In search of human oligodendroglia for myelin repair

Neuroscience Letters 456 (2009) 112–119

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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet

Review

In search of human oligodendroglia for myelin repair Delphine Buchet a,b , Anne Baron-Van Evercooren a,b,c,∗ a

INSERM, U975, Paris, F-75013, France Université Pierre et Marie Curie-Paris6, UMR S975. Paris, F-75013, France c AP-HP, Groupe hospitalier Pitié-Salpêtrière, Fédération de Neurologie, Paris, F-75013 France b

a r t i c l e

i n f o

Article history: Received 25 July 2008 Received in revised form 15 August 2008 Accepted 4 September 2008 Keywords: Neural progenitor cells Human oligodendrocytes Cell therapy Intracerebral grafting Myelin repair

a b s t r a c t Cell therapy appears as an exciting strategy for myelin repair in pathologies where oligodendrocytes are deficient or impaired, such as leucodystrophies and multiple sclerosis. Many studies indicate that several types of rodent cells, including neural stem and progenitor cells, play a beneficial role after grafting and induce functional recovery in animal models of myelin disorders. However, the difficulties to commit human neural stem cells towards the oligodendroglial lineage have long hampered human cell-based therapy for these diseases. In this review, we present recent advances in the field and discuss the various strategies that helped overcome the challenge of human oligodendroglial differentiation. © 2009 Elsevier Ireland Ltd. All rights reserved.

Contents Oligodendrocyte development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Isolation and derivation of oligodendroglial cells from the human foetal CNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Isolation and derivation of oligodendroglial cells from human ES cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . How to identify human myelin in vivo? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

During the past 20 years, cell therapy has emerged as one of the most promising strategies for the treatment of a wide range of CNS diseases, including myelin disorders such as leucodystrophies or multiple sclerosis. Remyelination through engraftment has been explored in various animal models of white matter diseases [4], such as rodent mutants, experimental autoimmune encephalomyelitis (EAE), as well as mechanical or chemical demyelination of the brain and spinal cord. Myelinogenic cells of multiple origins have been proposed as candidates for myelin repair, including the most immature pluripotent embryonic stem (ES) or induced pluripotent stem (iPS) cells [47,1b], “neurally” committed multipotent neural stem/progenitor cells [22,37], olfactory ensheathing cells [30,78] and Schwann cells [57,107]. All of these rodent cell types gave rise to promising results in terms of inte-

∗ Corresponding author at: Laboratoire des Affections de la Myéline et des Canaux Ioniques Musculaires, INSERM UMR S 546, 105 Bd de l’hôpital, 75013 Paris, France. E-mail address: [email protected] (A. Baron-Van Evercooren). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.09.086

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gration into the host tissue, differentiation, extent of myelination and eventual behavioural improvement, leading to the idea that cell therapy stands indeed as a suitable strategy for some of these disorders. As a natural follow-up, attempts have been made to isolate myelinating cells from human tissues. In the present review, we will focus on human CNS-derived myelinogenic cells, namely oligodendroglia and their derivatives. We will underline similarities and differences between rodent and human oligodendrocyte development, and discuss the difficulties to identify, isolate and derive human oligodendroglia from different sources, as well as their outcome after grafting into models of myelin disorders. Oligodendrocyte development During rodent CNS development, oligodendrocytes emerge around E12.5 and reach full maturity after birth, when myelination proceeds. In the spinal cord, the majority of oligodendrocytes arise from a very restricted ventral area called the pMN domain,

