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World J Stem Cells. 2022 Jan 26; 14(1): 140.

Medical Physiology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt

Oral Pathology Department, Faculty of Dentistry/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt

Human Anatomy and Embryology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt

Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt

Medical Physiology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt

Histology and Cell Biology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt

Medical Biochemistry Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt

Medical Biochemistry Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt

Medical Physiology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt

Forensic Medicine and Clinical toxicology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt

Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt

Histology and Cell Biology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt. ge.ude.demxela@annahem.awdar

Radwa A Mehanna, Medical Physiology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt;

Supported by Science and Technology Development Fund, No. 28932; and Cardiovascular Research, Education, Prevention Foundation, CVREP - Dr. Wael Al Mahmeed Grant.

Corresponding author: Radwa A Mehanna, MD, PhD, Academic Research, Professor, Executive President, Medical Physiology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Al Khartoum Square, Azareeta, Alexandria 21500, Egypt. ge.ude.demxela@annahem.awdar

Received 2021 Feb 26; Revised 2021 Jul 2; Accepted 2022 Jan 6.

Regenerative medicine is the field concerned with the repair and restoration of the integrity of damaged human tissues as well as whole organs. Since the inception of the field several decades ago, regenerative medicine therapies, namely stem cells, have received significant attention in preclinical studies and clinical trials. Apart from their known potential for differentiation into the various body cells, stem cells enhance the organ's intrinsic regenerative capacity by altering its environment, whether by exogenous injection or introducing their products that modulate endogenous stem cell function and fate for the sake of regeneration. Recently, research in cardiology has highlighted the evidence for the existence of cardiac stem and progenitor cells (CSCs/CPCs). The global burden of cardiovascular diseases morbidity and mortality has demanded an in-depth understanding of the biology of CSCs/CPCs aiming at improving the outcome for an innovative therapeutic strategy. This review will discuss the nature of each of the CSCs/CPCs, their environment, their interplay with other cells, and their metabolism. In addition, important issues are tackled concerning the potency of CSCs/CPCs in relation to their secretome for mediating the ability to influence other cells. Moreover, the review will throw the light on the clinical trials and the preclinical studies using CSCs/CPCs and combined therapy for cardiac regeneration. Finally, the novel role of nanotechnology in cardiac regeneration will be explored.

Keywords: Cardiac stem and progenitor cells, Cardiac stem cells secretome, Cardiac stem cells niche and metabolism, Nanotechnology, Clinical trials, Combined therapy

Core Tip: With the growing evidence for the existence of regenerating cardiac stem and progenitor cells, studies to evaluate their therapeutic potential have received increasing attention. Although pre-clinical research and clinical trials have demonstrated promising results, yet the latter were often inconsistent in many aspects thus imposing the need for deeper exploration of the molecular biology and relevant pathways regulating cardiogenesis and cardiac muscle repair. This review gives an insight into cardiac stem and progenitor cells regarding their embryological origin, populations, niche, secretome, and metabolism. It overviews the current preclinical research, including medical nanotechnology, and the clinical trials generally applied for cardiac regeneration.

Cardiovascular diseases are the leading cause of death globally, as stated by the latest report 2019 for the World Health Organization, with 17.9 million deaths per year, accounting for 31% of all deaths worldwide.

The heart is one of the least proliferative organs in the human body, and its minimal regenerative capacity has been dogma for decades. Such dogma has been led by the belief that the heart cannot regenerate from ischemic damage. The absence of primary tumors in the heart has further supported the notion of low proliferation. In an alleged post-mitotic organ, it has been debatable whether cardiac cells repair through activation of resident cardiac stem cells (CSCs) and cardiac progenitor cells (CPCs) or by the proliferation of pre-existing cardiomyocytes (CMs). In 2009, Bergmann et al[1] were the first to refute that notion and have reported that the heart can in fact self-renew. Based on the results obtained from their carbon-14-labelled DNA study to track CMs, Bergmann et al[1] stated that about 50% of CMs renew over the lifespan of an adult. Hsieh et al[2] provided further evidence for the origin of newly generated CMs from progenitor cells in an alpha myosin heavy chain (MHC) transgenic model. They estimated that approximately 15% of CMs can regenerate in adult hearts following ischemic damage. With progression of research, lineage tracing of regenerated cardiac tissue confirmed that the newly regenerated CMs develop from a non-CM and possibly from stem cells (SCs)[2].

Further studies have revealed various CSC/CPC candidates that are morphologically and functionally distinct from each other yet act in a complementary fashion and contribute to the regeneration process. This complex cell aggregation is known as the CSC niche that has been a challenge to characterize and locate anatomically[3].

SC applications have been under intensive research interest since the early 20th century. Many types have been isolated, starting from the embryonic, amniotic, and cord blood mesenchymal stem cells (MSCs) and passing through the adult SCs till the induced pluripotent SCs (iPSCs). Adult MSCs are undifferentiated cells with the same potentials as progenitor cells regarding the ability to differentiate into all three germ layer cells[4]. Exogenous MSCs from various sources, including bone marrow, adipose tissue, umbilical cord, placenta, and amniotic fluid[5], have shown promising results in the treatment of cardiovascular diseases. However, the outcome of CSC therapy has shown superior results in experimental studies but to a lesser extent in human clinical trials[6]. The applications of SC therapy for cardiovascular regeneration still hold a plethora of queries to be answered as well as commandment of the molecular and signaling features for CSCs in order to standardize this therapy. Among the aspects that need optimization are the types of SCs and supporting cells to be used, the number of cells, the route of injection, the frequency, and best timing for transplantation. Standardization requires an advanced understanding of the full biological features of CSCs.

SC therapy in cardiac regeneration has dual beneficiary actions. Primarily, the transplanted exogenous SCs would directly differentiate into CMs. Concomitantly, SCs activate the endogenous progenitors through their rich secretome of extracellular vesicles, immunomodulatory and growth factors, protein, and nucleic acid families[7]. These paracrine factors act to activate resident SCs and enhance vascularization to potentiate cardiac repair.

This review aims to provide insight into CSCs/CPCs regarding their embryological origin, populations, niche, metabolism, secretome, and therapeutic potentials. Also discussed is the interplay of nanotechnology with SCs in several aspects, including differentiation, tracking, imaging, and assisted therapy, showing the prospects and limitations of nanoparticle (NP)-based cardiac therapy. Finally, preclinical trials and ongoing, completed, and future clinical trials using CSCs and combined therapy are shown to delineate the potential applications in treating cardiac disease.

The heart is formed of a wide range of cell types originating from the mesodermal precursor cells. They include CMs and endocardial cells forming the inner layer, while epicardial-derived cells (EPDCs) and smooth muscle cells (SMCs) are found on the external layer. Differentiation of the mesodermal cells is initiated by the T-box transcriptional factors Brachyury (Bry) and Eomes. Bry+ cells differentiate into insulin gene enhancer protein islet-1 (ISL1) and T-box transcription factor 5 (TBX5) expressing cells, while Eomes induce expression of mesoderm posterior 1 (MESP1). MESP1+ cells are identified before the first heart field (FHF) and the second heart field (SHF) separations, so MESP1 serves as an indicator of early CPCs for both heart fields[8]. Chemokine receptor type 4 (CXCR4), fetal liver kinase 1 (FLK-1), and platelet derived growth factor receptor A are other surface markers that coincide with MESP1 and are used in combination to isolate CPCs[9,10].

In addition, a novel cell surface marker known as G protein-coupled receptor lysophosphatidic acid receptor 4 is specific to CPCs and determines its functional significance. Interestingly, its transient expression peaks in cardiac progenitors after 3 to 7 d of human (h)PSCs differentiation toward cardiac lineage, then it declines. In vivo, lysophosphatidic acid receptor 4 shows high expression in the initial stages of embryonic heart development and decreases throughout development[11].

The FHF cells are the firstly differentiated myocardial cells that are derived from cells in the anterior lateral plate mesoderm; they give rise to the left ventricle, partially some of the right ventricle population, sinoatrial node, atrioventricular node, and both atria[12]. Meanwhile, the SHF cells originate from the pharyngeal mesoderm to the posterior side of the heart and further divide into anterior and posterior SHF. They contribute to the right ventricle, atria, and the cardiac outflow tract (OFT) formation. Addition of the SHF-derived CMs to the ventricles depend on myocyte enhancer factor 2C (MEF2C). It has been found that MEF2C null mice die at 9.5-d post conception with severe heart defects due to failure of heart looping[13]. In OFT formation, two waves of SHF progenitors and their derivatives have been identified, making a differential contribution to the aorta and pulmonary artery. The early wave of cells is favorably directed to the aorta, while the second wave of cells contributes to the pulmonary artery. Phosphoinositide-dependent kinase-1 critically regulates the second wave of cells, and its deletion results in pulmonary stenosis[14]. The epicardium of the heart is formed of a transient proepicardial organ. Proepicardium is formed from homeobox protein NKx2.5 (NKx2.5) and ISL1+ cells. After epicardial formation, subepicardial mesenchymal space is formed by epithelial to mesenchymal cell transformation of the epicardial cells[15] (Figure ).

Embryonic cardiac progenitors, Brachyury-positive mesoderm precursors and Pax3+ neural crest cells. Brachyury (Bry+) mesoderm precursors give rise to the mesoderm posterior 1+ primordial precursors, which are the origin of the first heart field, second heart field, and proepicardial progenitors, each population of which is responsible for the development of different parts in the heart. Pax3+ neural crest cells are responsible for the development of vascular smooth muscle, outflow tract, valves and the conductive system. Progenitors are tagged with their specific markers. Created with BioRender.com. CPC: Cardiac progenitor cell; LT: Left; RT: Right; FHF: First heart field; SHF: Second heart field; OFT: Outflow tract.

The differentiation in the posterior SHF is regulated by Hoxb1 gene. Stimulation of Hoxb1 in embryonic stem cells (ESCs) halts cardiac differentiation, while Hoxb1-deficiency shows premature cardiac differentiation in embryos. Moreover, an atrioventricular septal defect develops as a result of ectopic differentiation in the posterior SHF of embryos deficient in Hoxb1 and its paralog Hoxa1[16].

Multiple signaling pathways have essential roles in cardiogenesis with a sequential arrangement. The transforming growth factor- (TGF-) superfamily, retinoic acid, Hedgehog, Notch, Wnt, and fibroblast growth factors (FGFs) pathways comprise the chief signaling pathways involved in cardiac development. These pathways, along with transcription factors and epigenetic regulators, regulate cardiac progenitors specification, proliferation, and differentiation into the different cardiac cell lineages[17].

The TGF- superfamily members consist of over 30 structurally associated polypeptide growth factors including nodal and bone morphogenetic proteins (BMP)[18].

Nodal signaling is vital for the formation of sinoatrial node. Nodal inhibition during the cardiac mesoderm differentiation stage downregulates PITX2c, a transcription factor recognized to inhibit the formation of the sinoatrial in the left atrium during cardiac development[19]. Moreover, nodal signaling is dispensable for initiation of heart looping; however, it regulates asymmetries that result in a helical shape at the heart tube poles[20].

BMP signaling, as a member of TGF-, has an important role in the different stages of heart development including the OFT formation, endocardium, and lastly the epicardium. The cardiac neural crest cells have a crucial role in normal cardiovascular development. They give rise to the vascular smooth muscle of the pharyngeal arch arteries, OFT septation, valvulogenesis, and development of the cardiac conduction system[21] (Figure ). The role of BMP in OFT septation mainly depends on their gradient signaling, which arranges neural crest cell aggregation along the OFT; this Dullard-mediated tuning of BMP signaling ensures the fine timed zipper-like closure of the OFT by the neural crest cells[22]. Furthermore, the BMP signaling promotes the development of endocardial cells (ECs) from hPSC-derived cardiovascular progenitors[23]. It is also integrated with Notch signaling for influencing the proepicardium formation, where overexpression of Notch intracellular receptor in the endothelium enhances BMP expression and increases the number of phospho-Smad1/5+ cells for enhancing the formation of the proepicardium[24].

Retinoic acid signaling plays a role in heart development. It is a key factor for efficient lateral mesoderm differentiation into atrial-like cells in a confined time frame. The structural, electrophysiological, and metabolic maturation of CMs are significantly influenced by retinoic acid[25]. However, it is reported that retinoic acid receptor agonists transiently enhance the proliferation of human CPCs at the expense of terminal cardiac differentiation[26].

The downregulation of the retinoic acid responsive gene, ripply transcriptional repressor 3 (RIPPLY3), within the SHF progenitors by histone deacetylase 1 is required during OFT formation[27].

Hedgehog signaling has a role in OFT morphogenesis. Lipoprotein-related protein 2 (LRP2) is a member of the LDL receptor gene family, a class of multifunctional endocytic receptors that play crucial roles in embryonic development. LRP2 is expressed in the anterior SHF cardiac progenitor niche, which leads to the elongation of the OFT during separation into aorta and pulmonary trunk. Loss of LRP2 in mutant mice results in depleting a pool of sonic hedgehog-dependent progenitor cells in the anterior SHF as they migrate into the OFT myocardium due to premature differentiation into CMs. This depletion results in aberrant shortening of the OFT[28].

Four Notch receptors (Notch1Notch4) and five structurally similar Notch ligands [Delta-like (DLL) 1, DLL3, DLL4, Jagged1, and Jagged2] have been detected in mammals[29]. Activation of Notch signaling enhances CM differentiation from human PSCs. However, the CMs derived from Notch-induced cardiac mesoderm are developmentally immature[30]. In vivo, the Notch pathway plays a significant role in CPC biology. An arterial-specific Notch ligand known as DLL4 is expressed by SHF progenitors at critical time-points in SHF biology. The DLL4-mediated Notch signaling is a crucial requirement for maintaining an adequate SHF progenitor pool, in a way that DLL4 knockout results in decreased proliferation and increased apoptosis. Reduced SHF progenitor pool leads to an underdeveloped OFT and right ventricle[31].

The Wnt signaling pathway has an essential role in many developmental stages of embryogenesis. The Wnt family consists of 19 distinct Wnt proteins and other 10 types of Frizzled receptors. On the basis of their primary functions, the Wnt and Frizzled receptors are divided into two major classes, which are the canonical and non-canonical Wnt pathways[32]. Accumulating evidence suggests a role for the dynamic balance between canonical and non-canonical Wnt signaling in cardiac formation and differentiation. Wnt/-catenin signaling is required for proper mesoderm formation and proliferation of CMs but needs to be low for terminal differentiation and cardiac specification. In contrast, for cardiac specification in murine and human ESCs, non-canonical -catenin independent Wnt signaling is essential, while the non-canonical Wnt signaling is necessary for terminal differentiation later in development[33].

The activation of non-canonical Wnt is non-catenin-independent, and the downstream proteins involve several kinases, including protein kinase C, calcium/ calmodulin-dependent kinase, and Jun N terminal kinase (JNK). Wnt11 enhances angiogenesis and improves cardiac function through non-canonical Wnt-protein kinase C-Jun N terminal kinase dependent pathways in myocardial infarction (MI)[34]. In hypoxia, Wnt11 expression preserves the integrity of mitochondrial membrane and facilitates the release of insulin growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF), thus protecting CMs against hypoxia[35]. Canonical dependent Wnt signaling, Wnt 3 Ligand, favors the pacemaker lineage, while its suppression promotes the chamber CM lineage[36].

The regenerative capacity of most organs is contingent on the adult SC populations that exist in their niches and are activated by injury. Adult SC populations vary greatly in their molecular marker expression profile and hence in their possible role in regenerative medicine. The transcriptome is a representation of the gene read-outs, the cellular state, and is imperative for studying all genetic disease and biological processes. The genome-wide profiling using novel sequencing technology has made transcriptome research accessible.

Receptor tyrosine kinase (RTK) c-KIT (also referred to as SC factor receptor or CD117)-expressing CPCs are mainly located in the atria and the ventricular apex, comprising most of the ventricular and atrial myocardium[37]. c-KIT+ cells also express the cardiac transcription factors NKx2.5, GATA binding protein 4 (GATA4), and MEF2C but are negative for the hematopoietic markers CD45, CD3, CD34, CD19, CD16, CD20, CD14, and CD56[38,39]. SC factor ligand attaches to the c-KIT receptor and activates the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and p38 mitogen-activated protein kinase (MAPK) signaling pathways[40]. Both PI3K/AKT and MAPK pathways control various CPCs functions like self-renewal, proliferation, migration, and survival[41]. During embryonic development and the early post-natal time, c-KIT+ CPCs contribute to the generation of new CMs. Such capacity declines in the adult heart with only a few new CMs originating from CPCs[42]. In a rat MI model, the c-KIT+ CPCs have migrated through the collagen type I and type III matrices into the infarcted area. The transplanted CPCs have shown overexpressed matrix metalloproteinases (MMPs; MMP2, MMP9, and MMP14) that degrade extracellular matrix (ECM), concluding that c-KIT+ CPCs hold an invasive capacity[43]. Transplanted CPCs (c-KIT+ CPCs and cardiospheres) also show an endogenous proliferative potential in vivo and additionally activate endogenous CPCs[44].

Stem cell antigen 1 (SCA-1) expressing CPC population exists predominantly in the atrium, intra-atrial septum, and atrium-ventricular boundary and dispersed inside the epicardial layer of adult hearts[45]. SCA-1 is a cell surface protein of the lymphocyte antigen-6 (Ly6) gene family, which has roles in cell survival, proliferation, and differentiation[46]. A population of SCA-1+ cells from murine adult myocardium hold a telomerase activity comparable to that of a neonatal heart. This SCA-1+ population is different from hematopoietic SCs as they lack CD45, CD34, c-KIT, LIM domain only 2, GATA2, VEGF receptor 1, and T-cell acute lymphoblastic leukemia 1/SC leukemia proteins. SCA-1+ cells are also distinct from endothelial progenitor cells and express cardiac lineage transcriptional factors such as GATA4, MEF2C, and translation elongation factor 1 yet lack transcripts for cardiomyocytic structural genes such as BMP1r1 and -, -MHC[47,48]. Although this population exhibits the endothelial marker CD31, it is suggested to be due to the contaminating endothelial CD31+/SCA-1+ cells. In vitro studies have revealed that 5-azacytidine (5-aza), a demethylating agent, pushed SCA-1+ cells to differentiate into CMs[48,49]. Further studies have isolated SCA-1+ cells that lack CD31 and CD45 markers, referring to them as lineage negative (Lin). The SCA-1+/Lin cells display a mesenchymal cell-surface profile (CD34, CD29+, CD90+, CD105+, and CD44+) and are able to differentiate, to a certain extent, into CMs and endothelial and smooth muscle-like cells[50,51].

Human SCA-1+-like cells also express early cardiac transcription factors (GATA4, MEF2C, insulin gene enhancer protein ISL-1, and Nkx-2.5) and can differentiate into contractile CMs[52]. Although a human ortholog of the SCA-1 protein has not been yet identified, an anti-mouse SCA-1 antibody is used to isolate SCA-1+-like cells from the adult human heart.

MESP1 expressing cells mainly contribute to the mesoderm and to the myocardium of the heart tube during development[53]. Transient expression of MESP1 seems to accelerate and enhance the appearance of cardiac progenitor. However, homologous disruption of the MESP1 gene has resulted in aberrant cardiac morphogenesis. MESP1 interacts with the promoter area of main cardiac transcription factors, including heart and neural crest derivatives expressed 2, Nkx2-5, myocardin, and GATA4[54]. These factors induce fibroblasts to express a full battery of cardiac genes, form sarcomeres, develop CM-like electrical activity, and in a few cases elicit beating activity[55]. Several studies have shown that the addition of MESP1 could enhance the efficacy of direct reprogramming of fibroblasts into CMs[56,57]. The transdifferentiation of fibroblasts to CMs via MESP1 suggests that MESP1 chiefly modulates the gene regulatory network for cardiogenesis[52].

Kinase insert domain receptor (KDR), also known as Flk-1, is one of the earliest discovered cardiogenic progenitor cell markers acting during the early stages of cardiac development in human[58]. Nelson et al[59] have reported that Flk-1 has a distinctive transcriptome that has been evident at day 6, immediately after gastrulation but prior to the expression of the cardiac transcription factors. KDR+ population lack the pluripotent octamer-binding transcription factor 4, sex determining region Y-Box transcription factor (SOX) 2, and endoderm SOX17 markers. On the other hand, KDR+ CPCs have shown a noteworthy upregulation in SOX7, a vasculogenic transcription factor, overlapping with the emergence of primordial cardiac transcription factors GATA4, myocardin, and NKx2.5. Moreover, KDR subpopulations that overexpress SOX7 are associated with a vascular phenotype rather than a cardiogenic phenotype. These outcomes offer insights for refining the therapeutic regenerative interventions.

The FHF cells express hyperpolarization activated cyclic nucleotide gated potassium channel 4 and TBX5, while SHF progenitors express TBX1, FGF 8, FGF10, and sine oculis homeobox2 (Figure ). Cells from the SHF exhibit high proliferative and migratory capacities and are mostly responsible for the elongation and winding of the heart tube. Moreover, SHF cells differentiate to CMs, SMCs, fibroblasts, and endothelial cells (ECs) along their journey in the heart tube to form the right ventricle, right ventricular OFT, and most of the atria[60,61]. However, FHF cells hold less proliferative and migratory potentials and differentiate predominantly to CMs that form the left ventricle and small parts of the atria[62]. The cells of the cardiac crescent, theoretically the progeny of FHF CPCs, are terminally differentiated cells expressing the markers of CMs, such as actin alpha cardiac muscle 1 and myosin light chain 7[63,64], hence they are unlikely to be multipotent progenitors. Therefore, it is difficult to identify FHF before Nkx2.5 and TBX5 expressions. Conversely, multipotent SHF CPCs were validated with a clonal tracing experiment and identified by ISL1 expression[65]. However, ISL1 expression is not specific for SHF and has been proposed to represent only the developmental stages[66]. Tampakakis et al[67] generated ESCs by using hyperpolarization activated cyclic nucleotide gated potassium channel 4-green fluorescent protein and TBX1-Cre; Rosa-red fluorescent protein reporters of the FHF and the SHF respectively, and also by using live immunostaining of the cell membrane CXCR4, a SHF marker and the reporters. The ESC-derived progenitor cells have shown functional properties and transcriptome similar to their in vivo equivalents. Thus, chamber-specific cardiac cells have been generated for modelling of heart diseases in vitro.

The EPDCs are important as a signaling source for heart development, cardiac regeneration, and post-MI heart repair. Throughout the development of the heart in mice, EPDCs aid in the formation of various cardiac cell types and secrete paracrine factors for myocardial maturation[68]. In the adult heart, EPDCs are normally dormant and become stimulated following myocardial injury. Transcriptional analysis of the EPDCs derived from human (h)iPSCs cells have revealed several markers of EPDCs including Wilms tumor protein 1, endoglin, thymus cell antigen 1, and aldehyde dehydrogenase 1 family member A2[69] (Figure ). Following MI in mice, EPDCs undergo an epithelial-to-mesenchymal transition, with overexpression of Wilms tumor protein 1, and differentiate mainly into SMCs/fibroblasts[70,71]. EPDC-secreted paracrine factors include VEGF-A, FGF2, and PDGF-C, which support the growth of blood vessels, protect the myocardium, and recover cardiac functions in an acute MI-mouse model[70].

