Cardiomyogenic Differentiation Potential Research Articles

Alteration of Cardiac Progenitor Cell Potency in GRMD Dogs

Among the animal models of Duchenne muscular dystrophy (DMD), the Golden Retriever muscular dystrophy (GRMD) dog is considered the best model in terms of size and pathological onset of the disease. As in human patients presenting with DMD or Becker muscular dystrophies (BMD), the GRMD is related to a spontaneous X-linked mutation of dystrophin and is characterized by myocardial lesions. In this respect, GRMD is a useful model to explore cardiac pathogenesis and for the development of therapeutic protocols. To investigate whether cardiac progenitor cells (CPCs) isolated from healthy and GRMD dogs may differentiate into myocardial cell types and to test the feasibility of cell therapy for cardiomyopathies in a preclinical model of DMD, CPCs were isolated from cardiac biopsies of healthy and GRMD dogs. Gene profile analysis revealed an active cardiac transcription network in both healthy and GRMD CPCs. However, GRMD CPCs showed impaired self-renewal and cardiac differentiation. Population doubling and telomerase analyses highlighted earlier senescence and proliferation impairment in progenitors isolated from GRMD cardiac biopsies. Immunofluorescence analysis revealed that only wt CPCs showed efficient although not terminal cardiac differentiation, consistent with the upregulation of cardiac-specific proteins and microRNAs. Thus, the pathological condition adversely influences the cardiomyogenic differentiation potential of cardiac progenitors. Using PiggyBac transposon technology we marked CPCs for nuclear dsRed expression, providing a stable nonviral gene marking method for in vivo tracing of CPCs. Xenotransplantation experiments in neonatal immunodeficient mice revealed a valuable contribution of CPCs to cardiomyogenesis with homing differences between wt and dystrophic progenitors. These results suggest that cardiac degeneration in dystrophinopathies may account for the progressive exhaustion of local cardiac progenitors and shed light on cardiac stemness in physiological and pathological conditions. Furthermore, we provide essential information that canine CPCs may be used to alleviate cardiac involvement in a large preclinical model of DMD.

Mesenchymal stromal cells (MSCs): science and f(r)iction

Due to their multi-lineage differentiation capacity, support of haematopoiesis, immunomodulation and secretion of proregenerative factors, mesenchymal stem/stromal cells (MSCs) are in the focus of intense research since decades. The literature is replete with reports on their potential in preclinical model systems. However, the heterogeneity of the primary cell population as starting material and the diverse protocols for isolation and cultivation are hampering progress in their clinical application. Consensus on common standards and harmonised isolation and characterisation protocols are important to ensure safety and efficacy. This review focuses on the recent scientific evidence of clinically relevant properties and on the speculative cardiomyogenic and hepatic differentiation potential of MSCs. Special emphasis is put on the importance of standardisation and harmonisation in clinical-scale manufacturing.

Bone Marrow-Derived Human Mesenchymal Stem Cells Express Cardiomyogenic Proteins But Do Not Exhibit Functional Cardiomyogenic Differentiation Potential

Despite their paracrine activites, cardiomyogenic differentiation of bone marrow (BM)-derived mesenchymal stem cells (MSCs) is thought to contribute to cardiac regeneration. To systematically evaluate the role of differentiation in MSC-mediated cardiac regeneration, the cardiomyogenic differentiation potential of human MSCs (hMSCs) and murine MSCs (mMSCs) was investigated in vitro and in vivo by inducing cardiomyogenic and noncardiomyogenic differentiation. Untreated hMSCs showed upregulation of cardiac tropopin I, cardiac actin, and myosin light chain mRNA and protein, and treatment of hMSCs with various cardiomyogenic differentiation media led to an enhanced expression of cardiomyogenic genes and proteins; however, no functional cardiomyogenic differentiation of hMSCs was observed. Moreover, co-culturing of hMSCs with cardiomyocytes derived from murine pluripotent cells (mcP19) or with murine fetal cardiomyocytes (mfCMCs) did not result in functional cardiomyogenic differentiation of hMSCs. Despite direct contact to beating mfCMCs, hMSCs could be effectively differentiated into cells of only the adipogenic and osteogenic lineage. After intramyocardial transplantation into a mouse model of myocardial infarction, Sca-1(+) mMSCs migrated to the infarcted area and survived at least 14 days but showed inconsistent evidence of functional cardiomyogenic differentiation. Neither in vitro treatment nor intramyocardial transplantation of MSCs reliably generated MSC-derived cardiomyocytes, indicating that functional cardiomyogenic differentiation of BM-derived MSCs is a rare event and, therefore, may not be the main contributor to cardiac regeneration.

