Cardiomyocyte differentiation of perinatally­derived mesenchymal stem cells.

Coronary heart disease is major cause of mortality worldwide and several risk factors have been shown to play a role in its pathogenesis, including smoking, obesity, hypertension and hypercholesterolemia. A number of therapeutic methods have been developed to improve the quality of patients' lives, including stem cell therapy using mesenchymal stem cells (MSCs). Perinatal sources, including the placenta (PL) and umbilical cord (UC), are rich sources of MSCs and have been identified as a potential source of cells for therapeutic use. Their role in cardiogenic differentiation is also of contemporary medical interest. The present study demonstrated the induced differentiation of MSCs obtained from the UC, PL and Wharton's jelly (WJ) into cardiomyocytes, using 10 µM 5­azacytidine. The characteristics of the MSCs from each source were studied and their morphology was compared. An immunofluorescence analysis for the cardiac­specific markers, GATA4 and Troponin T (TnT), was performed and tested positive in all sources. The expression of the cardiac­specific genes, Nkx2.5, α­cardiac actin and TnT, was analyzed by real­time RT­PCR and presented as fold change increases. The expression of each of the markers was observed to be higher in the 5­azacytidine­treated MSCs. The differences in expression among the sources of treated MSCs was as follows: TnT had the highest level of expression in the bone marrow (BM) MSCs; α­cardiac actin had the highest level of expression in the PLMSCs; and all the genes were expressed at significantly high levels in the WJMSCs compared with the control group. The present study showed the ability of alternative perinatally­derived MSCs to differentiate into cardiomyocyte­like cells and how this affects the therapeutic use of these cells.

Cardiogenic and myogenic gene expression in mesenchymal stem cells after 5-azacytidine treatment.

OBJECTIVE: 5-Azacytidine is a hypomethylating agent that is used for the treatment of myelodysplastic syndrome. This histone modifier is widely employed and plays a nonspecific role in influencing the differentiation capability of stem cells. The ability of bone marrow mesenchymal stem cells to differentiate into cardiomyocyte- and myocyte-like cells after exposure to 3 different doses of 5-azacytidine has been evaluated and compared. The aim of the study was to optimize the effective dose of 5-azacytidine for promoting the cardiomyocyte and myocyte differentiation capabilities of human mesenchymal stem cells (MSCs). MATERIALS AND METHODS: Human bone marrow aspirations were collected from healthy donors. MSCs were used for the study of mesodermal differentiation. MSCs were cultured to promote osteoblast differentiation and adipocyte differentiation. The evaluation of osteogenic or adipogenic properties was then performed through immunocytochemical staining. BMMSCs were trypsinized into single-cell suspensions and then prepared for flow cytometric analysis. The MSCs were treated with 5, 10, or 15 µM 5-azacytidine for 24 h and then cultured for 3 weeks. Total RNA was extracted from untreated and 5-azacytidine-treated cells. Troponin T and GATA4 antibodies were used as cardiogenic markers, whereas myogenin and MyoD antibodies were used as myocyte markers. RESULTS: The morphology and growth rate of MSCs that were treated with any of the 3 doses of 5-azacytidine were similar to the morphology and growth rate of control MSCs. An immunofluorescence analysis examining the expression of the cardiac-specific markers GATA4 and troponin T and the skeletal muscle-specific markers MyoD and myogenin revealed that cells treated with 15 µM 5-azacytidine were strongly positive for these markers. Real-time RT-PCR results were examined; these amplifications indicated that there were higher expression levels of cardiac- and skeletal muscle-specific mRNAs in MSCs treated with 15 µm 5-azacytidine than in MSCs that had either been treated with lower doses of 5-azacytidine or left untreated. CONCLUSION: MSCs treated with 5-azacytidine demonstrated the capacity to differentiate into both cardiomyocytes and skeletal myocytes, and 15 µM 5-azacytidine could be the optimal dose of this drug. Other promoting factors should be examined to investigate the possibility of promoting the differentiation of MSCs into specific cell types. CONFLICT OF INTEREST: None declared.

Alginate microcapsule as a 3D platform for propagation and differentiation of human embryonic stem cells (hESC) to different lineages.

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche(1-3). The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment(4) such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures(2). These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded(5). Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions(2) to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes(1). We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, >100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

In vitro vessel-forming capacity of endothelial progenitor cells in high glucose conditions.

