While many studies have utilized siRNA as a tool

While many studies have utilized siRNA as a tool for studying ESC differentiation, there are limited data reporting the presence or absence of off-target siRNA effects on differentiation outcome. Nonviral transfection of off-target siRNA molecules has been found to induce adipogenesis in human mesenchymal stem pppa cost (Xu et al., 2007). Hence, it is important to measure the effects that transfection or transduction may induce in ESC differentiation protocols. We found that siRNA-mediated knockdown of constitutively expressed RFP was highly efficient and induced no notable effects on endoderm differentiation. Cells were transfected with the siRNA molecules at Day 1 of differentiation and exhibited significant RFP knockdown at Day 6. This assay provides a siRNA screening platform that can potentially be utilized for differentiation pathway analysis. In an effort to demonstrate the efficiency and power of this 96-well assay system, we conducted a growth factor screening assay utilizing a single 96-well plate and analyzed 32 conditions in duplicate. We then extended the capabilities of this small-scale system to analyze time-course gene expression profiles for four of these conditions, again, within a single 96-well plate. bFGF in combination with activin A or BMP4 drove differentiation and reduced pluripotency. In particular, the greatest number of CXCR4+KDR– cells was induced by low to high activin A with no BMP4. Gene profiles over the 6-day time period for the activin A+BMP4 conditions confirmed that these CXCR4+KDR– cells were characteristic of definitive endoderm. These results correspond to previous studies that found increased CXCR4 expression and increased definitive endoderm yields by differentiating cells under high activin A containing SFD conditions (D\'Amour et al., 2005; Yao et al., 2006). Our results are similar to those of Vallier et al. in which high activin A in combination with bFGF and BMP4 induced mesendoderm (Vallier et al., 2009). Several studies have previously reported that activin A+bFGF blocks differentiation and maintains pluripotency (Vallier et al., 2009; Xiao et al., 2006), whereas our results demonstrated increased endoderm production. Our protocol includes a BMP4 exposure step at Day 0–1 to serve as a “jumpstart” to differentiation. The decrease in pluripotent marker expression in subsequent activin A + bFGF SFD conditions is likely attributable to this step. Activin A and BMP4 exhibited a competitive effect on mesoderm marker expression. The presence of activin A alone eliminated PDGFRα expression at Day 6, although all samples demonstrated transcript expression through the Day 2–4 primitive streak stage. This PDGFRα reducing effect of activin A has been previously observed (Vallier et al., 2009). Similarly, activin A reduced KDR+SSEA-3– expression, with highest expression levels seen under high BMP4 SFD conditions with no activin A. These results also corroborate previous studies that have induced mesoderm and vascular progenitors through BMP4 activation (Park et al., 2004; Bai et al., 2010) and noted reduced KDR expression with increasing activin A supplementation (Yang et al., 2008; Nostro et al., 2008; Vallier et al., 2009). The addition of VEGF to SFD had no notable effect on surface marker expression at Day 6. However, CD34 transcript expression increased and PDGFRα expression decreased, indicating a role in inducing early mesoderm cells toward a hematopoietic lineage. Indeed, several studies have utilized VEGF to induce mesendoderm or mesoderm progenitors toward a hematopoietic or cardiovascular state (Pick et al., 2007; Yang et al., 2008; Narazaki et al., 2008), and VEGF inclusion has been shown to improve subsequent hematopoietic blast colony formation (Nostro et al., 2008).
Materials and methods
Acknowledgments
Introduction Optimistic views of regenerative medicine have envisioned the use of stem cells as eventually curative for many types of human disorders, including ischemic heart disease, Parkinsonism, diabetes, and many other degenerative or genetically deficient disease conditions. The initiation of the first FDA-approved clinical trial using human ESC-derived cells (Alper, 2009) has raised the anxiety of many scientists who fear that should this trial fail, it could endanger public acceptance and respectability of the entire field of embryonic stem cell research. Potential tumorigenicity of donor cells is a major concern. Adding to this unease is the recent report that a child with ataxia telangiectasia developed multifocal tumors of the brain and spinal cord 4years after treatment with human neural stem cells (Amariglio et al., 2009) originating from at least two donors, even though the cells were relatively freshly derived from chromosomally normal fetuses.