Side population (SP) cells have been detected in the heart and other various tissues and hold enhanced stem and progenitor cell activity[72]. SP cells, when stained in vitro, hold the ability to flush out the DNA Hoechst dye from their nuclei[73]. Gene expression profiling of SP cells after MI has revealed a downregulation of Wnt-related signals coupled with increased SP cell proliferation. This has been validated in vitro by treatment of isolated SP cells with canonical Wnt agonists or recombinant Wnt, where the proliferation of SP cells has been repressed with partial arresting the G1 cell cycle phase[74]. Consistent with this observation, delivery of secreted Frizzled-related proteins (SFRP; the Wnt antagonizer) improves post-MI remodeling[75,76].

SP cells can be identified by surface marker adenosine triphosphate (ATP) binding cassette subfamily G member 2 (ABCG2), also referred to as the breast cancer resistance protein1[77]. ABCG2+ cells have been also observed in the adult heart and can differentiate in vitro into CMs[78]. When SP cells have been injected into the injured hearts of rats, they have been recruited to the injured regions, where they differentiate into CMs, ECs, and SMCs, suggesting that they may be endogenous SP cells[79]. However, ABCG2CreER based genetic lineage tracing has demonstrated that ABCG2+ cells could only differentiate into the multiple cardiac cell lineages during the embryonic stages but not in adulthood[80,81]. The combination of ABCG2+ cells with pre-existing CMs is more likely to stimulate CM proliferation rather than differentiation into CMs directly[82]. Therefore, genetic fate mapping investigations have disproved the SP cells property of the adult endogenous ABCG2+ SP and their in vivo renewing myogenic ability[83].

Cardiospheres contain a combination of stromal, mesenchymal, and progenitor cells that are isolated from cultures of human heart biopsy[39,84]. They represent a niche-like environment, with cardiac-committed cells in the center and supporting cells in the periphery of the spherical cluster[85]. The cardiosphere-derived cells (CDCs) were originally isolated from mouse heart explants and human ventricular biopsies based on their ability to form three-dimensional (3D) spheroids in suspension cultures[86]. CDCs have grabbed much attention due to their proliferation and differentiation abilities by inherent stimulation of cardio-specific differentiation factors [GATA4, MEF2C, Nkx2.5, heart and neural crest derivatives expressed 2, and cardiac troponin T (TNNT2)] using a clustered regularly interspaced short palindromic repeat/dead Cas9 (CRISPR/dCas9) assisted transcriptional enhancement system[87,88]. Sano et al[89] have postulated that the CRISPR/dCas9 system may provide a proficient method of modifying TNNT2 gene activation in SCs. Consequently, CRISPR/dCas9 can improve the therapeutic outcomes of patients with ischemic heart disease by enhancing the transplanted CDCs differentiation capacity within the ischemic myocardium. Heart tissue is usually obtained by endomyocardial biopsy or during open cardiac surgery and grown in explants to form CDCs. CDCs have shown a superior myogenic differentiation potential, angiogenesis, and paracrine factor secretion as compared to other cell types. In heart failure animal models, the injected CDCs potentially differentiated into CMs and vascular cells. Additionally, CDCs have diminished unfavorable remodeling and infarct size, and hence improve cardiac function[90]. Accordingly, cardiospheres and CDCs may be some of the most promising sources of CPCs for cardiac repair.

The niche in the heart integrates several heterogeneous cell types, including CSCs, progenitors, fibroblasts, SMCs, CMs, capillaries, and supporting telocytes (TCs)[91], together with the junctions and cementing ECM that hold the niche together. Such architectural arrangement is essential for protection against external damaging stimuli and for preserving the stemness of the CSCs (Figure ). Without the niche microenvironment, CSCs lose their stemness and initiate differentiation eventually, leading to the exhaustion of the CSC pool. Similarly, in vitro studies require feeder layers and cytokines supplements in the culture media to ensure that SCs remain in their undifferentiated state[37].

Invivo arrangement of the central cardiac stem cells and the surrounding cells that comprise the niche (right side) and the in vitro derived cardio spheres (left side). The key delineates the types of cells identified in the niche and cardio spheres. Created with BioRender. CSC: Cardiac stem cell.

In vitro studies have recapitulated the niche theory using cardiospheres, which are 20150 m spheres (Figure ) of cells generated from the explant outgrowth of heart tissues[92,93]. Cardiospheres consist of CSCs in the core and cells committed to the cardiac lineage such as myofibroblasts, while vascular SMCs and ECs form the outer layer of the spheres. The 3D structure of cardiospheres protects the interiorly located CSCs from oxidative stress as well as maintain their stemness and function[84].

Accurate anatomical identification of CSCs in vivo remains a challenge due to the lack of basal-apical anatomical orientation as seen in epithelial organs such as the intestines[94]. Moreover, the heart does not comprise a specific compartment, where cells form a well-defined lining as seen in the bone marrow osteoblasts[95]. The adult heart epicardial lining anatomically contains several classes of niches, which are not limited to the sub epicardium[96] but dispersed throughout the myocardium, more in the atria and apex away from hemodynamic stress[97]. Some niches have been described in the atrio-ventricular junction of adult mouse and rat hearts[98] and interestingly in the human hearts[99]. The young mouse heart has been studied morphometrically to identify the location of CSCs niche and has been defined as a randomly positioned ellipsoid structure consisting of cellular and extracellular components. Within the niches, undifferentiated CSCs are usually assembled together with early committed cells that express c-KIT on surface, Nkx2.5 in the nucleus, and the contractile protein -sarcomeric actin in the cytoplasmic[97].

CSCs niche consists of clusters of c-kit+, MDR1+, and Sca-1+ cells[98] but lack the expression of the transcription factors and cytoplasmic or membrane proteins of cardiac cells[99,100]. Cardiac c-kit+/CD45- cells comprise about 1% of the CSC niche[97], are self-renewing clonogenic, and possess a cardiac multilineage differentiation potential comprise[101].

Within the niche, gap junctions (connexins) and (cadherins) connect SCs to their supporting cells, myocytes/fibroblasts. Conversely, ECs and SMCs do not act as supporting cells. Hence, the communication between CSCs with CMs and fibroblasts has been investigated by using in vitro assays[102]. The transmission of dyes via gap junctions between CSCs and CMs or fibroblasts was demonstrated previously and verified the functional coupling of these three cell populations[97]. In addition, micro ribonucleic acid (miRNA-499) translocates from CMs to CSCs comprising to the initiation of lineage specification and formation of myocytes[103].

Identification of SC niches is contingent upon the fulfillment of explicit criteria, including the recognition and determination of the affixing of SCs to their supporting cells as well as assuring the existence of an ancestor-progeny association[104]. Chemical and physical signals modulate the behavior of SCs within the niche. Amongst these signals are cytokines, cell surface adhesion molecules, shear forces, oxygen tension, innervation, and ions that serve as major determinants of SCs function[97]. Cell-to-cell signaling mediates the fate of SCs within the niches to promote self-renewal and favors their migration and differentiation. The fine-tuned crosstalk between SCs and their supporting cells regulates the state of the niche regarding quiescence or activity[105].

CSC niches, similar to the bone marrow, characteristically live in low oxygen tension, which favors a quiescent primitive state for SCs[106]. The longstanding perpetuation of the CSC niche requires a hypoxic environment, while physiological normoxia could be required for active cardiomyogenesis[107]. Hypoxic c-KIT+ CSCs within niches have been found throughout the myocardium, especially at the atria and apex. Throughout all ages, bundles of CSCs with low oxygen content coexist with normoxic CSCs niches. Hypoxic CSCs, especially in the atria, are quiescent cells undergoing cell cycle arrest and cannot divide. Normoxic CSCs are pushed into intense proliferation and differentiation with continuous telomere erosion, resulting finally in dysfunctional aged CMs[108]. Additionally, Nkx2.5 and GATA4 expressions are only restricted to the normoxic CSC niche. A balance between the hypoxic and normoxic niche is essential for the preservation of the CSC compartment and for the maintenance of myocardial homeostasis during the organ lifespan. Some factors such as aging cause an imbalance by expanding the hypoxic quiescent CSCs so that less pools of cycling CSCs maintain cell turnover[100]. Hypoxic cardiac niches are abundant in the epicardium and subepicardium in an adult mouse heart, which also fosters a metabolically distinctive population of glycolytic progenitor cells[109].

The pool of CSCs seems to be heterogeneous, incorporating quiescent and actively proliferating cells, migratory and adherent cells, uncommitted and early committed cells, with young and senescent cells. Additional surface epitopes remain to be disclosed to classify pools of CSCs holding specific properties. Surface Notch1 expression distinguishes multipotent CSCs that are poised for lineage commitment, while c-Met and ephrin type-A receptor 2 receptors reveal cells with particular migratory potential out of the niche area. A specific compartment of CSCs, expressing IGF-1 receptor, can be stimulated to regenerate damaged myocardium, while those expressing IGF-2 receptor hold higher probability for senescence and apoptosis. Although this arrangement of cells seems to equip properly the CSC with homeostasis regulation, it does not effectively protect against aging or ischemic injury of the heart[100].

Circulatory angiogenic cells (CACs) are endothelial progenitor cells involved in vasculogenesis, angiogenesis, and stimulating myocardial repair, mainly through paracrine action. Latham et al[110] demonstrated that conditioned medium from CACCSC co-cultures exhibited greatly mobilized CACs, with induction of tubule formation in human umbilical vein endothelial cells, mainly through the upregulation of the angiogenic factors angiogenin, stromal cell-derived factor 1 (SDF-1), and VEGF. Moreover, administration of CACs and CSCs in infarcted hearts of non-obese/severe combined immunodeficient mice restored substantially the left ventricular ejection fraction (LVEF), with reduction of scar formation as revealed by echocardiography. Successful yet modest SMCs, ECs, and CM differentiation has been also reported.

Pericytes (also called Rouget cells, mural cells, or perivascular mesenchymal precursor cells) are mesodermal cells that border the endothelial lining. They are highly proliferative cells and express neural/glial antigen 2, SOX-2, PDGFR-, CD34, and several mesenchymal markers such as CD105, CD90, and CD44. It was previously reported that the transplantation of saphenous vein-derived pericytes (SVPs) into an ischemic limb of an immunodeficient mice restored the local circulatory network via angiogenesis[111]. Moreover, treatment with SVP reduced fibrotic scar, CM death, and vascular permeability in a mouse model of MI via miRNA-132 facilitated angiogenesis[112]. Avolio et al[113] were the first to describe the relationship between SVP and the endogenous CSCs. Combined CSC and SVP transplantation in the infarcted myocardium of severe combined immunodeficient/Beige-immunodeficient mice showed similar results to treatment with CSCs or SVP cells per se, regarding scar size and ventricular function, indicating that SVPs alone are as potent as CSCs.

TCs represent a recently described cell population in the stromal spaces located in many organs, including the heart. They are broadly dispersed throughout the heart and comprise a network in the three cardiac layers, heart valves, and in CSC niches. TCs have been documented also in primary culture from heart tissues[114,115]. The ratio of cardiac TCs (0.5%-1%) exceeds that of CSCs. Although they still represent a minute portion of human cardiac interstitial cells, their extremely long and extensive telopodes allow them to occupy more surface area, forming a 3D platform probably that extends to support other cells[116]. The telopodes act as tracks for the sliding of precursor cells towards mature CMs and their integration into heart architecture[91]. TCs form a tandem with CSCs/CPCs in niches, where they communicate through direct physical contact by atypical junctions or indirect paracrine signaling[115].

TC-CSC co-culturing have suggested that TCs and CSCs act synergistically to control the level of secreted proteins, as shown by the increased levels of monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein1 and 2 (MIP-1 and MIP-2), and interleukin (IL)-13. Whereas, the level of IL-2 decreased compared to the monoculture of CSCs or TCs. IL-6 found in TC culture is behind the upregulation of these chemokines. Chemokines elucidated the role of TCs in directing the formation of CMs. Within the context, MIP-1 and MCP-1 play roles in the formation of SMCs in the airway. Additionally, MCP-1 is also involved in mouse skeletal muscle regeneration by recruiting macrophages. The enhancement of MCP-1 secretion serves as an activator of another cell population, primarily macrophages, which are generally involved in such processes[117].

IL-6 also activates downstream signaling pathways and contributes to cardioprotection and vessel formation in the heart through activation of gp130/signal transducer and activator of transcription 3. The Gp130/signal transducer and activator of transcription 3 is essential for the commitment of cardiac SCA-1+ cells into endothelial lineage[118].

Furthermore, IL-6 targets VEGF and hepatocyte growth factor (HGF) genes. VEGF has a mitogenic effect on CMs[119]. It is known to mobilize bone marrow-derived mesenchymal stem cells (BM-MSCs) into the peripheral blood in MI patients[120]. HGF and its receptor (c-Met) are also involved in cardiogenesis, as it is expressed early during cardiac development[121]. The level of HGF mRNA is normally low in the heart, but it is upregulated for at least 14 d after ischemic insult in rats, enhancing CMs survival under ischemic conditions[122,123]. Moreover, it has the potential to generate an adhesive micro-environment for SCs, as demonstrated in a study of transplantation of HGF transfected BM-MSCs in the infarcted myocardium[124]. HGF is also a powerful angiogenic agent, conducting its mitogenic and morphogenic effects through the expression of its specific receptor in various types of cells, including myocytes. Moreover, HGF exerts antifibrotic and antiapoptotic effects on the myocardium[125,126].

Transcriptomic analysis also has disclosed that TCs express pro-angiogenic miRNAs including let-7e, miRNA-21, miRNA-27b, miRNA-126, miRNA-130, miRNA-143, miRNA-503, and miRNA-100[127]. The TCs and CSCs interact in vitro forming atypical junctions, such as puncta adherentia and stromal synapses. The puncta adherentia consists of cadherincatenin clusters. It controls the symmetry of division by facilitating the proper positioning of centrosomes. Therefore, an increased number of CSCs has been reported to be encountered in the presence of cardiac TCs[128,129].

The paracrine potential of CSCs/CPCs has been recently under focus. CSC-derived cytokines and growth factors include epidermal growth factor (EGF), HGF, IGF-1, IGF-2, IL-6, IL-1, and TGF-1[130,131]. Exosomes appear to harbor relevant reparative signals, which mechanistically underlie the beneficial effects of CSCs transplantation[132].

Structurally, exosomes are lipid bilayer nano-sized organelles, 20-150 nm in diameter, secreted from all cell types, and function as intercellular communicators. Exosomes are highly heterogenic in content, and this stems from the unique packaging process that occurs inside progenitor and SCs. Exosomes carry lipids, proteins, and nucleic acids, with an abundance of miRNAs that hold profound post-transcriptional gene regulatory effects[133].

Amongst the distinctive protein content of cardiac exosomes are the chaperone proteins heat shock protein (HSP) 70 and HSP60. The HSP70 and HSP60, which under normal conditions assist in protein folding processes and deter misfolding and protein aggregation under pathological states induced by stress, also play major roles in apoptosis[134]. Circulating exosomes from healthy individuals have been found to activate cardioprotective pathways in CMs via HSP70 through extracellular signal-regulated kinase and HSP27 phosphorylation[135].

The exosome protein cargo of CPCs is distinct from BM-MSCs, fibroblasts, and other sources as it contains ample amounts of the pregnancy-associated plasma protein-A (PAPP-A). PAPP-A is present on the surface of human exosomes and interacts with IGF binding proteins (IGFBPs) to release IGF-1[136]. The cardioprotective role of CPCs-exosomes has been proven experimentally in in vitro ischemia/reperfusion and MI models and on CMs apoptosis to surpass that of BM-MSC-exosomes owing to their rich content of PAPP-A[137].

Like all exosomes, mouse CPCs-derived exosomes are positive for the surface markers CD63, CD81, and CD9, TSG-101, and Alix, however, they express a high-level of GATA4-responsive-miRNA-451. MiRNA-451 has been shown to inhibit CM apoptosis in an acute mouse myocardial ischemia-reperfusion model through inhibition of the caspases 3/7. The expression of miRNA-21 in the mouse CPCs-exosomes additionally justifies their CM protection against oxidative stress and antiapoptotic effects via inhibition of programmed cell death protein 4 (PDCD4)[138]. Human CPCs-exosomes are enriched with miRNA-210, miRNA-132, and miRNA-146a-3p, which account for the diminished CM apoptosis, enhanced angiogenesis, and improved LVEF[139]. MiRNA-146a-5p is the most highly upregulated miRNA in human CPCs-exosomes and targets genes involved in inflammatory and cell death pathways[137].

The CDCs contain CD34+ stromal cells of cardiac origin and are multipotent and clonogenic but not self-renewing[140]. CDCs secrete exosomes that induce cardiomyogenesis and angiogenesis, regulate the immune response, downgrade fibrosis, and improve the overall cardiac function[141,142]. Moreover, CDCs homogeneously express CD105 but not CD45 or other hematopoietic markers. They also exhibit a high expression of miRNA-126[143]. Circulating miRNA-126 may participate in cardiac repair during acute MI and has been demonstrated to be downregulated in heart damage[144].

While exosomes are constitutively secreted, changes in the surrounding microenvironment, such as hypoxia, can induce modifications in CPCs- and CM- derived extracellular vesicles. Hypoxic CMs secrete large extracellular vesicles containing long noncoding RNA neat 1 (LNCRNA NEAT1), which is transcriptionally regulated under basal conditions by p53, while during hypoxia it is regulated by the hypoxia inducible factor 2A. An uptake of the hypoxic CM-derived extracellular vesicles by fibroblasts can prompt the expression of profibrotic genes[145]. Oxidative stress may also induce the release of cardiac CPCs exosomes, which in turn inhibit apoptosis when taken up by H9C2 (rat cardiomyoblast cell line)[132]. Furthermore, oxidative stress stimulates secretion of miRNA-21 rich exosomes, which could inhibit H9C2 apoptosis by targeting PDCD4 and hence can be accounted as a new method to treat ischemia-reperfusion[138].

Intercellular communication via exosomes occurs as part of various biological processes, including immune modulation, vasculogenesis, transport of genetic materials, and pathological conditions such as inflammation, apoptosis, and fibrosis, which can lead to cardiovascular disease when altered[146]. Hence, isolation and analysis of cardiac exosomes contents, mainly miRNA and proteins, could offer diagnostic information for several cardiovascular diseases[147] (Figure ).

Schematic diagram elucidating the diverse exosomal contents that serve as biomarkers for several cardiovascular diseases. Created with BioRender.com. HSP: Heat shock protein; lncRNA: Long non-coding RNA; miR: MicroRNA.

Functionally, exosomes mediate several intra-cardiac inter-cellular communications such as:

CPC-CM crosstalk through factors, such as miRNA-146a and PAPP-A, which activate extracellular signal-regulated kinases 1/2 pathway and inhibit apoptosis[139].

CPC-macrophage (M1) crosstalk via miRNA-181b and Y-RNA fragment transforms M1 to M2 macrophages with attenuated proinflammatory cytokines and increased IL-10[148,149] (Figure ).

Possible cardiac reparative effects of cardiac stem cell/cardiosphere-derived cell-derived exosomes in myocardial ischemia and ischemia/reperfusion injury. Created with BioRender.com. CSC: Cardiac stem cell; IL: Interleukin; IR: Ischemia/reperfusion; miRNA: MicroRNA; PI3K: Phosphoinositide 3-kinase; SDF-1: Stromal cell-derived factor 1; VEGF: Vascular endothelial growth factor.

CPC-fibroblast interaction via exosomes primes the fibroblasts and increases expression of VEGF and SDF-1. Experimental injection of fibroblasts primed with CPCs-exosomes into the myocardium of a MI model proved to reduce infarct size and improve cardiac function. In addition, cardiosphere-isolated exosomes have been used to prime inert fibroblasts, leading to an intensification of their angiogenic, cardiomyogenic, antifibrotic, and collective regenerative effects[150] (Figure ).

CPC-self regulatory mechanisms: Exosomes derived from CPCs may play critical roles in maintaining the self-renewal state of CPCs themselves and balance their differentiation, i.e. preserve their stemness[151] (Figure ). The CPC-derived exosomes activate the endogenous CPCs by transferring signal molecules directly within their niche[152].

CPC-derived exosomes release various RNA species in the extracellular space, modulating endogenous SC plasticity and tissue regeneration through their cytoprotective, immunomodulatory, pro-angiogenic, and anti-apoptotic actions[153].

Fibroblasts and pericytes interact after transdifferentiating to myofibroblasts and deposit ECM causing cardiac fibrosis. These fibrotic changes are usually induced by cardiac damage and lead to scar formation. Exosomes serve as messengers for cell-to-cell communication during cardiac fibrosis[154]. Molecular mechanisms of cardiac fibrosis are primarily related to TGF- pathways, IL-11 signaling pathway, nuclear factor- pathway, and Wnt pathways[155]. Accordingly, the bioactive substances targeted at these pathways could hypothetically be applied in the treatment of cardiac fibrosis. Wnt3a, being highly expressed in exosomes, could activate the Wnt/-catenin pathway in cardiac fibroblasts by restricting GSK3 activation[156]. Moreover, tumor necrosis factor contained in exosomes can be transferred between cardiac myocytes. In general activation/inhibition of the exosomes conveying remodeling substance secretion or uptake can control the myocardial remodeling and repair following MI[154,157].

The highlighted complex cell-to-cell communication from endogenous or exogenous CSCs provides an optimal microenvironment for resident CPC proliferation and differentiation (Figure ), rendering the environment receptive to transplanted CPCs. This adaptation is promoted through activation of pro-survival kinases, leading to the induction of a glycolytic switch in recipient CPCs[158].

Data from experimental models suggest that the exosomal component of the CPC secretome can fully recapitulate the effects of cellular therapy on ischemic and non-ischemic heart models[140]. In an ischemia-reperfusion injury rat model, Ciullo and partners[159] have shown that the systemic injection of exosomes (genetically manipulated to overexpress CXCR4ExoCXCR4) improve cardiac function. Additionally, expression of hypoxia-inducible factor 1 (HIF-1) in the infarcted myocardium is upregulated through the stimulation of SDF-1. The latter is one of the CXC chemokine family overexpressed in heart post-MI that readily attaches to the CXCR4 receptor and acts as a potent chemoattractant for CXCR4 expressing circulating progenitor cells. The ExoCXCR4 are more bioactive in the infarcted zone than naturally occurring exosomes injected via tail-vein, confirming their superior homing and cardioprotective properties in the damaged heart.

