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Efficient Generation of Myelinating Oligodendrocytes from Primary Progressive Multiple Sclerosis Patients by Induced Pluripotent Stem Cells

Summary Multiple sclerosis (MS) is a chronic demyelinating disease of unknown etiology that affects the CNS. While current therapies are primarily directed against the immune system, the new challenge is to address progressive MS with remyelinating and neuroprotective strategies. Here, we develop a highly reproducible protocol to efficiently derive oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes from induced pluripotent stem cells (iPSCs). Key elements of our protocol include adherent cultures, dual SMAD inhibition, and addition of retinoids from the beginning of differentiation, which lead to increased yields of OLIG2 progenitors and high numbers of OPCs within 75 days. Furthermore, we show the generation of viral and integration-free iPSCs from primary progressive MS (PPMS) patients and their efficient differentiation to oligodendrocytes. PPMS OPCs are functional, as demonstrated by in vivo myelination in the shiverer mouse. These results provide encouraging advances toward the development of autologous cell therapies using iPSCs.


INTRODUCTION
Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the CNS that is distinguished by recurrent episodes of focal inflammatory demyelination and consequent neurological symptoms (relapsing remitting MS [RRMS]). Although relapses usually resolve in spontaneous remission, RRMS can evolve with time into a secondary progressive form characterized by irreversible accumulation of disabilities. Furthermore, patients affected by the most severe primary progressive form (PPMS) experience a steady neurological decline from the onset of the disease (Antel et al., 2012). Currently available treatments targeting the immune system are highly effective at reducing or even stopping the intermittent episodes of inflammation, but they do not influence the course of progressive MS. Therapeutic options for PPMS patients are limited to symptomatic treatments and the long-term prognosis is generally poor (Rice et al., 2013). Clearly, the unsolved challenge in the MS field is to develop neuroprotective and remyelinating strategies for the treatment of progressive MS patients (Hauser et al., 2013). The generation of patient-specific cells from induced pluripotent stem cells (iPSCs) or somatic cell nuclear transfer has recently emerged as a promising strategy for the development of autologous cell therapies (Goldman et al., 2012;Yamada et al., 2014). iPSC-derived oligodendrocyte progenitor cells (OPCs) were shown to successfully remyelinate and rescue a hypomyelinated mouse model, raising the possibility of future clinical trials (Wang et al., 2013).
However, oligodendrocyte differentiation protocols are still inefficient and require over 120 days in culture. Therefore, an improved protocol that can generate large numbers of purified OPCs in a relatively short time is highly desirable. Moreover, this protocol should be reproducible and highly efficient among different iPSC lines, including those derived from MS patients. We have pioneered the efficient and robust generation of iPSC-derived OPCs from PPMS patients. Our protocol recapitulates the major steps of oligodendrocyte differentiation from neural stem cells to OLIG2 + progenitors and finally to O4 + OPCs in a significantly shorter time than the 120-150 days required by the most recently published protocols (Wang et al., 2013;Stacpoole et al., 2013). Furthermore, O4 + OPCs were able to differentiate into MBP + mature oligodendrocytes in vitro and to myelinate axons in vivo when injected into immunocompromised shiverer (shi/shi) mice. No abnormal growths were observed. Our results provide a proof of concept that transplantation of iPSC-derived, patient-specific cells for remyelination is technically feasible.

