E-cadherin-expressing feeder cells promote neural lineage restriction of human embryonic stem cells.

Human embryonic stem cells (hESCs) represent a promising source of tissues of different cell lineages because of their high degree of self-renewal and their unique ability to give rise to most somatic cell lineages. In this article, we report on a new approach to differentiate hESCs into neural stem cells that can be differentiated further into neuronal restricted cells. We have rapidly and efficiently differentiated hESCs into neural stem cells by presenting the cell adhesion molecule, E-cadherin, to undifferentiated hESCs via E-cadherin transfected fibroblast monolayers. The neural restricted progenitor cells rapidly express nestin and beta-III-tubulin, but not glial fibrillary acidic protein (GFAP) during the 1-week E-cadherin induction phase, suggesting that E-cadherin promotes rapid neuronal differentiation. Further, these cells are able to achieve enhanced neuronal differentiation with the addition of exogenous growth factors. Cadherin-induced hESCs show a loss in Oct4 and nestin expression associated with positive staining for vimentin, neurofilament, and neural cell adhesion molecule. Moreover, blocking by functional E-cadherin antibody and failure of paracrine stimulation suggested that direct E-cadherin engagement is necessary to induce neural restriction. By providing hESCs with molecular cues to promote differentiation, we are able to utilize a specific cell-cell adhesion molecule, E-cadherin, to influence the nature and degree of neural specialization.

Expedited growth factor-mediated specification of human embryonic stem cells toward the hepatic lineage.

Human embryonic stem cells (hESCs) have the potential to be a promising source of liver cells, hepatocytes, for regenerative medicine given their unlimited proliferative and pluripotent differentiative capacity. However, the inefficient embryoid body process and limited understanding of molecular signals potentiating cell-specific differentiation plague the use of hESCs as a hepatic source. In this study, we describe an efficient growth factor-based process for directed differentiation of hESCs that bypasses embryoid body development. The system involves adherent hESC culture exposure to activin A treatment followed by incorporation of various growth factor combinations composed of dexamethasone, oncostatin M, hepatocyte growth factor, and Wnt3A. The hESC-derived hepatocyte-like cells resulting from optimal growth factor combinations exhibit characteristic hepatocyte morphology, express hepatocyte markers, and possess hepatospecific functional activity. The differentiated cultures express hepatic-related genes shown by reverse transcription-polymerase chain reaction and immunofluorescence analysis revealed binucleated cells with coexpression of albumin/cytokeratin 18. Furthermore, the hESC-derived hepatocyte-like cells exhibit functional hepatic characteristics, such as indocyanine green uptake and release, albumin secretion, and inducible cytochrome P450 activity. This directed differentiation of adherent hESCs offers an efficient process to produce hepatocyte-like cells in vitro for hepatocyte differentiation studies and organotypic cultures for diagnostic and therapeutic applications.

Enhanced differentiation of embryonic stem cells using co-cultivation with hepatocytes.

We examined the effects of co-cultivated hepatocytes on the hepatospecific differentiation of murine embryonic stem (ES) cells. Utilizing an established mouse ES cell line expressing high or low levels of E-cadherin, that we have previously shown to be responsive to hepatotrophic growth factor stimulation (Dasgupta et al., 2005. Biotechnol Bioeng 92(3):257-266), we compared co-cultures of cadherin-expressing ES (CE-ES) cells with cultured rat hepatocytes, allowing for either paracrine interactions (indirect co-cultures) or both juxtacrine and paracrine interactions (direct co-cultures, random and patterned). Hepatospecific differentiation of ES cells was evaluated in terms of hepatic-like cuboidal morphology, heightened gene expression of late maturation marker, glucose-6-phosphatase in relation to early marker, alpha-fetoprotein (AFP), and the intracellular localization of albumin. Hepatocytes co-cultured with growth factor primed CE-ES cells markedly enhanced ES cell differentiation toward the hepatic lineage, an effect that was reversed through E-cadherin blockage and inhibited in control ES cells with reduced cadherin expression. Comparison of single ES cell cultures versus co-cultures show that direct contact co-cultures of hepatocytes and CE-ES cells maximally promoted ES cell commitment towards hepatodifferentiation, suggesting cooperative effects of cadherin-based juxtacrine and paracrine interactions. In contrast, E-cadherin deficient mouse ES (CD-ES) cells co-cultured with hepatocytes failed to show increased G6P expression, confirming the role of E-cadherin expression. To establish whether albumin expression in CE-ES cells was spatially regulated by co-cultured hepatocytes, we co-cultivated CE-ES cells around micropatterned, pre-differentiated rat hepatocytes. Albumin localization was enhanced "globally" within CE-ES cell colonies and was inhibited through E-cadherin antibody blockage in all but an interfacial band of ES cells. Thus, stem cell based cadherin presentation may be an effective tool to induce hepatotrophic differentiation by leveraging both distal/paracrine and contact/juxtacrine interactions with primary cells of the liver.