Cardiomyogenic Potential Research Articles

Optimizing Cardiomyocyte Differentiation: Comparative Analysis of Bone Marrow and Adipose-Derived Mesenchymal Stem Cells in Rats Using 5-Azacytidine and Low-Dose FGF and IGF Treatment.

Mesenchymal stem cells (MSCs) exhibit multipotency, self-renewal, and immune-modulatory properties, making them promising in regenerative medicine, particularly in cardiovascular treatments. However, optimizing the MSC source and induction method of cardiac differentiation is challenging. This study compares the cardiomyogenic potential of bone marrow (BM)-MSCs and adipose-derived (AD)-MSCs using 5-Azacytidine (5-Aza) alone or combined with low doses of Fibroblast Growth Factor (FGF) and Insulin-like Growth Factor (IGF). BM-MSCs and AD-MSCs were differentiated using two protocols: 10 μmol 5-Aza alone and 10 μmol 5-Aza with 1 ng/mL FGF and 10 ng/mL IGF. Morphological, transcriptional, and translational analyses, along with cell viability assessments, were performed. Both the MSC types exhibited similar morphological changes; however, AD-MSCs achieved 70-80% confluence faster than BM-MSCs. Surface marker profiling confirmed CD29 and CD90 positivity and CD45 negativity. The differentiation protocols led to cell flattening and myotube formation, with earlier differentiation in AD-MSCs. The combined protocol reduced cell mortality in BM-MSCs and enhanced the expression of cardiac markers (MEF2c, Troponin I, GSK-3β), particularly in BM-MSCs. Immunofluorescence confirmed cardiac-specific protein expression in all the treated groups. Both MSC types exhibited the expression of cardiac-specific markers indicative of cardiomyogenic differentiation, with the combined treatment showing superior efficiency for BM-MSCs.

Ascorbic acid and salvianolic acid B enhance the valproic acid and 5-azacytidinemediated cardiac differentiation of mesenchymal stem cells.

Cardiovascular diseases remain a major cause of death globally. Cardiac cells once damaged, cannot resume the normal functioning of the heart. Bone marrow derived mesenchymal stem cells (BM-MSCs) have shown the potential to differentiate into cardiac cells. Epigenetic modifications determine cell identity during embryo development via regulation of tissue specific gene expression. The major epigenetic mechanisms that control cell fate and biological functions are DNA methylation and histone modifications. However, epigenetic modifiers alone are not sufficient to generate mature cardiac cells. Various small molecules such as ascorbic acid (AA) and salvianolic acid B (SA) are known for their cardiomyogenic potential. Therefore, this study is aimed to examine the synergistic effects of epigenetic modifiers, valproic acid (VPA) and 5-azacytidine (5-aza) with cardiomyogenic molecules, AA and SA in the cardiac differentiation of MSCs. BM-MSCs were isolated, propagated, characterized, and then treated with an optimized dose of VPA or 5-aza for 24h. MSCs were maintained in a medium containing AA and SA for 21 days. All groups were assessed for the expression of cardiac genes and proteins through q-PCR and immunocytochemistry, respectively. Results show that epigenetic modifiers VPA or 5-aza in combination with AA and SA significantly upregulate the expression of cardiac genes MEF2C, Nkx2.5, cMHC, Tbx20, and GATA-4. In addition, VPA or 5-aza pretreatment along with AA and SA enhanced the expression of the cardiac proteins connexin-43, GATA-4, cTnI, and Nkx2.5. These findings suggest that epigenetic modifiers valproic acid and 5-azacytidine in combination with ascorbic acid and salvianolic acid B promote cardiac differentiation of MSCs. This pretreatment strategy can be exploited for designing future stem cell based therapeutic strategies for cardiovascular diseases.

Amniotic Fluid Mesenchymal Stromal Cells Derived from Fetuses with Isolated Cardiac Defects Exhibit Decreased Proliferation and Cardiomyogenic Potential.