Retinoic Acid Is Critical for Efficient Differentiation of PSCs to Oligodendrocytes
We aimed to develop an efficient differentiation protocol that recapitulates the critical developmental stages of oligodendrocyte specification as it occurs in the spinal cord. In this process, PAX6 + neural stem cells give rise to OLIG2 + progenitors, which become committed to the oligodendrocyte lineage by coexpressing NKX2.2 (pre-OPCs). They then differentiate to early OPCs by upregulating SOX10 and PDGFRa, followed by late OPCs expressing the sulfated glycolipid antigen recognized by the O4 antibody, and finally mature to myelin basic protein (MBP) + oligodendrocytes (Hu et al., 2009a). We utilized an OLIG2-GFP knockin human embryonic stem cell (hESC) reporter line to track the OLIG2 + progenitors by live fluorescent imaging (Liu et al., 2011). First, we induced PAX6 + cells using dual inhibition of SMAD signaling in adherent cultures (Chambers et al., 2009). Next, to mimic the embryonic spinal cord environment, we applied different concentrations of retinoic acid (RA) and/or sonic hedgehog (SHH) at various times and quantified the OLIG2-GFP expression by flow cytometry ( Figure 1A). Application of 100 nM RA from the beginning of induction generated 40.6% of OLIG2 + progenitors, whereas addition of SHH at 100 ng/ml from day 8 increased the yield to 57.7% ( Figure 1B). Interestingly, cells without exogenous SHH during the first 12 days showed an upregulation of SHH mRNA ( Figure S1A available online) and differentiated to O4 + cells, although at a lower efficiency compared with cells treated with SHH ( Figure S1B). We then replaced the recombinant human SHH protein with the smoothened agonist (SAG), which increased the yield further to 70.1% OLIG2 + progenitors ( Figure 1B). At day 12, cells were detached for sphere aggregation. The minimum number of cells required to form a sphere was 100, and we noted that the majority of the cells in the spheres were GFP + . To investigate this further, we sorted d12 cultures for GFP and observed that only GFP + cells formed aggregates, whereas the GFP À population did not ( Figure 1C). This suggests that the aggregation step alone provides enrichment for the OLIG2 + population.
Next, we validated the initial steps toward the generation of OLIG2 + progenitors by differentiating a second hESC line (RUES1) and comparing the transcript levels of PAX6, OLIG2, and NKX2.2 by quantitative RT-PCR (qRT-PCR). The upregulation of these transcription factors followed a temporal pattern similar to that of the OLIG2-GFP line, with PAX6 induction around day7, OLIG2 peak around day 13, and sustainably high levels of NKX2.2 after day 10 ( Figure 1D). Based on these results, we used the nongenetically modified RUES1 line to develop the following steps of the protocol from OLIG2 + progenitors to MBP + mature oligodendrocytes (Figure 2A). PAX6 + cells arose at day 7, and by day 12 they were arranged into multilayered structures ( Figures 2B and 2C). From day 12 to day 30 the cells were grown as spheres, and they were then plated onto poly-L-ornithine/laminin-coated dishes for the remainder of the differentiation protocol.

Oligodendrocytes Can Be Efficiently Generated from PPMS-iPSC Lines
To determine whether this protocol could be applied to iPSC lines from subjects with PPMS, we obtained skin biopsies from four PPMS patients. Fibroblast cultures were established from the biopsies and iPSCs were generated using a cocktail of modified mRNAs (Warren et al., 2010) together with a cluster of miRNAs to improve the reprogramming efficiency (StemGent). From day 12 to day 15 of reprogramming, TRA-1-60 + colonies ( Figure S3A), identified by live staining, were picked, expanded, and characterized by immunofluorescence for pluripotency markers ( Figure S3B).  Stem Cell Reports

iPSC-Derived Oligodendrocytes from MS Patients
Expression profiling for seven pluripotency genes confirmed that all four iPSC lines exhibited a profile comparable to that of a reference hESC line and divergent from the parental fibroblasts ( Figure S3C). All iPSC lines displayed a normal karyotype ( Figure S3D) and were able to differentiate into cell types of the three germ layers, both in vitro via spontaneous embryoid body differentiation ( Figure S3E) and in vivo via teratoma assay ( Figure S3F).
Next, we assessed whether the oligodendrocyte differentiation protocol was reproducible with our PPMS-iPSC lines. All iPSC lines tested were found to perform similarly to the RUES1 line ( Figures 3A-3I). The protocol was greatly reproducible and highly efficient as calculated by the frequency of sorted O4 + OPCs, with up to 70% O4 + cells from RUES1 and 43.6%-62.1% from the PPMS-iPSC lines. Additionally, we found that the O4 + fraction contained a subpopulation of cells double positive with PDGFRa (Figure 3J). O4 + cells could be easily purified by fluorescenceactivated cell sorting (FACS), frozen, and thawed without losing their morphology ( Figure S4D).

