Regulation of human oligodendrocyte progenitor fate by transcription factors
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Human oligodendrocyte progenitor cell (OPC) specification and differentiation occurs slowly and limits the potential for cell-based treatment of demyelinating disease. To unveil the transcriptional regulation of OPC fate commitment from neural stem/progenitor cell (NPC), we first performed a series of studies to purify primary human NPCs and OPCs. In chapter 3 , we investigated the potential overlap of NPC and OPC surface antigens using multicolor fluorescence-activated cell sorting (FACS) for CD133/PROM1, A2B5, and CD140a/PDGFRα antigens. We found that, firstly, A2B5 could not be used with CD133 to purify fetal human OPCs. Secondly, CD133 + CD140a - cells lacked the capacity to generate oligodendrocytes but were highly enriched for neurosphere initiating cells and were multipotent. Thirdly, both CD133 + CD140a + and CD133 - CD140a+ cells were OLIG2-expressing OPCs capable of oligodendrocyte differentiation, but formed neurospheres with lower efficiency and were largely restricted to glial fate. Finally, further gene expression analysis confirmed the stem cell nature of CD133 + CD140a - cells and the glia progenitor nature of D133 + CD140a + and CD133 - CD140a + cells. In chapter 4 , we identified 8 OPC-specific transcription factors and tested the hypothesis that over-expressing individual OPC-specific transcription factor would accomplish OPC fate specification from the NPCs. We over-expressed each transcription factor in NPCs via lentivirus and examined OPC marker expression, the proliferation and differentiation ability of induced OPCs in vitro and in vivo. Among all the transcription factors tested, ASCL1, SOX10, and NKX2.2 was sufficient to induce Sox10 enhancer activity, OPC marker mRNA and protein expression. However, only the transcriptome of SOX10-infected NPCs was induced to a human OPC-like gene expression signature. Furthermore, only SOX10 promoted oligodendrocyte commitment, and did so at quantitatively equivalent levels to native OPCs. In xenografts of shiverer/rag2 animals, SOX10 increased the rate of mature oligodendrocyte differentiation and axon ensheathment. Thus, SOX10 appears to be the principle and rate-limiting regulator of myelinogenic fate from human NPCs. Chapter 5 focuses on another human OPC-specific transcription factor PRRX1, which was identified in chapter 4. Compared to other well studied transcription factors, very little is known for the role of PRRX1 in OPCs. Therefore, we performed both in vitro and in vivo studies to examine whether PRRX1 regulates human OPC migration, proliferation and differentiation. Our preliminary studies suggested that in vitro over-expression of PRRX1a or 1b inhibited OPC migration in the transwell assay. Additionally, knocking down PRRX1 expression slightly increased OPC migration. Specifically, PRRX1 decreased primary human OPC proliferation after 24 hours BrdU treatment. Both PRRX1a and 1b promoted OPC differentiation to O4 + immature oligodendrocytes, without affecting astrocyte generation. In xenografts of shiverer/rag2 animals, human OPCs over-expressing PRRX1a exhibited significantly less rostral-dorsal migration than the mCherry control, which is consistent with the in vitro result. Further studies concerning cell differentiation and earlier time points of xenografts will be evaluated in the near future.