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under the influence of Sonic Hedgehog (Shh). A second wave of oligodendrocyte specification onsets at E15 in more dorsal areas by trans-differentiation of radial glia and independently from Shh signalling [15,29]. In the telencephalon, three distinct waves of oligodendrogenesis were described. A first wave takes place at E12.5 in the most ventral medial ganglionic eminence (MGE), followed by a second wave in the lateral ganglionic eminence (LGE) a few days later, around E15. These oligodendrocyte progenitor cells (OPCs) migrate dorsally and colonize the cortex around E18. The third wave of oligodendrogenesis takes place in the cortex itself after birth [52,75]. The progression along the oligodendroglial lineage occurs through a series of differentiation steps that can be schematically divided into four stages: early OPC, late OPC (or preoligodendrocyte), immature (or pre-myelinating) oligodendrocyte and mature (or myelinating) oligodendrocyte. Each of these stages is identifiable according to morphological features and specific patterns of marker expression. Early OPCs are small round bipolar cells with high proliferative and migratory potentials that express the transcription factors Olig1/2 [58,106], Nkx2.2 [34,105] and Sox10 [54], the membrane ganglioside A2B5 [25] and the alpha subunit of the platelet-derived growth factor receptor (PDGF-R␣) [68], closely followed by chondroitin sulphate proteoglycan NG2 [64] and DM20, an isoform of the proteolipid protein PLP [92]. Late OPCs extend multipolar short processes and start to express, in addition to early OPC markers, the transcription factor Sox17 [83] and a sulfatide recognized by the O4 antibody [84], which expression persists until the pre-myelinating stage. Pre-myelinating oligodendrocytes are post-mitotic cells characterized by long ramified branches and expression of galactocerebroside (GalC), 2 ,3 cyclic nucleotide 3 phosphodiesterase (CNPase), DM20/PLP [100] and APC (adenomatous polyposis coli [7]). These cells reach the mature oligodendrocyte stage by extending myelin membranes that form compact enwrapping sheaths around axons and by expressing myelin proteins such as CNPase, MAG, MOG, PLP and MBP. The well established expression of surface markers and/or transcription factors during rodent development has been transposed to humans to characterize the emergence of oligodendrocytes within the developing embryonic/foetal CNS, emphasizing numerous highly conserved mechanisms between the two species. As in rodents, Olig2 is expressed on each side of the ventral cord in the pMN domain as soon as 5 weeks of gestation (wg) [45]. Despite the early expression of Olig2, PDGF-R␣+ /O4+ OPCs are not detected before 6.5 wg, when they form clusters on each side of the ventral cord. By that time Shh is highly expressed in the floor plate [41], suggesting a conserved role of this morphogen in the emergence of ventral OPCs from rodents to humans. At 7.5 wg, OPCs disperse throughout the ventral and lateral white matter, where they start to differentiate into post-mitotic GalC+ pre-oligodendrocytes. At 10.5 wg, oligodendrocytes are detected in the dorsal spinal cord, suggesting that OPCs either migrate from the ventral parts of the CNS or emerge in dorsal parts, as established in rodents [52]. As observed in rodent spinal cord [61], human foetal oligodendrocytes mature along a rostro-caudal gradient, which correlates with the initial closure of the neural tube at the cervical level [19,41]. Although MBP transcripts are expressed in the human spinal cord as soon as 6.5 wg, the first myelinated segments appear around 11 wg, suggesting a clear dissociation between myelin protein expression and onset of myelination. In the human forebrain, Olig1- and Olig2-expressing cells are detected in ventral parts of the telencephalon and diencephalon at 5–5.5 wg [45], before PDGF-R␣ expressing OPCs appear. Thus, as in the spinal cord, Olig transcription factor expression precedes that of early OPC markers. In addition, as in rodents, homeodomain transcription factor expression suggests that telencephalic oligodendrocytes may have three distinct origins [72]. Two distinct

