Cardiomyocytes derived from human embryonic stem (ES) cells are a potential source for cell-based therapy for heart diseases. We studied the effect of bone morphogenetic protein (BMP)-4 in the presence of fetal bovine serum (FBS) on cardiac induction from human H1 ES cells during embryoid body (EB) development. Suspension culture for 4 days with 20% FBS produced the best results for the differentiation of early mesoderm and cardiomyocytes. The addition of Noggin reduced the incidence of beating EBs from 23.6% to 5.3%, which indicated the involvement of BMP signaling in the spontaneous cardiac differentiation. In this condition, treatment with 12.5–25 ng/ml BMP-4 during the 4-day suspension optimally promoted the cardiomyocyte differentiation. The incidence of beating EBs at 25 ng/ml BMP-4 reached 95.8% on day 6 of expansion and then plateaued until day 20. In real-time PCR analysis, the cardiac development-related genes MESP1 and Nkx2.5 were upregulated in the EB outgrowths by 25 ng/ml BMP-4. The activation of BMP signaling in EBs was confirmed by the increase in the phosphorylation of Smad1/5/8 and by the nuclear localization of phospho-Smad1/5/8 and Smad4. The addition of 150 ng/ml Noggin considerably decreased the incidence of beating EBs and Nkx2.5 expression, and Noggin alone increased Nestin expression and neural differentiation in EB outgrowths. The cardiomyocytes induced by 25 ng/ml BMP-4 showed proper cell biological characteristics and a course of differentiation as judged from isoproterenol administration, gene expression, protein assay, immunoreactivity, and subcellular structures. No remarkable change in the extent of apoptosis and proliferation in the cardiomyocytes was observed by BMP-4 treatment. These findings showed that BMP-4 in combination with FBS at the appropriate time and concentrations significantly promotes cardiomyocyte induction from human ES cells.
Human mesenchymal stem cells (hMSCs) that can differentiate into chondrocytes are a potential autologous cell source for repair of damaged tissue. Current methods usually induce the formation of all three chondrocyte phenotypes, hyaline, fibrous, and elastic, without the ability to selectively induce only one of them. By controlling the size of hMSC cell clusters, it may be possible to direct differentiation more uniformly toward hyaline chondrocytes. We designed new cell culture platforms containing microwells of different diameters. The platforms and wells were composed of a zirconia ceramics substratum. hMSCs briefly adhered to the substratum before releasing and entering the microwells. The physical restraints imposed by the microwells enabled hMSC clusters to homogenously differentiate into hyaline chondrocyte-like cells. Chondrogenic aggregates in microwells expressed the hyaline chondrocyte-specific genes Col II, aggrecan (ACAN), and cartilage oligomeric protein (COMP). The cultures also produced hyaline chondrocyte-specific matrix proteins Col II and ACAN homogenously throughout the aggregates. In contrast, chondrogenesis in pellet cultures was heterogeneous with the expression of nonhyaline chondrocyte genes CD105, Col X, and Col I. In these pellet cultures, hyaline and nonhyaline chondrocyte-specific matrix proteins were distributed heterogeneously. Thus, this novel ceramic microwell substratum technology efficiently directed the differentiation of hyaline chondrocyte-like cells from hMSCs. These results indicate that there is a close relationship between hMSC cluster size regulation in the microwells and differentiation tendency. This microwell culture differentiation method will provide a valuable experimental system for both experimental and potential clinical studies.
To induce hepatocytes from human embryonic stem (hES) cells easily and effectively, a simple suspension culture method that separates ES colonies with a scraper and transfers them into newly developed, nonadherent MPC (2-methacryloyloxyethyl phosphorylcholine) plates, and the staged-additional cocktail method, including growth factors, cytokines, and Lanford serum-free medium, were developed and evaluated mainly by morphological analysis. The formed embryoid bodies (EBs) showed compact cellular agglomeration until day 4 and later formed coeloms in their interior. RT-PCR (reverse transcriptase-polymerase chain reaction) analysis showed that they are gene markers of the three germ layers. Mesenchymal cells with rough endoplasmic reticulum (rER) and extracellular matrix (ECM), and without junctions, were recognized in the interior of the EBs by transmission electron microscopy (TEM) in addition to epithelial cells. When they were stimulated by the staged-additional cocktail, they expressed albumin-positive immunoreactivity, indocyanine green (ICG) uptake, and typical ultrastructures of the hepatocytes, including bile canaliculi. These results indicate that these combined methods promote EB formation and hepatocyte differentiation from hES cells.
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