Gallet et al[160] postulated the safety and efficiency of CDC-derived exosomes in acute and chronic myocardial injury animal models. Within the context of experimental research to validate the paracrine hypothesis for CDCsderived exosomes, it has been proven that human CDC-exosomes can recapitulate CDC therapy and boost cardiac function post-MI in pig models. Intramyocardial injection of human CDC-exosomes has resulted in higher exosome retention and efficacy as compared to intracoronary injection, with great reduction of scar size and increased ejection fraction. This indicates that the route of administration is imperative for full functional capacity of the exosomes. Subsequently, the researchers have devised a randomized preclinical study by means of a NOGA-guided intramyocardial exosome injection. Decreased collagen content in the infarct and border zone and increased neovascularization and Ki67+ CMs are indicative of the reparative functions of CDC-exosomes. Notably, human CDC-exosomes have shown a lack of an immune reaction, as seen by the lack of inflammatory reactions or CM necrosis in pig models. These observations strongly support the view that CDC-exosomes are ready to be tested in clinical trials.

Similar promising outcomes were observed in a Duchenne muscular dystrophy model (mdx), in which intramyocardial injection of CDC-exosomes efficiently recapitulated the effects of CDC injection on cardiac function, leading to recovery of movement. Administration of CPC-derived exosomes has resulted in transient restoration of partial expression of full-length dystrophin in mdx mice[161]. Further studies assessed the therapeutic potential of CPC-exosomes in a doxorubicin cardiotoxicity model and non-ischemic heart disease[162]. In addition, two concluded phase I clinical trials in patients with heart failure and revealed the capacity of CDCs to enhance cardiac function by reducing ventricular remodeling and scar formation. Despite receiving a single injection at the beginning of the study, the improvement in cardiac function was noted after the 1-year follow-up. This finding consequently leads to the proposition that transplanted CDCs mainly have imposed their actions at the site of injury by secreting paracrine factors including exosomes. In other words, CDC-exosomes achieved a biphasic beneficiary regenerative effect involving acute cardio protection coupled with long-term stimulation of endogenous cardiac repair[163].

While the fetal heart obtains most of its ATP supply via glycolysis[164], the adult heart relies mainly on fatty acid oxidation to fulfill the contracting myocardium high energy demand[164,165]. The loss of the regenerative phenotype is related to the oxidative metabolism of glucose and fatty acids[166,167] and is mediated by various physiological changes including increased workload and the demand for growth, which cannot be solely met by glycolysis[168,169], as well as postnatal increase in both circulating levels of free fatty acids and blood oxygen levels[164,165]. Studies have shown the involvement of the HIF-1 signaling pathway[170], peroxisome proliferator-activated receptor (PPAR)[171], and peroxisome proliferator-activated receptor coactivator-1 (PGC-1) in the switch toward oxidative metabolism[172], which is accompanied by dramatic increase in the number of mitochondria in CMs[173].

Notably, similar metabolic reprogramming occurs during differentiation from cardiac SCs to CMs[167]. Studies reported that after differentiation into CMs, there is an increase in the mitochondrial number and activity[174], increased oxidative metabolism[175], and increased respiratory capacity resulting in an increased adenosine diphosphate:ATP ratio[173] after differentiation into CMs.

The fact of the various metabolic changes that accompany the transition from glycolysis to fatty acids oxidation affect cardiac cell maturation[164,167] has mandated the consideration of substrate composition in cardiac differentiation protocols[167].

A study by Malandraki-Miller et al[176] investigated the effect of fatty acid supplementation, which mimics the metabolic switch from glucose to fatty acid oxidation, on adult cardiac progenitors. The study used radiolabeled substrate consumption for metabolic flux to investigate the role of the PPAR/PGC-1 axis during metabolic maturation. Oleic acid stimulated the PPAR pathway, enhanced the maturation of the cardiac progenitor, and increased the expression of MHC and connexin after differentiation. Moreover, total glycolytic metabolism, mitochondrial membrane potential, the expression of glucose, and fatty acid transporter increased. The recorded results contributed greatly in highlighting the role of fatty acids and PPAR in CPC differentiation.

Another study by Correia et al[177] has linked substrate utilization and functional maturation of CMs via studying the effect of the metabolic shift from glucose to galactose and fatty acid-containing medium in the maturation of hPSCs-derived CMs (hPSCs-CMs). The shift accelerated hPSC-CM maturation into adult-like CMs with higher oxidative metabolism, mature transcriptional signatures, higher myofibril density, improved calcium influx, and enhanced contractility. Galactose improved total oxidative capacity with reduction of fatty acid oxidation, thereby protecting the cells from lipotoxicity.

In CDCs, oxidative metabolism and cell differentiation reciprocally affect each other. In vitro cultures for CDCs revealed a PPAR agonist that triggers fatty acid oxidation. Metabolic changes have been characterized as the CDC differentiated towards a cardiac phenotype. Addition of a PPAR agonist at the onset of differentiation has induced a switch towards oxidative metabolism, as shown by changes in gene expression with decreasing glycolytic flux and increasing oxidation of glucose and palmitate. Undifferentiated CDCs have generated high levels of ATP from glycolysis and from oxidation of acetoacetate. Upon differentiation, oxidative metabolism of glucose and fatty acids is upregulated with decreased oxidation of acetoacetate, a metabolic phenotype similar to that of the adult heart[178].

Taken together, the metabolic hallmarks of differentiated CMs vary from their undifferentiated SCs. Energy substrate metabolism during cardiac development and differentiation shows gradual decrease in the contribution of glycolysis to ATP synthesis with simultaneous increase in fatty aciddependent mitochondrial respiration[179].

Common methods for the investigation of substrate metabolism include the measurement of metabolic fluxes using radio-labeled substrates, such as D-U-14C-glucose[180,181] as well as measurement of mitochondrial oxygen consumption rate and extracellular acidification rate using the XF Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, United States)[182,183].

Recently, a detailed protocol for metabolic characterization of hiPSCs-CMs has been developed. The hiPSCs are obtained from adult somatic cells via novel cell reprogramming approaches, followed by differentiation to CMs. The novel in vitro cardiac cellular model provided new insights into studying cardiac disease mechanisms and therapeutic potentials. The characterization protocol measures small metabolites and combines gas- and liquid-chromatography-mass spectrometry metabolic profiling, lactate/pyruvate, and glucose uptake assays as important tools[184]. Integration between the implemented assays has provided complementary metabolic characteristics besides the already established electrophysiological and imaging techniques, such as monitoring ion channel activities[185], measurement of action potentials, changes in Ca+2 fluxes[186], and mitochondria viability and apoptosis[187].

An alternative pathway for glucose metabolism in CMs involves the entry of glucose-6-phosphate (G6P) in the pentose phosphate pathway, with resultant generation of reduced nicotinamide adenine dinucleotide phosphate (NADPH)[188]. Reduced NADPH helps to regenerate reduced glutathione and thus acts protectively against reactive oxygen species induced cell injury.

The cardioprotective role of the pentose/G6P/NADPH/glutathione pathway has been emphasized by Jain et al[189] who demonstrated that G6P dehydrogenase (G6PD) lacking mice have more severe heart damage induced by the myocardial ischemia reperfusion injury in Langendorff-perfused hearts as compared with wild-type mice.

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Cardiac stem cells: Current knowledge and future prospects

Cardiac Stem Cells Comments Off on Cardiac stem cells: Current knowledge and future prospects | April 13th, 2023

Cardiovascular diseases represent the world's leading cause of death. In this heterogeneous group of diseases, ischemic cardiomyopathies are the most devastating and prevalent, estimated to cause 17.9 million deaths per year. Despite all biomedical efforts, there are no effective treatments that can replace the myocytes lost during an ischemic event or progression of the disease to heart failure. In this context, cell therapy is an emerging therapeutic alternative to treat cardiovascular diseases by cell administration, aimed at cardiac regeneration and repair. In this review, we will cover more than 30 years of cell therapy in cardiology, presenting the main milestones and drawbacks in the field and signaling future challenges and perspectives. The outcomes of cardiac cell therapies are discussed in three distinct aspects: The search for remuscularization by replacement of lost cells by exogenous adult cells, the endogenous stem cell era, which pursued the isolation of a progenitor with the ability to induce heart repair, and the utilization of pluripotent stem cells as a rich and reliable source of cardiomyocytes. Acellular therapies using cell derivatives, such as microvesicles and exosomes, are presented as a promising cell-free therapeutic alternative.

Keywords: Cardiac stem cell; Cardiovascular diseases; Cell therapy; Pluripotent stem cells; Progenitor cardiac cells; Stem cell.

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Stem cell therapies in cardiac diseases: Current status and future ...

Cardiac Stem Cells Comments Off on Stem cell therapies in cardiac diseases: Current status and future … | April 13th, 2023

The limited regenerative capacity of the heart is a major factor in heart failure and death. Once cardiac cells are diseased, its hard for them to heal like your body would with a cut. Studying how the heart forms in fetuses and then matures is a natural step for researchers interested in generating and regenerating heart cells. Theyre also investigating the effect of stem cell-derived cardiac cells on repairing damaged hearts and their potential to treat heart muscle diseases.

Cardiovascular progenitor cells (CPCs), a type of heart cell, are called building blocks because theyre used to form the heart during fetal development. They hold tremendous therapeutic potential because of their unique ability to develop into several different heart cell types. Researchers are studying how CPC cells can renew themselves in mice. Theyre studying whether this renewal also occurs in humans and whether this is useful for repairing damaged hearts.

Because CPCs regenerate, scientists may be able to grow them in a dish. Its not as easy to grow cells in a lab as it is in the body they often have developmental arrest and dont mature. However, a recent discovery of the pathways that lead a fetal cell into an adult cell will enable researchers to recreate adult heart tissue in the lab, which holds tremendous potential for new heart disease treatment.

Videos Heart tissue grown in a dish

Heart tissue grown in a dish from mouse cardiac progenitor cells (CPCs). The CPCs, and the tissue they built, were engineered to produce a red protein.

View labs and centersAdult Cardiac Catheterization LabCiccarone CenterChulan Kwon LabHeart and Vascular InstituteCardiovascular Stem Cell Program

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Stem Cell and Regenerative Biology | Johns Hopkins Heart and Vascular ...

Cardiac Stem Cells Comments Off on Stem Cell and Regenerative Biology | Johns Hopkins Heart and Vascular … | April 13th, 2023

Reparative stem cells have the capability to restore function to damaged tissue by renewing cell growth (shown in green) in cardiac cells destroyed by heart disease.

Approximately 28 million Americans have been diagnosed with heart disease. Traditional medical therapies are not able to fully address the burden of disease, and the shortage of organs for transplantation remains a key barrier more than 117,000 people are on the national transplant list.

This unmet need drives Mayo Clinic researchers to make new discoveries to accelerate regenerative solutions into clinical trials and rapidly provide new hope to patients who can't currently be treated.

Cardiac regeneration is a broad effort that aims to repair irreversibly damaged heart tissue with cutting-edge science, including stem cell and cell-free therapy. Reparative tools have been engineered to restore damaged heart tissue and function using the body's natural ability to regenerate. Working together, patients and providers are finding regenerative solutions that restore, renew and recycle patients' own reparative capacity. Through the vision and generous support of Russ and Kathy Van Cleve, strong efforts are underway to develop discoveries that will have a global impact on ischemic heart disease.

Mayo Clinic researchers are leading efforts in translating new knowledge into applicable therapeutics through a multidisciplinary community of practice. As technology evolves, it offers the potential to regenerate cardiac tissue from noncardiac sources and ultimately provide personalized products and services to people with cardiovascular disease.

The overarching vision for the cardiac regeneration program at Mayo Clinic is to develop new therapies to cure ischemic heart disease. Mayo researchers are developing products for clinical testing that span the disease spectrum, including the following areas:

More information about cardiac regenerative medicine research at Mayo Clinic is on the Van Cleve Cardiac Regenerative Medicine Program website.

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Center for Regenerative Biotherapeutics - Cardiac Regeneration

Cardiac Stem Cells Comments Off on Center for Regenerative Biotherapeutics – Cardiac Regeneration | April 13th, 2023

MAGENTA THERAPEUTICS, INC. MANAGEMENT'S DISCUSSION AND ANALYSIS OF FINANCIAL CONDITION AND RESULTS OF OPERATIONS (form 10-K)  Marketscreener.com

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MAGENTA THERAPEUTICS, INC. MANAGEMENT'S DISCUSSION AND ANALYSIS OF FINANCIAL CONDITION AND RESULTS OF OPERATIONS (form 10-K) - Marketscreener.com

Cardiac Stem Cells Comments Off on MAGENTA THERAPEUTICS, INC. MANAGEMENT’S DISCUSSION AND ANALYSIS OF FINANCIAL CONDITION AND RESULTS OF OPERATIONS (form 10-K) – Marketscreener.com | March 25th, 2023

CAREDX, INC. MANAGEMENT'S DISCUSSION AND ANALYSIS OF FINANCIAL CONDITION AND RESULTS OF OPERATIONS (form 10-K)  Marketscreener.com

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CAREDX, INC. MANAGEMENT'S DISCUSSION AND ANALYSIS OF FINANCIAL CONDITION AND RESULTS OF OPERATIONS (form 10-K) - Marketscreener.com

Cardiac Stem Cells Comments Off on CAREDX, INC. MANAGEMENT’S DISCUSSION AND ANALYSIS OF FINANCIAL CONDITION AND RESULTS OF OPERATIONS (form 10-K) – Marketscreener.com | March 1st, 2023

A Possible Connection between Mild Allergic Airway Responses and Cardiovascular Risk Featured in Toxicological Sciences  Newswise

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A Possible Connection between Mild Allergic Airway Responses and Cardiovascular Risk Featured in Toxicological Sciences - Newswise

Cardiac Stem Cells Comments Off on A Possible Connection between Mild Allergic Airway Responses and Cardiovascular Risk Featured in Toxicological Sciences – Newswise | February 4th, 2023

A heart surgeon, Professor Massimo Caputo from the Bristol Heart Institute has stated he "saved the life" of a baby by carrying out a "world-first" operation using stem cells from placentas.

Professor Massimo Caputo used pioneering stem cell injections to correct baby Finley's heart defect and says he now hopes to develop the technology so children born with congenital cardiac disease won't need much surgical operations.

Finley was born with the main arteries in his heart positioned the wrong way round and at just four days old had his first open-heart surgery at Bristol Royal Hospital for Children

Unfortunately the surgery did not solve the issue and his heart function deteriorated significantly, with the left side of the heart suffering from a severe lack of blood flow.

His mother, Melissa, from Corsham, in Wiltshire, said: "We were prepared from the start that the odds of him surviving were not good.

"After 12 hours, Finley finally came out of surgery but he needed a heart and lung bypass machine to keep alive, and his heart function had deteriorated significantly."

After weeks in intensive care it looked like there was no way to treat Finley's condition and he was reliant on drugs to keep his heart going.

But a new procedure was tried, involving stem cells from a placenta bank.

Prof Caputo injected the cells directly into Finley's heart in the hope they would help damaged blood vessels grow.

The so-called "allogeneic" cells were grown by scientists at the Royal Free Hospital in London, and millions of them were injected into Finley's heart muscle.

Allogeneic cells have the ability to grow into tissue that is not rejected and in Finley's case, have regenerated damaged heart muscle.

"We weaned him from all the drugs he was on, we weaned him from ventilation," said Prof Caputo.

"He was discharged from ITU and is now a happy growing little boy."

Finley is now aged two years.

Using a bio-printer, a stem cell scaffold is made to repair abnormalities to valves in blood vessels, and to mend holes between the two main pumping chambers of the heart.

In cardiac surgery, artificial tissue is normally used on babies for cardiac repairs, but it can fail and it does not grow with the heart, so as the children grow, they require more operations.

A child might therefore have to go through the same heart operation multiple times throughout its childhood but Prof Caputo and his team say the stem cell technology could save the UK government an estimated 30,000 for every operation no longer needed.

Dr Stephen Minger, an expert in stem cell biology and director of SLM Blue Skies Innovations Ltd said;

"Most studies that I am aware of in adults with heart dysfunction or failure show only minimal therapeutic benefit with stem cell infusion.

"I'm happy that the clinical team will go on to do a standard clinical trial which should tell us if this was a 'one-off' success and also give us some better understanding of mechanisms behind this."

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Baby's life saved by surgeon who carried out world's first surgery ...

Cardiac Stem Cells Comments Off on Baby’s life saved by surgeon who carried out world’s first surgery … | December 25th, 2022

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Cardiac Stem Cells Comments Off on An organoid model of colorectal circulating tumor cells with stem cell … | December 25th, 2022

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the potential to differentiate into various types of cells including skeletal muscle cells. The approach of converting ESCs/iPSCs into skeletal muscle cells offers hope for patients afflicted with the skeletal muscle diseases such as the Duchenne muscular dystrophy (DMD). Patient-derived iPSCs are an especially ideal cell source to obtain an unlimited number of myogenic cells that escape immune rejection after engraftment. Currently, there are several approaches to induce differentiation of ESCs and iPSCs to skeletal muscle. A key to the generation of skeletal muscle cells from ESCs/iPSCs is the mimicking of embryonic mesodermal induction followed by myogenic induction. Thus, current approaches of skeletal muscle cell induction of ESCs/iPSCs utilize techniques including overexpression of myogenic transcription factors such as MyoD or Pax3, using small molecules to induce mesodermal cells followed by myogenic progenitor cells, and utilizing epigenetic myogenic memory existing in muscle cell-derived iPSCs. This review summarizes the current methods used in myogenic differentiation and highlights areas of recent improvement.

Duchenne muscular dystrophy (DMD) is a genetic disease affecting approximately 1 in 3500 male live births [1]. It results in progressive degeneration of skeletal muscle causing complete paralysis, respiratory and cardiac complications, and ultimately death. Normal symptoms include the delay of motor milestones including the ability to sit and stand independently. DMD is caused by an absence of functional dystrophin protein and skeletal muscle stem cells, as well as the exhaustion of satellite cells following many rounds of muscle degeneration and regeneration [2]. The dystrophin gene is primarily responsible for connecting and maintaining the stability of the cytoskeleton of muscle fibers during contraction and relaxation. Despite the low frequency of occurrence, this disease is incurable and will cause debilitation of the muscle and eventual death in 20 to 30 year olds with recessive X-linked form of muscular dystrophy. Although there are no current treatments developed for DMD, there are several experimental therapies such as stem cell therapies.

Skeletal muscle is known to be a regenerative tissue in the body. This muscle regeneration is mediated by muscle satellite cells, a stem cell population for skeletal muscle [3, 4]. Although satellite cells exhibit some multipotential differentiation capabilities [5], their primary differentiation fate is skeletal muscle cells in normal muscle regeneration. Ex vivo expanded satellite cell-derived myoblasts can be integrated into muscle fibers following injection into damaged muscle, acting as a proof-of-concept of myoblast-mediated cell therapy for muscular dystrophies [69]. However, severe limitations exist in relation to human therapy. The number of available satellite cells or myoblasts from human biopsies is limited. In addition, the poor cell survival and low contribution of transplanted cells have hindered practical application in patients [6, 8, 9]. Human-induced pluripotent stem cells (hiPSCs) are adult cells that have been genetically reprogrammed to an embryonic stem cell- (ESC-) like state by being forced to express genes and factors important for maintaining the defining properties of ESCs. hiPSCs can be generated from a wide variety of somatic cells [10, 11]. They have the ability to self-renew and successfully turn into any type of cells. With their ability to capture genetic diversity of DMD in an accessible culture system, hiPSCs represent an attractive source for generating myogenic cells for drug screening.

The ESC/iPSC differentiation follows the steps of embryonic development. The origin of skeletal muscle precursor cells comes from the mesodermal lineage, which give rise to skeletal muscle, cardiac muscle, bone, and blood cells. Mesoderm subsequently undergoes unsegmented presomitic mesoderm followed by segmented compartments termed somites from anterior to caudal direction. Dermomyotome is an epithelial cell layer making up the dorsal part of the somite underneath the ectoderm. Dermomyotome expresses Pax3 and Pax7 and gives rise to dermis, skeletal muscle cells, endothelial cells, and vascular smooth muscle [12]. Dermomyotome also serves as a tissue for secreted signaling molecules to the neural tube, notochord, and sclerotome [13, 14]. Upon signals from the neural tube and notochord, the dorsomedial lip of dermomyotome initiates and expresses skeletal muscle-specific transcription factors such as MyoD and Myf5 to differentiate into myogenic cells termed myoblasts. Myoblasts then migrate beneath the dermomyotome to form myotome. Eventually, these myoblasts fuse with each other to form embryonic muscle fibers. ESCs/iPSCs mimic these steps toward differentiation of skeletal muscle cells. Many studies utilize methods of overexpression of muscle-related transcription factors such as MyoD or Pax3 [15], or the addition of small molecules which activate or inhibit myogenic signaling during development. Several studies show that iPSCs retain a bias to form their cell type of origin due to an epigenetic memory [1619], although other papers indicate that such epigenetic memory is erased during the reprogramming processes [2022]. Therefore, this phenomenon is not completely understood at the moment. In light of these developments, we have recently established mouse myoblast-derived iPSCs capable of unlimited expansion [23]. Our data demonstrates that these iPSCs show higher myogenic differentiation potential compared to fibroblast-derived iPSCs. Thus, myogenic precursor cells generated from human myoblast-derived iPSCs expanded ex vivo should provide an attractive cell source for DMD therapy. However, since DMD is a systemic muscle disease, systemic delivery of myoblasts needs to be established for efficient cell-based therapy.

During developmental myogenesis, presomitic mesoderm is first formed by Mesogenin1 upregulation, which is a master regulator of presomitic mesoderm [24]. Then, the paired box transcription factor Pax3 gene begins to be expressed from presomitic mesoderm to dermomyotome [25]. Following Pax3 expression, Pax7 is also expressed in the dermomyotome [26], and then Myf5 and MyoD, skeletal muscle-specific transcription factor genes, begin to be expressed in the dorsomedial lip of the dermomyotome in order to give rise to myoblasts which migrate beneath the dermomyotome to form the myotome. Subsequently, Mrf4 and Myogenin, other skeletal muscle-specific transcription factor genes, followed by skeletal muscle structural genes such as myosin heavy chain (MyHC), are expressed in the myotome for myogenic terminal differentiation (Figure 1) [27, 28]. Pax3 directly and indirectly regulates Myf5 expression in order to induce myotomal cells. Dorsal neural tube-derived Wnt proteins and floor plate cells in neural tube and notochord-derived sonic hedgehog (Shh) positively regulate myotome formation [13, 29]. Neural crest cells migrating from dorsal neural tubes are also involved in myotome formation: Migrating neural crest cells come across the dorsomedial lip of the dermomyotome, and neural crest cell-expressing Delta1 is transiently able to activate Notch1 in the dermomyotome, resulting in conversion of Pax3/7(+) myogenic progenitor cells into MyoD/Myf5(+) myotomal myoblasts [30, 31]. By contrast, bone morphogenetic proteins (BMPs) secreted from lateral plate mesoderm are a negative regulator for the myotome formation by maintaining Pax3/Pax7(+) myogenic progenitor cells [29, 32]. Pax3 also regulates cell migration of myogenic progenitor cells from ventrolateral lip of dermomyotome to the limb bud [33]. Pax3 mutant mice lack limb muscle but trunk muscle development is relatively normal [34]. Pax3/Pax7 double knockout mice display failed generation of myogenic cells, suggesting that Pax3 and Pax7 are critical for proper embryonic myogenesis [35]. Therefore, both Pax3 and Pax7 are also considered master transcription factors for the specification of myogenic progenitor cells. Importantly, MyoD was identified as the first master transcription factor for myogenic specification since MyoD is directly able to reprogram nonmuscle cell type to myogenic lineage when overexpressed [3638]. In addition, genetic ablation of MyoD family gene(s) via a homologous gene recombination technique causes severe myogenic developmental or regeneration defects [3945]. Finally, genetic ablation of combinatory MyoD family genes demonstrates that MyoD/:Myf5/:MRF4/ mice do not form any skeletal muscle during embryogenesis, indicating the essential roles in skeletal muscle development of MyoD family genes [28, 46]. It was proven that Pax3 also possesses myogenic specification capability since ectopic expression of Pax3 is sufficient to induce myogenic programs in both paraxial and lateral plate mesoderm as well as in the neural tube during chicken embryogenesis [47]. In addition, genetic ablation of Pax3 and Myf5 display complete defects of body skeletal muscle formation during mouse embryogenesis [48]. Finally, overexpression of Pax7 can convert CD45(+)Sca-1(+) hematopoietic cells into skeletal muscle cells [49]. From these notions, overexpression of myogenic master transcription factors such as MyoD or Pax3 has become the major strategy for myogenic induction in nonmuscle cells, including ES/iPSCs.