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ventral populations may originate in the MGE and LGE, respectively, before migrating laterally and dorsally toward the cortical SVZ and a third population may originate in the cortex itself. Between 17 and 24 wg, migratory early OPCs are found in a gradient from the cortical SVZ towards the cortical plate, while late OPCs accumulate in the transient sub-plate zone [46] and predominate in the sub-cortical white matter between 23 and 32 wg [3]. Although MBP expression can be detected in occasional pre-myelinating oligodendrocytes within the emerging white matter at midgestation [46], the first myelinated segments appear only around 30 wg, thus underlining again a clear dissociation between MBP expression and the onset of myelination. Altogether, these results highlight numerous similarities between rodent and human CNS development. However, major differences were also underlined, most of which may be related to the extended duration of human CNS development and to the requirement to generate larger numbers of cells within the human CNS. First, while myelination takes place after birth in rodents, it starts during gestation in humans, around the 30th week of gestation [3]. Moreover, Olig transcription factor expression (e.g. lineage specification) precedes the emergence of PDGF-R␣+ OPCs in both the spinal cord and telencephalon, and cells expressing MBP are detected several weeks before myelinated fibre tracts can be observed in these regions. Although the sequence of events may be the same in both species, the duration of human CNS development renders these mechanisms easier to observe, with a clear delay between transcription factor expression and OPC emergence on one hand, and MBP expression and myelination process on the other hand. Isolation and derivation of oligodendroglial cells from the human foetal CNS In 1987, Gumpel and colleagues provided the first evidence for extensive myelination of the rodent CNS by grafted human foetal cells. CNS fragments, obtained after elective abortions during the first trimester of gestation, were transplanted into the newborn MBP-deficient shiverer mouse brain and gave rise to extensive MBP expression throughout the parenchyma [40]. Moreover, these fragments were frozen for long periods of time without losing their myelinogenic potential [79]. These pioneering data demonstrated the ability of human cells to survive, migrate and integrate as functional oligodendrocytes into the dysmyelinated environment. However, grafting CNS fragments raised several technical issues. First, the complexity and heterogeneity of CNS fragments, together with the variability introduced by donor age and CNS region, rendered grafted cell characterization difficult. Second, the limited amount of human tissue obtained after abortion raised the necessity of cell expansion. Therefore, the main challenges in recent years resided in standardizing cell transplantation and designing protocols for successful amplification of pro-myelinating cells. The discovery of multipotent neural stem cells in the rodent developing CNS with the ability to self-renew in vitro [74] raised the hope that such cells could also be isolated from the human foetal CNS. Their inherent ability to self-renew and differentiate into the three major cell types of the CNS, namely neurons, astrocytes and oligodendrocytes, has made of them an appealing cellular tool for CNS repair including myelin repair. The first isolation of neural stem/progenitor cells (NPCs) from the foetal CNS was described in 1995 by Buc-Caron [11]. Cells were amplified as adherent monolayers [11,16,18,19,85–87] or floating neurospheres [28,60,94,95,103], in the presence of bFGF with or without EGF and LIF, without losing their tripotent differentiation ability [85]. In vitro, oligodendrocyte maturation followed the same sequential pathway as rat cells (Fig. 1), with the successive appearance

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Fig. 1. Four step-maturation of neural stem cells towards the oligodendroglial lineage. Oligodendrocyte progenitor cells (OPCs), pre-oligodendrocytes, immature oligodendrocytes and mature (myelinating) oligodendrocytes can be distinguished by their increasingly complex morphology, by their proliferating, migrating and myelinating potentials, as well as by their expression of a wide range of well defined markers. (A–E) illustrate oligodendroglial maturation of human neural stem cells. (A–D) Neural cells isolated from an 8.5-week-old human embryo display various marker expression that identify different maturation states. (E) Mature MBP-positive oligodendrocytes can be isolated from the adult human temporal white matter.