Amniotic fluid mesenchymal stromal cells (AF-MSCs) represent an autologous cell source to ameliorate congenital heart defects (CHDs) in children. The AF-MSCs, having cardiomyogenic potential and being of fetal origin, may reflect the physiological and pathological changes in the fetal heart during embryogenesis. Hence, the study of defects in the functional properties of these stem cells during fetal heart development will help obtain a better understanding of the cause of neonatal CHDs. Therefore, in the present study, we compared the proliferative and cardiomyogenic potential of AF-MSCs derived from ICHD fetuses (ICHD AF-MSCs) with AF-MSCs from structurally normal fetuses (normal AF-MSCs). Compared to normal AF-MSCs, the ICHD AF-MSCs showed comparable immunophenotypic MSC marker expression and adipogenic and chondrogenic differentiation potential, with decreased proliferation, higher senescence, increased expression of DNA-damaged genes, and osteogenic differentiation potential. Furthermore, the expression of cardiac progenitor markers (PDGFR-α, VEGFR-2, and SSEA-1), cardiac transcription factors (GATA-4, NKx 2-5, ISL-1, TBX-5, TBX-18, and MeF-2C), and cardiovascular markers (cTNT, CD31, and α-SMA) were significantly reduced in ICHD AF-MSCs. Overall, these results suggest that the AF-MSCs of ICHD fetuses have proliferation defects with significantly decreased cardiomyogenic differentiation potential. Thus, these defects in ICHD AF-MSCs highlight that the impaired heart development in ICHD fetuses may be due to defects in the stem cells associated with heart development during embryogenesis.

Intramyocardial delivery of human cardiac stem cell spheroids with enhanced cell engraftment ability and cardiomyogenic potential for myocardial infarct repair

Strategies for stem cell-based cardiac regeneration and repair are key issues for the ischemic heart disease (IHD) patients with chronic complications related to ischemic necrosis. Cardiac stem cells (CSCs) have demonstrated high therapeutic efficacy for IHD treatment owing to their specific cardiac-lineage commitment. The therapeutic potential of CSCs could be further enhanced by designing a cellular spheroid formulation. The spheroid culture condition of CSCs was optimized to ensure regulated size and minimal core necrosis in the spheroids. The CSC spheroids revealed mRNA profiles of the factors related to cardiac regeneration, angiogenesis, anti-inflammatory, and cardiomyocyte differentiation with a higher expression level than the CSCs. Intramyocardially delivered CSC spheroids in the rat IHD model resulted in a significant increase in retention rate by 1.82-fold (day 3) and 1.98-fold (day 14) compared to CSCs. Endothelial cell differentiation and neovascularization of the engrafted CSC spheroids were noted in the infarcted myocardium. CSC spheroids significantly promoted cardiac regeneration: i.e., decreased infarction and fibrotic area (11.22% and 4.18%) and increased left ventricle thickness (0.62 mm) compared to the untreated group. Cardiac performance was also improved by 2.04-fold and 1.44-fold increase in the ejection fraction and fractional shortening, respectively. Intramyocardial administration of CSC spheroids might serve as an advanced therapeutic modality with enhanced cell engraftment and regenerative abilities for cardiac repair after myocardial infarction.

Single-cell transcriptomics defines heterogeneity of epicardial cells and fibroblasts within the infarcted murine heart.

In the adult heart, the epicardium becomes activated after injury, contributing to cardiac healing by secretion of paracrine factors. Here, we analyzed by single-cell RNA sequencing combined with RNA in situ hybridization and lineage tracing of Wilms tumor protein 1-positive (WT1+) cells, the cellular composition, location, and hierarchy of epicardial stromal cells (EpiSC) in comparison to activated myocardial fibroblasts/stromal cells in infarcted mouse hearts. We identified 11 transcriptionally distinct EpiSC populations, which can be classified into three groups, each containing a cluster of proliferating cells. Two groups expressed cardiac specification markers and sarcomeric proteins suggestive of cardiomyogenic potential. Transcripts of hypoxia-inducible factor (HIF)-1α and HIF-responsive genes were enriched in EpiSC consistent with an epicardial hypoxic niche. Expression of paracrine factors was not limited to WT1+ cells but was a general feature of activated cardiac stromal cells. Our findings provide the cellular framework by which myocardial ischemia may trigger in EpiSC the formation of cardioprotective/regenerative responses.