The overexpression of MyoD approach to induce myogenic cells from mESCs was first described by Dekel et al. in 1992. This has been a standard approach for the myogenic induction from pluripotent stem cells (Table 1). Ozasa et al. first utilized Tet-Off systems for MyoD overexpression in mESCs and showed desmin(+) and MyHC(+) myotubes in vitro [50]. Warren et al. transfected synthetic MyoD mRNA in to hiPSCs for 3 days, which resulted in myogenic differentiation (around 40%) with expression of myogenin and MyHC [51]. Tanaka et al. utilized a PiggyBac transposon system to overexpress MyoD in hiPSCs. The PiggyBac transposon system allows cDNAs to stably integrate into the genome for efficient gene expression. After integration, around 70 to 90% of myogenic cells were induced in hiPSC cultures within 5 days [52]. This study also utilized Miyoshi myopathy patient-derived hiPSCs for the MyoD-mediated myogenic differentiation. Miyoshi myopathy is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in dysferlin gene. The patient-derived hiPSC-myogenic cells will be able to provide the opportunity for therapeutic drug screening. Abujarour et al. also established a model of patient-derived skeletal muscle cells which express NCAM, myogenin, and MyHC by doxycycline-inducible overexpression of MyoD in DMD patient-derived hiPSCs [53]. Interestingly, MyoD-induced iPSCs also showed suppression of pluripotent genes such as Nanog and a transient increase in the gene expression levels of T (Brachyury T), Pax3, and Pax7, which belong to paraxial mesodermal/myogenic progenitor genes, upstream genes of myogenesis. It is possible that low levels of MyoD activity in hiPSCs may initially suppress their pluripotent state while failing to induce myogenic programs, which may result in transient paraxial mesodermal induction. Supporting this idea, BAF60C, a SWI/SNF component that is involved in chromatin remodeling and binds to MyoD, is required to induce full myogenic program in MyoD-overexpressing hESCs [54]. Overexpression of MyoD alone in hESC can only induce some paraxial mesodermal genes such as Brachyury T, mesogenin, and Mesp1 but not myogenic genes. Co-overexpression of MyoD and BAF60C was now able to induce myogenic program but not paraxial mesodermal gene expression, indicating that there are different epigenetic landscapes between pluripotent ESCs/iPSCs and differentiating ESC/iPSCs in which MyoD is more accessible to DNA targets than those in pluripotent cells. The authors then argued that without specific chromatin modifiers, only committed cells give rise to myogenic cells by MyoD. These results strongly indicate that nuclear landscapes are important for cell homogeneity for the specific cell differentiation in ESC/iPSC cultures. Similar observations were seen in overexpression of MyoD in P19 embryonal carcinoma stem cells, which can induce paraxial mesodermal genes including Meox1, Pax3, Pax7, Six1, and Eya2 followed by muscle-specific genes. However, these MyoD-induced paraxial mesodermal genes were mediated by direct MyoD binding to their regulatory regions, which was proven by chromatin immunoprecipitation (ChIP) assays, indicating the novel role for MyoD in paraxial mesodermal cell induction [55].

hESCs/iPSCs have been differentiated into myofibers by overexpression of MyoD, and this method is considered an excellent in vitro model for human skeletal muscle diseases for muscle functional tests, therapeutic drug screening, and genetic corrections such as exon skipping and DNA editing. Shoji et al. have shown that DMD patient-derived iPSCs were used for myogenic differentiation via PiggyBac-mediated MyoD overexpression. These myogenic cells were treated with morpholinos for exon-skipping strategies for dystrophin gene correction and showed muscle functional improvement [56]. Li et al. have shown that patient-derived hiPSC gene correction by TALEN and CRISPR-Cas9 systems, and these genetically corrected hiPSCs were used for myogenic differentiation via overexpression of MyoD [57]. This work also revealed that the TALEN and CRISPR-Cas9-mediated exon 44 knock-in approach in the dystrophin gene has high efficiency in gene-editing methods for DMD patient-derived cells in which the exon 44 is missing in the genome.

Along this line of the strategy, Darabi et al. first performed overexpression of Pax3 gene, which can be activated by treatment with doxycycline in mESCs, and showed efficient induction of MyoD/Myf5(+) skeletal myoblasts in EB cultures [15]. Upon removing doxycycline, these myogenic cells underwent MyHC(+) myotubes. However, teratoma formation was observed after EB cell transplantation into cardiotoxin-injured regenerating skeletal muscle in Rag2/:C/ immunodeficient mice [15]. This indicates that myogenic cell cultures induced by Pax3 in mESCs still contain some undifferentiated cells which gave rise to teratomas. To overcome this problem, the same authors separated paraxial mesodermal cells from Pax3-induced EB cells by FACS using antibodies against cell surface markers as PDGFR(+)Flk-1() cell populations. After cell sorting, isolated Pax3-induced paraxial mesodermal cells were successfully engrafted and contributed to regenerating muscle in mdx:Rag2/:C/ DMD model immunodeficient mice without any teratoma formations. Darabi et al. also showed successful myogenic induction in mESCs and hES/iPSCs by overexpression of Pax7 [58, 59]. Pax3 and Pax7 are not only expressed in myogenic progenitor cells. They are also expressed in neural tube and neural crest cell-derived cells including a part of cardiac cell types in developmental stage, suggesting that further purification to skeletal muscle cell lineage is crucial for therapeutic applications for muscle diseases including DMD.

Taken together, overexpression of myogenic master transcription factors such as MyoD or Pax3/Pax7 is an excellent strategy for myogenic induction in hESCs and hiPSCs, which can be utilized for in vitro muscle disease models for their functional test and drug screening. However, for the safe stem cell therapy, it is essential to maintain the good cellular and genetic qualities of hESC/hiPSC-derived myogenic cells before transplantation. Therefore, random integration sites of overexpression vectors for myogenic master transcription factors and inappropriate expression control of these transgenes may diminish the safety of using these induced myogenic cells for therapeutic stem cell transplantation.

Stepwise induction protocols utilizing small molecules and growth factors have been established as alternative myogenic induction approaches and a more applicable method for therapeutic situations. As described above, during embryonic myogenesis, somites and dermomyotomes receive secreted signals such as Wnts, Notch ligands, Shh, FGF, BMP, and retinoic acid (RA) with morphogen gradients from surrounding tissues in order to induce the formation of myogenic cells (Figure 2). The canonical Wnt signaling pathway has been shown to play essential roles in the development of myogenesis. In mouse embryogenesis, Wnt1 and Wnt3a secreted from the dorsal neural tube can promote myogenic differentiation of dorsomedial dermomyotome via activation of Myf5 [31, 32, 60]. Wnt3a is able to stabilize -catenin which associates with TCF/LEF transcription factors that bind to the enhancer region of Myf5 during myogenesis [61]. Other Wnt proteins, Wnt6 and Wnt7a, which emerge from the surface ectoderm, induce MyoD [62]. BMP functions as an inhibitor of myogenesis by suppression of some myogenic gene expressions. In the lateral mesoderm, BMP4 is able to increase Pax3 expression which delays Myf5 expression in order to maintain an undifferentiated myogenic progenitor state [63]. Therefore, Wnts and BMPs regulate myogenic development by antagonizing each other for myogenic transcription factor gene expression [64, 65]. Wnt also induces Noggin expression to antagonize BMP signals in the dorsomedial lip of the dermomyotome [66]. In this region, MyoD expression level is increased, which causes myotome formation. Notch signaling plays essential roles for cell-cell communication to specify the different cells in developmental stages. During myotome formation, Notch is expressed in dermomyotome, and Notch1 and Notch2 are expressed in dorsomedial lip of dermomyotome. Delta1, a Notch ligand, is expressed in neural crest cells which transiently interact with myogenic progenitor cells in dorsomedial lip of dermomyotome via Notch1 and 2. This contact induces expression of the Myf5 or MyoD gene in the myogenic progenitor cells followed by myotome formation. The loss of function of Delta1 in the neural crest displays delaying skeletal muscle formation [67]. Knockdown of Notch genes or use of a dominant-negative form of mastermind, a Notch transcriptional coactivator, clearly shows dramatically decrease of Myf5 and MyHC(+) myogenic cells. Interestingly, induction of Notch intracellular domain (NICD), a constitutive active form of Notch, can promote myogenesis, while continuous expression of NICD prevents terminal differentiation. Taken together, transient and timely activation of Notch is crucial for myotome formation from dermomyotome [30].

Current studies for myogenic differentiation of ESCs/iPSCs have utilized supplementation with some growth factors and small molecules, which would mimic the myogenic development described above in combination with embryoid body (EB) aggregation and FACS separation of mesodermal cells (Table 2). To induce paraxial mesoderm cells from mESCs, Sakurai et al. utilized BMP4 in serum-free cultures [68]. Three days after treatment with BMP4, mESCs could be differentiated into primitive streak mesodermal-like cells, but the continuous treatment with BMP4 turned the ESCs into osteogenic cells. Therefore, they used LiCl after treatment with BMP4 to enhance Wnt signaling, which is able to induce myogenic differentiation. After treatment with LiCl, PDGFR(+) E-cadherin() paraxial mesodermal cells were sorted by FACS. These sorted cells were cultured with IGF, HGF, and FGF for two weeks in order to induce myogenic differentiation. Hwang et al. have shown that treatment with Wnt3a efficiently promotes skeletal muscle differentiation of hESCs [69]. hESCs were cultured to form EB for 9 days followed by differentiation of EBs for additional 7 days, and then PDGFR(+) cells were sorted by FACS. These PDGFR(+) cells were cultured with Wnt3a for additional 14 days. Consequently, these Wnt3a-treated cells display significantly increased myogenic transcription factors and structural proteins at both mRNA and protein levels. An interesting approach to identify key molecules that induce myogenic cells was reported by Xu et al. [70]. They utilized reporter systems in zebrafish embryos to display myogenic progenitor cell induction and myogenic differentiation in order to identify small compounds for myogenic induction. Myf5-GFP marks myogenic progenitor cells, while myosin light polypeptide 2 (mylz2)-mCherry marks terminally differentiated muscle cells. They found that a mixed cocktail containing GSK3 inhibitor, bFGF, and forskolin has the potential to induce robust myogenic induction in hiPSCs. GSK3 inhibitors act as a canonical Wnt signaling activator via stabilizing -catenin protein, which is crucial for inducing mesodermal cells. Forskolin activates adenylyl cyclase, which then stimulates cAMP signaling. cAMP response element-binding protein (CREB) is able to stimulate cell proliferation of primary myoblasts in vitro, suggesting that the forskolin-cAMP-CREB pathway may help myogenic cell expansion [71], However the precise mechanisms for CREB-mediated myogenic cell expansion remain unclear. The adenylyl cyclase signaling cascade leads to CREB activation [71]. During embryogenesis, phosphorylated CREB has been found at dorsal somite and dermomyotome. CREB gene knockout mice display significantly decreased Myf5 and MyoD expressions in myotomes. While activation of Wnt1 or Wnt7a promotes Pax3, Myf5, and MyoD expressions, inhibition of CREB eliminates these Wnt-mediated myogenic gene expressions without altering the Wnt canonical pathway, suggesting that CREB-induced myogenic activation may be mediated through noncanonical Wnt pathways. Several groups also utilized GSK3 inhibitors for inducing mesodermal cells from ESCs and iPSCs [72, 73]. These mesodermal cell-like cells were expanded by treatment with bFGF, and then ITS (insulin/transferrin/selenite) or N2 medium were used to induce myogenic differentiation. Finally, bFGF is a stimulator for myogenic cell proliferation. Caron et al. demonstrated that hESCs treated with GSK3 inhibitor, ascorbic acid, Alk5 inhibitor, dexamethasone, EGF, and insulin generated around 80% of Pax3(+) myogenic precursor cells in 10 days [74]. Treatment with SB431542, an inhibitor of Alk4, 5, and 7, PDGF, bFGF, oncostatin, and IGF was able to induce these Pax3(+) myogenic precursor cells into around 5060% of MyoD(+) myoblasts in an additional 8 days. For the final step, treatment with insulin, necrosulfonamide, an inhibitor of necrosis, oncostatin, and ascorbic acid was able to induce these myoblasts into myotubes in an additional 8 days. Importantly, the same authors utilized ESCs from human facioscapulohumeral muscular dystrophy (FSHD) to demonstrate the myogenic characterization after myogenic induction by using the protocol described above. Hosoyama et al. have shown that hESCs/iPSCs with high concentrations of bFGF and EGF in combination with cell aggregation, termed EZ spheres, efficiently give rise to myogenic cells [75]. After 6-week culture, around 4050% of cells expressed Pax7, MyoD, or myogenin. However, the authors also showed that EZ spheres included around 30% of Tuj1(+) neural cells. Therefore, the authors discussed the utilization of molecules for activation of mesodermal and myogenic signaling pathways such as BMPs and Wnts.

Taken together, it is likely that the induced cell populations from ESCs/iPSCs may contain other cell types such as neural cells or cardiac cells because neural cells share similar transcription factor gene expression with myogenic cells such as Pax3, and cardiac cells also develop from mesodermal cells. To overcome this limitation, Chal et al. treated ESCs/iPSCs with BMP4 inhibitor, which prevents ESCs/iPSCs from differentiating into lateral mesodermal cells [76, 77]. To identify what genes are involved in myogenic differentiation in vivo, they performed a microarray analysis which compared samples of dissected fragments in mouse embryos, which are able to separate tail bud, presomitic mesoderm, and somite regions. From microarray data, the authors focused on Mesogenin1 (Msgn1) and Pax3 genes. Importantly, they utilized three lineage tracing reporters, Msgn1-repV (Mesogenin1-Venus) marking posterior somitic mesoderm, Pax3-GFP marking anterior somitic mesoderm and myogenic cells, and Myog-repV (Myogenin-Venus) marking differentiated myocytes, allowing the authors to readily detect different differentiation stages during ESC/iPSC cultures. Treatment with GSK3 inhibitors and then BMP inhibitors in ESC cultures induced Msgn1(+) somitic mesoderm with 45 to 65% efficiencies, Pax3(+) anterior somitic mesoderm with 30 to 50% efficiencies, and myogenin(+) myogenic cells with 25 to 30% efficiencies. Furthermore, the authors examined differentiation of mdx ESCs into skeletal muscle cells and revealed abnormal branching myofibers. Current protocols were also published and described more details for hiPSC differentiation [77].

Some nonmuscle cell populations such as mesoangioblasts have the potential to differentiate into skeletal muscle [6]. Mesoangioblasts were originally isolated from embryonic mouse dorsal aorta as vessel-associated pericyte-like cells, which have the ability to differentiate into a myogenic lineage in vitro and in vivo [6, 78]. Mesoangioblasts possess an advantage for the clinical cell-based treatment because they can be injected through an intra-arterial route to systemically deliver cells, which is crucial for therapeutic cell transplantation for muscular dystrophies [79]. Tedesco et al. successfully generated human iPSC-derived mesoangioblast-like stem/progenitor cells called HIDEMs by stepwise protocols without FACS sorting [80, 81]. They displayed similar gene expression profiles as embryonic mesoangioblasts. However, HIDEMs do not spontaneously differentiate into skeletal muscle cells, and thus, the authors utilized overexpression of MyoD to differentiate into skeletal muscle cells. Similar to mesoangioblasts, HIDEM-derived myogenic cells could be delivered to injured muscle via intramuscular and intra-arterial routes. Furthermore, HIDEMs have been generated from hiPSCs derived from limb-girdle muscular dystrophy (LGMD) type 2D patients and used for gene correction and cell transplantation experiments for the potential therapeutic application.

Myogenic precursor cells derived from ESCs/iPSCs by various methods may contain nonmuscle cells. Therefore, further purification is mandatory for therapeutic applications. Barberi et al. isolated CD73(+) multipotent mesenchymal precursor cells from hESCs by FACS, and these cells underwent differentiation into fat, cartilage, bone, and skeletal muscle cells [82]. Barberi et al. also demonstrated that hESCs cultured on OP9 stroma cells generated around 5% of CD73(+) adult mesenchymal stem cell-like cells [83]. After FACS, these CD73(+) mesenchymal stem cell-like cells were cultured with ITS medium for 4 weeks and then gave rise to NCAM(+) myogenic cells. After FACS sorting, these NCAM(+) myogenic cells were purified by FACS and transplanted into immunodeficient mice to show their myogenic contribution to regenerating muscle.

It has been shown that many genes are associated with myogenesis. In addition, exhaustive analysis, such as microarray, RNA-seq, and single cell RNA-seq supplies much gene information in many different stages. Chal et al. showed key signaling factors by microarray from presomitic somite, somite, and tail bud cells [76]. They found that initial Wnt signaling has important roles for somite differentiation. Furthermore, mapping differentiated hESCs by single cell RNA-seq analysis is useful to characterize each differentiated stage [84].

As shown above, cell sorting of mesodermal progenitor cells, mesenchymal precursor cells, or myogenic cells is a powerful tool to obtain pure myogenic populations from differentiated pluripotent cells. Sakurai et al. have been able to induce PDGFR(+)Flk-1() mesodermal progenitor cells by FACS followed by myogenic differentiation [85]. Chang et al. and Mizuno et al. have been able to sort SMC-2.6(+) myogenic cells from mouse ESCs/iPSCs [86, 87]. These SMC-2.6(+) myogenic cells were successfully engrafted into mouse regenerating skeletal muscle. However, this SMC-2.6 antibody only recognizes mouse myogenic cells but not human myogenic cells [86, 88]. Therefore, Borchin et al. have shown that hiPSC-derived myogenic cells differentiated into c-met(+)CXCR4(+)ACHR(+) cells, displaying that over 95% of sorted cells are Pax7(+) myogenic cells [72]. Taken together, current myogenic induction protocols utilizing small molecules and growth factors, with or without myogenic transcription factors, have been largely improved in the last 5 years. It is crucial to standardize the induction protocols in the near future to obtain sufficient myogenic cell conversion from pluripotent stem cells.

Recent work demonstrated that cells inherit a stable genetic program partly through various epigenetic marks, such as DNA methylation and histone modifications. This cellular memory needs to be erased during genetic reprogramming, and the cellular program reverted to that of an earlier developmental stage [16, 22, 89]. However, iPSCs retaining an epigenetic memory of their origin can readily differentiate into their original tissues [1619, 90100]. This phenomenon becomes a double-edged sword for the reprogramming process since the retention of epigenetic memory may reduce the quality of pluripotency while increasing the differentiation efficiency into their original tissues. DNA methylation levels are relatively low in the pluripotent stem cells compared to the high levels of DNA methylation seen in somatic cells [101]. Global DNA demethylation is required for the reprogramming process [102]. In the context of these observations, recent work demonstrates that activation-induced cytidine deaminase AID/AICDA contributing to the DNA demethylation can stabilize stem-cell phenotypes by removing epigenetic memory of pluripotent genes. This directly deaminates 5-methylcytosine in concert with base-excision repair to exchange cytosine in genomic DNA [103]. MicroRNA-155 has been identified as a key player for the retention of epigenetic memory during in vitro differentiation of hematopoietic progenitor cell-derived iPSCs toward hematopoietic progenitors [104]. iPSCs that maintained high levels of miR-155 expression tend to differentiate into the original somatic population more efficiently.

Recently, we generated murine skeletal muscle cell-derived iPSCs (myoblast-derived iPSCs) [23] and compared the efficiency of differentiation of myogenic progenitor cells between myoblast-derived iPSCs and fibroblast-derived iPSCs. After EB cultures, more satellite cell/myogenic progenitor cell differentiation occurred in myoblast-derived iPSCs than that in fibroblast-derived-iPSCs (unpublished observation and Figure 3), suggesting that myoblast-derived iPSCs are potential myogenic and satellite cell sources for DMD and other muscular dystrophy therapies (Figure 4). We also noticed that MyoD gene suppression by Oct4 is required for reprogramming in myoblasts to produce iPSCs (Figure 3) [23]. During overexpression of Oct4, Oct4 first binds to the Oct4 consensus sequence located in two MyoD enhancers (a core enhancer and distal regulatory region) [105107] preceding occupancy at the promoter in myoblasts in order to suppress MyoD gene expression. Interestingly, Oct4 binding to the MyoD core enhancer allows for establishment of a bivalent state in MyoD promoter as a poised state, marked by active (H3K4me3) and repressive (H3K27me3) modifications in fibroblasts, one of the characteristics of stem cells (Figure 3) [23, 108]. It should be investigated whether the similar bivalent state is also established in Oct4-expressing myoblasts during reprogramming process from myoblasts to pluripotent stem cells. It remains to be elucidated whether Oct4-mediated myogenic repression only relies on repression of MyoD expression or is just a general phenomenon of functional antagonism between Oct4 and MyoD on activation of muscle genes. Nevertheless, myoblast-derived iPSCs will enable us to produce an unlimited number of myogenic cells, including satellite cells that could form the basis of novel treatments for DMD and other muscular dystrophies (Figure 4).

There are pros and cons of transgene-free small molecule-mediated myogenic induction protocols. In the transgene-mediated induction protocols, integration of the transgene in the host genome may lead to risk for insertional mutagenesis. To circumvent this issue, there is an obvious advantage for transgene-free induction protocols. Some key molecules such as Wnt, FGF, and BMP have used signaling pathways to induce myogenic differentiation of ES/iPSCs. However, these molecules are also involved in induction of other types of cell lineages, which makes it difficult for ES/iPSCs to induce pure myogenic cell populations in vitro. By contrast, transgene-mediated myogenic induction is able to dictate desired specific cell lineages. In any case, it is necessary to intensively investigate these myogenic induction protocols for the efficient and safe stem cell therapy for patients.

