FGF-4 increases in vitro expansion rate of human adult bone marrow-derived mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (MSCs) exhibit limited in vitro growth. Fibroblast growth factors (FGFs) elicit a variety of biological responses, such as cell proliferation, differentiation and migration. FGF-4 represents one of the FGFs with the highest cell mitogenic activity. We studied the effect of FGF-4 on MSCs growth and pluripotency. MSCs duplication time (Td) was significantly reduced with FGF-4 compared to controls (2.2 +/- 0.2 vs. 4.1 +/- 0.2 days, respectively; p = 0.03) while BMP-2 and SCF-1 did not exert a significant growth effect. MSC expression of surface markers, differentiation into adipogenic and osteogenic lineages, and baseline expression of cardiomyogenic genes were unaffected by FGF-4. In summary, exogenous FGF-4 increases the rate at which MSC proliferate and has no significant effect on MSC pluripotency.     

Brief report: benchmarking human pluripotent stem cell markers during differentiation into the three germ layers unveils a striking heterogeneity: all markers are not equal

Pluripotent stem cells (PSC) are functionally characterized by their capacity to differentiate into all the cell types from the three germ layers. A wide range of markers, the expression of which is associated with pluripotency, has been used as surrogate evidence of PSC pluripotency, but their respective relevance is poorly documented. Here, we compared by polychromatic flow cytometry the kinetics of loss of expression of eight widely used pluripotency markers (SSEA3, SSEA4, TRA-1-60, TRA-1-81, CD24, OCT4, NANOG, and alkaline phosphatase [AP]) at days 0, 5, 7, and 9 after induction of PSC differentiation into cells representative of the three germ layers. Strikingly, each marker showed a different and specific kinetics of disappearance that was similar in all the PSC lines used and for all the induced differentiation pathways. OCT4, SSEA3, and TRA-1-60 were repeatedly the first markers to be downregulated, and their expression was completely lost at day 9. By contrast, AP activity, CD24, and NANOG proteins were still detectable at day 9. In addition, we show that differentiation markers are coexpressed with pluripotency markers before the latter begin to disappear. These results suggest that OCT4, SSEA3, and TRA-1-60 might be better to trace in vitro the emergence of pluripotent cells during reprogramming.     

Epigenetic signatures and temporal expression of lineage-specific genes in hESCs during differentiation to hepatocytes in vitro

Embryonic stem cells (ESCs) maintain unique epigenetic states to maintain their pluripotency. Differentiation of ESCs into specialized cell types requires changes in these epigenetic states. However, the dynamics of epigenetic marks found in hESCs during differentiation are poorly understood. Here, we report the variation in the dynamics of epigenetic modifications associated with the expression of lineage-specific genes during differentiation of hESCs to hepatocytes in vitro. The promoter regions of pluripotency marker genes characterized by permissive histone marks such as trimethylation of H3 at lysine 4 (H3K4me3) and acetylation of H3 at lysine 9 (H3K9ac) in hESCs were instead enriched with repressive histone marks such as dimethylation of H3 at lysine 9 (H3K9me2), trimethylation of H3 at lysine 9 (H3K9me3) and trimethylation of H3 at lysine 27 (H3K27me3) during differentiation to hepatocytes. Interestingly, expression of definitive endoderm marker genes containing bivalent and non-bivalent domains may be modulated by a marked reduction in H3K27me3 and a significant enhancement of permissive marks such as H3K4me3 and H3K9ac during hESC differentiation. Expression of hepatocyte marker genes regulated by histone modifications was similar to that of pluripotency marker genes. Our findings provide insight into the epigenetic mechanisms regulating expression of developmental genes. Of particular interest, they may be differentially regulated either in a bivalent or non-bivalent domain manner during hESC differentiation.     

Transduction of Wnt11 promotes mesenchymal stem cell transdifferentiation into cardiac phenotypes

Transplantation of mesenchymal stem cells (MSCs) has emerged as a potential treatment for ischemic heart repair. Previous studies have suggested that Wnt11 plays a critical role in cardiac specification and morphogenesis. In this study, we examined whether transduction of Wnt11 directly increases MSC differentiation into cardiac phenotypes. MSCs harvested from rat bone marrow were transduced with both Wnt11 and green fluorescent protein (GFP) (MSC(Wnt11)) using the murine stem cell virus (pMSCV) retroviral expression system; control cells were only GFP-transfected (MSC(Null)). Compared with control cells, MSC(Wnt11) was shown to have higher expression of Wnt11 by immunofluorescence, real-time polymerase chain reaction, and western blotting. MSC(Wnt11) shows a higher expression of cardiac-specific genes, including GATA-4, brain natriuretic peptide (BNP), islet-1, and α-actinin, after being cultured with cardiomyocytes (CMs) isolated from ventricles of neonatal (1-3 day) SD rats. Some MSC(Wnt11) were positive for α-actinin when MSCs were cocultured with native CMs for 7 days. Electron microscopy further confirmed the appearance of sarcomeres in MSC(Wnt11). Connexin 43 was found between GFP-positive MSCs and neonatal rat CMs labeled with red fluorescent probe PKH26. The transdifferentiation rate was significantly higher in MSC(Wnt11) than in MSC(Null), as assessed by flow cytometry. Functional studies indicated that the differentiation of MSC(Wnt11) was diminished by knockdown of GATA-4 with GATA-4-siRNA. Transduction of Wnt11 into MSCs increases their differentiation into CMs by upregulating GATA-4.     

