Analysis of the factors that limit the ability of feeder cells to maintain the undifferentiated state of human embryonic stem cells.

Human embryonic stem cell (hESC) culture is routinely performed using inactivated mouse embryonic fibroblasts (MEFs) as a feeder cell layer (FL). Although these cells maintain pluripotency of hESCs, the molecular basis for this is unknown. Objectives of this study were to determine whether timing between MEF inactivation and their use as a FL influenced hESC growth and differentiation, and to begin defining the mechanism(s) involved. hESCs were plated on MEFs prepared 1 (MEF-1), 4 (MEF-4), and 7 (MEF-7) days earlier. hESC colony morphology and Oct3/4 expression levels were evaluated to determine the influence of different FLs. Significant enhancement of hESC growth (self-renewal) was observed on MEF-1 compared with MEF-4 and/or MEF-7. Conditioned media (CM) collected from MEF-1 supported significantly better hESC growth in a FL-free system compared to MEF-7 CM. Effects of MEFs on hESC growth were not caused by differences in cell density or viability, although indications of apoptosis were observed in MEF-7. Scanning electron microscopy demonstrated that MEF-7 were morphologically distinct from MEF-1 and MEF-4. Microarray analysis identified 19 genes related to apoptosis with significantly different levels of expression between MEF-1 and MEF-7. Several differentially expressed RNAs had gene ontology classifications associated with extracellular matrix (ECM) structural constituents and growth factors. Because members of Wnt signaling pathway were identified in the array analysis, we examined the ability of the Wnt1 CM and secreted frizzled-related proteins to affect hESC growth and differentiation. The addition of Wnt1 CM to both MEF-1 and MEF-7 significantly increased the number of undifferentiated colonies, while the addition of Sfrps promoted differentiation. Together, these results suggest that microenvironment, ECM, and soluble factors expressed by MEF-1 are significantly better at maintaining self-renewal and pluripotency of hESCs. Our findings have important implications in the optimization of hESC culture when MEFs are used as FL or CM is used in FL-free culture.

Transplacental RNAi: deciphering gene function in the postimplantation-staged embryo.

RNAi offers the opportunity to examine the role in postimplantation development of genes that cause preimplantation lethality and to create allelic series of targeted embryos. We have delivered constituitively expressed short hairpin (sh) RNAs to pregnant mice during the early postimplantation period of development and observed gene knockdown and defects that phenocopy the null embryo. We have silenced genes that have not yet been "knocked out" in the mouse (geminin and Wnt8b), those required during earlier cleavage stages of development (nanog), and genes required at implantation (Bmp4, Bmp7) singly and in combination (Bmp4 + Bmp7), and obtained unique phenotypes. We have also determined a role in postimplantation development of two transcripts identified in a differential display RT-PCR screen of genes induced in ES cells by noggin exposure, Aggf1 and an Est (GenBank AK008955). Systemic delivery of shRNAs provides a valuable approach to gene silencing in the embryo.

Gene silencing using RNA interference in embryonic stem cells.

Pluripotent embryonic stem (ES) cells are an important model system to examine gene expression and lineage segregation during differentiation. One powerful approach to target and inhibit gene expression, RNAi, has been applied to ES cells with the goal of teasing out the cascades of gene expression/repression that shape the early embryo. In this chapter, we describe the current understanding of the mechanisms of gene silencing by small hairpin RNAs, as well as controls and caveats to using this approach in ES cells. A consideration of synthetic vs plasmid-based RNAi vectors, design of targeting constructs, transfection of ES cells, and flow sorting of targeted cells is followed by methods for the analysis of phenotype and behavior of targeted cell populations using immunohistochemistry, reverse transcriptase polymerase chain reaction, Western blotting, and scanning electron microscopy.