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

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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78130/1/scd.2008.0010.pd

Tri-lineage Differentiation of Embryonic Stem Cells: Role of Wnt Signaling.

Since embryonic stem cells (ESC) and the epiblast share a gene expression profile and an attenuated cell cycle, ESC are an attractive model system to study lineage choice at gastrulation. We have employed mouse ESC as a model system to examine the role of Wnt signaling in the transition from pluripotency to neural precursors and have identified a new function for the Geminin gene in mesendodermal differentiation. Wnt signaling is required for differentiation of both epiblast and ESC to the mesendodermal lineage. To test the hypothesis that blocking Wnt signaling in the absence of BMP signaling would promote differentiation of mESC to neural precursors, we developed a mESC line that inducibly expresses a dominant negative Tcf4 (dnTcf4) protein to block canonical Wnt signaling. Cells expressing the dnTcf4 protein differentiated largely to Sox3 positive neural precursors but were unable to progress to beta-III tubulin positive neurons unless Wnt signaling was de-repressed. This cell line will provide an important resource for other investigators studying the role of Wnt signaling in embryonic development. Geminin has been described as a bi-functional protein that inhibits endoreduplication during cell division and promotes neural differentiation by inhibiting BMP signaling. Geminin also binds transcription factors and chromatin remodeling proteins in the nucleus to inhibit their function. In mESC over-expression of Geminin induced mesendodermal differentiation and an epithelial to mesenchymal transition (EMT) via increasing Wnt signaling. We hypothesize that Geminin binds to Groucho/TLE proteins in the nucleus thereby dis-inhibiting Wnt target gene transcritption by Tcf/Lef proteins. Germinin is frequently over-expressed in cancer cells and EMT is an integral part of cancer metastasis. These observations make Geminin an attractive candidate for cancer therapy; reduction of Geminin protein could reduce metastasis as well as induce cell death due to endoreduplication. Overall, this work demonstrates that Wnt signaling is required for mesendodermal differentiation, EMT, and the transition from proliferative neural precursor to primitive neurons during lineage allocation of mESC.Ph.D.Cell and Developmental BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/77753/1/nslawny_1.pd