Ethical and moral issues rule out the use of human induced pluripotent stem cells (iPSCs) in chimera studies that would determine the full extent of their reprogrammed state, instead relying on less rigorous assays such as teratoma formation and differentiated cell types. To date, only mouse iPSC lines are known to be truly pluripotent. However, initial mouse iPSC lines failed to form chimeric offspring, but did generate teratomas and differentiated embryoid bodies, and thus these specific iPSC lines were not completely reprogrammed or truly pluripotent. Therefore, there is a need to address whether the reprogramming factors and process used eventually to generate chimeric mice are universal and sufficient to generate reprogrammed iPSC that contribute to chimeric offspring in additional species. Here we show that porcine mesenchymal stem cells transduced with 6 human reprogramming factors (POU5F1, SOX2, NANOG, KLF4, LIN28, and C-MYC) injected into preimplantation-stage embryos contributed to multiple tissue types spanning all 3 germ layers in 8 of 10 fetuses. The chimerism rate was high, 85.3% or 29 of 34 live offspring were chimeras based on skin and tail biopsies harvested from 2- to 5-day-old pigs. The creation of pluripotent porcine iPSCs capable of generating chimeric offspring introduces numerous opportunities to study the facets significantly affecting cell therapies, genetic engineering, and other aspects of stem cell and developmental biology.

Human embryonic stem cells (hESC) have the potential to produce all of the cells in the body. They are able to self-renew indefinitely, potentially making them a source for large-scale production of therapeutic cell lines. Here, we developed a monolayer differentiation culture that induces hESC (WA09 and BG01) to form epithelial sheets with mesodermal gene expression patterns (BMP4, RUNX1, and GATA4). These E-cadherin+ CD90low cells then undergo apparent epithelial-mesenchymal transition for the derivation of mesenchymal progenitor cells (hESC-derived mesenchymal cells [hES-MC]) that by flow cytometry are negative for hematopoietic (CD34, CD45, and CD133) and endothelial (CD31 and CD146) markers, but positive for markers associated with mesenchymal stem cells (CD73, CD90, CD105, and CD166). To determine their functionality, we tested their capacity to produce the three lineages associated with mesenchymal stem cells and found they could form osteogenic and chondrogenic, but not adipogenic lineages. The derived hES-MC were able to remodel and contract collagen I lattice constructs to an equivalent degree as keloid fibroblasts and were induced to express alpha-smooth muscle actin when exposed to transforming growth factor (TGF)-beta1, but not platelet derived growth factor-B (PDGF-B). These data suggest that the derived hES-MC are multipotent cells with potential uses in tissue engineering and regenerative medicine and for providing a highly reproducible cell source for adult-like progenitor cells.

Availability of human embryonic stem cells (hESC) has enhanced human neural differentiation research. The derivation of neural progenitor (NP) cells from hESC facilitates the interrogation of human embryonic development through the generation of neuronal subtypes and supporting glial cells. These cells will likely lead to novel drug screening and cell therapy uses. This review will discuss the current status of derivation, maintenance and further differentiation of NP cells with special emphasis on the cellular signaling involved in these processes. The derivation process affects the yield and homogeneity of the NP cells. Then when exposed to the correct environmental signaling cues, NP cells can follow a unique and robust temporal cell differentiation process forming numerous phenotypes. J. Cell. [Carpenter et al., 2001;Keirstead et al., 2005;Shin et al., 2006;Hong et al., 2008] and has been the topic of recent reviews [Zhang, 2004;Wilson and Stice, 2006;Cai and Grabel, 2007;Robertson et al., 2008]. While differentiation is directed towards the neural lineage, lack of an optimal protocol implies generation of other lineage cells as contaminants. Thus, one major challenge in the field is to generate a homogeneous and renewable, easy to culture, neural progenitor (NP) cell population committed to the neural lineage, capable of serving as an unlimited lineage-restricted cell source for replacement therapy and/or for other studies. Producing NP cell populations from hESC, specific to different regions of nervous system still remains elusive. Therefore, it is critical to optimally differentiate hESC to NP cells, their maintenance as a self-renewing population, and further to generate highly pure population of regionally specified cell types in culture. Cell signaling plays an important role in each process. Recent advances covering these issues of hESC biology for generation of NP cells are the focus of this review. PRINCIPLE OF NEURAL INDUCTION IN hESCMaintenance of pluripotent state allows hESC to multiply continually in culture while preventing differentiation to other lineage cells. Any disruption of the pluripotency supporting system (for example, by withdrawing key factor(s) from the medium or forcing the hESC to grow in suspension, etc.) promotes spontaneous differentiation to several lineages. Due to the stochastic nature of spontaneously differentiating hESC, many other cell types are also generated; therefore, spontaneous differentiation is not an efficient method for generating neural cells. Thus, it is imperative to make use of certain factors that direct hESC differentiation specifically to neural lineage. Addition of instructive factors and removal of preventive factors form the basis of directed differentiation resulting in an improved NP cell yield. Once the hESC differentiate into NP cells, the newly formed NP cells maintain expression of SOX2 and begin expressing other neuroepithelial markers, such as Nestin, SOX1, SOX3, PSA-NCAM, and MUSASHI-1 (Fig. 1). Formation of ''neural rosettes'' is...

No abstract