Cardio-myogenic differentiation of human amniotic fluid colony derived stem cells in the form of embryonic body-like structure

To investigate cardio-myogenic differentiation potential of human amniotic fluid colony derived stem cells (HAFCDSC) in the form of embryonic body (EB)-like structure in vitro. HAFCDSC were isolated from second trimester amniotic fluid which was backup of amniocentesis specimens. The forth passage of HAFCDSC were cultured by hanging-drop preparation in complete medium for 5 days to form EB-like structures followed by inducing medium in regular tissue culture dishes for 2 weeks (experiment group). The EB-like structures cultured in complete medium were served as control group. Cardiomyocyte phenotypes were detected by RT-PCR and immunofluorescence staining. HAFCDSC could form EB-like structures 24 h after hanging drop culture, and the diameter of EB-like structures gradually increased with culture duration and the mean diameter of EB-like structures reached 237.3 ± 26.7 µm on 5(th) day. The formation rate of EB-like structures was 93.5%. RT-PCR analysis showed EB-like structures expressed stem cell related mRNA. Immunofluorescence staining demonstrated that some cardiomyocyte phenotypes such as Desmin, α-Actinin and Tn I were expressed by HAFCDSCs in EB-like structures after cardio-myogenic induction. Cardiomyocytes specific mRNA including Desmin, α-Actin, Tn I, Tn T, T-box and NK2.5 were also transcripted as detected by RT-PCR. No positive results was found in control group. HAFCDSC can form uniform EB-like structures after hanging drop culture for 5 days. HAFCDSC differentiate into cardiomyocyte-like cells through the form of EB-like structures after induction by 5-Aza.

Composite Cell Sheets

The safety and efficacy of myocardial regeneration using embryonic stem cells are limited by the risk of teratoma and the high rate of cell death. To address these issues, we developed a composite construct made of a sheet of adipose tissue-derived stroma cells and embryonic stem cell-derived cardiac progenitors. Ten Rhesus monkeys underwent a transient coronary artery occlusion followed, 2 weeks later, by the open-chest delivery of the composite cell sheet over the infarcted area or a sham operation. The sheet was made of adipose tissue-derived stroma cells grown from a biopsy of autologous adipose tissue and cultured onto temperature-responsive dishes. Allogeneic Rhesus embryonic stem cells were committed to a cardiac lineage and immunomagnetically sorted to yield SSEA-1(+) cardiac progenitors, which were then deposited onto the cell sheet. Cyclosporine was given for 2 months until the animals were euthanized. Preimplantation studies showed that the SSEA-1(+) progenitors expressed cardiac markers and had lost pluripotency. After 2 months, there was no teratoma in any of the 5 cell-treated monkeys. Analysis of >1500 histological sections showed that the SSEA-1(+) cardiac progenitors had differentiated into cardiomyocytes, as evidenced by immunofluorescence and real-time polymerase chain reaction. There were also a robust engraftment of autologous adipose tissue-derived stroma cells and increased angiogenesis compared with the sham animals. These data collected in a clinically relevant nonhuman primate model show that developmentally restricted SSEA-1(+) cardiac progenitors appear to be safe and highlight the benefit of the epicardial delivery of a construct harboring cells with a cardiomyogenic differentiation potential and cells providing them the necessary trophic support.

Abstract 3420: Potential of Human Mesenchymal Stem Cells to Differentiate into Cardiomyocytes Depends on Donor Age and Donor Tissue

Treatment of damaged myocardium using autologous adult stem cells is under intense investigation as this may regenerate lost myocardium, thereby improving cardiac function. Initial cardiac cell therapy trials using various adult stem cell types have shown beneficial effects. However in contrast to earlier animal studies no true, quantitative reconstitution of myocardium has been observed. We hypothesized that the cardiomyogenic potential of stem cells depends on donor age and cell source. We used an in vitro differentiation protocol that employs co-culture of enhanced green fluorescent protein-labeled human mesenchymal stem cells (hMSCs) with neonatal rat cardiomyocytes (nrCMCs) for 13 days, without additional chemical or mutagenic stimulation. hMSCs were either derived from adult patients, or from human fetal tissue (gestational age 18 –22 weeks) or post natally collected umbilical cord blood. Differentiation of hMSCs was assessed by the presence of a properly developed sarcomere using immunological labeling for cardiac troponin I, myosin heavy chain and actinin. Functionality of newly differentiated cardiomyocytes was assessed by the presence of a functional calcium transient in conjunction with myocyte contraction. Mesenchymal stem cells isolated from clinically relevant sources (bone marrow, fat, and synovium) of adult patients are unable to adopt a cardiomyocyte phenotype upon co-incubation with nrCMCs. In contrast, MSCs from fetal donor material all give rise to functional cardiomyocytes, although with varying efficiency (umbilical cord blood: 6.8%±3.0, bone marrow: 0.4%±1.4, liver: 1.8%±0.8, lung: 1.4%±1.3, amnion: 9.3%±5.0 [mean±SD, n=3]). Using short hairpin RNA-mediated knockdown of connexin 43 (Cx43) gene expression, we found that sarcomeric development (organization) depends on the presence of Cx43 in the differentiating stem cells. Both donor age and the tissue source of human MSCs critically influence their cardiomyogenic differentiation potential. These results suggest that these non-embryonic, fetal cells may potentially be clinically useful for cardiac cell therapy.