PPMS-Derived Late OPCs Myelinate Axons in the Mouse Brain
To verify that OPCs obtained through our protocol were functionally myelinogenic, we injected d75 FACS-purified O4 + cells into the forebrain of neonatal, immunocompromised shiverer mice (10 5 cells/animal; Figure S4A). The injected cells were depleted of any contaminant iPSCs, as shown by flow-cytometry analysis of the pluripotency markers SSEA4 and TRA-1-60 ( Figure S4B). However, we still purified our cultures before in vivo transplantation to retain the potential for translation to clinical studies. Cells were frozen, thawed, and allowed to recover for 24-48 hr before transplantation ( Figure S4C). Animals were sacrificed at 12-16 weeks, at which point human hNA + cells were distributed throughout the corpus callosum and forebrain white matter. The density of hNA + cells in the corpus callosum was 34,400 ± 3,090 cells/mm 3 at 12 weeks and approximately double that by 16 weeks. We did not observe the presence of cell clusters or overt tumorigenesis, and the proliferative fraction of engrafted hNA + cells was 17% at 12 weeks and decreased to only 8% Ki67 + at 16 weeks when only 5% of cells were PCNA + ( Figure 4H). Importantly, more than 80% of hNA + cells in the corpus callosum coexpressed OLIG2 protein, suggesting that the engrafted cells were restricted to the oligodendrocyte lineage ( Figure 4I). Furthermore, human MBP + oligodendrocytes were found diffusely throughout engrafted corpus callosum at 12 and 16 weeks ( Figure 4A). At 16 weeks, 31% ± 3% of host mouse axons were ensheathed within the engrafted mouse corpus callosum ( Figure 4B).
We then asked whether PPMS-derived OPCs could form compact myelin. Transmission electron microscopy on 16-week-old corpus callosum revealed mature compact myelin with the presence of alternating major dense and intraperiod lines ( Figures 4C and 4D), whereas uninjected shiverer/rag2 mice possessed thin and loosely wrapped myelin (data not shown). Likewise, the thickness of myelin ensheathment, as assessed by g-ratio measurement, reflected a restoration of normal myelin in several callosal axons.
At 12 weeks, transplanted cells remained as NG2 + OPCs in the corpus callosum ( Figure 4E), and by 16 weeks they started to migrate to the overlying cerebral cortex (Figure 4F). Very few O4-sorted cells underwent differentiation as hGFAP + astrocytes and the majority of hGFAP + cells were localized to the subventricular zone and around the ventricles ( Figure 4G), suggesting that the local environment may induce astrocytic differentiation in these regions. Similarly, hNESTIN-expressing cells were rarely found in the corpus callosum and likewise concentrated in the subventricular zone (data not shown). Importantly, bIII-TUBULIN + neurons were not detected in any of the engrafted animals. Taken together, our data demonstrate that PPMS-derived, O4-sorted cells were capable of achieving mature oligodendrocyte differentiation in vivo and forming dense compact myelin resembling normal myelin in the brain.

DISCUSSION
In this work, using a fast and highly reproducible protocol, we demonstrated efficient in vivo myelination of neurons by iPSC-derived OPCs from PPMS patients. A previous report on MS-derived iPSCs showed that oligodendrocytes could be differentiated in vitro from an integrating, retrovirally reprogrammed iPSC line from one 35-year-old RRMS patient (Song et al., 2012); here, we generated four integration-free iPSC lines from PPMS patients of both sexes and with ages ranging from 50 to 62 years.
Since most protocols for oligodendrocyte differentiation have been optimized using only one or two hESC lines and their reproducibility with iPSC lines is controversial (Alsanie et al., 2013), we tested our protocol with two hESC and four hiPSC lines derived from PPMS patients. Previous  Stem Cell Reports iPSC-Derived Oligodendrocytes from MS Patients work has elegantly shown that iPSC-derived OPCs from healthy controls were able to rescue a mouse model of hypomyelination, although differentiation to the O4 stage was rather inefficient and required more than 120 days (Wang et al., 2013). We provide an improved differentiation protocol and further proof that patient-specific iPSC lines can be successfully used to generate oligodendrocytes. We obtained 44%-70% O4 + cells in all lines after 75 days of differentiation, compared with the minimum of 120 days required according to previous reports (Wang et al., 2013;Stacpoole et al., 2013). There are several critical differences between our approach and previously published protocols. First, we began neural induction with dual SMAD inhibition in adherent as opposed to suspension cultures (Nistor et al., 2005;Hu et al., 2009b;Wang et al., 2013). Using this approach, we started with only 10,000 cells/cm 2 of iPSCs at day 0 and achieved a great expansion of neural progenitors, ultimately generating an abundance of human OPCs. The use of RA and SHH as caudalizing and ventralizing patterning agents recapitulates the signals that are present around the pMN domain of the spinal cord, from which motor neurons and oligodendrocytes are believed to arise (Hu et al., 2009a). The high efficiency in the generation of OLIG2 + cells at d12 can be explained by the synergistic effect of activin/nodal receptor kinase inhibition, BMP4 inhibition, and RA and SHH signaling (Patani et al., 2011;Miller et al., 2004). The optimal concentration of RA in our hands is 100 times less than the concentration commonly used by other groups (Nistor et al., 2005;Izrael et al., 2007;Gil et al., 2009;Hu et al., 2009b). Surprisingly, induction with RA alone (without exogenous SHH) generated a large population of OLIG2 + cells. While the combination of RA and fibroblast growth factor (FGF) signaling is known to promote OLIG2 expression during chicken development and has been used for in vitro differentiation of both hESCs and hiPSCs (Novitch et al., 2003;Nistor et al., 2005;Pouya et al., 2011), we achieved OLIG2 induction in the absence of any exogenous FGF in our culture conditions. We show that RA, synergistically with the dual inhibition of SMAD proteins, upregulates OLIG2 (Figure S1C), possibly by stimulating the endogenous expression of SHH ( Figure S1A). We confirmed that SAG is an efficient replacement for SHH and indeed showed superior efficacy in our hands (Stacpoole et al., 2013). The addition of HGF to the medium, although not essential, appeared to slightly improve the differentiation efficiency (data not shown; Hu et al., 2009c). Finally, the transition from adherent cultures to spheres proved to be a critical step to enrich the OLIG2 + population and possibly restrict differentiation to the oligodendrocyte lineage.
In human development, OPCs are characterized by PDGFRa and NG2 expression, followed by expression of O4 (Jakovcevski et al., 2009). Under our culture conditions, by day 75, most of the O4 + cells had lost PDGFRa, but retained NG2 expression. At this stage, we did not observe any residual pluripotent cells in culture. Our study differs from recent work in that our iPSC lines were derived from PPMS patients and the cells used for in vivo transplantation were sorted using the late-OPC marker O4 to maximally restrict the differentiation potential. Despite these differences, the PPMS-derived, O4 + -sorted OPCs exhibited a similar engraftment efficiency, mitotic fraction, and proportion of host ensheathed axons while generating fewer GFAP + astrocytes compared with the unsorted iPSC-derived OPCs reported previously. Taken together, our data suggest that PPMS-derived OPCs performed in vivo at least as efficiently as healthy iPSC-derived cells (Wang et al., 2013).
These proof-of-principle experiments establish that our OPC induction protocol can generate myelinogenic oligodendrocytes from patient samples and may be useful for the development of autologous cell-replacement therapies for MS in the future.
iPSC technology is also emerging as a tool for developing new drugs and gaining insight into disease pathogenesis (Han et al., 2011). Our differentiation protocol will aid the development of high-throughput in vitro screens for compounds that promote myelination (Lee et al., 2013). Furthermore, the PPMS iPSC lines described here provide an additional resource for investigating the process of neurodegeneration in MS. Future studies comparing PPMS iPSCs and appropriate healthy control iPSC lines will be needed to shed light on the potential intrinsic differences among patient-derived oligodendrocytes.