of proliferative PDGF-aR+ early OPCs (1DIV), O4+ /PLP-DM20+ late OPCs (3 and 7 DIV), GalC+ pre-oligodendrocytes (mostly after 7 DIV) and MBP+ mature oligodendrocytes (mostly after 3 weeks) [60,103]. However, human foetal NPC cultures gave rise to very low proportions of oligodendroglial cells, which furthermore took twice as long as their rodent counterparts to differentiate. Moreover, the ability of human NPCs to generate oligodendrocytes decreased with time in culture, especially when grown in the presence of bFGF alone [19,103]. Candidate soluble factors known to efficiently promote oligodendroglial cell proliferation/specification in rodents were tested for their ability to enhance human NPC-derived oligodendrogenesis [19,60,69]. While T3 treatment of neural spheres increased the rate of OPC formation and the complexity of their shape, PDGF treatment did not potentiate this effect [60]. Until now, successful generation of oligodendroglia from foetal CNS was not achieved by means of “epigenetic” stimulation, suggesting that human NPCs/OPCs have distinct signalling requirements to their rodent counterparts with respect to the generation of oligodendrocytes in vitro. Consistent with the low oligodendroglial potential of human cells in vitro, transplantation experiments performed into the embryonic rat ventricle [10], new-born mouse or rat CNS [27,28,76], or intact adult rodent brain [12,26,33,77] showed that, although human NPCs responded to environmental cues and assumed neuronal and astrocytic phenotypes in neurogenic and non-neurogenic areas of the brain, respectively, they generated very limited numbers of myelinating oligodendrocytes in vivo. Moreover, when

grafted into the non-lesioned new-born or adult CNS [26,27], a consistent proportion of human foetal NPCs remained undifferentiated after 14 and 20 weeks, respectively, suggesting that immature human NPCs take a much longer time than their rodent counterparts to differentiate. However, after grafting into the contused rat spinal cord, homogeneous populations of human neural stem cells prospectively isolated on the basis of their CD133 expression differentiated into neurons and glia, including APC+ mature oligodendrocytes, and induced locomotor improvement [21]. Moreover, myelin ensheathment was confirmed by electron microscopy within three different environments, the shiverer new-born brain and adult crushed spinal cord, and the nude mouse contused spinal cord. This study therefore raised several new and important findings. First, although grafted cells were homogeneous populations of uncommitted neural stem cells, they differentiated rapidly (4–6 weeks) within the contused spinal cord, suggesting that human neural stem cells respond to local cues, and/or that the lesioned environment may act as an accelerator of differentiation. Second, it suggested that the type of lesion may be determinant for the commitment of the cells since human foetal NPCs did not give rise to oligodendrocytes 6 weeks after transplantation into the X-irradiated EB demyelinated rat spinal cord [19], while they did within the contused rat spinal cord [21]. The extent of myelination remained somehow surprising since spinal cord injury is known to promote BMP release [80,81] and since these molecules are known to promote astrocyte or Schwann cell differentiation from CNS stem cells [39,50,59,70,71]. This study

