Differentiation of oligodendrocyte progenitor cells from dissociated monolayer and feeder-free cultured pluripotent stem cells

<div><p>Oligodendrocytes myelinate axons and form myelin sheaths in the central nervous system. The development of therapies for demyelinating diseases, including multiple sclerosis and leukodystrophies, is a challenge because the pathogenic mechanisms of disease remain poorly understood. Primate pluripotent stem cell-derived oligodendrocytes are expected to help elucidate the molecular pathogenesis of these diseases. Oligodendrocytes have been successfully differentiated from human pluripotent stem cells. However, it is challenging to prepare large amounts of oligodendrocytes over a short amount of time because of manipulation difficulties under conventional primate pluripotent stem cell culture methods. We developed a proprietary dissociated monolayer and feeder-free culture system to handle pluripotent stem cell cultures. Because the dissociated monolayer and feeder-free culture system improves the quality and growth of primate pluripotent stem cells, these cells could potentially be differentiated into any desired functional cells and consistently cultured in large-scale conditions. In the current study, oligodendrocyte progenitor cells and mature oligodendrocytes were generated within three months from monkey embryonic stem cells. The embryonic stem cell-derived oligodendrocytes exhibited <i>in vitro</i> myelinogenic potency with rat dorsal root ganglion neurons. Additionally, the transplanted oligodendrocyte progenitor cells differentiated into myelin basic protein-positive mature oligodendrocytes in the mouse corpus callosum. This preparative method was used for human induced pluripotent stem cells, which were also successfully differentiated into oligodendrocyte progenitor cells and mature oligodendrocytes that were capable of myelinating rat dorsal root ganglion neurons. Moreover, it was possible to freeze, thaw, and successfully re-culture the differentiating cells. These results showed that embryonic stem cells and human induced pluripotent stem cells maintained in a dissociated monolayer and feeder-free culture system have the potential to generate oligodendrocyte progenitor cells and mature oligodendrocytes <i>in vitro</i> and <i>in vivo</i>. This culture method could be applied to prepare large amounts of oligodendrocyte progenitor cells and mature oligodendrocytes in a relatively short amount of time.</p></div

OPCs from A3 exhibit myelinogenic potency <i>in vitro</i>.

<p>OPCs from human iPSCs were co-cultured with rat primary DRG neurons for 45 days (A) and 70 days (B). Neurons and differentiated oligodendrocytes immunostained with NF (green) and MBP (red), respectively. Scale bars = 20 μm.</p

OPCs and oligodendrocytes derived from the monkey ESCs CMK6_SFF.

<p>(A) Protocol for differentiation into OPCs and oligodendrocytes. (B–D) Immunostained CMK6_SFF before differentiation. Cultured cells in Step 0 immunostained with Nanog (green) and Hoechst (blue, B). Cells stained with Oct4 (red) and Hoechst (blue, C). Sample from panel C also stained with Sox2 (green, D). (E–G) Immunostained CMK6_SFF from Step 1. Cells from day 6 immunostained with Sox2 (green) and Hoechst (blue, E). Cells stained with Pax6 (green), Sox1 (red), and Hoechst (blue, F), as well as Nkx2.2 (green), Olig2 (red), and Hoechst (blue, G). (H–J) Immunostained CMK6_SFF from Step 2. Cells were cryopreserved for 581 days and the cells from day 59 immunostained with Nkx2,2 (green), Olig2 (red), and Hoechst (blue, H). Cells stained with PDGFRα (green) and Hoechst (blue, I), as well as with A2B5 (green), Olig2 (red), and Hoechst (blue, J). (K–M) Immunostained CMK6_SFF from Step 3. Cells from day 73 immunostained with O4 (green) and MBP (red). Scale bars = 50 μm.</p

CMK6_SFF-derived OPCs exhibit myelinogenic potency <i>in vitro</i>.

<p>OPCs from monkey ESCs co-cultured with rat primary DRG neurons for 45 days. (A, B) Neurons and differentiated oligodendrocytes immunostained with NF (green) and MBP (red), respectively. Scale bars = 30 μm. (C) Western blotting analysis of MBP protein and CNPase protein in rat primary DRG neurons without (lane 1) or co-cultured with OPCs (lane 2).</p

OPCs and oligodendrocytes derived from the human iPSCs A3.

<p>(A) Protocol for differentiation into OPCs and oligodendrocytes. (B–D) Immunostained A3 prior to differentiation. Cultured cells from Step 0 immunostained with Nanog (red) and Hoechst (blue, B), as well as with SSEA-4 (green) and Hoechst (blue, C). Cells stained with Sox2 (green), Oct4 (red), and Hoechst (blue, D). (E–G) Immunostained A3 from Step 1. Cells from day 9 immunostained with Sox2 (green) and Hoechst (blue, E). Cells stained with Pax6 (green), Sox1 (red), and Hoechst (blue, F), as well as with Nkx2.2 (green), Olig2 (red), and Hoechst (blue, G). (H–J) Immunostained A3 from Step 2. Cells were cryopreserved for 90 days and the cells from day 85 immunostained with Nkx2,2 (green), Olig2 (red), and Hoechst (blue, H). Cells stained with PDGFRα (green) and Hoechst (blue, I), as well as with A2B5 (green), and Hoechst (blue, J). (K–M) Immunostained A3 from Step 3. Cells from day 99 immunostained with O4 (green) and MBP (red). Scale bars = 50 μm.</p

Transplanted CMK6_SFF-derived OPCs differentiate into oligodendrocytes <i>in vivo</i>.

<p>Distribution of CMK6_SFF-derived cells transplanted into the corpus callosum (CC) of the cerebral cortex (Cx) in neonatal mice. (A) Sections stained with HuN (green) in the CC and Cx (B). The same section immunostained with MBP (red) and DAPI (blue). (C) Enlarged image of B. The dotted lines show needle insertion locations. Scale bars = 50 μm. (D-G) High-magnification images with boxed region in C. Arrowheads show HuN-positive cells co-stained with MBP. Scale bars = 10 μm. (H-J) Immunostained contralateral cerebral cortex with MBP (red), HuN (green) and DAPI (blue). (K) Immunostained cerebellum sections. Enlarged images of MBP (L), HuN (M), and overlay (N). Spinal cord section (O–Q). Scale bars = 50 μm.</p