Subjects
Skin biopsies were obtained from deidentified PPMS patients at the Tisch Multiple Sclerosis Research Center of New York, upon institutional review board approval (BRANY) and receipt of informed consent. All four patients were diagnosed with PPMS according to the standard diagnostic criteria. All patients were Caucasian.
73 (E), a cropped image of O4 + cells to highlight the ramified processes (F), MBP + mature oligodendrocytes at low (G)

Cell Lines
Three hESC lines and four hiPSC lines were used for the study. RUES1 and HUES 45 are both NIH-approved hESC lines. The OLIG2-GFP reporter line is derived from the BG01 hESC line and was a gift from Dr. Ying Liu (University of Texas Health Science Center at Houston). Four iPSC lines were derived in our laboratory from skin biopsies of PPMS patients through the mRNA/miRNA method (StemGent).

Oligodendrocyte Differentiation Protocol
hESCs and hiPSCs were induced to neural differentiation through dual SMAD inhibition together with 100 nM all-trans RA. At day 8, SAG (1 mM) was added to the medium, and at day 12, adherent cells were lifted and seeded in low-attachment plates to favor sphere aggregation. Spheres were cultured in the presence of RA and SAG. At day 30, spheres were plated into poly-L-ornithine/laminin-coated dishes and cells were allowed to migrate out of the sphere. At this stage, PDGF medium was used to promote OPC formation, and from day 75 onward, glial medium was used to drive oligodendrocyte maturation. For the qRT-PCR primer sequences and media compositions used, see Tables S1 and S2, respectively.
Transplantation into Shiverer 3 Rag2 À/À Mice All experiments using shiverer/rag2 mice (a gift from Dr. Steven A. Goldman, University of Rochester; Windrem et al., 2008) were performed according to protocols approved by the University at Buffalo Institutional Animal Care and Use Committee. Injections were performed as previously described (Sim et al., 2011) and the animals were sacrificed 12-16 weeks later. Cryopreserved coronal sections were cut and immunohistochemistry was performed as described previously (Sim et al., 2011). For transmission electron microscopy, tissue was processed as described previously (Sim et al., 2002). For a list of the primary antibodies used, see Table S3.