In type 2 diabetes, the impairment of vascular repair processes and angiogenesis are due to endothelial progenitor cell (EPC) dysfunction. In this study, we established a quantitative methodology to assess EPC function by using an in vitro 5-(6)-carboxyfluorescein diacetate succinimidyl ester-labeling vessel formation assay. The EPCs were cultured in three different glucose concentrations (100, 189.5, and 295.5 mg/dl of D: -glucose) representing normal control and diabetes with either good or poor glycemic control, respectively. We found that the in vitro vessel-forming capacity was impaired in EPCs cultured in high glucose concentrations compared to normal control (43.4 ± 0.8% and 34.7 ± 0.7% vs. 50.8 ± 2.1%). We further studied expression of various genes involved in vessel formation. There was a lower level of angiopoietin 1 gene expression in EPCs cultured in high glucose concentrations. The addition of recombinant angiopoietin 1 significantly increased the vessel-forming capacity of EPCs cultured in high glucose concentration (35.3 ± 2.0% to 48.8 ± 2.7%), whereas the addition of angiopoietin 2 (a competitive inhibitor of angiopoietin 1) impaired the vessel-forming capacity of EPCs cultured in normal glucose concentration (51.8 ± 1.3% to 41.3 ± 0.6%). We conclude that the in vitro vessel-forming capacity of EPCs cultured in high glucose concentration is impaired due to low levels of angiopoietin 1.

CD14-/CD34+ is the founding population of umbilical cord blood-derived endothelial progenitor cells and angiogenin1 is an important factor promoting the colony formation.

The origin of endothelial progenitor cells (EPCs) in umbilical cord blood (UCB) is unknown. In this study, we explored the origin of UCB-derived EPCs by culturing CD14+ or CD14- subpopulation separately and co-culturing these two subpopulations either with or without transwells. We found no colony formation with CD14+ or CD14- subpopulation alone, but there were EPC colonies observed in direct co-cultures of both subpopulations. Transwell culture system was used to further study the effect of cytokines on EPC colony formation. We observed the presence of EPC colonies derived from CD14- subpopulation in the presence of CD14+ subpopulation in the upper compartment whereas there was no colony generated from CD14+ subpopulation with CD14- subpopulation in the upper compartment. Therefore, CD14- subpopulation is likely to be the origin of EPCs and EPC colony derivation requires cytokines released from CD14+ subpopulation. We further characterized the founding population of UCB-derived EPCs by separating CD14- subpopulation into CD14-/CD34+ and CD14-/CD34- subpopulations. There were colonies observed only in co-cultures of CD14+ with CD14-/CD34+ subpopulation but not with CD14-/CD34- subpopulation either with or without transwells. We screened 42 cytokines involving in angiogenesis using an ELISA array in the supernatant collected from CD14+ compared to CD14- subpopulations. We found consistently the presence of angiogenin1 in the supernatant of CD14+ subpopulation but not in that of CD14- subpopulation. The addition of angiogenin1 in culture of CD14- subpopulation yielded EPC colonies. We conclude that UCB-derived EPCs are confined to CD14-/CD34+ subpopulation and angiogenin1 released from CD14+ subpopulation may be an important factor promoting the EPC colony formation.

Alginate microcapsule for propagation and directed differentiation of hESCs to definitive endoderm.

Human embryonic stem cells (hESCs) are potential renewable sources of cells in replacement therapies for many diseases including type 1 diabetes. We have established a three dimensional (3D) model to culture and differentiate hESCs that are encapsulated in calcium alginate microcapsules. This system promotes cellular interactions that are essential for both maintaining pluripotency and differentiation. This 3D model also provides opportunity to separate out hESCs from fibroblasts used as feeder layer during culture. In this study, we compared the viability and proliferation of the encapsulated hESCs cultured in serum replacement (SR) medium, human fetal fibroblast-conditioned medium (hFF-CM), in the presence and absence of Y-27632, a ROCK inhibitor. Treatment of hESCs with Y-27632 promoted cell survival, cell cluster formation and proliferation rate in both SR medium and hFF-CM. These encapsulated hESC clusters were then directly differentiated to definitive endoderm cells that expressed mesendoderm (Brachyury 70-fold), definitive endoderm (SOX17>300-fold, FOXA2>800-fold, and CXCR4>100-fold) and primitive gut tube (HNF1beta>120-fold) as compared to the undifferentiated hESCs. These data show that microcapsules can be used for differentiation of hESCs into definitive endoderm in 3D and could have potential application for immune-isolation and prevention of teratomas formation of hESCs during transplantation.