For skeletal muscle diseases, patient-derived hiPSCs, which possess the ability to differentiate into myogenic progenitor cells followed by myotubes, can be a useful tool for drug screening and personalized medicine in clinical practice. However, there are still limitations for utilizing hiPSC-derived myogenic cells for regenerative medicine. For cell-based transplantation therapies such as a clinical situation, animal-free defined medium is essential for stem cell culture and skeletal muscle cell differentiation. Therefore, such animal-free defined medium needs to be established for optimal myogenic differentiation from hiPSCs. Gene correction in DMD patient iPSCs by TALENs and CRISPR-Cas9 systems are promising therapeutic approaches for stem cell transplantation. However, there are still problems for DNA-editing-mediated stem cell therapy such as safety and efficacy. Since iPSC-derived differentiated myotubes do not proliferate, they are not suited for cell transplantation. Therefore, a proper culture method needs to be established for hiPSCs in order to maintain cells in proliferating the myogenic precursor cell stage in vitro in order to expand cells to large quantities of transplantable cells for DMD and other muscular dystrophies. For other issues, it is essential to establish methods to separate ES/iPSC-derived pure skeletal muscle precursor cells from other cell types for safe stem cell therapy that excludes tumorigenic risks of contamination with undifferentiated cells. In the near future, these obstacles will be taken away for more efficient and safe stem cell therapy for DMD and other muscular dystrophies.

The authors declare that they have no conflicts of interest.

This work was supported by the NIH R01 (1R01AR062142) and NIH R21 (1R21AR070319). The authors thank Conor Burke-Smith and Neeladri Chowdhury for critical reading.

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Skeletal Muscle Cell Induction from Pluripotent Stem Cells

Cardiac Stem Cells Comments Off on Skeletal Muscle Cell Induction from Pluripotent Stem Cells | December 1st, 2022

Specific location in the body containing stem cells

Stem-cell niche refers to a microenvironment, within the specific anatomic location where stem cells are found, which interacts with stem cells to regulate cell fate.[1] The word 'niche' can be in reference to the in vivo or in vitro stem-cell microenvironment. During embryonic development, various niche factors act on embryonic stem cells to alter gene expression, and induce their proliferation or differentiation for the development of the fetus. Within the human body, stem-cell niches maintain adult stem cells in a quiescent state, but after tissue injury, the surrounding micro-environment actively signals to stem cells to promote either self-renewal or differentiation to form new tissues. Several factors are important to regulate stem-cell characteristics within the niche: cellcell interactions between stem cells, as well as interactions between stem cells and neighbouring differentiated cells, interactions between stem cells and adhesion molecules, extracellular matrix components, the oxygen tension, growth factors, cytokines, and the physicochemical nature of the environment including the pH, ionic strength (e.g. Ca2+ concentration) and metabolites, like ATP, are also important.[2] The stem cells and niche may induce each other during development and reciprocally signal to maintain each other during adulthood.

Scientists are studying the various components of the niche and trying to replicate the in vivo niche conditions in vitro.[2] This is because for regenerative therapies, cell proliferation and differentiation must be controlled in flasks or plates, so that sufficient quantity of the proper cell type are produced prior to being introduced back into the patient for therapy.

Human embryonic stem cells are often grown in fibroblastic growth factor-2 containing, fetal bovine serum supplemented media. They are grown on a feeder layer of cells, which is believed to be supportive in maintaining the pluripotent characteristics of embryonic stem cells. However, even these conditions may not truly mimic in vivo niche conditions.

Adult stem cells remain in an undifferentiated state throughout adult life. However, when they are cultured in vitro, they often undergo an 'aging' process in which their morphology is changed and their proliferative capacity is decreased. It is believed that correct culturing conditions of adult stem cells needs to be improved so that adult stem cells can maintain their stemness over time.[citation needed]

A Nature Insight review defines niche as follows:

"Stem-cell populations are established in 'niches' specific anatomic locations that regulate how they participate in tissue generation, maintenance and repair. The niche saves stem cells from depletion, while protecting the host from over-exuberant stem-cell proliferation. It constitutes a basic unit of tissue physiology, integrating signals that mediate the balanced response of stem cells to the needs of organisms. Yet the niche may also induce pathologies by imposing aberrant function on stem cells or other targets. The interplay between stem cells and their niche creates the dynamic system necessary for sustaining tissues, and for the ultimate design of stem-cell therapeutics ... The simple location of stem cells is not sufficient to define a niche. The niche must have both anatomic and functional dimensions."[3]

Though the concept of stem cell niche was prevailing in vertebrates, the first characterization of stem cell niche in vivo was worked out in Drosophila germinal development.

By continuous intravital imaging in mice, researchers were able to explore the structure of the stem cell niche and to obtain the fate of individual stem cells (SCs) and their progeny over time in vivo. In particular in intestinal crypt,[4] two distinct groups of SCs have been identified: the "border stem cells" located in the upper part of the niche at the interface with transit amplifying cells (TAs), and "central stem cells" located at the crypt base. The proliferative potential of the two groups was unequal and correlated with the cells' location (central or border). It was also shown that the two SC compartments acted in accord to maintain a constant cell population and a steady cellular turnover. A similar dependence of self-renewal potential on proximity to the niche border was reported in the context of hair follicle, in an in vivo live-imaging study.[5]

This bi-compartmental structure of stem cell niche has been mathematically modeled to obtain the optimal architecture that leads to the maximum delay in double-hit mutant production.[6] They found that the bi-compartmental SC architecture minimizes the rate of two-hit mutant production compared to the single SC compartment model. Moreover, the minimum probability of double-hit mutant generation corresponds to purely symmetric division of SCs with a large proliferation rate of border stem cells along with a small, but non-zero, proliferation rate of central stem cells.[citation needed]

Stem cell niches harboring continuously dividing cells, such as those located at the base of the intestinal gland, are maintained at small population size. This presents a challenge to the maintenance of multicellular tissues, as small populations of asexually dividing individuals will accumulate deleterious mutations through genetic drift and succumb to mutational meltdown.[7] Mathematical modeling of the intestinal gland reveals that the small population size within the stem cell niche minimizes the probability of carcinogenesis occurring anywhere, at the expense of gradually accumulated deleterious mutations throughout organismal lifetimea process that contributes to tissue degradation and aging.[8] Therefore, the population size of the stem cell niche represents an evolutionary trade-off between the probability of cancer formation and the rate of aging.

Germline stem cells (GSCs) are found in organisms that continuously produce sperm and eggs until they are sterile. These specialized stem cells reside in the GSC niche, the initial site for gamete production, which is composed of the GSCs, somatic stem cells, and other somatic cells. In particular, the GSC niche is well studied in the genetic model organism Drosophila melanogaster and has provided an extensive understanding of the molecular basis of stem cell regulation.[citation needed]

In Drosophila melanogaster, the GSC niche resides in the anterior-most region of each ovariole, known as the germarium. The GSC niche consists of necessary somatic cells-terminal filament cells, cap cells, escort cells, and other stem cells which function to maintain the GSCs.[9] The GSC niche holds on average 23 GSCs, which are directly attached to somatic cap cells and Escort stem cells, which send maintenance signals directly to the GSCs.[10] GSCs are easily identified through histological staining against vasa protein (to identify germ cells) and 1B1 protein (to outline cell structures and a germline specific fusome structure). Their physical attachment to the cap cells is necessary for their maintenance and activity.[10] A GSC will divide asymmetrically to produce one daughter cystoblast, which then undergoes 4 rounds of incomplete mitosis as it progresses down the ovariole (through the process of oogenesis) eventually emerging as a mature egg chamber; the fusome found in the GSCs functions in cyst formation and may regulate asymmetrical cell divisions of the GSCs.[11] Because of the abundant genetic tools available for use in Drosophila melanogaster and the ease of detecting GSCs through histological stainings, researchers have uncovered several molecular pathways controlling GSC maintenance and activity.[12] [13]

The Bone Morphogenetic Protein (BMP) ligands Decapentaplegic (Dpp) and Glass-bottom-boat (Gbb) ligand are directly signalled to the GSCs, and are essential for GSC maintenance and self-renewal.[14] BMP signalling in the niche functions to directly repress expression of Bag-of-marbles (Bam) in GSCs, which is up-regulated in developing cystoblast cells.[15] Loss of function of dpp in the niche results in de-repression of Bam in GSCs, resulting in rapid differentiation of the GSCs.[10] Along with BMP signalling, cap cells also signal other molecules to GSCs: Yb and Piwi. Both of these molecules are required non-autonomously to the GSCs for proliferation-piwi is also required autonomously in the GSCs for proliferation.[16] In the germarium, BMP signaling has a short-range effect, therefore the physical attachment of GSCs to cap cells is important for maintenance and activity.[citation needed]

The GSCs are physically attached to the cap cells by Drosophila E-cadherin (DE-cadherin) adherens junctions and if this physical attachment is lost GSCs will differentiate and lose their identity as a stem cell.[10] The gene encoding DE-cadherin, shotgun (shg), and a gene encoding Beta-catenin ortholog, armadillo, control this physical attachment.[17] A GTPase molecule, rab11, is involved in cell trafficking of DE-cadherins. Knocking out rab11 in GSCs results in detachment of GSCs from the cap cells and premature differentiation of GSCs.[18] Additionally, zero population growth (zpg), encoding a germline-specific gap junction is required for germ cell differentiation.[19]

Both diet and insulin-like signaling directly control GSC proliferation in Drosophila melanogaster. Increasing levels of Drosophila insulin-like peptide (DILP) through diet results in increased GSC proliferation.[20] Up-regulation of DILPs in aged GSCs and their niche results in increased maintenance and proliferation.[21] It has also been shown that DILPs regulate cap cell quantities and regulate the physical attachment of GSCs to cap cells.[21]

There are two possible mechanisms for stem cell renewal, symmetrical GSC division or de-differentiation of cystoblasts. Normally, GSCs will divide asymmetrically to produce one daughter cystoblast, but it has been proposed that symmetrical division could result in the two daughter cells remaining GSCs.[22][23] If GSCs are ablated to create an empty niche and the cap cells are still present and sending maintenance signals, differentiated cystoblasts can be recruited to the niche and de-differentiate into functional GSCs.[24]

As the Drosophila female ages, the stem cell niche undergoes age-dependent loss of GSC presence and activity. These losses are thought to be caused in part by degradation of the important signaling factors from the niche that maintains GSCs and their activity. Progressive decline in GSC activity contributes to the observed reduction in fecundity of Drosophila melanogaster at old age; this decline in GSC activity can be partially attributed to a reduction of signaling pathway activity in the GSC niche.[25][26] It has been found that there is a reduction in Dpp and Gbb signaling through aging. In addition to a reduction in niche signaling pathway activity, GSCs age cell-autonomously. In addition to studying the decline of signals coming from the niche, GSCs age intrinsically; there is age-dependent reduction of adhesion of GSCs to the cap cells and there is accumulation of Reactive Oxygen species (ROS) resulting in cellular damage which contributes to GSC aging. There is an observed reduction in the number of cap cells and the physical attachment of GSCs to cap cells through aging. Shg is expressed at significantly lower levels in an old GSC niche in comparison to a young one.[26]

Males of Drosophila melanogaster each have two testes long, tubular, coiled structures and at the anterior most tip of each lies the GSC niche. The testis GSC niche is built around a population of non-mitotic hub cells (a.k.a. niche cells), to which two populations of stem cells adhere: the GSCs and the somatic stem cells (SSCs, a.k.a. somatic cyst stem cells/cyst stem cells). Each GSC is enclosed by a pair of SSCs, though each stem cell type is still in contact with the hub cells. In this way, the stem cell niche consists of these three cell types, as not only do the hub cells regulate GSC and SSC behaviour, but the stem cells also regulate the activity of each other. The Drosophila testis GSC niche has proven a valuable model system for examining a wide range of cellular processes and signalling pathways.[27]

The process of spermatogenesis begins when the GSCs divide asymmetrically, producing a GSC that maintains hub contact, and a gonialblast that exits the niche. The SSCs divide with their GSC partner, and their non-mitotic progeny, the somatic cyst cells (SCCs, a.k.a. cyst cells) will enclose the gonialblast. The gonialblast then undergoes four rounds of synchronous, transit-amplifying divisions with incomplete cytokinesis to produce a sixteen-cell spermatogonial cyst. This spermatogonial cyst then differentiates and grows into a spermatocyte, which will eventually undergo meiosis and produce sperm.[27]

The two main molecular signalling pathways regulating stem cell behaviour in the testis GSC niche are the Jak-STAT and BMP signalling pathways. Jak-STAT signalling originates in the hub cells, where the ligand Upd is secreted to the GSCs and SSCs.[28][29] This leads to activation of the Drosophila STAT, Stat92E, a transcription factor which effects GSC adhesion to the hub cells,[30] and SSC self-renewal via Zfh-1.[31] Jak-STAT signalling also influences the activation of BMP signalling, via the ligands Dpp and Gbb. These ligands are secreted into the GSCs from the SSCs and hub cells, activate BMP signalling, and suppress the expression of Bam, a differentiation factor.[32] Outside of the niche, gonialblasts no longer receive BMP ligands, and are free to begin their differentiation program. Other important signalling pathways include the MAPK and Hedgehog, which regulate germline enclosure [33] and somatic cell self-renewal,[34] respectively.

The murine GSC niche in males, also called spermatogonial stem cell (SSC) niche, is located in the basal region of seminiferous tubules in the testes. The seminiferous epithelium is composed of sertoli cells that are in contact with the basement membrane of the tubules, which separates the sertoli cells from the interstitial tissue below. This interstitial tissue comprises Leydig cells, macrophages, mesenchymal cells, capillary networks, and nerves.[35]

During development, primordial germ cells migrate into the seminiferous tubules and downward towards the basement membrane whilst remaining attached to the sertoli cells where they will subsequently differentiate into SSCs, also referred to as Asingle spermatogonia.[35][36] These SSCs can either self-renew or commit to differentiating into spermatozoa upon the proliferation of Asingle into Apaired spermatogonia. The 2 cells of Apaired spermatogonia remain attached by intercellular bridges and subsequently divide into Aaligned spermatogonia, which is made up of 416 connected cells. Aaligned spermatogonia then undergo meiosis I to form spermatocytes and meiosis II to form spermatids which will mature into spermatozoa.[37][38] This differentiation occurs along the longitudinal axis of sertoli cells, from the basement membrane to the apical lumen of the seminiferous tubules. However, sertoli cells form tight junctions that separate SSCs and spermatogonia in contact with the basement membrane from the spermatocytes and spermatids to create a basal and an adluminal compartment, whereby differentiating spermatocytes must traverse the tight junctions.[35][39] These tight junctions form the blood testis barrier (BTB) and have been suggested to play a role in isolating differentiated cells in the adluminal compartment from secreted factors by the interstitial tissue and vasculature neighboring the basal compartment.[35]

The basement membrane of the seminiferous tubule is a modified form of extracellular matrix composed of fibronectin, collagens, and laminin.[35] 1- integrin is expressed on the surface of SSCs and is involved in their adhesion to the laminin component of the basement membrane although other adhesion molecules are likely also implicated in the attachment of SSCs to the basement membrane.[40] E cadherin expression on SSCs in mice, unlike in Drosophila, have been shown to be dispensable as the transplantation of cultured SSCs lacking E-cadherin are able to colonize host seminiferous tubules and undergo spermatogenesis.[41] In addition the blood testis barrier provides architectural support and is composed of tight junction components such as occludins, claudins and zonula occludens (ZOs) which show dynamic expression during spermatogenesis.[42] For example, claudin 11 has been shown to be a necessary component of these tight junctions as mice lacking this gene have a defective blood testis barrier and do not produce mature spermatozoa.[40]

GDNF (Glial cell-derived neurotrophic factor) is known to stimulate self-renewal of SSCs and is secreted by the sertoli cells under the influence of gonadotropin FSH. GDNF is a related member of the TGF superfamily of growth factors and when overexpressed in mice, an increase in undifferentiated spermatogonia was observed which led to the formation of germ tumours.[35][40] In corroboration for its role as a renewal factor, heterozygous knockout male mice for GDNF show decreased spermatogenesis that eventually leads to infertility.[40] In addition the supplementation of GDNF has been shown to extend the expansion of mouse SSCs in culture. However, the GDNF receptor c-RET and co-receptor GFRa1 are not solely expressed on the SSCs but also on Apaired and Aaligned, therefore showing that GDNF is a renewal factor for Asingle to Aaligned in general rather than being specific to the Asingle SSC population. FGF2 (Fibroblast growth factor 2), secreted by sertoli cells, has also been shown to influence the renewal of SSCs and undifferentiated spermatogonia in a similar manner to GDNF.[35]

Although sertoli cells appear to play a major role in renewal, it expresses receptors for testosterone that is secreted by Leydig cells whereas germ cells do not contain this receptor- thus alluding to an important role of Leydig cells upstream in mediating renewal. Leydig cells also produce CSF 1 (Colony stimulating factor 1) for which SSCs strongly express the receptor CSF1R.[37] When CSF 1 was added in culture with GDNF and FGF2 no further increase in proliferation was observed, however, the longer the germ cells remained in culture with CSF-1 the greater the SSC density observed when these germ cells were transplanted into host seminiferous tubules. This showed CSF 1 to be a specific renewal factor that tilts the SSCs towards renewal over differentiation, rather than affecting proliferation of SSCs and spermatogonia. GDNF, FGF 2 and CSF 1 have also been shown to influence self-renewal of stem cells in other mammalian tissues.[35]

Plzf (Promyelocytic leukaemia zinc finger) has also been implicated in regulating SSC self-renewal and is expressed by Asingle, Apaired and Aaligned spermatogonia. Plzf directly inhibits the transcription of a receptor, c-kit, in these early spermatogonia. However, its absence in late spermatogonia permits c-kit expression, which is subsequently activated by its ligand SCF (stem cell factor) secreted by sertoli cells, resulting in further differentiation. Also, the addition of BMP4 and Activin-A have shown to reduce self-renewal of SSCs in culture and increase stem cell differentiation, with BMP4 shown to increase the expression of c-kit.[37]

Prolonged spermatogenesis relies on the maintenance of SSCs, however, this maintenance declines with age and leads to infertility. Mice between 12 and 14 months of age show decreased testis weight, reduced spermatogenesis and SSC content. Although stem cells are regarded as having the potential to infinitely replicate in vitro, factors provided by the niche are crucial in vivo. Indeed, serial transplantation of SSCs from male mice of different ages into young mice 3 months of age, whose endogenous spermatogenesis had been ablated, was used to estimate stem cell content given that each stem cell would generate a colony of spermatogenesis.[35][43] The results of this experiment showed that transplanted SSCs could be maintained far longer than their replicative lifespan for their age. In addition, a study also showed that SSCs from young fertile mice could not be maintained nor undergo spermatogenesis when transplanted into testes of old, infertile mice. Together, these results points towards a deterioration of the SSC niche itself with aging rather than the loss of intrinsic factors in the SSC.[43]

Vertebrate hematopoietic stem cells niche in the bone marrow is formed by cells subendosteal osteoblasts, sinusoidal endothelial cells and bone marrow stromal (also sometimes called reticular) cells which includes a mix of fibroblastoid, monocytic and adipocytic cells (which comprise marrow adipose tissue).[1]

The hair follicle stem cell niche is one of the more closely studied niches thanks to its relative accessibility and role in important diseases such as melanoma. The bulge area at the junction of arrector pili muscle to the hair follicle sheath has been shown to host the skin stem cells which can contribute to all epithelial skin layers. There cells are maintained by signaling in concert with niche cells signals include paracrine (e.g. sonic hedgehog), autocrine and juxtacrine signals.[44] The bulge region of the hair follicle relies on these signals to maintain the stemness of the cells. Fate mapping or cell lineage tracing has shown that Keratin 15 positive stem cells' progeny participate in all epithelial lineages.[45] The follicle undergoes cyclic regeneration in which these stem cells migrate to various regions and differentiate into the appropriate epithelial cell type. Some important signals in the hair follicle stem cell niche produced by the mesenchymal dermal papilla or the bulge include BMP, TGF- and Fibroblast growth factor (FGF) ligands and Wnt inhibitors.[46] While, Wnt signaling pathways and -catenin are important for stem cell maintenance,[47] over-expression of -catenin in hair follicles induces improper hair growth. Therefore, these signals such as Wnt inhibitors produced by surrounding cells are important to maintain and facilitate the stem cell niche.[48]

Intestinal organoids have been used to study intestinal stem cell niches. An intestinal organoid culture can be used to indirectly assess the effect of the manipulation on the stem cells through assessing the organoid's survival and growth. Research using intestinal organoids have demonstrated that the survival of intestinal stem cells is improved by the presence of neurons and fibroblasts,[49] and through the administration of IL-22.[50]

Cardiovascular stem cell niches can be found within the right ventricular free wall, atria and outflow tracks of the heart. They are composed of Isl1+/Flk1+ cardiac progenitor cells (CPCs) that are localized into discrete clusters within a ColIV and laminin extracellular matrix (ECM). ColI and fibronectin are predominantly found outside the CPC clusters within the myocardium. Immunohistochemical staining has been used to demonstrate that differentiating CPCs, which migrate away from the progenitor clusters and into the ColI and fibronectin ECM surrounding the niche, down-regulate Isl1 while up-regulating mature cardiac markers such as troponin C.[51] There is a current controversy over the role of Isl1+ cells in the cardiovascular system. While major publications have identified these cells as CPC's and have found a very large number in the murine and human heart, recent publications have found very few Isl1+ cells in the murine fetal heart and attribute their localization to the sinoatrial node,[52] which is known as an area that contributes to heart pacemaking. The role of these cells and their niche are under intense research and debate.[citation needed]

Neural stem cell niches are divided in two: the Subependymal zone (SEZ) and the Subgranular zone (SGZ).