Cardio Phenotypic Potential of Mesenchymal Stem Cells.

Cell therapy is being investigated as a powerful intervention to ameliorate the consequences of coronary artery disease. Among the different stem cell options, mesenchymal stem cells (MSCs) are particularly attractive due to their high availability, as well as immune-privileged status. However, it is still unclear whether mesenchymal stem cells can acquire cardiomyogenic characteristics after they are transplanted to the myocardium. In this article, we outline protocols that illustrate the plasticity of MSCs and their ability to acquire cardiogenic characteristics when they are in an ischemic-like environment, as typically encountered after transplantation into the ischemic heart. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Isolation of mesenchymal stem cells (MSCs) Support Protocol 1: Characterization of MSCs by flow cytometry Basic Protocol 2: Isolation of neonatal cardiomyoctes (NCMs) Support Protocol 2: Characterization of NCMs Basic Protocol 3: Cardiogenic plasticity of MSCs under ischemic-like conditions Support Protocol 3: Characterization of the cardiomyogenic potential of MSCs.

Decellularized Human Dermal Matrix as a Biological Scaffold for Cardiac Repair and Regeneration.

The complex and highly organized environment in which cells reside consists primarily of the extracellular matrix (ECM) that delivers biological signals and physical stimuli to resident cells. In the native myocardium, the ECM contributes to both heart compliance and cardiomyocyte maturation and function. Thus, myocardium regeneration cannot be accomplished if cardiac ECM is not restored. We hypothesize that decellularized human skin might make an easily accessible and viable alternate biological scaffold for cardiac tissue engineering (CTE). To test our hypothesis, we decellularized specimens of both human skin and human myocardium and analyzed and compared their composition by histological methods and quantitative assays. Decellularized dermal matrix was then cut into 600-μm-thick sections and either tested by uniaxial tensile stretching to characterize its mechanical behavior or used as three-dimensional scaffold to assess its capability to support regeneration by resident cardiac progenitor cells (hCPCs) in vitro. Histological and quantitative analyses of the dermal matrix provided evidence of both effective decellularization with preserved tissue architecture and retention of ECM proteins and growth factors typical of cardiac matrix. Further, the elastic modulus of the dermal matrix resulted comparable with that reported in literature for the human myocardium and, when tested in vitro, dermal matrix resulted a comfortable and protective substrate promoting and supporting hCPC engraftment, survival and cardiomyogenic potential. Our study provides compelling evidence that dermal matrix holds promise as a fully autologous and cost-effective biological scaffold for CTE.

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential.

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Thus, the purpose of this protocol is to prepare and mature the target cell population through electromechanical stimulation, recapitulating physiological parameters. Cardiac tissue engineering is evolving toward more biological approaches, and strategies based on biophysical stimuli, thus, are gaining momentum. The device developed for this purpose is unique and allows individual or simultaneous electrical and mechanical stimulation, carefully characterized and validated. In addition, although the methodology has been optimized for this stimulator and a specific cell population, it can easily be adapted to other devices and cell lines. The results here offer evidence of the increased cardiac commitment of the cell population after electromechanical stimulation. Electromechanically stimulated cells show an increased expression of main cardiac markers, including early, structural, and calcium-regulating genes. This cell conditioning could be useful for further regenerative cell therapy, disease modeling, and high-throughput drug screening.

Cardiac Sca-1+ Cells Are Not Intrinsic Stem Cells for Myocardial Development, Renewal, and Repair.