Cardiomyogenic differentiation potential of human adipose precursor cells

DNA demethylation agent 5-azacytidine has been widely described in literature as an effective chemical stimulus used to promote cardiomyogenic differentiation in various cell types, ranging from embryonic stem cells, P19 cells, bone marrow-derived mesenchymal stem cells, and recently to adipose-derived stem cells. The purpose of this study was to examine the effects of 5-azacytidine on human adipose precursor cell differentiation along the cardiomyogenic lineage.

Sca-1 expression is associated with decreased cardiomyogenic differentiation potential of skeletal muscle-derived adult primitive cells

Adult stem cells from skeletal muscle (SM) have been shown to differentiate into multiple lineages. The impact of stem cell antigen-1 (Sca-1) expression on cardiomyogenic differentiation potential of SM-derived primitive cells remains unknown. Cardiomyogenic differentiation was induced in freshly isolated or culture-expanded Sca-1 +/CD45 −/c-kit −/Thy-1 + (SM+) and Sca1 −/CD45 −/c-kit −/Thy-1 + (SM−) cells isolated from SM of C57BL/6 mice. Expression of mRNA of cardiac-specific antigens and those associated with pluripotency was examined by real-time RT-PCR. Phenotypic analysis of expanded cells was performed during each passage by flow cytometry. Cardiomyocytic differentiation in vitro was verified by morphologic analysis, immunocytochemistry, and contractile properties. In freshly isolated cells, compared with unfractionated SM-derived cells as well as SM+ cells, mRNA expression of cardiac-specific antigens and those associated with cellular pluripotency was greater in SM− cells. Compared with SM− cells, SM+ cells exhibited greater expansion capacity. Freshly isolated SM− cells exhibited greater cardiac differentiation potential compared with freshly isolated SM+ cells (21.8 ± 0.3% of SM− cells positive for cardiac markers vs. 9.1 ± 0.7% of SM+ cells, P = 0.00009). Differentiated SM− cells acquired a cardiomyocytic phenotype and exhibited spontaneous rhythmic contractions in vitro. The number of Sca-1 + cells in the SM− population increased markedly with time (0.9 ± 0.1% in freshly isolated cells vs. 11.9 ± 0.9% after the first passage vs. 99.0 ± 0.6% after the second passage). This increase in Sca-1 expression was associated with a marked decline in the expression of cardiac markers following differentiation induction in culture-expanded SM− cells (21.8 ± 0.3% in unexpanded cells vs. 16.6 ± 1.3% after the first passage vs. 6.0 ± 0.5% after the second passage, P = 0.00001 vs. unexpanded cells). In contrast, the SM+ cells did not exhibit any consistent pattern in either phenotypic or differentiation capability with expansion. We conclude that SM− cells are inherently predisposed to undergo cardiac differentiation and are enriched in markers of pluripotency. While both Sca-1 + and Sca-1 − primitive cells from SM can undergo cardiac differentiation, Sca-1 − cells exhibit greater cardiomyogenic potential, and the appearance of Sca-1 during expansion is associated with a decline in cardiac differentiation plasticity.

GATA4 and the Two Sides of Gene Expression Reprogramming

See related article, pages 837–845 During embryonic development as well as in postnatal life, cardiac cells are continuously exposed to a host of extracellular signals to which they adapt and respond by activating intracellular signaling pathways and modulating the expression of nuclear genes. In the adult heart, the most common and easily recognized response to stressful stimuli is hypertrophy of cardiomyocytes and remodeling of the entire organ.1 This reactive growth response requires de novo synthesis of contractile and structural proteins, obtained through a complex series of events culminating in gene expression reprogramming.2 GATA4 is a zinc-finger transcription factor highly expressed in cardiomyocytes at different developmental stages where it regulates the transcription of several structural and regulatory genes, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), α- and β-myosin heavy chain (MHC).3 In human hearts, GATA4 has also been demonstrated to be a critical regulator of cardiac development, as shown by the association between GATA4 mutations and the presence of congenital cardiac malformations.4 The specific effects of GATA4 deletion during embryonic age have been extensively investigated in genetically modified mouse models.5–7 Mice homozygous for a GATA4 null allele6 or homozygous GATA4-deficient mice (GATA4−/−)5 die early in development because of abnormal …