The SEZ is a thin area beneath the ependymal cell layer that contains three types of neural stem cells: infrequently dividing neural stem cells (NSCs), rapidly dividing transit amplifying precursors (TaPs) and neuroblasts (NBs). The SEZ extracellular matrix (ECM) has significant differences in composition compared to surrounding tissues. Recently, it was described that progenitor cells, NSCs, TaPs and NBs were attached to ECM structures called Fractones.[53] These structures are rich in laminin, collagen and heparan sulfate proteoglycans.[54] Other ECM molecules, such as tenascin-C, MMPs and different proteoglycans are also implicated in the neural stem cell niche.[55]

Cancer tissue is morphologically heterogenous, not only due to the variety of cell types present, endothelial, fibroblast and various immune cells, but cancer cells themselves are not a homogenous population either.[citation needed]

In accordance with the hierarchy model of tumours, the cancer stem cells (CSC) are maintained by biochemical and physical contextual signals emanating from the microenvironment, called the cancer stem cell niche.[56] The CSC niche is very similar to normal stem cells niche (embryonic stem cell (ESC), Adult Stem Cell ASC) in function (maintaining of self-renewal, undifferentiated state and ability to differentiate) and in signalling pathways (Activin/Noda, Akt/PTEN, JAK/STAT, PI3-K, TGF-, Wnt and BMP).[57] It is hypothesized that CSCs arise form aberrant signalling of the microenvironment and participates not only in providing survival signals to CSCs but also in metastasis by induction of epithelial-mesenchymal transition (EMT).[citation needed]

Hypoxic condition in stem cell niches (ESC, ASC or CSC) is necessary for maintaining stem cells in an undifferentiated state and also for minimizing DNA damage via oxidation. The maintaining of the hypoxic state is under control of Hypoxia-Inducible transcription Factors (HIFs).[58] HIFs contribute to tumour progression, cell survival and metastasis by regulation of target genes as VEGF, GLUT-1, ADAM-1, Oct4 and Notch.[57]

Hypoxia plays an important role in the regulation of cancer stem cell niches and EMT through the promotion of HIFs.[59] These HIFs help maintain cancer stem cell niches by regulating important stemness genes such as Oct4, Nanog, SOX2, Klf4, and cMyc.[60][61] HIFs also regulate important tumor suppressor genes such as p53 and genes that promote metastasis.[62][63] Although HIFs increase the survival of cells by decreasing the effects of oxidative stress, they have also been shown to decrease factors such as RAD51 and H2AX that maintain genomic stability.[64] In the hypoxic condition there is an increase of intracellular Reactive Oxygen Species (ROS) which also promote CSCs survival via stress response.[65][66] ROS stabilizes HIF-1 which promotes the Met proto-oncogene, which drives metastasis or motogenic escape in melanoma cells.[67] All of these factors contribute to a cancer stem cell phenotype which is why it is often referred to as a hypoxic stem cell niche. Hypoxic environments are often found in tumors where the cells are dividing faster that angiogenesis can occur. It is important to study hypoxia as an aspect of cancer because hypoxic environments have been shown to be resistant to radiation therapy.[68] Radiation has been shown to increase the amounts of HIF-1.[69] EMT induction by hypoxia though interactions between HIF-1 and ROS is crucial for metastasis in cancers such as melanoma. It has been found that many genes associated with melanoma are regulated by hypoxia such as MXI1, FN1, and NME1.[70]

Epithelialmesenchymal transition is a morphogenetic process, normally occurs in embryogenesis that is "hijacked" by cancer stem cells by detaching from their primary place and migrating to another one. The dissemination is followed by reverse transition so-called Epithelial-Mesenchymal Transition (EMT). This process is regulated by CSCs microenvironment via the same signalling pathways as in embryogenesis using the growth factors (TGF-, PDGF, EGF), cytokine IL-8 and extracellular matrix components. These growth factors' interactions through intracellular signal transducers like -catenin has been shown to induce metastatic potential.[71][72] A characteristic of EMT is loss of the epithelial markers (E-cadherin, cytokeratins, claudin, occluding, desmoglein, desmocolin) and gain of mesenchymal markers (N-cadherin, vimentin, fibronectin).[73]

There is also certain degree of similarity in homing-mobilization of normal stem cells and metastasis-invasion of cancer stem cells. There is an important role of Matrix MetalloProteinases (MMP), the principal extracellular matrix degrading enzymes, thus for example matrix metalloproteinase-2 and 9 are induced to expression and secretion by stromal cells during metastasis of colon cancer via direct contact or paracrine regulation. The next sharing molecule is Stromal cell-Derived Factor-1 (SDF-1).[73][74]

The EMT and cancer progression can be triggered also by chronic inflammation. The main roles have molecules (IL-6, IL-8, TNF-, NFB, TGF-, HIF-1) which can regulate both processes through regulation of downstream signalling that overlapping between EMT and inflammation.[57] The downstream pathways involving in regulation of CSCs are Wnt, SHH, Notch, TGF-, RTKs-EGF, FGF, IGF, HGF.

NFB regulates the EMT, migration and invasion of CSCs through Slug, Snail and Twist. The activation of NFB leads to increase not only in production of IL-6, TNF- and SDF-1 but also in delivery of growth factors.

The source of the cytokine production are lymphocytes (TNF-), Mesenchymal Stem Cells (SDF-1, IL-6, IL8).

Interleukin 6 mediates activation of STAT3. The high level of STAT3 was described in isolated CSCs from liver, bone, cervical and brain cancer. The inhibition of STAT3 results in dramatic reduction in their formation. Generally IL-6 contributes a survival advantage to local stem cells and thus facilitates tumorigenesis.[57]

SDF-1 secreted from Mesenchymal Stem Cells (MSCs) has important role in homing and maintenance of Hematopoietic Stem Cell (HSC) in bone marrow niche but also in homing and dissemination of CSC.[74]

Hypoxia is a main stimulant for angiogenesis, with HIF-1 being the primary mediator. Angiogenesis induced by hypoxic conditions is called an "Angiogenic switch". HIF-1 promotes expression of several angiogenic factors: Vascular Endothelial Growth Factor (VEGF), basic Fibroblast Growth Factor (bFGF), Placenta-Like Growth Factor (PLGF), Platelet-Derived Growth Factor (PDGF) and Epidermal Growth Factor. But there is evidence that the expression of angiogenic agens by cancer cells can also be HIF-1 independent. It seems that there is an important role of Ras protein, and that intracellular levels of calcium regulate the expression of angiogenic genes in response to hypoxia.[73]

The angiogenic switch downregulates angiogenesis suppressor proteins, such as thrombospondin, angiostatin, endostatin and tumstatin. Angiogenesis is necessary for the primary tumour growth.[citation needed]

During injury, support cells are able to activate a program for repair, recapitulating aspects of development in the area of damage. These areas become permissive for stem cell renewal, migration and differentiation. For instance in the CNS, injury is able to activate a developmental program in astrocytes that allow them to express molecules that support stem cells such as chemokines i.e. SDF-1[75] and morphogens such as sonic hedgehog.[76]

It is evident that biophysio-chemical characteristics of ECM such as composition, shape, topography, stiffness, and mechanical strength can control the stem cell behavior. These ECM factors are equally important when stem cells are grown in vitro. Given a choice between niche cell-stem cell interaction and ECM-stem cell interaction, mimicking ECM is preferred as that can be precisely controlled by scaffold fabrication techniques, processing parameters or post-fabrication modifications. In order to mimic, it is essential to understand natural properties of ECM and their role in stem cell fate processes. Various studies involving different types of scaffolds that regulate stem cells fate by mimicking these ECM properties have been done.[2])

[77]

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Stem-cell niche - Wikipedia

Cardiac Stem Cells Comments Off on Stem-cell niche – Wikipedia | December 1st, 2022

A protein that helps make neurons also works to reprogram scar tissue cells into heart muscle cells, especially in partnership with a second protein, according to a study led by Li Qian, PhD, at the UNC School of Medicine.

CHAPEL HILL, N.C. Scientists at the UNC School of Medicine have made a significant advance in the promising field of cellular reprogramming and organ regeneration, and the discovery could play a major role in future medicines to heal damaged hearts.

In a study published in the journal Cell Stem Cell, scientists at the University of North Carolina at Chapel Hill discovered a more streamlined and efficient method for reprogramming scar tissue cells (fibroblasts) to become healthy heart muscle cells (cardiomyocytes). Fibroblasts produce the fibrous, stiff tissue that contributes to heart failure after a heart attack or because of heart disease. Turning fibroblasts into cardiomyocytes is being investigated as a potential future strategy for treating or even someday curing this common and deadly condition.

Surprisingly, the key to the new cardiomyocyte-making technique turned out to be a gene activity-controlling protein called Ascl1, which is known to be a crucial protein involved in turning fibroblasts into neurons. Researchers had thought Ascl1 was neuron-specific.

Its an outside-the-box finding, and we expect it to be useful in developing future cardiac therapies and potentially other kinds of therapeutic cellular reprogramming, said study senior author Li Qian, PhD, associate professor in the UNC Department of Pathology and Lab Medicine and associate director of the McAllister Heart Institute at UNC School of Medicine.

Scientists over the last 15 years have developed various techniques to reprogram adult cells to become stem cells, then to induce those stem cells to become adult cells of some other type. More recently, scientists have been finding ways to do this reprogramming more directly straight from one mature cell type to another. The hope has been that when these methods are made maximally safe, effective, and efficient, doctors will be able to use a simple injection into patients to reprogram harm-causing cells into beneficial ones.

Reprogramming fibroblasts has long been one of the important goals in the field, Qian said. Fibroblast over-activity underlies many major diseases and conditions including heart failure, chronic obstructive pulmonary disease, liver disease, kidney disease, and the scar-like brain damage that occurs after strokes.

In the new study, Qians team, including co-first-authors Haofei Wang, PhD, a postdoctoral researcher, and MD/PhD student Benjamin Keepers, used three existing techniques to reprogram mouse fibroblasts into cardiomyocytes, liver cells, and neurons. Their aim was to catalogue and compare the changes in cells gene activity patterns and gene-activity regulation factors during these three distinct reprogrammings.

Unexpectedly, the researchers found that the reprogramming of fibroblasts into neurons activated a set of cardiomyocyte genes. Soon they determined that this activation was due to Ascl1, one of the master-programmer transcription factor proteins that had been used to make the neurons.

Since Ascl1 activated cardiomyocyte genes, the researchers added it to the three-transcription-factor cocktail they had been using for making cardiomyocytes, to see what would happen. They were astonished to find that it dramatically increased the efficiency of reprogramming the proportion of successfully reprogrammed cells by more than ten times. In fact, they found that they could now dispense with two of the three factors from their original cocktail, retaining only Ascl1 and another transcription factor called Mef2c.

In further experiments they found evidence that Ascl1 on its own activates both neuron and cardiomyocyte genes, but it shifts away from the pro-neuron role when accompanied by Mef2c. In synergy with Mef2c, Ascl1 switches on a broad set of cardiomyocyte genes.

Ascl1 and Mef2c work together to exert pro-cardiomyocyte effects that neither factor alone exerts, making for a potent reprogramming cocktail, Qian said.

The results show that the major transcription factors used in direct cellular reprogramming arent necessarily exclusive to one targeted cell type.

Perhaps more importantly, they represent another step on the path towards future cell-reprogramming therapies for major disorders. Qian says that she and her team hope to make a two-in-one synthetic protein that contains the effective bits of both Ascl1 and Mef2c, and could be injected into failing hearts to mend them.

Cross-lineage Potential of Ascl1 Uncovered by Comparing Diverse Reprogramming Regulatomes was co-authored by Haofei Wang, Benjamin Keepers, Yunzhe Qian, Yifang Xie, Marazzano Colon, Jiandong Liu, and Li Qian. Funding was provided by the American Heart Association and the National Institutes of Health (T32HL069768, F30HL154659, R35HL155656, R01HL139976, R01HL139880).

Media contact: Mark Derewicz, 919-923-0959

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Scientists Discover Protein Partners that Could Heal Heart Muscle | Newsroom - UNC Health and UNC School of Medicine

Cardiac Stem Cells Comments Off on Scientists Discover Protein Partners that Could Heal Heart Muscle | Newsroom – UNC Health and UNC School of Medicine | October 13th, 2022

ReportLinker

Abstract: Whats New for 2022?? Global competitiveness and key competitor percentage market shares. Market presence across multiple geographies - Strong/Active/Niche/Trivial.

New York, Oct. 10, 2022 (GLOBE NEWSWIRE) -- Reportlinker.com announces the release of the report "Global Induced Pluripotent Stem Cell (iPSC) Industry" - https://www.reportlinker.com/p05798831/?utm_source=GNW

Online interactive peer-to-peer collaborative bespoke updates

Access to our digital archives and MarketGlass Research Platform

Complimentary updates for one yearGlobal Induced Pluripotent Stem Cell ((iPSC) Market to Reach $0 Thousand by 2027- In the changed post COVID-19 business landscape, the global market for Induced Pluripotent Stem Cell ((iPSC) estimated at US$1.4 Billion in the year 2020, is projected to reach a revised size of US$0 Thousand by 2027, growing at a CAGR of -100% over the analysis period 2020-2027. Vascular Cells, one of the segments analyzed in the report, is projected to record a -100% CAGR and reach US$0 Thousand by the end of the analysis period. Taking into account the ongoing post pandemic recovery, growth in the Cardiac Cells segment is readjusted to a revised -100% CAGR for the next 7-year period.- The U.S. Market is Estimated at $629.2 Million, While China is Forecast to Grow at -100% CAGR- The Induced Pluripotent Stem Cell ((iPSC) market in the U.S. is estimated at US$629.2 Million in the year 2020. China, the world`s second largest economy, is forecast to reach a projected market size of US$0 Thousand by the year 2027 trailing a CAGR of -100% over the analysis period 2020 to 2027. Among the other noteworthy geographic markets are Japan and Canada, each forecast to grow at -100% and -100% respectively over the 2020-2027 period. Within Europe, Germany is forecast to grow at approximately -100% CAGR.Neuronal Cells Segment to Record -100% CAGR- In the global Neuronal Cells segment, USA, Canada, Japan, China and Europe will drive the -100% CAGR estimated for this segment. These regional markets accounting for a combined market size of US$188.9 Million in the year 2020 will reach a projected size of US$0 Thousand by the close of the analysis period. China will remain among the fastest growing in this cluster of regional markets.

Select Competitors (Total 51 Featured)Axol Bioscience Ltd.Cynata Therapeutics LimitedEvotec SEFate Therapeutics, Inc.FUJIFILM Cellular Dynamics, Inc.NcardiaPluricell BiotechREPROCELL USA, Inc.Sumitomo Dainippon Pharma Co., Ltd.Takara Bio, Inc.Thermo Fisher Scientific, Inc.ViaCyte, Inc.

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

I. METHODOLOGY

II. EXECUTIVE SUMMARY

1. MARKET OVERVIEWInfluencer Market InsightsImpact of Covid-19 and a Looming Global RecessionInduced Pluripotent Stem Cells (iPSCs) Market Gains fromIncreasing Use in Research for COVID-19Studies Employing iPSCs in COVID-19 ResearchStem Cells, Application Areas, and the Different Types: A PreludeApplications of Stem CellsTypes of Stem CellsInduced Pluripotent Stem Cell (iPSC): An IntroductionProduction of iPSCsFirst & Second Generation Mouse iPSCsHuman iPSCsKey Properties of iPSCsTranscription Factors Involved in Generation of iPSCsNoteworthy Research & Application Areas for iPSCsInduced Pluripotent Stem Cell ((iPSC) Market: Growth Prospectsand OutlookDrug Development Application to Witness Considerable GrowthTechnical Breakthroughs, Advances & Clinical Trials to SpurGrowth of iPSC MarketNorth America Dominates Global iPSC MarketCompetitionRecent Market ActivitySelect Innovation/AdvancementInduced Pluripotent Stem Cell (iPSC) - Global Key CompetitorsPercentage Market Share in 2022 (E)Competitive Market Presence - Strong/Active/Niche/Trivial forPlayers Worldwide in 2022 (E)

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

3. MARKET TRENDS & DRIVERSEffective Research Programs Hold Key in Roll Out of AdvancediPSC TreatmentsInduced Pluripotent Stem Cells: A Giant Leap in the TherapeuticApplicationsResearch Trends in Induced Pluripotent Stem Cell SpaceWorldwide Publication of hESC and hiPSC Research Papers for thePeriod 2008-2010, 2011-2013 and 2014-2016Number of Original Research Papers on hESC and iPSC PublishedWorldwide (2014-2016)Concerns Related to Embryonic Stem Cells Shift the Focus ontoiPSCsRegenerative Medicine: A Promising Application of iPSCsInduced Pluripotent: A Potential Competitor to hESCs?Global Regenerative Medicine Market Size in US$ Billion for2019, 2021, 2023 and 2025Global Stem Cell & Regenerative Medicine Market by Product(in %) for the Year 2019Global Regenerative Medicines Market by Category: Breakdown(in %) for Biomaterials, Stem Cell Therapies and TissueEngineering for 2019Pluripotent Stem Cells Hold Significance for CardiovascularRegenerative MedicineLeading Causes of Mortality Worldwide: Number of Deaths inMillions & % Share of Deaths by Cause for 2017Leading Causes of Mortality for Low-Income and High-IncomeCountriesGrowing Importance of iPSCs in Personalized Drug DiscoveryPersistent Advancements in Genetics Space and Subsequent Growthin Precision Medicine Augur Well for iPSCs MarketGlobal Precision Medicine Market (In US$ Billion) for the Years2018, 2021 & 2024Increasing Prevalence of Chronic Disorders Supports Growth ofiPSCs MarketWorldwide Cancer Incidence: Number of New Cancer CasesDiagnosed for 2012, 2018 & 2040Number of New Cancer Cases Reported (in Thousands) by CancerType: 2018Fatalities by Heart Conditions: Estimated Percentage Breakdownfor Cardiovascular Disease, Ischemic Heart Disease, Stroke,and OthersRising Diabetes Prevalence Presents Opportunity for iPSCsMarket: Number of Adults (20-79) with Diabetes (in Millions)by Region for 2017 and 2045Aging Demographics Add to the Global Burden of ChronicDiseases, Presenting Opportunities for iPSCs MarketExpanding Elderly Population Worldwide: Breakdown of Number ofPeople Aged 65+ Years in Million by Geographic Region for theYears 2019 and 2030Growth in Number of Genomics Projects Propels Market GrowthGenomic Initiatives in Select CountriesNew Gene-Editing Tools Spur Interest and Investments inGenetics, Driving Lucrative Growth Opportunities for iPSCs:Total VC Funding (In US$ Million) in Genetics for the Years2014, 2015, 2016, 2017 and 2018Launch of Numerous iPSCs-Related Clinical Trials Set to BenefitMarket GrowthNumber of Induced Pluripotent Stem Cells based Studies bySelect Condition: As on Oct 31, 2020iPSCs-based Clinical Trial for Heart DiseasesInduced Pluripotent Stem Cells for Stroke Treatment?Off-the-shelf? Stem Cell Treatment for Cancer Enters ClinicalTrialiPSCs for Hematological DisordersMarket Benefits from Growing Funding for iPSCs-Related R&DInitiativesStem Cell Research Funding in the US (in US$ Million) for theYears 2016 through 2021Human iPSC Banks: A Review of Emerging Opportunities and DrawbacksHuman iPSC Banks Worldwide: An OverviewCell Sources and Reprogramming Methods Used by Select iPSC BanksInnovations, Research Studies & Advancements in iPSCsKey iPSC Research Breakthroughs for Regenerative MedicineResearchers Develop Novel Oncogene-Free and Virus-Free iPSCProduction MethodScientists Study Concerns of Genetic Mutations in iPSCsiPSCs Hold Tremendous Potential in Transforming Research EffortsResearchers Highlight Potential Use of iPSCs for DevelopingNovel Cancer VaccinesScientists Use Machine Learning to Improve Reliability of iPSCSelf-OrganizationSTEMCELL Technologies Unveils mTeSR? PlusChallenges and Risks Related to Pluripotent Stem CellsA Glance at Issues Related to Reprogramming of Adult Cells toiPSCsA Note on Legal, Social and Ethical Considerations with iPSCs

4. GLOBAL MARKET PERSPECTIVETable 1: World Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Geographic Region -USA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld Markets - Independent Analysis of Annual Sales in US$Thousand for Years 2020 through 2025 and % CAGR

Table 2: World 5-Year Perspective for Induced Pluripotent StemCell (iPSC) by Geographic Region - Percentage Breakdown ofValue Sales for USA, Canada, Japan, China, Europe, Asia-Pacificand Rest of World Markets for Years 2021 & 2025

Table 3: World Recent Past, Current & Future Analysis forVascular Cells by Geographic Region - USA, Canada, Japan,China, Europe, Asia-Pacific and Rest of World Markets -Independent Analysis of Annual Sales in US$ Thousand for Years2020 through 2025 and % CAGR

Table 4: World 5-Year Perspective for Vascular Cells byGeographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

Table 5: World Recent Past, Current & Future Analysis forCardiac Cells by Geographic Region - USA, Canada, Japan, China,Europe, Asia-Pacific and Rest of World Markets - IndependentAnalysis of Annual Sales in US$ Thousand for Years 2020 through2025 and % CAGR

Table 6: World 5-Year Perspective for Cardiac Cells byGeographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

Table 7: World Recent Past, Current & Future Analysis forNeuronal Cells by Geographic Region - USA, Canada, Japan,China, Europe, Asia-Pacific and Rest of World Markets -Independent Analysis of Annual Sales in US$ Thousand for Years2020 through 2025 and % CAGR

Table 8: World 5-Year Perspective for Neuronal Cells byGeographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

Table 9: World Recent Past, Current & Future Analysis for LiverCells by Geographic Region - USA, Canada, Japan, China, Europe,Asia-Pacific and Rest of World Markets - Independent Analysisof Annual Sales in US$ Thousand for Years 2020 through 2025 and% CAGR

Table 10: World 5-Year Perspective for Liver Cells byGeographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

Table 11: World Recent Past, Current & Future Analysis forImmune Cells by Geographic Region - USA, Canada, Japan, China,Europe, Asia-Pacific and Rest of World Markets - IndependentAnalysis of Annual Sales in US$ Thousand for Years 2020 through2025 and % CAGR

Table 12: World 5-Year Perspective for Immune Cells byGeographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

Table 13: World Recent Past, Current & Future Analysis forOther Cell Types by Geographic Region - USA, Canada, Japan,China, Europe, Asia-Pacific and Rest of World Markets -Independent Analysis of Annual Sales in US$ Thousand for Years2020 through 2025 and % CAGR

Table 14: World 5-Year Perspective for Other Cell Types byGeographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

Table 15: World Recent Past, Current & Future Analysis forCellular Reprogramming by Geographic Region - USA, Canada,Japan, China, Europe, Asia-Pacific and Rest of World Markets -Independent Analysis of Annual Sales in US$ Thousand for Years2020 through 2025 and % CAGR

Table 16: World 5-Year Perspective for Cellular Reprogrammingby Geographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

Table 17: World Recent Past, Current & Future Analysis for CellCulture by Geographic Region - USA, Canada, Japan, China,Europe, Asia-Pacific and Rest of World Markets - IndependentAnalysis of Annual Sales in US$ Thousand for Years 2020 through2025 and % CAGR

Table 18: World 5-Year Perspective for Cell Culture byGeographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

Table 19: World Recent Past, Current & Future Analysis for CellDifferentiation by Geographic Region - USA, Canada, Japan,China, Europe, Asia-Pacific and Rest of World Markets -Independent Analysis of Annual Sales in US$ Thousand for Years2020 through 2025 and % CAGR

Table 20: World 5-Year Perspective for Cell Differentiation byGeographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

Table 21: World Recent Past, Current & Future Analysis for CellAnalysis by Geographic Region - USA, Canada, Japan, China,Europe, Asia-Pacific and Rest of World Markets - IndependentAnalysis of Annual Sales in US$ Thousand for Years 2020 through2025 and % CAGR

Table 22: World 5-Year Perspective for Cell Analysis byGeographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

Table 23: World Recent Past, Current & Future Analysis forCellular Engineering by Geographic Region - USA, Canada, Japan,China, Europe, Asia-Pacific and Rest of World Markets -Independent Analysis of Annual Sales in US$ Thousand for Years2020 through 2025 and % CAGR