For more than a decade, Sca-1+ cells within the mouse heart have been widely recognized as a stem cell population with multipotency that can give rise to cardiomyocytes, endothelial cells, and smooth muscle cells in vitro and after cardiac grafting. However, the developmental origin and authentic nature of these cells remain elusive. Here, we used a series of high-fidelity genetic mouse models to characterize the identity and regenerative potential of cardiac resident Sca-1+ cells. With these novel genetic tools, we found that Sca-1 does not label cardiac precursor cells during early embryonic heart formation. Postnatal cardiac resident Sca-1+ cells are in fact a pure endothelial cell population. They retain endothelial properties and exhibit minimal cardiomyogenic potential during development, normal aging and upon ischemic injury. Our study provides definitive insights into the nature of cardiac resident Sca-1+ cells. The observations challenge the current dogma that cardiac resident Sca-1+ cells are intrinsic stem cells for myocardial development, renewal, and repair, and suggest that the mechanisms of transplanted Sca-1+ cells in heart repair need to be reassessed.

Fractionation of embryonic cardiac progenitor cells and evaluation of their differentiation potential

Mid-gestation mouse ventricles (E11.5) contain a larger number of Nkx2.5+ cardiac progenitor cells (CPCs). The proliferation rates are consistently higher in CPCs compared to myocyte population of developing ventricles. Recent studies suggested that CPCs are an ideal donor cell type for replacing damaged tissue in diseased hearts. Thus, the ability to isolate and expand CPCs from embryos or stem cell cultures could be useful for cell fate studies and regenerative therapies. Since embryonic CPCs possess fewer mitochondria compared to cardiomyocytes, we reasoned that CPCs can be fractionated using a fluorescent mitochondrial membrane potential dye (TMRM) and these cells may retain cardiomyogenic potential even in the absence of cardiomyocytes (CMs). FACS sorting of TMRM stained embryonic ventricular cells indicated that over 99% of cells in TMRM high fraction stained positive for sarcomeric myosin (MF20) and all of them expressed Nkx2.5. Although majority of cells present in TMRM low fraction expressed Nkx2.5, very few cells (~1%) stained positive for MF20. Further culturing of TMRM low cells over a period of 48 h showed a progressive increase in MF20 positive cells. Additional analyses revealed that MF20 negative cells in TMRM low fraction do not express markers for endothelial cells (vWF, CD31) or smooth muscle cells (SM myosin). Treatment of TMRM low cells with known cardiogenic factors DMSO and dynorphin B significantly increased the percentage of MF20+ cells compared to untreated cultures. Collectively, these studies suggest that embryonic CPCs can be separated as a TMRM low fraction and their differentiation potential can be enhanced by exogenous addition of known cardiomyogenic factors.

Cardiomyogenic Heterogeneity of Clonal Subpopulations of Human Bone Marrow Mesenchymal Stem Cells

We have evaluated the cardiomyogenic potential of clonal populations of human bone marrow mesenchymal stem cells (BM-MSC). Four rapidly proliferating clones of BM-MSC were obtained from the BM of a healthy donor which were then treated with 5-azacytidine and evaluated for the expression of GATA-4, NKx-2.5, FOG-2, TDGF-1, β-MHC, MEF2D and NPPA genes and cTnT, Desmin and β-MHC proteins. Of the four clones (i) Clone-1 had high expression of GATA-4 (1.89 fold (p<0.05), Nkx2.5 (2.29 fold; p<0.05), FOG2 (2.76 fold; p<0.05), TDGF1 (6.97 fold, p<0.005), βMHC (10.22 fold; p<0.005), MEF-2D (1.91 fold; p<0.005) and NPPA (1.65 fold; p<0.005); (ii) clone-2 had up-regulation of Nkx2.5 (1.98 fold; p<0.05) but down-regulation of rest of the genes; (iii) clone-3 had up-regulation of Nkx2.5 (2.11 fold; p<0.05), TDGF1 (1.88 fold; p<0.05), MEF-2D (1.30 fold; p<0.05) and NPPA (1.21 fold; p<0.05), down regulation of GATA-4 and Fog-2 but no change in βMHC gene; and (iv) clone-4 had up-regulation of MEF-2D (1.17 fold; p<0.05) and down regulation of GATA-4, Nkx2.5 but no change in other genes compared to untreated cells of the clones. At the protein level, clone-1 expressed cTnT, Desmin, and βMHC; clone-2 Desmin only while clones-3 and 4 each expressed cTnT, Desmin, and βMHC. Our data shows that BM-MSC are a heterogenous population of stem cells with sub-populations exhibiting a marked difference in the expression of cardiac markers both at gene and protein levels. This highlights that administering selected sub-populations of BM-MSC with a cardiomyogenic potential may be more efficacious than whole population of cells for cardiac regeneration.

