Human embryonic stem cell-derived OPCs
Derivation of OPCs
Figure 1 Timeline of oligodendrocyte differentiation from pluripotent human embryonic stem cells, showing the expression of relevant cellular markers at each stage.
OL development and myelination and tightly regulated processes in the human central nervous system and thorough understanding of these in vivo developmental biology has helped us design efficient processes to derive OPCs with myelinating potential from pluripotent stem cells in the laboratory.
Development & myelination
OL development and myelination is regulated by extrinsic and intrinsic factors. Extrinsic factors include extracellular ligands, secreted molecules and neuronal activity. In fact, OLs have been shown to be sensitive to neuronal electrical activity and this could be a potential way for an axon to induce an OL to myelinate it.
Phase contrast images of various stages of oligodendrocyte differentiation. A. Pluripotent human embryonic stem cells on MEFs, B. EBs one day after being generated by the scraping method, C. NPCs sprouting from the periphery of a plated EB on matrigel after 24 hrs, D. OPC 9 days after 2 expansions in OPC inducing medium, and OLs one week after being exposed to OL inducing media.
Intrinsic factors include transcription factors, chromatin remodeling and microRNAs. MicroRNA regulation was reviewed in detail because these nucleic acids are easily synthesized and transfected into the cell in vitro; hence, knowledge of the mechanisms by which they regulate myelination could be used to create OPCs faster and with greater myelination potential for use in acute SCI patients.
Figure 3 Indirect immunofluorescence microscopy to examine differentiation of human ESCs into the oligodendrocyte lineage. The nuclei are stained blue by DAPI. Nestin (green) is shown for neural progenitors while NG2 (red), PDGFRa (green) O4 (red), RIP (red) and NOGO-a (green) are expressed by 5-day old oligodendrocyte progenitor cells.
The method for derivation of OPCs from human embryonic stem (hES) cells relies on the generation of embryoid bodies, which are subsequently differentiated to neural progenitors (NP), glial progenitors (GPs) and finally OPCs. Differentiation from hES colony state to OPC state took approximately thirty days. OPCs could be maintained in a proliferative state for around twenty days before spontaneously maturing to branched OLs.
Based on observations in our laboratory, an extensive list of intra- and extra-cellular markers has been identified to identify various stages of OL maturation from the pluripotent state. Some of these included A2B5 and SOX10 for the NP stage, PDGFR-a, OLIG1, OLIG2 for GPs, O4, O1, CNPASE for OPs and MBP, MOG, MAG as markers of myelin production.