ESCs are generally isolated from the inner cell masses (ICMs) of blastocysts, which consist of pluripotent cell populations that are able to generate the primitive ectoderm during embryogenesis (Fig. 1). More specifically, in normal embryonic development, the primitive ectoderm gives rise during the gastrulating process to the primary germ layers, including ectoderm, mesoderm, and endoderm. These three germ layers might subsequently generate a variety of organized tissue structures involving complex epithelial-mesenchymal interactions. Similarly, the injection of ESC-derived progenitors into severe combined immunodeficient (SCID) mice might also result in the formation of teratomas corresponding to the complex structures containing the differentiated cell types from three germ layers [21, 22]. Moreover, ESCs can generate multiple cell progenitors that express the specific markers of three germ layers in vitro, including endoderm (α-fetoprotein and α1-antitrypsin), mesoderm (ζ-globin, enolase, kallikrein, cartilage matrix protein, myosin heavy chain, and muscle actin), and ectoderm (68-kDa neurofilament, class III β-tubulin, and keratin) [21, 22]. In this matter, the ESCs, when cultured in suspension in vitro, are able to spontaneously form embryoid bodies (EBs) that consist of spheres containing a variety of more differentiated progenitor cell types. The partial disaggregating and subculture of EBs might allow for the isolation and differentiation of a particular progenitor cell type that might be isolated on the basis of the expression of specific cellular markers. More particularly, the use of specific growth factors or cytokines during the outgrowth of EBs in culture in vitro might induce their differentiation into the specific cell lineages (Fig. 1) [21, 22, 42, 43]. For instance, it has been reported that the nerve growth factor (NGF) and hepatocyte growth factor (HGF) might induce the differentiation of ESC progenitors into cells from three embryonic germ layers, whereas EGF, basic fibroblast growth factor (bFGF, also known as fibroblast growth factor 2 [FGF-2]), retinoic acid (RA), and bone morphogenic protein-4 (BMP-4) instead generate the progenitors expressing the ectodermal and mesodermal markers . Furthermore, the differentiation of hESCs in the presence of the transforming growth factor-β (TGF-β) family-related protein, activin A, and a weak serum level might also generate a population containing up to 80% endodermal cells whose progenitors might be further enriched by using the cell-surface receptor CXCR4 . More recently, the selective differentiation of hESCs into neural, definitive endoderm/pancreatic, and cardiac progenitors has also been performed on a Matrigel-coated surface under chemically defined conditions by using Noggin, activin A, and activin A plus BMP, respectively . Among the ESC progenitors, there are the hematopoietic cell lineages, neuron-like cells, glial progenitors, dendritic cells, cardiomyocytes, skin cells, lung alveoli, hepatocytes, pancreatic islet-like cells, osteoblasts, chondrocytes, adipocytes, muscle cells, endothelial cells, and retinal cells (Table 1) [16, 19, 21, 22, 42, 43, 46, , , –50]. More recently, in vitro derivation of hESCs into large amounts of functional osteogenic cells has also been performed without the intermediate step of the EB formation, by separation of the hESC population into single cells . However, mouse and human ESCs express different marker profiles and might respond differently to certain growth factors, giving rise to distinct cell lineage progenitors. Therefore, these interspecies differences underline the importance of further establishing the particular differentiation pathways of hESC-derived progenitors for their clinical applications in humans.
One of the critical steps in the purification procedure appears to be the enrichment of EB-derived progenitor cells by the elimination of pluripotent and undifferentiated stem cells. Indeed, the elimination of undifferentiated stem cells that may form teratomas or teratocarcinomas in vivo appears to be essential for generating transplantable sources of differentiated stem cell progenitors for the treatment of diverse disorders [51, –53]. As a matter of fact, it has been reported that the presence of less than 0.2% of specific markers of undifferentiated ESCs Oct-4 and SSEA-1 in the preparation of insulin-producing β-cells, which was detected in graft tissues by different techniques, including flow cytometry and immunohistochemistry, was sufficient to generate teratomas in vivo . Therefore, the purification stages for the enrichment of differentiated ESC progenitors and the elimination of pluripotent and undifferentiated ESCs are essential for the development of safe ESC-based therapies. In this matter, it has been reported that the enrichment of nestin-positive neuroprogenitors in EB-derived cell populations and grafts in vivo may be performed by the elimination of pluripotent and proliferative Oct-4+/prostate apoptosis response-4+ (PAR-4+) ESCs . In fact, the selective apoptotic death of residual Oct-4+/PAR-4+ ESCs might be induced by the overexpression of PAR-4 or the treatment of these cells with ceramide analogs such as N-oleoyl serinol (S18), which also activate the PAR-4 pathway . The activation of the PAR-4 pathway might lead to the downregulation of several survival cascades that are mediated through the atypical protein kinase Cζ, mitogen-activated protein kinase (MAPK), and nuclear factor-κB (NF-κB). Thus, the use of ceramide analogs might result in the selective elimination of the pluripotent and teratogenic Oct-4+/PAR-4+ ESCs during neural differentiation, and thereby prevent teratoma formation after the transplantation of EB-derived progenitors in brain. Moreover, the elimination of Oct-4+ undifferentiated ESCs from the EBs, which has been performed by Percoll centrifugation and magnetic cell sorting, also produced a transplantable source of hepatocytes that formed no teratomas in vivo .