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iPSC-Progeny Aid Remyelination

“Human iPSC-Derived Oligodendrocyte Progenitor Cells Can Myelinate and Rescue a Mouse Model of Congenital Hypomyelination”

Human embryonic stem cells (hESCs) (Hu et al, Izrael et al and Keirstead et al) and foetal and adult human brain tissue (Dietrich et al, Roy et al and Windrem et al 2004) have both been used as sources of human glial progenitor cells capable of oligodendrocytic maturation and myelination for cell-based repair of demyelinated lesions. The problem of immune rejection, however, looms over these strategies. However, a recent study from the laboratory of Steven A. Goldman at the Center for Translational Neuromedicine and Department of Neurology, University of Rochester Medical Center, NY, USA has demonstrated that oligodendrocyte progenitor cells (OPCs) can be effectively generated from patient-specific skin-derived human induced pluripotent stem cells (hiPSCs) which can differentiate into myelinogenic oligodendrocytes, and also astrocytes, which can mediate myelination in the brain in a mouse model of congenital hypomyelination (Wang et al); altogether outlining a new resource of less immunogenic cells for this approach.

hESCs and iPSCs derived from fibroblasts and keratinocytes were assessed using an optimised protocol for the production of glial progenitor cells (Hu et al and Izrael et al). This six-stage OPC differentiation protocol of 110-150 days efficiently generated human OPCs from hESCs and hiPSCs alike. Astroglia were also produced, first appearing at day 70 and becoming abundant by the end of the differentiation period, as were oligodendrocytes (OLs), which were produced by the withdrawal of gliogenic growth factors to half-normal levels. OPCs were isolated from hiPSC cultures using A2B5, CD140a, CD9, and O4 which serially identify OPCs at different stages of lineage restriction, giving an enriched population equivalent to between 30 and 40% of total cell number.

Co-culture of isolated OPCs with human fetal cortical neurons isolated from 20 week gestational age foetal brain demonstrated abundant myelin basic protein (MBP)-positive processes that contacted neighbouring axons and initiated ensheathment. However, myelination was not definitively observed and so an in vivo approach was next utilised. OPCs were transplanted into the corpus callosum of newborn homozygous shiverer immunodeficient mice (an autosomal recessive mutant mouse with myelin deficiency) and were analysed 3 to 4.5 months later and demonstrated that progeny from hiPSCs could robustly myelinate processes within recipient brains, with high donor cell densities and widespread dispersal of grafted cells observed throughout forebrain white matter.   Indeed, the density of cells and levels of myelination were equal to or higher to that observed when using OPCs derived from second-trimester fetal brain tissue (Sim et al, Windrem et al 2004 and Windrem et al 2008). While OL differentiation from OPCs was obviously evident, progenitors also persisted and, additionally, astrocytic differentiation was observed. Assessment of any functional response to OPCs in the shiverer mice was next assessed using a using a five-site forebrain and brainstem injection protocol that achieves whole-neuraxis engraftment via transplanted OPCs. Control uninjected mice had an average lifespan of 141 days but, encouragingly, 19 of the 22 implanted mice lived longer than the control mouse with the greatest longevity. Injected mice who survived beyond 6 months exhibited substantial myelination of brain, brainstem, and cerebellar axons; indeed the level of myelination was greater in hiPSC-OPC-engrafted mice than in mice previously engrafted with fetal-human-tissue derived OPCs (Windrem et al 2008). Microscopic analysis found that only in the injected mouse brains, callosa were densely myelinated by mature compact myelin characterized by concentrically organized major dense lines and interlaminar tight junctions. Additionally, reconstitution of the nodes of Ranvier (gaps formed between the myelin sheaths to aid action potential conductance) in the injected mice suggests that hiPSC-derived OLs generate the cues necessary for the nodal organization of axonal proteins.

Finally, concerns over tumourigenesis were appraised; initially through protein mRNA analysis of pluripotency-associated markers. At late stages of the in vitro OPC protocol, no detectable OCT4, NANOG, or SSEA4 protein was observed and mRNA transcripts of OCT4 and hTERT were essentially undetectable. In vivo, even though NES or SOX2 expression in donor-derived cells suggested the persistence of progenitors, no persistent expression of OCT4, NANOG, or SSEA4 was detectable. As expected, no evidence of teratoma formation was observed in any mice and for any hiPSC line used; even for those mice that survived to 9 months. However, hiPSCs differentiated to early stages of the protocol then transplanted did show some tumour formation by 3 months, suggesting that the differentiation state of these cells was key to their tumourigenic capacity.

Overall, this exciting study demonstrates the ability to produce large amounts OPCs from patient-specific hiPSCs whose capacity for differentiation and myelination compares favourably to tissue derived fetal human glial progenitors, and with no evidence of tumourigenesis. Together, these results suggest that these cells could be used to provide myelinogenic autografts in disorders such as multiple sclerosis and traumatic demyelination, while the observed gliogenesis may also be of use for treating human glial pathologies.

 

References

  • Dietrich, J. (2002). Characterization of A2B5+ glial precursor cells from cryopreserved human fetal brain progenitor cells. Glia 40, 65–77.
  • Hu, B.Y. (2009). Human oligodendrocytes from embryonic stem cells: conserved SHH signaling networks and divergent FGF effects. Development 136, 1443–1452.
  • Hu, B.Y. et al (2009). Differentiation of human oligodendrocytes from pluripotent stem cells. Nat. Protoc. 4, 1614–1622.
  • Izrael, M. et al (2007). Human oligodendrocytes derived from embryonic stem cells: Effect of noggin on phenotypic differentiation in vitro and on myelination in vivo. Mol. Cell. Neurosci. 34, 310–323.
  • Keirstead, H.S. et al (2005). Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. J. Neurosci. 25, 4694–4705.
  • Roy, N.S. et al (1999). Identification, isolation, and promoter-defined separation of mitotic oligodendrocyte progenitor cells from the adult human subcortical white matter. J. Neurosci. 19, 9986–9995.
  • Sim, F.J. et al (2011). CD140a identifies a population of highly myelinogenic, migration-competent and efficiently engrafting human oligodendrocyte progenitor cells. Nat. Biotechnol. 29, 934–941.
  • Windrem, M.S. et al (2004). Fetal and adult human oligodendrocyte progenitor cell isolates myelinate the congenitally dysmyelinated brain. Nat. Med. 10, 93–97.
  • Windrem, M.S.et al. (2008). Neonatal chimerization with human glial progenitor cells can both remyelinate and rescue the otherwise lethally hypomyelinated shiverer mouse. Cell Stem Cell 2, 553–565.

Study originally appeared in Cell Stem Cell.

Stem Cell Correspondent Stuart P. Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.