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therefore indicates that signalling molecules other than BMPs may be involved in cell differentiation in vivo and may differ from one type of lesion to another. The difficulty to derive consistent amounts of oligodendroglial cells has long hampered successful remyelination strategies. This pleaded for the necessity to sort out human OPCs in order to improve their ability to produce myelin in vivo. This conjecture was admirably demonstrated by Goldman and colleagues [97] who prospectively isolated human foetal glial progenitors on the basis of surface marker expression. In doing so, they showed that human A2B5+ /PSA-NCAM− cells not only spread throughout the shiverer mouse neuraxis and formed a chimeric white matter with compact myelin after transplantation, but also induced normal node of Ranvier acquisition and restoration of transcallosal conduction velocities, thereby resulting in substantially improved clinical features and considerably prolonged survival of the transplanted animals. Similar A2B5+ /PSA-NCAM− progenitors were isolated from the adult human brain [96]. After transplantation into the newborn shiverer brain, they displayed limited migration potential and differentiated into mature MBP+ oligodendrocytes within 4 weeks, whereas foetal progenitors showed extended migration and delayed differentiation, with no MBP+ cells generated before 12 weeks post-grafting. Moreover, foetal progenitors also generated astrocytes in vivo [96], thereby displaying bipotential glial progenitor characteristics rather than committed OPC features, whereas adult cells did not. Although isolated on the same selection criteria, foetal and adult progenitors displayed very different features, raising the possibility that foetal glial progenitors progressively lose their plasticity with aging or that A2B5+ /PSA-NCAM− selection identifies two distinct populations in the foetus and in the adult. The use of a larger panel of markers should help improve the characterization of human glial progenitors. In summary, neural stem and progenitor cells represent a potential source of myelinogenic cells, provided that OPC enrichment can be achieved, either by exogenous stimulation or selection of cells of the appropriate lineage. The critical point for the achievement of successful therapies relies on the determination of the exact phenotype of the cells at the time of grafting. Regarding this aspect, foetal cells may not represent the ideal source for reproducible data, since donor/gestational age and CNS region give rise to phenotypic and/or functional heterogeneity [19,96]. Further phenotypic characterisation of these various populations should be undertaken. Moreover, one of the major hurdles for the use of CNS stem/progenitor cells for clinical applications relies on the limited availability of tissue and limited expansion abilities of the cells before they show signs of in vitro senescence [98]. Isolation and derivation of oligodendroglial cells from human ES cells Another possible source of oligodendroglia has been considered after the isolation of ES cell lines from supernumerary human blastocysts [91]. These pluripotent cells can be expanded in largescale cultures and have the inherent ability to give rise to all the cell types of the organism, including neural cells and theoretically oligodendrocytes. One of the major limitations for the therapeutic use of these cells is their ability to form teratomas when grafted at an undifferentiated state, due to the uncontrolled growth and differentiation [8,9]. Therefore, ES cells represent powerful tools for cell-based therapy, provided that they can be adequately geared towards the desired lineage. Given the large potential of these cells and the complexity of events leading to their maturation into multiple lineages, a large variety of protocols were described to control human ES (hES) cell differentiation into neuroectodermal cells. Exposure to bFGF

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and/or EGF in a defined medium containing insulin, transferrin and sodium selenite promotes expression of two neuroectodermal markers, nestin and musashi, as well as neurosphere formation [42,73,104]. This approach can be combined with BMP-antagonists such as Noggin to further increase the generation of neural cells [36,43,93]. A third strategy which relies on all-trans retinoic acid (ATRA) treatment [17] promotes neural commitment of hES cells and neurosphere expansion. However, as observed with foetal human NPCs, hES-derived neuroectodermal cells generate a limited amount of oligodendroglial cells in vitro, which are generally identified as OPCs or early multipolar pre-oligodendrocytes expressing O4 and NG2 and do not assume more differentiated phenotypes. Moreover, transplantation of hES-derived NPCs into new-born mice [104] or irradiated EB-demyelinated mouse spinal cord [48] provided evidence for neuron and astrocyte generation only. Therefore, as suggested for foetal NPCs [19], multipotent NPCs require ex vivo oligodendrocyte lineage commitment prior to transplantation. Protocols based on cell selection or further inductive treatment have thus been designed to enhance the proportion of oligodendrocytes and their maturation from hES-derived neural stem/progenitor cells. Nistor and colleagues succeeded in generating cultures highly enriched in OPCs (over 80% of O4+ cells) [65] by subjecting ATRAtreated hES cells to differential selection of oligodendrocyte-lineage cells in defined media followed by growth in Matrigel. Although these ES-derived OPCs exhibited myelinating capacities after grafting into the contused spinal cord [65] or shiverer mouse brain [51], they did not fully mature in vitro. Kang and colleagues [49] proposed another protocol to expand hES-derived NPCs and improve OPC generation. After derivation of nestin+ neuroectodermal cells through bFGF exposure, they sequentially supplemented the medium with EGF and PDGF, known to stimulate NPC [62] and OPC [5] proliferation, respectively. OPCs were identified by their expression of PDGF-R␣, A2B5 and NG2. Further exposure to T3, known to induce oligodendrocyte survival and differentiation [23], achieved maturation of the cells towards O4+ /MBP+ oligodendrocytes. Izrael and colleagues used a molecular approach based on the combined expression of Olig2, Nkx2.2 and Sox10 as a criterion to define oligodendroglial commitment [44] and showed that, unlike what was observed with mouse ES cells [101,102], exposure to high levels of bFGF is insufficient to induce the combination of the three transcription factor, Nkx2.2 being absent from their cultures. This observation further supports the idea that protocols found to be efficient for mouse ES-derived oligodendrocyte production do not always apply to human cells and highlights differences between mice and human ES cells. However, ATRA exposure was found critical for Nkx2.2 induction and, once this transcription factor was expressed, Noggin-driven inhibition of BMPs induced Sox10 expression, a key event for the maturation of hES-NSC toward MBP+ oligodendrocytes. Most interestingly, the authors also showed that Noggin critically stimulated O4+ OPCs prior to grafting and considerably enhanced their myelinating potential within the shiverer brain. In conclusion, hES cells represent an appealing alternative to the use of human foetal NPCs, because they can be expanded in high scale cultures without undergoing senescence. Moreover, after amplification, these cells can be oriented towards the appropriate cell lineage and OPCs can be further grown in vitro, a step that could not be achieved with human foetal NPCs. However, one of the crucial points for safe use of hES cells resides in the development of culture protocols implemented with defined or human-derived products only [47]. While the above studies provided proof of concept for the feasability to derive OPCs from ES cells with remyelination capacities, their ability to sustain long-term remyelination leading to functional recovery remains to be addressed.

