Promoting human embryonic stem cell renewal or differentiation by modulating Wnt signal and culture conditions

Abstract

We previously showed that Wnt3a could stimulate human embryonic stem (hES) cell proliferation and affect cell fate determination. In the absence of feeder cell-derived factors, hES cells cultured under a feeder-free condition survived and proliferated poorly. Adding recombinant Wnt3a in the absence of feeder cell derived-factors stimulated hES cell proliferation but also differentiation. In the present study, we further extended our analysis to other Wnt ligands such as Wnt1 and Wnt5a. While Wnt1 displayed a similar effect on hES cells as Wnt3a, Wnt5a had little effect in this system. Wnt3a and Wnt1 enhanced proliferation of undifferentiated hES cells when feeder-derived self-renewal factors and bFGF are also present. To explore the possibility to promote the proliferation of undifferentiated hES cells by activating the Wnt signaling, we overexpressed Wnt3a or Wnt1 gene in immortalized human adult fibroblast (HAFi) cells that are superior in supporting long-term growth of undifferentiated hES cells than primary mouse embryonic fibroblasts. HAFi cells with or without a Wnt transgene can be propagated indefinitely. Over-expression of the Wnt3a gene significantly enhanced the ability of HAFi feeder cells to support the undifferentiated growth of 3 different hES cell lines we tested. Co-expression of three commonly-used drug selection genes in Wnt3a-overpressing HAFi cells further enabled us to select rare hES clones after stable transfection or transduction. These immortalized engineered feeder cells (W3R) that co-express growth-promoting genes such as Wnt3a and three drug selection genes should empower us to efficiently make genetic modified hES cell lines for basic and translational research.

De NovoMyocardial Regeneration: Advances and Pitfalls

“…These data were later supported by other research groups (14,67,73,86,88,113). Several studies have independently described cardiac stem=progenitor cell isolation from various species, including murine, rat, feline, pig, dog, and human, that display stem= progenitor cell characteristics (5,18,60,73,88,117).…”

“…Various strategies have been successfully adopted to address these issues. Genetic selection of differentiated ES cells or their partial differentiation into cardiomyocytes or endothelial cells in vitro before engraftment has given encouraging results in experimental animal models (13,14,34,76). Further insight from the heart development studies and discovery of the signaling pathways involved in ES cell differentiation will facilitate isolation of the pure population of cardiomyocytes, thereby limiting the risk of teratoma formation.…”

Proliferation and Pluripotency of Human Embryonic Stem Cells Maintained on Type I Collagen

“…sFRP-1 contains a domain homologous to the cysteine-rich domain of Fzd and will bind to WNT, inhibiting WNT's interaction with Fzd and blocking activation of the b-catenin pathway [60]. This pathway is known to have a key role in stem cell proliferation and maintenance of pluripotency through activation of NANOG and other factors such as OCT4 [61,[63][64][65][66][67][68][69], although increased WNT signaling has also been associated with hESC differentiation [70]. Thus, disruption of this pathway is a plausible mechanism for sFRP-1's inhibitory activity in our hESC growth assays.…”

Efficient Derivation and Genetic Modifications of Human Pluripotent Stem Cells on Engineered Human Feeder Cell Lines

“…The TW3R cell line was created by transducing TW2R with a lentiviral vector expressing puromycin resistance (Puro R ) gene. The retroviral and lentiviral vectors were previously described [18].…”

“…This underlies the need to develop systems that can sufficiently support human iPSC clonal growth and have the potential to be compatible with clinical production under current Good Manufacturing Practice (cGMP) standards. We have previously demonstrated that human adult marrow stromal cells [also called mesenchymal stem cells (MSCs)] could efficiently support undifferentiated growth of hESCs [16] and that selfrenewal of hESCs can be promoted by modulating the Wnt signaling pathway [17,18]. It has also been reported that MSCs facilitated the derivation of hESCs from cryopreserved poor-quality embryos [19].…”