Table 24: World 5-Year Perspective for Cellular Engineering byGeographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

Table 25: World Recent Past, Current & Future Analysis forOther Research Methods by Geographic Region - USA, Canada,Japan, China, Europe, Asia-Pacific and Rest of World Markets -Independent Analysis of Annual Sales in US$ Thousand for Years2020 through 2025 and % CAGR

Table 26: World 5-Year Perspective for Other Research Methodsby Geographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

Table 27: World Recent Past, Current & Future Analysis for DrugDevelopment & Toxicology Testing by Geographic Region - USA,Canada, Japan, China, Europe, Asia-Pacific and Rest of WorldMarkets - Independent Analysis of Annual Sales in US$ Thousandfor Years 2020 through 2025 and % CAGR

Table 28: World 5-Year Perspective for Drug Development &Toxicology Testing by Geographic Region - Percentage Breakdownof Value Sales for USA, Canada, Japan, China, Europe,Asia-Pacific and Rest of World for Years 2021 & 2025

Table 29: World Recent Past, Current & Future Analysis forAcademic Research by Geographic Region - USA, Canada, Japan,China, Europe, Asia-Pacific and Rest of World Markets -Independent Analysis of Annual Sales in US$ Thousand for Years2020 through 2025 and % CAGR

Table 30: World 5-Year Perspective for Academic Research byGeographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

Table 31: World Recent Past, Current & Future Analysis forRegenerative Medicine by Geographic Region - USA, Canada,Japan, China, Europe, Asia-Pacific and Rest of World Markets -Independent Analysis of Annual Sales in US$ Thousand for Years2020 through 2025 and % CAGR

Table 32: World 5-Year Perspective for Regenerative Medicine byGeographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

Table 33: World Recent Past, Current & Future Analysis forOther Applications by Geographic Region - USA, Canada, Japan,China, Europe, Asia-Pacific and Rest of World Markets -Independent Analysis of Annual Sales in US$ Thousand for Years2020 through 2025 and % CAGR

Table 34: World 5-Year Perspective for Other Applications byGeographic Region - Percentage Breakdown of Value Sales forUSA, Canada, Japan, China, Europe, Asia-Pacific and Rest ofWorld for Years 2021 & 2025

III. MARKET ANALYSIS

UNITED STATESInduced Pluripotent Stem Cell (iPSC) Market Presence - Strong/Active/Niche/Trivial - Key Competitors in the United Statesfor 2022 (E)Table 35: USA Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Cell Type - VascularCells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cellsand Other Cell Types - Independent Analysis of Annual Sales inUS$ Thousand for the Years 2020 through 2025 and % CAGR

Table 36: USA 5-Year Perspective for Induced Pluripotent StemCell (iPSC) by Cell Type - Percentage Breakdown of Value Salesfor Vascular Cells, Cardiac Cells, Neuronal Cells, Liver Cells,Immune Cells and Other Cell Types for the Years 2021 & 2025

Table 37: USA Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Research Method -Cellular Reprogramming, Cell Culture, Cell Differentiation,Cell Analysis, Cellular Engineering and Other Research Methods -Independent Analysis of Annual Sales in US$ Thousand for theYears 2020 through 2025 and % CAGR

Table 38: USA 5-Year Perspective for Induced Pluripotent StemCell (iPSC) by Research Method - Percentage Breakdown of ValueSales for Cellular Reprogramming, Cell Culture, CellDifferentiation, Cell Analysis, Cellular Engineering and OtherResearch Methods for the Years 2021 & 2025

Table 39: USA Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Application - DrugDevelopment & Toxicology Testing, Academic Research,Regenerative Medicine and Other Applications - IndependentAnalysis of Annual Sales in US$ Thousand for the Years 2020through 2025 and % CAGR

Table 40: USA 5-Year Perspective for Induced Pluripotent StemCell (iPSC) by Application - Percentage Breakdown of ValueSales for Drug Development & Toxicology Testing, AcademicResearch, Regenerative Medicine and Other Applications for theYears 2021 & 2025

CANADATable 41: Canada Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Cell Type - VascularCells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cellsand Other Cell Types - Independent Analysis of Annual Sales inUS$ Thousand for the Years 2020 through 2025 and % CAGR

Table 42: Canada 5-Year Perspective for Induced PluripotentStem Cell (iPSC) by Cell Type - Percentage Breakdown of ValueSales for Vascular Cells, Cardiac Cells, Neuronal Cells, LiverCells, Immune Cells and Other Cell Types for the Years 2021 &2025

Table 43: Canada Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Research Method -Cellular Reprogramming, Cell Culture, Cell Differentiation,Cell Analysis, Cellular Engineering and Other Research Methods -Independent Analysis of Annual Sales in US$ Thousand for theYears 2020 through 2025 and % CAGR

Table 44: Canada 5-Year Perspective for Induced PluripotentStem Cell (iPSC) by Research Method - Percentage Breakdown ofValue Sales for Cellular Reprogramming, Cell Culture, CellDifferentiation, Cell Analysis, Cellular Engineering and OtherResearch Methods for the Years 2021 & 2025

Table 45: Canada Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Application - DrugDevelopment & Toxicology Testing, Academic Research,Regenerative Medicine and Other Applications - IndependentAnalysis of Annual Sales in US$ Thousand for the Years 2020through 2025 and % CAGR

Table 46: Canada 5-Year Perspective for Induced PluripotentStem Cell (iPSC) by Application - Percentage Breakdown of ValueSales for Drug Development & Toxicology Testing, AcademicResearch, Regenerative Medicine and Other Applications for theYears 2021 & 2025

JAPANInduced Pluripotent Stem Cell (iPSC) Market Presence - Strong/Active/Niche/Trivial - Key Competitors in Japan for 2022 (E)Table 47: Japan Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Cell Type - VascularCells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cellsand Other Cell Types - Independent Analysis of Annual Sales inUS$ Thousand for the Years 2020 through 2025 and % CAGR

Table 48: Japan 5-Year Perspective for Induced Pluripotent StemCell (iPSC) by Cell Type - Percentage Breakdown of Value Salesfor Vascular Cells, Cardiac Cells, Neuronal Cells, Liver Cells,Immune Cells and Other Cell Types for the Years 2021 & 2025

Table 49: Japan Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Research Method -Cellular Reprogramming, Cell Culture, Cell Differentiation,Cell Analysis, Cellular Engineering and Other Research Methods -Independent Analysis of Annual Sales in US$ Thousand for theYears 2020 through 2025 and % CAGR

Table 50: Japan 5-Year Perspective for Induced Pluripotent StemCell (iPSC) by Research Method - Percentage Breakdown of ValueSales for Cellular Reprogramming, Cell Culture, CellDifferentiation, Cell Analysis, Cellular Engineering and OtherResearch Methods for the Years 2021 & 2025

Table 51: Japan Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Application - DrugDevelopment & Toxicology Testing, Academic Research,Regenerative Medicine and Other Applications - IndependentAnalysis of Annual Sales in US$ Thousand for the Years 2020through 2025 and % CAGR

Table 52: Japan 5-Year Perspective for Induced Pluripotent StemCell (iPSC) by Application - Percentage Breakdown of ValueSales for Drug Development & Toxicology Testing, AcademicResearch, Regenerative Medicine and Other Applications for theYears 2021 & 2025

CHINAInduced Pluripotent Stem Cell (iPSC) Market Presence - Strong/Active/Niche/Trivial - Key Competitors in China for 2022 (E)Table 53: China Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Cell Type - VascularCells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cellsand Other Cell Types - Independent Analysis of Annual Sales inUS$ Thousand for the Years 2020 through 2025 and % CAGR

Table 54: China 5-Year Perspective for Induced Pluripotent StemCell (iPSC) by Cell Type - Percentage Breakdown of Value Salesfor Vascular Cells, Cardiac Cells, Neuronal Cells, Liver Cells,Immune Cells and Other Cell Types for the Years 2021 & 2025

Table 55: China Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Research Method -Cellular Reprogramming, Cell Culture, Cell Differentiation,Cell Analysis, Cellular Engineering and Other Research Methods -Independent Analysis of Annual Sales in US$ Thousand for theYears 2020 through 2025 and % CAGR

Table 56: China 5-Year Perspective for Induced Pluripotent StemCell (iPSC) by Research Method - Percentage Breakdown of ValueSales for Cellular Reprogramming, Cell Culture, CellDifferentiation, Cell Analysis, Cellular Engineering and OtherResearch Methods for the Years 2021 & 2025

Table 57: China Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Application - DrugDevelopment & Toxicology Testing, Academic Research,Regenerative Medicine and Other Applications - IndependentAnalysis of Annual Sales in US$ Thousand for the Years 2020through 2025 and % CAGR

Table 58: China 5-Year Perspective for Induced Pluripotent StemCell (iPSC) by Application - Percentage Breakdown of ValueSales for Drug Development & Toxicology Testing, AcademicResearch, Regenerative Medicine and Other Applications for theYears 2021 & 2025

EUROPEInduced Pluripotent Stem Cell (iPSC) Market Presence - Strong/Active/Niche/Trivial - Key Competitors in Europe for 2022 (E)Table 59: Europe Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Geographic Region -France, Germany, Italy, UK and Rest of Europe Markets -Independent Analysis of Annual Sales in US$ Thousand for Years2020 through 2025 and % CAGR

Table 60: Europe 5-Year Perspective for Induced PluripotentStem Cell (iPSC) by Geographic Region - Percentage Breakdown ofValue Sales for France, Germany, Italy, UK and Rest of EuropeMarkets for Years 2021 & 2025

Table 61: Europe Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Cell Type - VascularCells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cellsand Other Cell Types - Independent Analysis of Annual Sales inUS$ Thousand for the Years 2020 through 2025 and % CAGR

Table 62: Europe 5-Year Perspective for Induced PluripotentStem Cell (iPSC) by Cell Type - Percentage Breakdown of ValueSales for Vascular Cells, Cardiac Cells, Neuronal Cells, LiverCells, Immune Cells and Other Cell Types for the Years 2021 &2025

Table 63: Europe Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Research Method -Cellular Reprogramming, Cell Culture, Cell Differentiation,Cell Analysis, Cellular Engineering and Other Research Methods -Independent Analysis of Annual Sales in US$ Thousand for theYears 2020 through 2025 and % CAGR

Table 64: Europe 5-Year Perspective for Induced PluripotentStem Cell (iPSC) by Research Method - Percentage Breakdown ofValue Sales for Cellular Reprogramming, Cell Culture, CellDifferentiation, Cell Analysis, Cellular Engineering and OtherResearch Methods for the Years 2021 & 2025

Table 65: Europe Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Application - DrugDevelopment & Toxicology Testing, Academic Research,Regenerative Medicine and Other Applications - IndependentAnalysis of Annual Sales in US$ Thousand for the Years 2020through 2025 and % CAGR

Table 66: Europe 5-Year Perspective for Induced PluripotentStem Cell (iPSC) by Application - Percentage Breakdown of ValueSales for Drug Development & Toxicology Testing, AcademicResearch, Regenerative Medicine and Other Applications for theYears 2021 & 2025

FRANCEInduced Pluripotent Stem Cell (iPSC) Market Presence - Strong/Active/Niche/Trivial - Key Competitors in France for 2022 (E)Table 67: France Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Cell Type - VascularCells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cellsand Other Cell Types - Independent Analysis of Annual Sales inUS$ Thousand for the Years 2020 through 2025 and % CAGR

Table 68: France 5-Year Perspective for Induced PluripotentStem Cell (iPSC) by Cell Type - Percentage Breakdown of ValueSales for Vascular Cells, Cardiac Cells, Neuronal Cells, LiverCells, Immune Cells and Other Cell Types for the Years 2021 &2025

Table 69: France Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Research Method -Cellular Reprogramming, Cell Culture, Cell Differentiation,Cell Analysis, Cellular Engineering and Other Research Methods -Independent Analysis of Annual Sales in US$ Thousand for theYears 2020 through 2025 and % CAGR

Table 70: France 5-Year Perspective for Induced PluripotentStem Cell (iPSC) by Research Method - Percentage Breakdown ofValue Sales for Cellular Reprogramming, Cell Culture, CellDifferentiation, Cell Analysis, Cellular Engineering and OtherResearch Methods for the Years 2021 & 2025

Table 71: France Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Application - DrugDevelopment & Toxicology Testing, Academic Research,Regenerative Medicine and Other Applications - IndependentAnalysis of Annual Sales in US$ Thousand for the Years 2020through 2025 and % CAGR

Table 72: France 5-Year Perspective for Induced PluripotentStem Cell (iPSC) by Application - Percentage Breakdown of ValueSales for Drug Development & Toxicology Testing, AcademicResearch, Regenerative Medicine and Other Applications for theYears 2021 & 2025

GERMANYInduced Pluripotent Stem Cell (iPSC) Market Presence - Strong/Active/Niche/Trivial - Key Competitors in Germany for 2022 (E)Table 73: Germany Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Cell Type - VascularCells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cellsand Other Cell Types - Independent Analysis of Annual Sales inUS$ Thousand for the Years 2020 through 2025 and % CAGR

Table 74: Germany 5-Year Perspective for Induced PluripotentStem Cell (iPSC) by Cell Type - Percentage Breakdown of ValueSales for Vascular Cells, Cardiac Cells, Neuronal Cells, LiverCells, Immune Cells and Other Cell Types for the Years 2021 &2025

Table 75: Germany Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Research Method -Cellular Reprogramming, Cell Culture, Cell Differentiation,Cell Analysis, Cellular Engineering and Other Research Methods -Independent Analysis of Annual Sales in US$ Thousand for theYears 2020 through 2025 and % CAGR

Table 76: Germany 5-Year Perspective for Induced PluripotentStem Cell (iPSC) by Research Method - Percentage Breakdown ofValue Sales for Cellular Reprogramming, Cell Culture, CellDifferentiation, Cell Analysis, Cellular Engineering and OtherResearch Methods for the Years 2021 & 2025

Table 77: Germany Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Application - DrugDevelopment & Toxicology Testing, Academic Research,Regenerative Medicine and Other Applications - IndependentAnalysis of Annual Sales in US$ Thousand for the Years 2020through 2025 and % CAGR

Table 78: Germany 5-Year Perspective for Induced PluripotentStem Cell (iPSC) by Application - Percentage Breakdown of ValueSales for Drug Development & Toxicology Testing, AcademicResearch, Regenerative Medicine and Other Applications for theYears 2021 & 2025

ITALYTable 79: Italy Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Cell Type - VascularCells, Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cellsand Other Cell Types - Independent Analysis of Annual Sales inUS$ Thousand for the Years 2020 through 2025 and % CAGR

Table 80: Italy 5-Year Perspective for Induced Pluripotent StemCell (iPSC) by Cell Type - Percentage Breakdown of Value Salesfor Vascular Cells, Cardiac Cells, Neuronal Cells, Liver Cells,Immune Cells and Other Cell Types for the Years 2021 & 2025

Table 81: Italy Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Research Method -Cellular Reprogramming, Cell Culture, Cell Differentiation,Cell Analysis, Cellular Engineering and Other Research Methods -Independent Analysis of Annual Sales in US$ Thousand for theYears 2020 through 2025 and % CAGR

Table 82: Italy 5-Year Perspective for Induced Pluripotent StemCell (iPSC) by Research Method - Percentage Breakdown of ValueSales for Cellular Reprogramming, Cell Culture, CellDifferentiation, Cell Analysis, Cellular Engineering and OtherResearch Methods for the Years 2021 & 2025

Table 83: Italy Recent Past, Current & Future Analysis forInduced Pluripotent Stem Cell (iPSC) by Application - DrugDevelopment & Toxicology Testing, Academic Research,Regenerative Medicine and Other Applications - IndependentAnalysis of Annual Sales in US$ Thousand for the Years 2020through 2025 and % CAGR

Table 84: Italy 5-Year Perspective for Induced Pluripotent StemCell (iPSC) by Application - Percentage Breakdown of ValueSales for Drug Development & Toxicology Testing, AcademicResearch, Regenerative Medicine and Other Applications for theYears 2021 & 2025

UNITED KINGDOMInduced Pluripotent Stem Cell (iPSC) Market Presence - Strong/Active/Niche/Trivial - Key Competitors in the United Kingdomfor 2022 (E)Table 85: UK Recent Past, Current & Future Analysis for InducedPluripotent Stem Cell (iPSC) by Cell Type - Vascular Cells,Cardiac Cells, Neuronal Cells, Liver Cells, Immune Cells andOther Cell Types - Independent Analysis of Annual Sales in US$Thousand for the Years 2020 through 2025 and % CAGR

Table 86: UK 5-Year Perspective for Induced Pluripotent StemCell (iPSC) by Cell Type - Percentage Breakdown of Value Salesfor Vascular Cells, Cardiac Cells, Neuronal Cells, Liver Cells,Immune Cells and Other Cell Types for the Years 2021 & 2025

Read the rest here:
Global Induced Pluripotent Stem Cell ((iPSC) Market to Reach $0 Thousand by 2027 - Yahoo Finance

Cardiac Stem Cells Comments Off on Global Induced Pluripotent Stem Cell ((iPSC) Market to Reach $0 Thousand by 2027 – Yahoo Finance | October 13th, 2022

Self-organizing lumps of human brain tissue grown in the laboratory have been successfully transplanted into the nervous systems of newborn rats in a step towards finding new ways to treat neuropsychiatric disorders.

The 3D organoids, developed from stem cells to resemble a simplified model of the human cortex, connected and integrated with the surrounding tissue in each rat's cortex to form a functional part of the rodent's own brain, displaying activity related to sensory perception.

This, according to a team of researchers led by neuroscientist Sergiu Paca of Stanford University, overcomes the limitations of dish-grown organoids, and gives us a new platform for modeling human brain development and disease in a living system.

"Most of the work that my lab has been doing has been motivated by this mission of trying to understand psychiatric disorders at the biological level so that we can actually find effective therapeutics," Paca explained in a press briefing.

"Many of these psychiatric conditions, such as autism and schizophrenia, are likely uniquely human, or at least, they are anchored in unique features of the human brain. And the human brain has certainly not been very accessible, which has precluded the progress we've been making in understanding the biology of these conditions."

In 2008, scientists made a breakthrough: brain cells grown from induced pluripotent stem cells. Mature cells harvested from adult humans were reverse engineered (or induced) to return them to the 'blank' state of stem cells the form cells take before they grow into cells with specializations, such as skin cells or cardiac cells.

These stem cells were then guided to develop into brain cells, which scientists cultivated to form lumps of brain-like tissue called organoids. These models of key areas of brain anatomy, such as the wrinkled outer cortex, could be used to study functions and development of brains up close.

As useful as they are, in vitro cortical organoids have limitations. Because they aren't connected to living systems, they don't complete maturation, depriving researchers of an opportunity to observe how they integrate with other major parts of a brain.

In addition, a brain organoid in a dish can't reveal the behavioral consequences of any defects scientists might identify. Since psychiatric disorders are defined by behavior, this stymies the ability to identify the physiological characteristics of these disorders.

In previous research, scientists have tried to overcome these hurdles by implanting human brain organoids into the brains of adult rats. Because of the developmental mismatch, the transplants didn't take: the developing neurons in the organoid couldn't form a strong connection with the fully developed network of an adult rat brain.

So Paca and his colleagues tried something else: grafting the human brain tissue onto the brains of newborn rats, whose own brains have not yet developed and matured.

Human cortical organoids were cultured in a dish, and then transplanted directly into the somatosensory cortex (the area of the brain responsible for receiving and processing sensory information) of rat pups just a few days old. These rats were then left to grow into adults for another 140 days (rats are fully sexually mature between 6 and 12 weeks).

Then, the scientists studied the rats. They had genetically engineered the organoids to respond to blue light simulation, activating neurons when blue light is shone on them. This stimulation on the human neurons was performed while the rats were being trained to lick a spout to receive water. Later, when the blue light was shone on the organoids, the rats would automatically lick displaying a response not seen in control groups.

This indicated that not only was the organoid functioning as part of the rat brain, it could help drive reward-seeking behavior.

Another group of neurons in the organoid showed activity when the scientist pushed the rats' whiskers evidence that the neurons can respond to sensory stimulation.

Brain cells cultivated from three human patients with a genetic disease called Timothy syndrome were also used for some of the organoids. Timothy syndrome affects the heart, digits and nervous system, and usually results in early death.

After the behavioral tests, the rats were euthanized and their brains extracted and dissected, allowing the researchers to observe the integration of the organoids on a cellular level. They found the organoid neurons grew much larger than any neurons grown in vitro, extending into the rats' brains and forming networks with the native rat neurons.

The neurons in the rats with Timothy syndrome transplants showed less elaborate shapes, and formed different synaptic connections with the surrounding brain tissue compared to control groups. This is a new discovery, and could not have been discovered in a brain organoid in a dish.

Although the platform still has some limitations, the team believes that it has the potential to become a powerful new tool for understanding brain development and disease.

"Overall, this in vivo platform represents a powerful resource to complement in vitro studies of human brain development and disease," the authors write in their paper.

"We anticipate that this platform will allow us to uncover new circuit-level phenotypes in patient-derived cells that have otherwise been elusive and to test novel therapeutic strategies."

The research has been published in Nature.

Originally posted here:
Scientists Spliced Human Brain Tissue Into The Brains of Baby Rats - ScienceAlert

Cardiac Stem Cells Comments Off on Scientists Spliced Human Brain Tissue Into The Brains of Baby Rats – ScienceAlert | October 13th, 2022

Tissue source and processing

Paired sections of tissue, including both artery and plaque, were recovered from the atherosclerotic core (AC) and proximally adjacent (PA) region of three patients with asymptomatic type VII calcified plaques who underwent carotid endarterectomy (Fig. S1a, TableS1). Due to the rich cellular composition of carotid artery and plethora of debris in plaque (i.e., lipid, fibrinogen, etc.), dissociation and generation of single-cell suspensions amenable to single-cell RNA sequencing were difficult. After tissue collection, enzymatic digestion, RBC lysis, and filtration were the initial steps required to generate single cells (see Methods and Fig. S1b). However, despite efficient enzymatic dissociation and significant filtering of our sample, we were still challenged by abundant plaque debris, which ultimately resulted in poor single-cell capture rates. In order to overcome this issue without isolating specific cell types through cell-marker antibody labeling, we devised a strategy to label all cells in the sample with a far-red excitation-emission live/dead cell nuclear stain (DRAQ5). All cells in the sample were stained, with debris being left unstained by the dye. Previous studies have used nuclear staining in library preparation and sequencing experiments to discriminate single versus doublet cells during cell sorting without adverse effects for downstream applications such as single-cell and bulk RNA sequencing17,18,19,20. Subsequently, DRAQ5+ cells were manually gated and sorted from the remainder of the debris using FACS. Cells isolated from the entire filtered sample represented <1% of the total particles in the sample (Figs. S2aS2f). Viability of remaining cells was assessed and was always >80% using this technique for cell separation (see Methods). The cells were then processed for single-cell sequencing.