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Fig. 2. Human-specific anti-NogoA antibody allows detection of human oligodendrocytes after grafting. Two months after grafting into the new-born shiverer mouse brain, hNPCs isolated from an 8.5-week-old foetus survive, migrate and give rise to myelinating oligodendrocytes that can be discriminated from the host by their expression of human NogoA (green, A and B) and MBP (red, A). Note that NogoA-positive cells also express Olig2 (red, B). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

How to identify human myelin in vivo? An additional challenge that has to be faced for human cellbased pre-clinical studies is the identification without reservation of the grafted cells and their distinction from host cells. Human cell tracking within the host parenchyma can be achieved through a variety of means. Cells can be detected using human-specific antibodies directed against nuclear or cytoplasmic antigens, such as human-nuclei antigen and human-mitochondria [51,95], or directed against lineage-specific markers such as human-specific anti-GFAP (SMI21) for astrocytes [12,90] and human-specific antiTau [66] for neurons. Until now, antibodies specifically directed against human oligodendrocytes and designed to distinguish human cells from the host tissue were not available. However, we have used a human-specific anti-NogoA antibody that allowed unambiguous detection of human pre-myelinating and myelinating oligodendrocytes after grafting into the mouse brain (Fig. 2). Cells can also be tracked by in situ hybridization directed against humanspecific Alu DNA repeated sequences [12,104]. Alternatively, cells can be tagged before transplantation, for example via viral vector transduction encoding fluorescent proteins [13,89]. Recently, non-invasive methods were developed to follow transplanted cells by magnetic resonance imaging, using superparamagnetic iron oxide particles to label the transplanted cells [6,14,31,63,67], thus allowing cell tracking over long periods of time. Although the latter method needs further improvement in terms of cell specificity, these tracing methods ease the identification of human cells grafted in rodents and provide means to address their potential for survival, migration and differentiation within the host CNS environment. However, when it comes to myelin detection, several technical hurdles can be faced. In genetic models such as the MBP-deficient shiverer mouse or PLP-deficient md rat, expression of the missing protein or enzyme can be attributed to the exogenous human cells, therefore allowing for both their tracking and determination of their differentiation potential. However, the demonstration of myelin protein expression by immunohistochemistry is not sufficient to demonstrate proper myelin ensheathment and compaction, or the functional benefits of the grafted cells. The only solid demonstration of myelin compaction relies on electron microscopy. In genetic models where myelin compaction fails, as it is the case in shiverer [56] or twitcher [53] mice, compact myelin can be attributed to exogenous myelinating cells in view of their ability to form an electron-dense line [21,24,35,40,44,65,96,97,99].

