In this study we investigated whether transgenic selection c

In this study, we investigated whether transgenic selection could help to enrich low-purity populations that commonly result from pMN differentiation protocols. We generated a new heterozygous “knock in” mouse ESC line (P-Olig2) where the protein-coding region in one allele of Olig2 was replaced with PAC, allowing for positive selection of Olig2+ pMNs during the differentiation. Olig2 expression was analyzed during directed differentiation of ESCs into pMNs using the Shh signaling agonist, purmorphamine (Sinha and Chen, 2006; Wu et al., 2004). Puromycin-treated SCR 7 were assessed for expression of pMN-specific markers and differentiation into pMN progeny, including MNs and oligodendrocytes. This study demonstrates the first use of puromycin resistance for positive selection of a specific population of neural progenitor cells.
Results
Discussion Current protocols for differentiation of mouse ESCs are often hindered by low efficiencies. Directed differentiation may lead to heterogeneous ESC-derived populations that must be further purified to obtain the desired lineages prior to cell culture studies or transplantation. In this study, we demonstrate that positive selection of Olig2+ pMNs through transgenic expression of the puromycin resistance enzyme PAC can provide a simple method for enrichment of pMNs. Purmorphamine exhibited a dose-dependent effect on pMN gene expression during directed differentiation of G-Olig2 ESCs. Greater concentrations of purmorphamine led to a significant increase in Olig2 expression. Conversely, expression of Dbx2 and Irx3 (more dorsal transcription factors) were reduced with increasing concentrations of purmorphamine. Nkx2.2, which is expressed in the more ventral p3 progenitor domain, was not detected in any of the conditions tested. Based on the gene expression data, directed differentiation appears to favor pMNs at the highest concentration of purmorphamine tested. However, this condition still resulted in a mixed cell population with nearly 40% of cells not expressing Olig2. This undesired population may include cells from neighboring progenitor domains that have been previously observed following directed differentiation of ESCs into pMNs (Wichterle et al., 2002). Differentiation of ESCs into cells from multiple spinal progenitor domains is potentially due to overlapping dependency on Shh signaling in the ventral neural tube or our inability to precisely control localized concentration of purmorphamine over the duration of the experiment. Furthermore, variations in the responsiveness of each cell to Shh signaling may attribute to heterogeneity. Positive selection of cardiomyocytes and endothelial cells through puromycin resistance has been previously shown using randomly inserted resistance cassettes containing a cell-type specific promoter (Marchetti et al., 2002; Kim and von Recum, 2009; Kolossov et al., 2006). By knocking in PAC expression, we preserve regulatory mechanisms for the native Olig2 gene. Expression of PAC in the final P-Olig2 cell line recapitulated expression of Olig2 in the native allele. Specificity of the PAC cassette driven by the native Olig2 GRE was shown by puromycin sensitivity. Cell death was induced in puromycin treated P-Olig2 ESCs within 48h which is similar to the time-course previously described for puromycin-induced cell death (Watanabe et al., 1995). Only when P-Olig2 ESCs were differentiated into pMNs using the 2−/4+ differentiation protocol did cells remain viable following puromycin treatment. Cell death was still observed in differentiated ESC cultures suggesting that only cells having expressed Olig2 show resistance. Consistent with this hypothesis, puromycin treatment removed all broad flat cells previously identified as Olig2−. In addition, puromycin killed all Olig2−/Oct4+ undifferentiated stem cells. The majority of remaining viable cells were positive for the transcription factors Olig2 or Hb9. These results demonstrate selective resistance in pMNs and their progeny.