Human iPSC-derived oligodendrocyte progenitor cells can myelinate and rescue a mouse model of congenital hypomyelination

Abstract

Neonatal engraftment by oligodendrocyte progenitor cells (OPCs) permits the myelination of the congenitally dysmyelinated brain. To establish a potential autologous source of these cells, we developed a strategy by which to differentiate human induced pluripotent stem cells (hiPSCs) into OPCs. From three hiPSC lines, as well as from human embryonic stem cells (hESCs), we generated highly enriched OLIG2(+)/PDGFRα(+)/NKX2.2(+)/SOX10(+) human OPCs, which could be further purified using fluorescence-activated cell sorting. hiPSC OPCs efficiently differentiated into both myelinogenic oligodendrocytes and astrocytes, in vitro and in vivo. Neonatally engrafted hiPSC OPCs robustly myelinated the brains of myelin-deficient shiverer mice and substantially increased their survival. The speed and efficiency of myelination by hiPSC OPCs was higher than that previously observed using fetal-tissue-derived OPCs, and no tumors from these grafts were noted as long as 9 months after transplant. These results suggest the potential utility of hiPSC-derived OPCs in treating disorders of myelin loss.

Figure 1. hiPSCs can be directed into OPC fate

(A) A schematic protocol for directed differentiation of hiPSC into OPCs. Embryoid bodies (EBs) were differentiated from undifferentiated hiPSC (Stage 1) from DIV 0-5. EBs were then differentiated as neuroepithelial (NE) cells in neural induction media (NIM; see Methods) with bFGF. (B-D) Undifferentiated hiPSCs (stage 1) and hiPSC colonies expressed the pluripotency markers SSEA-4 and OCT4. EBs (E) and neuroepithelial cells (F-G) could be generated from hiPSC (stages 2-3). hiPSC-derived EBs at this stage expressed the neuroepithelial markers PAX6 and SOX1. (H-I) OLIG2⁺/NKX2.2^- early GPCs appeared under the influence of RA and purmorphamine, a small molecule agonist of sonic hedgehog signaling. By stage 4, OLIG2 was expressed in early pre-OPCs, which then serially developed NKX2.2 expression. (J) OLIO2⁺/NKX2.2^- early pre-OPCs were differentiated into later-stage OLIG2⁺/NKX2.2⁺ pre-OPCs, when RA was replaced by bFGF at stage 5. (K-M) Pre-OPCs were further differentiated into bipotential OPCs in glial induction media (GIM; see Methods), supplemented with PDGF AA, T3, NT3 and IGF. Stage 6 was extended as long as 3- 4 months, so as to maximize the production of myelinogenic OPCs. By the time of transplant, these cells expressed not only OLIG2 and NKX2.2 (K), but also SOX10 (L-M) and PDGFRα (M). By the end of stage 6, hiPSC OPCs could be identified as OLIG2⁺/NKX2.2⁺/SOX10⁺/PDGFRα⁺. (N-P) In vitro terminal differentiation of hiPSC OPCs into human iPSC-derived oligodendrocytes (hiOLs), identified by O4⁺ (N) and MBP⁺ (O). Oligodendrocytes and GFAP⁺ astrocytes (P) arose with reduction in glial mitogens. Scale: B-N, P, 100 μm; O, 25 μm. See also Figure S1.

Figure 2. Both astrocytes and oligodendrocytes are efficiently generated from hiPSC OPCs

(A) Expression of neural markers during induction of oligodendroglial lineage hiPSC-derived neuroepithelial cells in stage 3, pre-OPCs in stages 4 and 5, and OPCs in stage 6. Cultures were immunostained for Pax6 and Sox1, or Olig2 and Nkx2.2, respectively. The proportion of immunopositive clusters for each marker set were scored, for each hiPSC line. At least 3 repeats in each group were performed; data are provided as means ± SEM. In stage 6, gliogenic clusters were dissociated to single cell suspensions and plated in glial induction media, resulting in the terminal differentiation of both astrocytes and myelinogenic oligodendrocytes. (B-C) GFAP⁺ astrocytes were evident in cultures of hiPSC OPCs by 95 DIV; C27 (B) and K04 (C) derived astrocytes are shown here. D, the proportion of GFAP⁺ astrocytes among all cultured cells at 95 DIV; the remainder expressed oligodendroglial lineage markers (see Figure S2I). Later in stage 6 (E-J, 160 DIV), hiPSC-derived OPCs differentiated as both O4⁺ (F-G) and myelin basic protein (MBP)⁺ (G) oligodendrocytes. (H-J) When co-cultured with human fetal cortical neurons, hiPSC OPCs derived from C27 (H), C14 (I), and K04 (J) hiPSCs all generated MBP⁺ myelinogenic oligodendrocytes that engaged neurofilament⁺ axons (MBP, red; neurofilament, green). Scale: 50 μm. See also Figure S2.