In animal models of chemical or mechanical demyelination, such as LPC or spinal cord contusion, spontaneous remyelination occurs and can be distinguished from non-affected myelin by G ratio determination. However, it is much more difficult to discriminate between endogenous and transplanted cell-derived exogenous remyelination. Although human cells can be easily tagged and/or traced by immunohistochemistry, the targeted proteins are generally excluded from compact myelin sheaths, rendering co-localisation of human myelin and human markers difficult to prove. Although technical progress has been made to enhance immuno-EM efficiency [38,82], a way to circumvent this hurdle would be to use X-irradiated EB-demyelinated mice or rats (reviewed in Refs. [4,20]), since irradiation hampers endogenous remyelination and allows for grafted cell discrimination at the lesion site. In any case, this model also comprises its own limitations since X-irradiation seems to hamper oligodendrocyte differentiation from the grafted NPCs [19,48] and to favour the differentiation of transplanted cells into Schwann cells [1,32,88]. Conclusion Although myelin formation by human cells was demonstrated more than 20 years ago [40], human cell-based therapy has long been hampered by several technical hurdles. First, the need for consistent amounts of available tissue has raised the necessity for in vitro amplification. Although amplification of human foetal NPCs could be achieved, their limited spontaneous generation of oligodendroglial cells in vitro and in vivo has made oligodendroglial commitment and/or selection an appealing strategy for myelin repair. However, due to human-specific signalling requirements and difficulties to characterise human oligodendrocytes in vitro, foetal NPC differentiation toward the oligodendroglial lineage still remains an unresolved issue. Oligodendrocyte commitment and amplification were however successfully achieved using pluripotent hES cells. Due to standardization and “humanization” of culture protocols, this cell source may represent a clinically compatible tool for myelin repair in the near future. Second, graft rejection has also represented a major hurdle for experimental human cell-based therapy. Nude and NOD-scid mice were extensively used and represent one of the most permissive environments for human cell survival. For studies in new-born or immunocompetent animals, poor success of human cell graft outcome was obtained unless animals were immunosuppressed by daily injections of ciclosporine. However, recently, the MBP shiverer