Suspension cultures of bone-marrow-derived mesenchymal stem cells: effects of donor age and glucose level

Both ageing and diabetes are associated with reduced numbers and functional viability of mesenchymal stem cells (MSCs) in vivo which in turn lead to degenerative pathologies of the musculoskeletal system. The overall aim of this study was to elucidate the effects of age and raised glucose levels on the proliferation and self-renewal of rat nonadherent bone marrow MSCs (Na-BM-MSCs) in suspension cultures. MSC cultures isolated from 3- and 12-month-old rats were maintained using the "pour-off" method for up to 14 days in media containing different glucose levels and the phenotype, growth characteristics, colony forming unit-fibroblastic (CFU-f) numbers, and pluripotency characteristics of these cells were determined. This study indicates that rat adult bone marrow harbors pluripotent Na-BM-MSCs that seem to be unaffected by ageing during in vitro expansion. The Na-BM-MSCs express the pluripotency markers Oct4, Sox2, and Nanog. It was found that culture in high-glucose-containing medium had a negative effect on colony formation and differentiation. In contrast to classical MSC cultures, the generation of colonies by Na-BM-MSCs in suspension culture was not reduced in the older animals. The Na-BM-MSCs were found to express the pluripotency markers Oct4, Sox2, and Nanog, suggesting a more primitive stage of differentiation as compared with adherent MSCs. These data indicate that rat adult bone marrow harbors a population of pluripotent Na-BM-MSCs that appear to be relatively unaffected by ageing during in vitro expansion in suspension.     

Embryonic NANOG Activity Defines Colorectal Cancer Stem Cells and Modulates through AP1- and TCF-dependent Mechanisms

Embryonic NANOG (NANOG1) is considered as an important regulator of pluripotency while NANOGP8 (NANOG-pseudogene) plays a role in tumorigenesis. Herein, we show NANOG is expressed from both NANOG1 and NANOGP8 in human colorectal cancers (CRC). Enforced NANOG1-expression increases clonogenic potential and tumor formation in xenograft models, although it is expressed only in a small subpopulation of tumor cells and is colocalized with endogenous nuclear β-catenin(High) . Moreover, single NANOG1-CRCs form spherical aggregates, similar to the embryoid body of embryonic stem cells (ESCs), and express higher levels of stem-like Wnt-associated target genes. Furthermore, we show that NANOG1-expression is positively regulated by c-JUN and β-catenin/TCF4. Ectopic expression of c-Jun in murine Apc(Min/+) -ESCs results in the development of larger xenograft tumors with higher cell density compared to controls. Chromatin immunoprecipitation assays demonstrate that c-JUN binds to the NANOG1-promoter via the octamer M1 DNA element. Collectively, our data suggest that β-Catenin/TCF4 and c-JUN together drive a subpopulation of CRC tumor cells that adopt a stem-like phenotype via the NANOG1-promoter. STEM Cells2012;30:2076-2087.     

CD49f enhances multipotency and maintains stemness through the direct regulation of OCT4 and SOX2

CD49f (integrin subunit α6) regulates signaling pathways in a variety of cellular activities. However, the role of CD49f in regulating the differentiation and pluripotency of stem cells has not been fully investigated. Therefore, in this study, human mesenchymal stem cells (hMSCs) were induced to form spheres under nonadherent culture conditions, and we found that the CD49f-positive population was enriched in MSC spheres compared with MSCs in a monolayer. The expression of CD49f regulated the ability of hMSCs to form spheres and was associated with an activation of the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway. Furthermore, the forced expression of CD49f modulated the proliferation and differentiation potentials of hMSCs through prolonged activation of PI3K/AKT and suppressed the level of p53. We showed that the pluripotency factors OCT4 and SOX2 were recruited to the putative promoter region of CD49f, indicating that OCT4 and SOX2 play positive roles in the expression of CD49f. Indeed, CD49f expression was upregulated in human embryonic stem cells (hESCs) compared with hMSCs. The elevated level of CD49f expression was significantly decreased upon embryoid body formation in hESCs. In hESCs, the knockdown of CD49f downregulated PI3K/AKT signaling and upregulated the level of p53, inducing differentiation into three germ layers. Taken together, our data suggest that the cell-surface protein CD49f has novel and dynamic roles in regulating the differentiation potential of hMSCs and maintaining pluripotency.     

3种不同来源CD146阳性间充质干细胞亚群的生物学特性比较

目的比较脐带间充质干细胞(UCMSCs)、脂肪间充质干细胞(AMSCs)以及骨髓间充质干细胞(BM-MSCs)中CD146^+细胞亚群的生物学特性。方法用磁珠分选法分选不同组织来源的间充质干细胞,获得高纯度CD146^+亚群;用流式细胞计量术分析表型;透射电子显微镜观察细胞结构;成脂诱导分化后油红O染色;RT-qPCR检测成脂相关基因LPL、C/EBPα和PPARγ表达;成骨诱导分化后ALP染色,检测成骨相关基因ALP、OPN和RUNX2表达;检测细胞干性基因及血管生成相关基因表达;检测细胞体外成管能力。结果3种组织来源CD146^+MSCs具有相似的形态,表达除CD106外相似的细胞表面标志分子。与其他两种细胞比较,CD146^+AMSCs表达更高的干性基因OCT-4、SOX2和NANOG。在相同的诱导时间,CD146^+UCMSCs成脂和成骨能力较其他两种来源的CD146^+MSCs弱。3种组织来源的CD146^+MSCs均可表达血管内皮相关刺激因子BFGF、VEGF、Ang-1和EGF,但CD146^+BM-MSCs具有更强的体外成管能力。结论3种不同来源的CD146^+MSC具有不同的生物学特性,为进一步研究不同组织来源的间充质干细胞的特定应用提供了基础。