hEB formation has been utilized as an initial step in a wide range of studies aimed at differentiating hESCs into a specific desired cell type. Alternative strategies, utilizing specific growth factor combinations and cell-cell induction systems, have enhanced differentiation of hEBs into a desired lineage. Schuldiner and colleagues were the first to examine the exposure of differentiating hESCs to growth factors.68 Five day-old-hEBs were plated on a Petri dish and grown in the presence of eight growth factors known for their ability to direct the differentiation of mESCs basic fibroblast growth factor (bFGF); transforming growth factor | (TGF|0; activin-A, bone morphogenic protein 4 (BMP-4); hepatocyte growth factor (HGF); epidermal growth factor (EGF); | nerve growth factor (|NGF); and retinoic acid.68 Differentiation was examined by the expression of 24 cell-specific molecular markers that cover all embryonic germ layers and 11 different tissues. The results showed that Activin-A and TGF|1 induced differentiation primarily into mesodermal cells; retinoic-acid, EGF, BMP-4 and bFGF induced differentiation into endodermal and mesodermal markers; and NGF and HGF allowed differentiation into three embryonic germ layers. None of the growth factors directed the differentiation exclusively to one cell type, indicating that multiple human cell types maybe enriched in vitro by specific factors.68
Detailed studies have examined the use of | nerve growth factor (bNGF) and retinoic acid (RA), showing an increase in the proportion of neuronal cells.69'10 Moreover, three protocols have been established for the in vitro differentiation, enrichment and transplantation of neural precursors11'12 One study showed that, upon aggregation to hEBs, differentiating hESCs formed large numbers of neural tube-like structures in the presence of FGF-2 which can be isolated by selective enzymatic digestion and further purified on the basis of differential adhesion.11 Withdrawal of FGF-2 resulted in their differentiation into neurons, astrocytes and oligodendrocytes.11 These progenitors further differentiated into both neurons and astrocytes after hES cell-derived neural precursors were transplanted into the neonatal mouse brain.11 Another protocol observed a neural precursor population in hEBs grown in suspension in serum-free conditions, in the presence of 50% conditioned medium from the human hepatocarcinoma cell line HepG2.12
Recently it was demonstrated that, using a protocol consisting of several steps, insulin-producing cells can be generated from hESCs.13 The procedure included the following phases: culturing and plating of hEBs in insulin-transferrin-selenium-fibronectin medium; supplementation of medium with N2, B27, and bFGF; reduction of glucose concentration in the medium, withdrawal of bFGF; addition of nicotinamide; and, finally, dissociation of the cells and re-growing them in suspension. This process resulted in the formation of clusters which exhibited higher insulin secretion and had longer durability than cells grown as monolayers. In addition to insulin, most cells also co-expressed glucagon or somatostatin, indicating similarity to immature pancreatic cells.73
Enrichment of cardiomyocytes was achieved when 4-6 day-old hEBs were treated with 5-aza-2'-deoxycytidine.74 Moreover, the differentiated cultures could be dissociated and separated via Percoll gradient density centrifuga-tion, which led to a population of 70% pure cardiomyocytes. Hematopoietic cells were also derived from hESCs by co-culturing them with murine bone marrow cell lines and yolk sac endothelial cell lines.75 Treating formed hEBs with hematopoietic cytokines (SCF, Flt3L, IL-3, IL-6 and G-CSF) demonstrated a six-fold increase in the proportion of CD45+ cells co-expressing CD34.63 The addition of these cytokines plus BMP-4 increased the frequency of CD45+CD34+ cells by six-fold. It seems that BMP-4 promotes the self-renewal of hESC-derived hemetopoietic progenitors.63 In a continuous study, it was further shown that vascular endothelial growth factor A (VEGF-A165) selectively promotes erythropoietic development from hESCs. Effects of VEGF-A165 were dependent on the presence of hematopoietic cytokines and BMP-4, and could be augmented by the addition of erythropoietin (EPO). Treatment of hEBs with VEGF-A165 increased the frequency of cells co-expressing CD34 and VEGF receptor 2 (KDR), as well as cells expressing erythroid markers. In addition to promoting erythropoietic differentiation from hESCs, the presence of VEGF-A165 enhanced the in vitro self-renewal potential of primitive hematopoietic cells capable of erythroid progenitor capacity.76
For the induction of trophblasts, hEBs were explanted into Matrigel "rafts" for up to 53 days.66 This resulted in small protrusions which appeared on the outer surface of hEBs, as well as an increase in secretion of hCG, progesterone and estradiol-17beta which remained dramatically elevated over the culture period. In comparison, hEBs maintained in suspension culture failed to demonstrate this elevation in hormone secretion.66
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