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mutation was backcrossed onto the NOD-Scid or RAG2 (−/−) backgrounds and serious improvement of cell survival was observed [21,97]. Finally, human cell-derived myelination of the CNS has been evaluated in a variety of dys/demyelination models developed in rodents, each of them representing a valuable tool to address specific questions. Although consistent migration of human OPCs and wide myelination was observed in the rodent CNS, there is a need to evaluate the potential of these cells in a CNS environment of larger size and philogenetically closer to humans. Models of demyelination were developed in the macaque spinal cord [2] and optic nerve [55], mimicking acute and chronic demyelination, respectively. Evaluating the behaviour of human NPCs/OPCs in such models may provide new and important insights into the ability of these cells to migrate over long distances and produce functional myelin in the appropriate environment. Acknowledgements We are grateful to current members of Anne Baron-Van Evercooren’s lab for helpful discussions. We also thank Dr. Corina Garcia for her critical reading of the manuscript and for the illustration of MBP-expressing human oligodendrocytes. Studies on human cells are supported by INSERM, ELA, NMSS (USA) and Contrat Interface INSERM/AP-HP. DB was supported by fellowships from APETREIMC, La Fondation Motrice and IRME. References [1] Y. Akiyama, O. Honmou, T. Kato, T. Uede, K. Hashi, J.D. Kocsis, Transplantation of clonal neural precursor cells derived from adult human brain establishes functional peripheral myelin in the rat spinal cord, Exp. Neurol. 167 (2001) 27–39. [1b] G. Amabile, A. Meissner, Induced pluripotent stem cells: current progress and potential for regenerative medicine, Trends Mol Med 15 (2009) 59–68. [2] C. Bachelin, F. Lachapelle, C. Girard, P. Moissonnier, C. Serguera-Lagache, J. Mallet, D. Fontaine, A. Chojnowski, E. Le Guern, B. Nait-Oumesmar, A. Baron-Van Evercooren, Efficient myelin repair in the macaque spinal cord by autologous grafts of Schwann cells, Brain 128 (2005) 540–549. [3] S.A. Back, N.L. Luo, N.S. Borenstein, J.M. Levine, J.J. Volpe, H.C. Kinney, Late oligodendrocyte progenitors coincide with the developmental window of vulnerability for human perinatal white matter injury, J. Neurosci. 21 (2001) 1302–1312. [4] A. Baron-Van Evercooren, W.F. Blakemore, Remyelination through engraftment, in: R. Lazzarini (Ed.), Myelin Biology and Disorders, vol. 1, Elsevier Academic Press, San Diego, 2004, pp. 143–161. [5] W. Baron, B. Metz, R. Bansal, D. Hoekstra, H. de Vries, PDGF and FGF-2 signaling in oligodendrocyte progenitor cells: regulation of proliferation and differentiation by multiple intracellular signaling pathways, Mol. Cell. Neurosci. 15 (2000) 314–329. [6] T. Ben-Hur, R.B. van Heeswijk, O. Einstein, M. Aharonowiz, R. Xue, E.E. Frost, S. Mori, B.E. Reubinoff, J.W. Bulte, Serial in vivo MR tracking of magnetically labeled neural spheres transplanted in chronic EAE mice, Magn. Reson. Med. 57 (2007) 164–171. [7] R.V. Bhat, K.J. Axt, J.S. Fosnaugh, K.J. Smith, K.A. Johnson, D.E. Hill, K.W. Kinzler, J.M. Baraban, Expression of the APC tumor suppressor protein in oligodendroglia, Glia 17 (1996) 169–174. [8] L.M. Bjorklund, R. Sanchez-Pernaute, S. Chung, T. Andersson, I.Y. Chen, K.S. McNaught, A.L. Brownell, B.G. Jenkins, C. Wahlestedt, K.S. Kim, O. Isacson, Embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model, Proc. Natl. Acad. Sci. U.S.A. 99 (2002) 2344–2349. [9] A. Brederlau, A.S. Correia, S.V. Anisimov, M. Elmi, G. Paul, L. Roybon, A. Morizane, F. Bergquist, I. Riebe, U. Nannmark, M. Carta, E. Hanse, J. Takahashi, Y. Sasai, K. Funa, P. Brundin, P.S. Eriksson, J.Y. Li, Transplantation of human embryonic stem cell-derived cells to a rat model of Parkinson’s disease: effect of in vitro differentiation on graft survival and teratoma formation, Stem Cells 24 (2006) 1433–1440. [10] O. Brustle, K. Choudhary, K. Karram, A. Huttner, K. Murray, M. Dubois-Dalcq, R.D. McKay, Chimeric brains generated by intraventricular transplantation of fetal human brain cells into embryonic rats, Nat. Biotechnol. 16 (1998) 1040–1044. [11] M.H. Buc-Caron, Neuroepithelial progenitor cells explanted from human fetal brain proliferate and differentiate in vitro, Neurobiol. Dis. 2 (1995) 37–47. [12] D. Buchet, M.H. Buc-Caron, O. Sabate, F. Lachapelle, J. Mallet, Long-term fate of human telencephalic progenitor cells grafted into the adult mouse brain: effects of previous amplification in vitro, J. Neurosci. Res. 68 (2002) 276–283.

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