Synergistic role of 5-azacytidine and ascorbic acid in directing cardiosphere derived cells to cardiomyocytes in vitro by downregulating Wnt signaling pathway via phosphorylation of β-catenin.

BackgroundCardiosphere derived cells (CDCs) represent a valuable source in stem cell based therapy for cardiovascular diseases, yet poor differentiation rate hinders the transplantation efficiency. The aim of this study is to check the ability of 5-Azacytidine (Aza) alone and in combination with ascorbic acid (Aza+AA) in delineating CDCs to cardiomyogenesis and the underlying Wnt signaling mechanism in induced differentiation.MethodsCDCs were treated with Aza and Aza+AA for a period of 14 days to examine the expression of cardiac specific markers and Wnt downstream regulators by immunofluorescence, real time PCR and western blot.ResultsResults revealed that Aza+AA induced efficient commitment of CDCs to cardiomyogenic lineage. Immunofluorescence analysis showed significant augment for Nkx 2.5, GATA 4 and α-Sarcomeric actinin markers in Aza+AA group than control group (p = 0.0118, p = 0.009 and p = 0.0091, respectively). Relative upregulation of cardiac markers, Nkx 2.5 (p = 0.0156), GATA 4 (p = 0.0087) and down regulation of Wnt markers, β-catenin (p = 0.0107) and Cyclin D1 (p = 0. 0116) in Aza+AA group was revealed by RNA expression analysis. Moreover, the Aza+AA induced prominent expression of GATA 4, α-Sarcomeric actinin and phospho β-catenin while non phospho β-catenin and Cyclin D1 expression was significantly suppressed as displayed in protein expression analysis. Generation of spontaneous beating in Aza+AA treated CDCs further reinforced that Aza+AA accelerates the cardiomyogenic potential of CDCs.ConclusionCombined treatment of Aza along with AA implicit in inducing cardiomyogenic potential of CDCs and is associated with down regulating Wnt signaling pathway. Altogether, CDCs represent a valuable tool for the treatment of cardiovascular disorders.

Depletion of Mageb16 induces differentiation of pluripotent stem cells predominantly into mesodermal derivatives

The Melanoma-associated Antigen gene family (MAGE) generally encodes for tumour antigens. We had identified that one of the MAGE gene members, Mageb16 was highly expressed in undifferentiated murine embryonic stem cells (ESCs). While the role of Mageb16 in stemness and differentiation of pluripotent stem cells is completely unknown, here, in our current study, we have demonstrated that Mageb16 (41 kDa) is distributed in cytosol and/or in surface membrane in undifferentiated ESCs. A transcriptome study performed at differentiated short hairpin RNA (shRNA)-mediated Mageb16 knockdown (KD) ESCs and scrambled control (SCR) ESCs until a period of 22 days, revealed that Mageb16 KD ESCs mainly differentiated towards cells expressing mesodermal and cardiovascular lineage - gene markers. Gene markers of other mesoderm-oriented biological processes such as adipogenesis, osteogenesis, limb morphogenesis and spermatogenesis were also significantly enriched in the differentiated Mageb16 KD ESCs. The expression levels of contractile genes were higher in differentiated Mageb16 KD ESCs when compared to differentiated SCR and wild ESCs, suggesting a higher cardiomyogenic potential of Mageb16 depleted ESCs. Further analysis indicates that regulative epigenetic networks and nucleocytoplasmic modifications induced by the depletion of Mageb16, may play a probable role in differentiation.

