CD133/CD140a-Based Isolation of Distinct Human Multipotent Neural Progenitor Cells and Oligodendrocyte Progenitor Cells

Abstract

The mechanisms underlying the specification of oligodendrocyte fate from multipotent neural progenitor cells (NPCs) in developing human brain are unknown. In this study, we sought to identify antigens sufficient to distinguish NPCs free from oligodendrocyte progenitor cells (OPCs). We investigated the potential overlap of NPC and OPC antigens using multicolor fluorescence-activated cell sorting (FACS) for CD133/PROM1, A2B5, and CD140a/PDGFaR antigens. Surprisingly, we found that CD133, but not A2B5, was capable of enriching for OLIG2 expression, Sox10 enhancer activity, and oligodendrocyte potential. As a subpopulation of CD133-positive cells expressed CD140a, we asked whether CD133 enriched bone fide NPCs regardless of CD140a expression. We found that CD133+ CD140a -cells were highly enriched for neurosphere initiating cells and were multipotent. Importantly, when analyzed immediately following isolation, CD133 + CD140a -NPCs lacked the capacity to generate oligodendrocytes. In contrast, CD133+ CD140a + cells were OLIG2-expressing OPCs capable of oligodendrocyte differentiation, but formed neurospheres with lower efficiency and were largely restricted to glial fate. Gene expression analysis further confirmed the stem cell nature of CD133 + CD140a -cells. As human CD133 + cells comprised both NPCs and OPCs, CD133 expression alone cannot be considered a specific marker of the stem cell phenotype, but rather comprises a heterogeneous mix of glial restricted as well as multipotent neural precursors. In contrast, CD133/CD140a-based FACS permits the separation of defined progenitor populations and the study of neural stem and oligodendrocyte fate specification in the human brain.

Transcription factor induction of human oligodendrocyte progenitor fate and differentiation

“…We chose CD140a/PDGFaR antigen because it represents an early and specific marker of human OPCs (5). Through a combination with CD133, we were able to distinguish CD133 + CD140a − NPCs from CD140a + OPCs (15). Genomic analysis of CD140a + OPCs relative to NPCs identified known OPC-specific TFs, including SOX10, OLIG2, NKX2.2, MYT1, ID2, and HES5 (23).…”

“…Cytometry and sorting were performed with a BD FACSAria cell sorter, as described previously (15). For CD133/CD140a, cells were stained with CD140a-PE (BD Pharmingen) and CD133-APC (Miltenyi Biotech).…”

“…committed OPCs defined by CD133 + CD140a + antigenicity (15) as well as with CD140a/O4-defined OPCs. We identified 12 TFs that were up-regulated during the specification of OPC fate and/or between O4 + oligodendrocyte-biased and bipotential CD140a/PDGFαR + OPCs (Fig.…”

“…We selected a species conserved SOX10 enhancer as an initial screen of OPC fate induction. We recently found that the Sox10-MCS5 enhancer is differentially active in human CD140a-defined OPCs compared with native CD133 + CD140a − NPCs (15), and can be used to identify human OPCs (18). At 24 h after isolation, NPCs were infected with a Sox10-MCS5:GFP reporter virus and a candidate TF or mCherry-expressing virus as a negative control.…”

“…Given the species differences in OPC gene expression (14), it is likely that the role of these TFs is subtly different in human cells. To select instructive TFs in an unbiased manner, we performed microarray analysis on antigenically defined human progenitors (15,16). We compared the transcriptional profile of CD140a and O4-defined OPCs with that of CD133 + CD140a − NPCs.…”