Differentiation of human oligodendrocytes from pluripotent stem cells

Abstract

We have developed a four-part protocol to differentiate human embryonic stem cells (hESCs) to oligodendrocyte progenitor cells (OPCs) according to developmental principles. In the first 2 weeks, hESCs are induced to differentiate into neuroepithelial cells, which form neural tube-like rosettes. In the following 10 d, these neuroepithelial cells are specified to OLIG2-expressing progenitors in the presence of retinoic acid (RA) and sonic hedgehog (SHH). Upon treatment with fibroblast growth factor 2 (FGF2) for another 10 d, these progenitors convert to OLIG2 and NKX2.2-expressing preOPCs. Finally, the pre-OPCs take 8-9 weeks to differentiate into OPCs, which express additional markers of oligodendrocytes, such as SOX10, platelet-derived growth factor receptor alpha (PDGFRα) and NG2. The unique aspects of the protocol are the use of FGF2 to promote the differentiation of gliogenic pre-OPCs in the third part and the removal of FGF2 during the transition of pre-OPCs to OPCs. This 3-month differentiation protocol consistently yields OPCs of high purity capable of producing myelin sheaths in vivo.

Specification of functional neurons and glia from human pluripotent stem cells

“…Since MBP is mutated in these mice, MBP is not expressed within their brain and there is no compacted myelin sheath surrounding the nerve fibers. The hPSC-derived OPCs, upon transplantation into the brains of shiverer mice, are able to differentiate into mature oligodendrocytes that can re-myelinate the hypo-myelinated nerve fibers, indicating these in vitro-produced cells have gained the authentic function of oligodendrocytes (Hu et al, 2009a(Hu et al, , 2009b (Fig. 2E).…”

Transcription factor induction of human oligodendrocyte progenitor fate and differentiation

“…In these studies, the rate of donor-derived myelination was dependent on both developmental stage and purity of the cell population. However, as human OPCs cannot be readily expanded following isolation, various approaches have been used to specify OPC fate from embryonic stem cells (7,8). Although these procedures are able to generate enriched cultures of antigenically defined OPCs, specification of platelet-derived growth factor α receptor (PDGFαR)-expressing OPCs from OLIG2 + neural stem/progenitor cells (NPCs) requires more than 8 wk in culture, and the resulting cells initiate myelination at a significantly slower rate than native PDGFαR/CD140a-defined OPCs directly isolated from fetal human brain (5,9).…”

A Modular Assembly of Spinal Cord–Like Tissue Allows Targeted Tissue Repair in the Transected Spinal Cord

“…Nestin immunoreactivity was assessed and confirmed for all the neurospheres. Oligodendrocyte precursor cells (OPCs) were obtained according to the published protocols with slight modifications 25, 33. Briefly, NSCs at passage 2 were plated on polylysine‐coated culture dishes and cultured with DMEM/F12 containing 10 ng mL −1 platelet‐derived growth factor AA (PDGF‐AA, Life Technologies, USA), 10 ng mL −1 bFGF (Life Technologies, USA), 30 ng mL −1 triiodothyronine (T3, Sigma‐Aldrich), and 1% fetal bovine serum (FBS, Gibco) for 3 days.…”