Single-cell analysis of the fate of c-kit-positive bone marrow cells

The plasticity of c-kit-positive bone marrow cells (c-kit-BMCs) in tissues different from their organ of origin remains unclear. We tested the hypothesis that c-kit-BMCs are functionally heterogeneous and only a subgroup of these cells possesses cardiomyogenic potential. Population-based assays fall short of identifying the properties of individual stem cells, imposing on us the introduction of single cell-based approaches to track the fate of c-kit-BMCs in the injured heart; they included viral gene-tagging, multicolor clonal-marking and transcriptional profiling. Based on these strategies, we report that single mouse c-kit-BMCs expand clonally within the infarcted myocardium and differentiate into specialized cardiac cells. Newly-formed cardiomyocytes, endothelial cells, fibroblasts and c-kit-BMCs showed in their genome common sites of viral integration, providing strong evidence in favor of the plasticity of a subset of BMCs expressing the c-kit receptor. Similarly, individual c-kit-BMCs, which were infected with multicolor reporters and injected in infarcted hearts, formed cardiomyocytes and vascular cells organized in clusters of similarly colored cells. The uniform distribution of fluorescent proteins in groups of specialized cells documented the polyclonal nature of myocardial regeneration. The transcriptional profile of myogenic c-kit-BMCs and whole c-kit-BMCs was defined by RNA sequencing. Genes relevant for engraftment, survival, migration, and differentiation were enriched in myogenic c-kit-BMCs, a cell subtype which could not be assigned to a specific hematopoietic lineage. Collectively, our findings demonstrate that the bone marrow comprises a category of cardiomyogenic, vasculogenic and/or fibrogenic c-kit-positive cells and a category of c-kit-positive cells that retains an undifferentiated state within the damaged heart.

Fractionation of embryonic cardiac progenitor cells and evaluation of their differentiation potential

Abstract Mid-gestation mouse ventricles (E11.5) contain a larger number of Nkx2.5+ cardiac progenitor cells (CPCs). The proliferation rates are consistently higher in CPCs compared to myocyte population of developing ventricles. Recent studies suggested that CPCs are an ideal donor cell type for replacing damaged tissue in diseased hearts. Thus, the ability to isolate and expand CPCs from embryos or stem cell cultures could be useful for cell fate studies and regenerative therapies. Since embryonic CPCs possess fewer mitochondria compared to cardiomyocytes, we reasoned that CPCs can be fractionated using a fluorescent mitochondrial membrane potential dye (TMRM) and these cells may retain cardiomyogenic potential even in the absence of cardiomyocytes (CMs). FACS sorting of TMRM stained embryonic ventricular cells indicated that over 99% of cells in TMRM high fraction stained positive for sarcomeric myosin (MF20) and all of them expressed Nkx2.5. Although majority of cells present in TMRM low fraction expressed Nkx2.5, very few cells (~1%) stained positive for MF20. Further culturing of TMRM low cells over a period of 48 h showed a progressive increase in MF20 positive cells. Additional analyses revealed that MF20 negative cells in TMRM low fraction do not express markers for endothelial cells (vWF, CD31) or smooth muscle cells (SM myosin). Treatment of TMRM low cells with known cardiogenic factors DMSO and dynorphin B significantly increased the percentage of MF20+ cells compared to untreated cultures. Collectively, these studies suggest that embryonic CPCs can be separated as a TMRM low fraction and their differentiation potential can be enhanced by exogenous addition of known cardiomyogenic factors.

