scientific Consortium Publish Data and New Recommendations for in Vitro Risk Assessment of the Cardiac Safety of… | April 8th, 2020