The analytical approach in this manuscript is depicted in Fig.1a. Generation of single cells from three patient-matched AC and PA samples (batched per patient on a single NextSeq flow cell) yielded 51,981 cells total, with an average of ~13,000 AC cells/patient and ~5000PA cells/patient. Cell number disparities are due to the difference in size of the AC vs PA tissue itself. Given the abundance of AC versus PA cells, down-sampling was performed to equalize the contribution of each sample and condition to the unsupervised discovery of cell types and to mitigate bias due to class imbalance. UMAP-based clustering (see Methods) of this down-sampled dataset reveals 15 distinct cell partitions (Fig. S1c, d), representing 17,100 cells total. In order to assign partitions to major cell types we examined genes expressed in >80% of cells per partition and at a mean expression count >2. A dotplot representing three marker genes selected for each partition is presented in (Fig. S1e). A comparison of VSMC marker genes used in our study with those in the literature15 is provided (Fig. S1f). Cell-type labels assigned to these 15 initial partitions based on these marker genes include: T-lymphocytes (2 partitions), macrophages, VSMCs (2 partitions), ECs (2 partitions), B-lymphocytes, natural killer T-cells, B1-lymphocytes, mast cells, lymphoid progenitors, plasmacytoid dendritic cells, and an unidentified partition (TableS2). Following doublet filtering using a marker-gene exclusion method (see Supplemental Methods), removal of partitions with too few cells for differential gene expression analysis (mast cells, lymphoid progenitors, plasmacytoid dendritic cells, and the unidentified partition), and merging of partitions assigned to the same cell-type, we assessed differential gene expression between AC and PA regions across the 6 remaining major cell types: macrophages, ECs, VSMCs, NKT cells, T- and B-lymphocytes (Fig.1bd, Fig. S4, Supplementary Data16). We performed a number of independent partitioning experiments using various algorithmic variations to confirm the reproducibility of these partitions and cell-label assignments (see Supplemental Methods).

a Schematic diagram of analytical steps from tissue dissociation to key driver analysis. b, c UMAP visualization of 6 major cell types following doublet removal via gene exclusion criteria (see Supplemental Methods), separated by anatomic location (b), and by cell type (c). d Dotplot depicting cell-type marker genes, resulting in the identification of macrophages, ECs, VSMCs, NKT cells, T- and B-Lymphocytes. Dot size depicts the fraction of cells expressing a gene. Dot color depicts the degree of expression of each gene. n=3 for PA and AC groups.

GWAS results have highlighted biological processes in the vessel wall as key drivers of coronary artery disease (CAD)21. Our prior work has demonstrated the vascular wall to be involved in the most impactful common genetic risk factor for CAD22. Our results here also demonstrate extensive differential expression in these cell types across anatomic locations compared to the remaining cell types. Therefore, we chose to focus our efforts on dissecting expression alterations in VSMCs and ECs in order to illuminate pathogenic genomic signatures within these cell-types. As above, each cell type is compared across anatomic location (Fig.2a, e), and the top differentially expressed genes are shown (Fig.3b, f), revealing interesting spatial and expression magnitude differences between AC and PA cells.

a, e UMAP visualization of VSMCs (a) and ECs (e), separated by anatomic location. b, f Volcano plots of the top differentially expressed genes in VSMCs (b) and ECs (f). Dotted lines represented q-value 0.5 and <0.5 corresponding to PA and AC cells, respectively. c, d UMAP visualization of the top 4 upregulated genes in AC VSMCs (c), and PA VSMCs (d). Gray-colored cells indicate 0 expression of designated gene, while color bar gradient indicates lowest (black) to highest (yellow) gene expression level. g, h UMAP visualization of the top 4 upregulated genes in AC ECs (g), and PA ECs (h). Color scheme is similar to the above-described parameters. VSMCs=3674 cells; ECs=2764 cells. n=3 for PA and AC groups.

a, b Normalized enrichment score (NES) ranking of all significant PA and AC Hallmarks generated from GSEA analysis of differentially expressed genes for VSMCs (a) and ECs (b) (FDR q-value<0.05). c Fully clustered on/off heatmap visualization of overlap between leading edge EMT hallmark genes generated by GSEA. Heatmaps are downsampled and represent 448 cells from each cell type and anatomic location (1792 total cells). A dotplot corresponding to gene expression levels for each cell type in the heatmap is included. Dot size depicts the fraction of cells expressing a gene. Dot color depicts the degree of expression of each gene. d Volcano plot of differentially expressed genes between the two groups of VSMCs in (c). Dotted lines represented q-value<0.01 and normalized effect >0.5 and <0.5. e, f Gene co-expression networks generated from VSMC Module 13 (d) and EC Module 1 (e) representing the EMT hallmark from GSEA analysis. Genes are separated by anatomic location (red=AC genes, cyan=PA genes), differential expression (darker shade=higher DE, gray=non-significantly DEGs), correlation with other connected genes (green line=positive correlation, orange line=negative correlation) and strength of correlation (connecting line thickness). Significantly DEGs (q<0.05) with high connectivity scores (>0.3) are denoted by a box instead of a circle. n=3 for PA and AC groups.

VSMCs generate three subclusters in the UMAP plot. A large fraction of PA VSMCs form a PA-specific VSMC subcluster. In contrast, AC VSMCs form 2 separate clusters both of which are intermingled with PA VSMCs. This suggests VSMCs occupy three major cell states, including one completely distinct PA subtype, and two that are predominantly AC VSMCs (Fig.2a). The top four upregulated genes in the AC are sparsely expressed and include SPP1, SFRP5, IBSP, and CRTAC1 (Fig.2b, c), while APOD, PLA2G2A, C3, and MFAP5 are upregulated in many PA VSMCs (Fig.2b, d).

The spatial clustering of upregulated genes in AC VSMCs suggests the presence of separate subpopulations of matrix-secreting VSMCs involved with ECM remodeling (Fig.2c). SPP1 (osteopontin) is a secreted glycoprotein involved in bone remodeling23 and has been implicated in atherosclerosis for inhibiting vascular calcification and inflammation in the plaque milieu24. IBSP (bone sialoprotein) is a significant component of bone, cartilage, and other mineralized tissues25. CRTAC1 is a marker to distinguish chondrocytes from osteoblasts and other mesenchymal stem cells26,27. These findings suggest the presence of cartilaginous and osseous matrix-secreting VSMCs in the AC region. SFRP5, an adipokine that is a direct WNT antagonist, reduces the secretion of inflammatory factors28 and is thought to exert favorable effects on the development of atherosclerosis29. The high expression of SFRP5 in the AC suggests a deceleration of these inflammatory processes in the core of the plaque, and an overall shift in the AC to calcification and matrix remodeling.

Conversely, the upregulated genes in PA VSMCs are more ubiquitously expressed by VSMCs in a PA-specific region of the UMAP plot (Fig.2d). C3 is highly differentially expressed in many PA cells (Fig.2d). Complement activation has long been appreciated for its role in atherosclerosis30, with maturation of plaque shown to be dependent, in part, on C3 opsonization for macrophage recruitment and stimulation of antibody responses31. Its predominance in our PA samples suggests complement activation in atherosclerosis is anatomically driven by VSMCs located adjacent to areas of maximal plaque build-up. PLA2G2A (phospholipase A2 group IIA), also selectively expressed by this group of cells, is pro-atherogenic, modulates LDL oxidation and cellular oxidative stress, and promotes inflammatory cytokine secretion32, further facilitating the inflammatory properties of this group of VSMCs. Full differential expression results for VSMCs are provided (Supplementary Data5).

Overall, we identify increased calcification and ECM remodeling by VSMCs in the AC versus pro-inflammatory signaling by VSMCs in the PA. These differences in biological processes are strongly supported further in the systems analyses below.

In contrast to VSMCs, for ECs we observe a more complete separation of cells into two distinct subgroups (Fig.2e). PA ECs significantly outnumber the AC ECs (2316 vs 448 cells, respectively), possibly due to intimal erosion and loss of endothelial cell layer integrity during advanced disease5,33,34,35 resulting in fewer captured ECs in the AC. Cellular transdifferentiation may also cause a subpopulation of ECs to lose common EC marker expression, resulting in lower numbers of ECs identified in AC compared to the PA counterpart. Histologic assessment of AC plaque collected from our patients supports the assertion of endothelial layer attenuation as the principal reason for lower AC EC capture (Fig. S3b, c). In contrast to VSMCs, there is a skew toward higher magnitude expression changes in AC ECs vs PA ECs. The top four upregulated genes are ITLN1, DKK2, F5, and FN1 in the AC and IL6, MLPH, HLA-DQA1, and ACKR1 in PA ECs (Fig.2g, h).

The upregulated genes in AC ECs again suggest a synthetic profile. ITLN (omentin) is an adipokine enhancing insulin-sensitivity in adipocytes36. Interestingly, circulating plasma omentin levels were shown to negatively correlate with carotid intima-media thickness37, inhibit TNF-induced vascular inflammation in human ECs38, and promote revascularization39, suggesting an anti-inflammatory and intimal repair role in AC ECs. DKK2 further indicates intimal repair as it stimulates angiogenesis in ECs40. The significant upregulation of FN1 (fibronectin) in this group further suggests active ECM remodeling and may serve as a marker for mesenchymal cells and EMT-related processes41.

Similarly to PA VSMCs, the upregulated genes in PA ECs suggest an overall inflammatory profile. Central players in inflammation and antigen presentation are upregulated specifically in PA ECs (Fig.2h). IL6, a key inflammatory cytokine associated with plaque42, is the most upregulated gene. Furthermore, ACKR1, highly upregulated in many PA ECs, binds and internalizes numerous chemokines and facilitates their presentation on the cell surface in order to boost leukocyte recruitment and augment inflammation43. Antigen presentation on ECs via HLA-DQA1 (MHC class II molecule) may support activation and exhaustion of CD4+ T-cells44,45 as previously described. Full differential expression results for ECs are provided (Supplementary Data6).

Overall, we identify two main EC subtypes: synthetic ECs in the AC that appear to participate in intimal repair, revascularization, and ECM modulation, and inflammatory ECs in the PA region that likely facilitate inflammation via antigen/chemokine presentation and recruitment of immune cells, including CD4+ T-cells. These differences in biological processes are strongly supported further in the systems analyses below.

In order to explore the anatomic differences for these cell types further, gene set enrichment analysis (GSEA) was used to asses hallmark processes most significantly altered in VSMCs and ECs (Fig.3a, b). Epithelial to mesenchymal transition (EMT), oxidative phosphorylation, and myogenesis gene upregulation were strongly enriched in both AC VSMCs and ECs, collectively suggesting an increase in cellular metabolic activity and proliferation. In contrast, a distinctly inflammatory profile was seen in PA VSMCs and ECs, with IFN gamma/alpha responses and TNFa signaling via NFkB dominating the enriched processes in these groups of cells. Because EMT and TNFa signaling were both shared and strongly enriched processes in the two cell types, the gene signatures associated with these hallmarks were further scrutinized through generation of heatmaps consisting of leading-edge differentially expressed genes from each hallmark process (EMTFig.3c, TNFa signaling via NFkBFig. S5a).

While overlapping at the hallmark level, separation of cells by cell type as well as anatomic location in the EMT hallmark heatmap suggests the overlapping processes are mediated by distinct gene sets in each cell type. Moreover, analysis of EMT hallmark genes further supports the presence of 2 cellular subtypes of AC VSMCs as they appear to cluster into two distinct groups of cells with dichotomous expression of contractile (MYL9, TPM2, TAGLN, FLNA) versus synthetic/EMT (POSTN, LUM, FBLN2, DCN, PCOLCE2, MGP, COL3A1) gene signatures (Fig.3c, d). These results indicate a group of VSMCs in the AC may perform the contractile functions of the blood vessel wall, while the other group of VSMCs may be involved with CTD and ECM remodeling. Furthermore, cells with an ACTA2+Thy1 gene signature in Fig.3c may be, in part, plaque-stabilizing myofibroblasts (orange line), indicating that these contractile cells may also have a large role in ECM remodeling.

In contrast to distinct subclustering of cells by EMT-related genes, there appears to be a common gradient of genes involved in inflammation and response to inflammation expressed throughout the atherosclerotic tissue, with higher levels of TNF-related inflammatory genes expressed in PA VSMCs and ECs compared to AC cells, indicating a predominance of inflammatory processes occurring in the PA region overall (Fig. S5a). Collectively these genes (EIF1, FOS, JUN, JUNB, ZFP36, PNRC1, KLF2, IER2, CEBPD, NFKBIA, GADD45B, EGR1, PPP1R15A, and SOCS3), in addition to IL6 expression in PA ECs, appear to coordinate the inflammatory response pathways in plaque and its adjacent structures. All cell types analyzed thus far are coordinated along this gradient of inflammation.

To further dissect VSMC and EC anatomical gene expression differences in order to identify candidate key genes driving the significant hallmark processes, we reconstructed gene co-expression networks using a partial correlation-based approach (see Methods), defined modules by clustering, and overlaid differential expression analysis results on these modules to identify those enriched in genes differentially expressed between AC and PA tissues.

Using this strategy, 31 and 39 distinct gene network modules were generated in our VSMC and EC datasets, respectively (see Supplemental Methods, Supplementary Data7, 8). Of these, 8 modules in VSMCs, and 5 modules in ECs were enriched with differentially expressed genes (p-value<0.05, Fishers exact test, see Methods). Furthermore, differentially expressed EMT-related hallmark genes overlapped significantly and specifically with a single VSMC and EC module. Differentially expressed TNFa signaling via NFkB-related hallmark genes also overlapped significantly with one VSMCs and EC module (p-value<0.05, Fishers exact test). No other hallmark processes overlapped with generated network modules.

The EMT gene signature generated from GSEA analysis of network modules and the robust upregulation of genes found in matrix-secreting cells in this cohort suggests the possibility of CTD occurring and/or completing in the atherosclerotic core. Therefore, in order to further characterize genes which may stimulate CTD in AC VSMCs and ECs we examined gene co-expression networks in conjunction with differential expression data from the modules enriched with EMT hallmark genes. In VSMCs we identified 9 genes (SPP1, IBSP, POSTN, MMP11, COL15A1, FN1, COL4A1, SMOC1, TIMP1) whose expression was significantly upregulated in AC cells and with strong network connectivity (see Methods). Among these genes we identify POSTN, SPP1, and IBSP as possible key gene drivers of CTD processes in AC VSMCs due to their strong central connectivity and high degree of differential expression in the network module (Fig.3e). POSTN (periostin) is expressed by osteoblasts and other connective tissue cell types involved with ECM maturation46 and stabilization during EMT in non-cardiac lineages47,48. POSTN, SPP1, and IBSP are highly interconnected in our network and likely serve as drivers of CTD by modulating other correlated genes such as TIMP1, VCAN, TPST2, SMOC1, MMP11, FN1, and COL4A1 (Fig.3e), all genes which are involved with cellular differentiation49 and extracellular matrix remodeling50,51.

In our EC network we identified 18 genes (ITLN1, FN1, OMD, S100A4, SCX, PRELP, GDF7, TMP2, SERPINE2, SLPI, HEY2, IGFBP3, FOXC2, RARRES2, PTGDS, TAGLN, LINC01235, and COL6A2) whose expression was significantly upregulated in AC cells and with strong network connectivity. Among these genes, we identify ITLN1, S100A4, and SCX as possible gene drivers of CTD in ECs associated with the AC (Fig.3f). ITLN1 (omentin) is highly upregulated in ECs associated with the atherosclerotic core, and network data indicate it is strongly correlated with genes involved with cellular proliferation and ECM modulation. ITLN is also strongly correlated to OGN (osteoglycin) which induces ectopic bone formation52, indicating that ITLN1 may modulate ECs with osteoblast-like features in the atherosclerotic core. SCX (scleraxin), a transcription factor that plays a critical role in mesoderm formation, and the development of chondrocyte lineages53, as well as regulating gene expression involved with ECM synthesis and breakdown in tenocytes54, is co-expressed with IL11RA, an interleukin receptor implicated in chondrogenesis55, as well as with a variety of genes involved with cellular development and modulation of ECM structures. Thus, SCX may modulate chondrocyte-like ECs in the AC. S100A4 is a calcium-binding protein that is highly expressed in smooth muscle cells of human coronary arteries during intimal thickening56, and silencing this gene in endothelial cells prevents endothelial tube formation and tumor angiogenesis in mice57. Co-expression with HEY2, a transcription factor involved with NOTCH signaling and critical for vascular development58, may indicate an important role in repair via re-endothelialization of plaque-denuded artery.

Next, genes critical to stimulating TNFa signaling via NFkB in PA VSMCs and ECs were evaluated. In the VSMC module we identified 14 genes (APOLD1, MT1A, ZFP36, EGR1, JUNB, FOSB, JUN, FOS, RERGL, MT1M, DNAJB1, CCNH, HSPA1B, and HSPA1A) whose expression was significantly upregulated in PA cells and with strong network connectivity. Among these genes we identify immediate-early (IE) genes ZFP36, EGR1, JUNB, FOSB, and FOS as critical response genes in this hallmark process. Importantly, the paired-sample study design in which AC and PA samples from the same patient are processed identically at the same time ensures that these IE genes preferentially upregulated in the PA region are critical for the inflammatory response and not an artifact of tissue processing stressors.

In the EC module we identified two genes (IER2 and FOS) whose expression was significantly increased in PA EC cells (Fig. S5e), and are highly correlated with other critical transcription factors in our network, including FOSB, JUNB, EGR1, and ZFP36, further supporting this group of genes importance in the TNFa signaling hallmark (Fig. S5d).

Finally, in order to identify and characterize refined subpopulations from each anatomic region, we selected the 7 VSMC and 5 EC differentially expressed modules described above and biclustered cells and genes (Fig.4a, d). The likely biological functions of these subpopulations were then inferred based on the genes differentially expressed and subsequent gene ontology enrichment analysis across these subpopulations. A continuous gene expression model, based on the fraction of AC cells per subpopulation, and subsequent gene ontology enrichment analysis was used to evaluate these cell subtype differences (Fig.4b, c, e, f).

a, d Biclustered heatmap visualization of all significant genes (q<0.05) from VSMC (a) and EC (d) modules enriched with differentially expressed genes. a 1224 VSMCs from each anatomic location (2448 cells total). Large color bar denotes PA (cyan) and AC (orange) VSMCs. Small color bar above denotes distinct cell subpopulations (blue, forest green, lime green, brown, purple, magenta, red). d 448 ECs from each anatomic location (896 cells total) in. Large color bar denotes PA (blue) and AC (red) ECs. Small color bar above denotes distinct cell subpopulations (cyan, green, magenta). A dotplot corresponding to gene expression levels for each cell subpopulation on the heatmap is included. Colored dots next to specific genes correspond to critical genes related to the designated cell subpopulation. Continuous gene expression based gene ontology enrichment analysis of biological function performed based on the fraction of AC cells per subpopulation of VSMCs (b, c) and ECs (e, f). n=3 for PA and AC groups.

We identified four cell subpopulations of VSMCs with some overlapping features in our analysis (Fig.4a). The four subpopulations appear to form a continuum of cell states, starting with a population that consists exclusively of PA VSMCs (Fig.4a, green bar), characterized by genes involved in recruitment of inflammatory mediators, with early signs of CTD. Specifically, C3 (opsonization and macrophage recruitment; normalized effect=6.5, q=1.74e07) is highly differentially expressed in this subpopulation and likely augments PA inflammation and macrophage recruitment. This group of VSMCs also shows evidence of early migratory and CTD-like qualities given the expression of FBN1, SEMA3C, HTRA3, and C1QTNF3, (normalized effect=2.77, 3.65, 4.0, 3.58, respectively; q=6.93e41, 1.25e20, 2.53e05, 0.00012, respectively) genes that are both highly differentially expressed in this cohort and with high signal strength in our networks (Fig.4a, Supplementary Data7). FBN1 (ECM component) is strongly correlated with TGFBR3, SEMA3C, and CD248 (modulators of EMT-like processes)59,60,61. Interestingly, this group of cells co-expresses IGSF10, a marker of early osteochondroprogenitor cells62, TMEM119 (bone formation and mineralization; promotes differentiation of myeloblasts into osteoblasts)63,64, and WNT11 (bone formation)65 (Supplementary Data7).

On the other end of this continuum, we identify a subpopulation of ~70% AC cells (Fig.4a, red bar) that have elevated expression of POSTN (osteoblasts; normalized effect=2.206, q=3.60e16), CRTAC1 (chrondrocytes; normalized effect=3.22, q=3.91e26), TNFRSF11B (bone remodeling; normalized effect=0.98, q=7.31e06)66, ENG (VSMC migration; normalized effect=0.87, q=1.41e13)67, COL4A2, and COL4A1 (cell proliferation, association with CAD; normalized effect=0.98, 1.03 and q=3.17e15, 5.68e11, respectively)68,69. Collectively, the differential gene expression data and the underlying biology behind our gene co-expression networks support this group of cells as likely representing synthetic osteoblast- and chondrocyte-like VSMCs which facilitate calcification and cartilaginous matrix-secretion and reside largely in the AC.

Furthermore, gene ontology enrichment analysis provides a clear progression from muscle system processes, extracellular structure reorganization, and catabolic processes enriched in the PA to processes involved with CTD such as ossification, fat cell differentiation, and regulation of cell motility, adhesion, and cellular transdifferentiation enriched in the AC (Fig.4b, c). The shift in cell states supports a continuum of cell state changes leading to increased CTD in the atherosclerotic core.

Overall, we observe three EC subpopulations. Like VSMCs, these cells display transitory properties as they move through a continuum of cell states (Fig.4d). First, there is a group comprised near exclusively of inflammatory PA ECs that is involved in recruitment of inflammatory mediators (Fig.4d, magenta bar). This group has a greater number of cells expressing immune genes such as the cluster of HLA genes, as well as CD74 (normalized effect=1.63, q=2.07e112), a gene which forms part of the invariant chain of the MHC II complex and is a receptor for the cytokine macrophage migration inhibitory factor (MIF)70. The upregulation of MHC class II complex in this subset of PA ECs complements our previous finding of CD4+T-cell recruitment to this subpopulation of PA ECs, leading to over-activation and exhaustion via antigen-persistence.

The next group of cells is intermediate in its composition of AC (67.5%) and PA (~32.5%) ECs with a mixed gene expression profile with characteristics similar to each of the other two groups of cells (Fig.4d, green bar), likely representing dysfunctional ECs that are in transition from the inflamed subtype to the CTD subtype described below.

The final group of cells is largely comprised of ECs from the AC (96.8%) (Fig.4d, cyan bar) and is largely devoid of endothelial-marker gene EMCN71 (normalized effect=0.86, q=1.17e09). Critical EMT genes identified earlier (ITLN1, SCX, and S100A4) are predominantly expressed in this large cluster of AC ECs alongside highly correlated genes OMD, OGN, and CRTAC1, again indicating that this population of ECs likely represents the main group of transdifferentiated ECs.

Gene ontology enrichment analysis further supports this shift in EC cell state from cells primarily involved with immune response (antigen processing and presentation, adaptive immune response, etc.) to cell states predominantly involved with proliferation, migration, vascular development, and angiogenesis (Fig.4e, f).

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