Surface Marker Analysis to Predict Successful Reprogramming to Pluripotency
Journal Title: Biomedical Journal of Scientific & Technical Research (BJSTR) - Year 2019, Vol 12, Issue 4
Abstract
Induced pluripotent stem cells (iPSCs) are produced by introduction of the defined factors (Oct4, Sox2, Klf4, c-Myc: OSKM), and exhibit infinitive self-renewal and pluripotency. However, iPSCs are not efficiently established, and the details of precursor cells toward iPSCs remain uncovered. Although we previously proposed a surface marker profile Sca1-CD34- as iPSC progenitors, several studies reported that SSEA1+ and CD44-CD54+ cell populations are also predictors for successful reprogramming. Here we examine the detailed correlation of surface marker expression profiles among Sca1, CD34, CD44, CD54, and SSEA1. Following OSKM-infection in mouse embryonic fibroblasts, cell surface marker genes, Sca1 and CD34 were upregulated. Fluorescence-activated cell sorting analysis showed that the highest incidence of iPSC colony formation was observed in Sca1-CD34- cells sorted in the early-to-mid phase of reprogramming, when SSEA1 is barely detected. In contrast, in the late phase, half of Sca1-CD34- population expressed SSEA1, whereas CD44- cells displayed the lower percentage of SSEA1-positive regardless of CD54 expression. In addition, favorable efficiency of iPSC induction from Sca1-CD34- cells was also observed in somatic cells reprogrammed by OSK and L-Myc instead of oncogenic c-Myc. Altogether, the surface marker profile Sca1-CD34- is a more sensitive early predictor for bona fide iPSC progenitors, and it may shed light on the molecular basis of successful reprogramming to iPSCs.Induced pluripotent stem cells (iPSCs) are generated from somatic cells by introduction of Yamanaka factors comprised of Oct4, Sox2, Klf4, and c-Myc (OSKM), and they are highly desired for applications in regenerative medicine, tailor-made therapy, and disease modeling [1,2]. iPSCs derived from patients’ somatic cells are thought to minimize graft rejection when they are implanted into the target tissue. In addition, it is not necessary to consider an ethical issue accompanied with embryonic stem cells. However, due to the low efficiency of iPSC formation and heterogeneity in the reprogramming process, it remains difficult to gain insights into the molecular basis of successful reprogramming to iPSCs. To address these issues above, we have recently revealed that a certain surface marker profile determines a cell population in which bona fide reprogramming progenitors is highly enriched [3]. We previously performed microarray analysis to identify surface marker profiles for bona fide iPSC progenitors [3]. Based on 886 genes registered as surface markers in a Gene Ontology database, we explored surface marker genes regulated by introduction of reprogramming OSKM in mouse embryonic fibroblasts (MEFs). There were 61 upregulated and 131 downregulated genes by OSKM introduction at the early phase of reprogramming. Among these candidate marker genes, we focused on stem cell antigen 1 (Sca1) and cluster of differentiation gene 34 (CD34), also known as marker genes of hematopoietic stem cells, and examined their involvement in successful reprogramming to iPSCs. Although expressions of Sca1 and CD34 are induced by OSKM at the early state of the reprogramming process, they are not detectable in undifferentiated iPSCs. Of note, we have recently revealed that Sca1-CD34- surface marker profile determines a cell population in which bona fide reprogramming progenitors is highly enriched (Kida et al.). Sca1-CD34- cells on day5, early-to-mid phase of reprogramming, efficiently produce iPSC colonies positive for Nanog, a specific marker of undifferentiated pluripotent stem cells. Thus, Sca1 and CD34 would be available to distinguish iPSC early progenitors. In contrast, cell populations apart from Sca1-CD34- cells may give rise to cell conversion to other cell types or undergo apoptotic cell death. We assessed, in the present study, further details of Sca1-CD34- population, and we showed that this pool of intermediate cells at the late stage revealed a high incidence of preiPSCs positive for stage-specific embryogenic antigen 1 (SSEA1), reported as another surface marker of iPSC progenitor. Our results also indicate that Sca1-CD34- profile is a useful early predictor for successful reprogramming to iPSCs
Authors and Affiliations
Tomoe Ueyama, Shu Nakao, Tasuku Tsukamoto, Dai Ihara, Yukihiro Harada, Yuka Akagi, Shohei Torii, Sae Nakagawa, Tomoaki Ishida, Takahiro Sogo, Teruhisa Kawamura
Keywords
Related Articles
- EP ID EP587425
- DOI 10.26717/BJSTR.2019.12.002283
- Views 200
- Downloads 0
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