Effects of donor age on human adipose-derived adherent stromal cells under oxidative stress conditions.

ObjectiveAdipose-derived stromal vascular fractions (SVFs) are heterogeneous complex populations of cells with therapeutic efficacy for tissue generation and vascular stabilization. SVFs have cardiomyogenic potential, and many researchers have examined the possibility of SVF transplantation for heart disease. In cell-based therapies, donor age affects the regenerative capability, cell yield, and differentiation potential of adult tissues; however, opposing or controversial results have been found in humans. We examined whether SVF transplantation into impaired heart tissue shows differential effects according to donor age.MethodsWe investigated differences in protein expression in human umbilical vein endothelial cells (HUVECs) co-cultured with adipose-derived adherent stromal cells (ADASs) from donors of different ages [>40-year-olds (40s group) and >60-year-olds (60s group)] under oxidative stress conditions.ResultsAlthough co-culturing HUVECs with ADASs ameliorated inflammation due to increased oxidative stress conditions, few differences were observed between the ADASs from the 40s and 60s groups. Moreover, the Database for Annotation, Visualization, and Integrated Discovery classification tool revealed differentially expressed genes in the Kyoto Encyclopedia of Genes and Genomes pathway associated with cytokine–cytokine receptor interaction in response to ADASs.ConclusionProtein expression profiles were unchanged in HUVECs induced by isolated ADASs from donors of different ages under oxidative stress conditions.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells.

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells. Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein α, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

Erratum to: Characteristics and Cardiomyogenic Potential of Rat Fetal Cardiac Progenitor Cells at Different Developmental Stage.

[This corrects the article DOI: 10.1007/s13770-016-0016-z.].

Abstract 198: Multipotent Placenta-derived Cdx2 Cells Possess in vitro and in vivo Cardiomyogenic Potential

Stem cell-based therapies for cardiac regeneration are of crucial importance and an ideal cell-type is yet to be established. We previously reported that fetal cells from placenta “home” to injured maternal heart and approximately 40% (40/100) of the migrating cells expressed homeodomain protein Cdx2. This interesting observation led us to hypothesize that placental Cdx2 could be a novel cell target for cardiac differentiation. To understand this phenomenon, we employed a cre-lox strategy that labeled Cdx2 cells in placenta with e-GFP and induced myocardial infarction (MI) in pregnant mice at mid-gestation. The maternal heart was analyzed 4 weeks post-MI for the presence of Cdx2-eGFP-derived cardiomyocytes. Additionally, Cdx2 cells were isolated from late-gestation placenta and assayed for cardiac differentiation in vitro followed by live cell imaging. Phenotypic and whole-cell proteomic analysis, clonal and vascular lineage differentiation and immune profiling were carried out subsequently. We observed that Cdx2 cells migrated to injured maternal hearts and differentiated into cardiomyocytes highlighting the functional significance of fetal-maternal stem cell transfer. Additionally, isolated Cdx2 cells from the late placenta differentiated into spontaneously beating cardiomyocytes and expressed structural proteins cardiac troponin T(cTnT), α-sarcomeric actinin and gap junction protein Cx43. These cells underwent clonal expansion and differentiated into endothelial and smooth muscle lineages in culture indicative of their multipotent nature. Low expression of MHC molecules and other components of the immune-response, infer that these cells possess the ability to evade host immune surveillance. Proteomic analysis demonstrated that 145 proteins were uniquely identified in the Cdx2 cells compared to embryonic stem cells. These protein networks reflected an increased activation of functions involving migration, fertility, homing, and chemotaxis. Our study is the first to demonstrate that Cdx2 may play a role in cardiac differentiation and delineate multipotent cells in placenta with an inherent “homing” ability. These findings point to a potential role for Cdx2 cells in cardiac regenerative therapies using allogeneic cells.