Figure 3. OPCs can be isolated from mixed hiPSC culture by CD140a- and CD9-directed FACS

(A) hiPSC OPC-derived oligodendrocytes were recognized and isolated by fluorescence-activated cell sorting (FACS) using monoclonal antibody O4, which recognizes oligodendrocytic sulfatide. The incidence of O4⁺ oligodendroglia varied across different hiPSC lines, from 4 to 12% (See Supplementary Table 1; n=4-7 experiments). (B) OPCs derived from hiPSCs (C27 and K04) were readily recognized with the cell surface marker A2B5. (C) OPCs derived from either hiPSCs (C27 and K04) or hESC (WA09/H9) were readily recognized with cell surface markers, PDGFRα (CD140a) and CD9, by FACS analysis. The relative proportions of CD140a, CD9 and CD140a/CD9 double-labeled cells varied across the different cell line-derived OPCs (n=4-7 experiments). See also Supplementary Table 1, Tables S1A and S1B.

Figure 4. hiPSCs migrate widely and differentiate as astroglia and myelinogenic oligodendrocytes

hiPSC OPCs generated from all 3 hiPSC lines migrated throughout the shiverer brain, engrafting most densely in white matter. Distributions of C27 (A) and K04 (B) hiPSC-derived OPCs shown (human nuclear antigen (hNA)⁺, red, mapped in Stereo Investigator). By 13 weeks of age, C27 hiPSC OPCs (C), K04 hiPSC OPCs (E, G), and C14 hiPSC OPCs (H) matured into myelin basic protein (MBP, green)-expressing oligodendroglia throughout the subcortical white matter, including callosal and capsular (C, E, H) as well as striatal (G) tracts. In these 13 week-old shiverer mouse recipients, C27 (D), K04 (F), and C14 (I) hiPSC-derived OPCs also differentiated as astroglia (human-specific GFAP, green), especially as fibrous astrocytes in the central white matter. Scale: C-I, 100 μm. See also Supplementary Table 2.

Figure 5. hiPSC OPCs robustly myelinate in vivo

Confocal images of the callosal and capsular white matter of mice engrafted with hiPSC OPCs derived from all 3 tested hiPSC lines demonstrate dense donor-derived myelination. A-D, C27-derived; E-H, K04; I-K, C14. A, G, and J show abundant, donor-derived myelin basic protein expression (MBP, green) by C27, K04 and C14 hiPSC OPCs (human nuclear antigen, hNA, red), respectively. Representative z-stacks of individual hNA⁺ oligodendrocytes are shown as asterisks in A and E. By the 19 week time-point assessed here, C27 (B), K04 (F, G), and C14 hiPSC oligodendroglia (J) robustly myelinated axons (neurofilament, NF, red). hiPSC-derived oligodendroglial morphologies exemplified in panels F (K04) and I (C14); F shows multi-axon myelination by single oligodendrocytes in the striatum. hiPSC OPCs also generated astroglia (C, C27; H, K04), which exhibited the complex fibrous morphologies typical of human astrocytes (human-specific GFAP, green). Many cells also remained as progenitors, immunostaining for NG2 (D, C27) and human-specific PDGFαR (K, C14). Scale: A, B, C, G, J, 50 μm; C, D, E, F, H, K, 20 μm; I, 10 μm; insets to A, E, 10 μm.

Figure 6. hiPSC OPCs myelinate widely to greatly extend the survival of hypomyelinated mice

A, Dot map indicating distribution of human iPSC-derived donor cells (C27) at 7 months of age, following neonatal engraftment in a shiverer mouse brain. Widespread dispersal and chimerization by hiPSC OPCs is evident (human nuclear antigen, red). B, hiPSC OPC-derived myelination in shiverer forebrain, at 7 months; section 1 mm lateral to A. MBP-immunoreactivity (green) is all donor-derived. C, D. Myelination in sagittal sections taken at different mediolateral levels from two additional 7 month-old mice, each engrafted with C27 hiPSC OPCs at birth. E, Kaplan-Meier plot of survival of C27 iPSC-OPC implanted (n=22) vs. saline-injected (n=19) control mice. Remaining engrafted mice sacrificed for electron microscopy at 9-10 months (≥270 days). Scale: A-B, 2 mm. See also Figure S3.

Figure 7. hiPSC-derived oligodendrocytes produce compact myelin and induce nodes of Ranvier

Representative electron microscopic images of sections through the corpus callosum (A-B) and ventral pons (C) of a 40-week-old shiverer mouse neonatally-engrafted with C27 hiPSC OPCs, showing donor-derived compact myelin with evident major dense lines, ensheathing mouse axons. D-G show higher power images of the corpus callosum, also at 40 weeks. D, The alternating major dense (arrowheads) and intraperiod lines, characteristic of mature myelin, are evident. F-G, myelin sheaths in the corpus callosum ensheathing central axons, their maturation manifested by parallel arrays of tight junctions (F, arrowhead) and major dense lines (G, arrowhead). This mature myelination permitted the organization of architecturally-appropriate nodes of Ranvier by hiPSC oligodendroglia. In H-I, nodal reconstitution in transplanted shiverers is demonstrated by immunostaining of oligodendrocytic paranodal Caspr protein (red), seen flanking nodes of Ranvier identified here by ßIV spectrin (green). An isolated node is shown in confocal cross-section in I. Scale: A-E, 200 nm; F-G, 100 nm; H-I, 5 μm. See also Figure S4.