Human embryonic stem cell-derived neurons establish region-specific, long-range projections in the adult brain
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While the availability of pluripotent stem cells has opened new prospects for generating neural donor cells for nervous system repair, their capability to integrate with adult brain tissue in a structurally relevant way is still largely unresolved. We addressed the potential of human embryonic stem cell-derived long-term self-renewing neuroepithelial stem cells (lt-NES cells) to establish axonal projections after transplantation into the adult rodent brain. Transgenic and species-specific markers were used to trace the innervation pattern established by transplants in the hippocampus and motor cortex. In vitro, lt-NES cells formed a complex axonal network within several weeks after the initiation of differentiation and expressed a composition of surface receptors known to be instrumental in axonal growth and pathfinding. In vivo, these donor cells adopted projection patterns closely mimicking endogenous projections in two different regions of the adult rodent brain. Hippocampal grafts placed in the dentate gyrus projected to both the ipsilateral and contralateral pyramidal cell layers, while axons of donor neurons placed in the motor cortex extended via the external and internal capsule into the cervical spinal cord and via the corpus callosum into the contralateral cortex. Interestingly, acquisition of these region-specific projection profiles was not correlated with the adoption of a regional phenotype. Upon reaching their destination, human axons established ultrastructural correlates of synaptic connections with host neurons. Together, these data indicate that neurons derived from human pluripotent stem cells are endowed with a remarkable potential to establish orthotopic long-range projections in the adult mammalian brain.
KeywordsNeural stem cells Transplantation Axon outgrowth Synaptogenesis
Recent progress with the derivation of neural stem cells from embryonic [1–3], fetal [4–6], and adult  sources provides interesting prospects for regenerative medicine . However, the capability of grafted neurons to integrate and in particular to establish appropriate long-range projections in the adult brain has been a matter of controversy. Pioneering studies employing primary fetal human donor cells [9, 10] showed a substantial capacity for axonal outgrowth from telencephalic transplantation sites. However, massive in vitro expansion of neural cells was in some studies associated with impaired axonal outgrowth [11–13], while other studies reported extensive or enhanced axonal outgrowth even after extensive pre-transplant in vitro proliferation of the donor cells [2, 14, 15]. Site of implantation, age of the transplant recipient, presence and extent of local lesions, and glial scaring are also considered to influence axonal outgrowth from grafted neurons . Park et al. found bioscaffolds highly effective to facilitate axonal growth from grafts after hypoxic injury, which was otherwise inhibited . Technically, labeling strategies sufficient for the detection of distant processes were not always applied, which might have led to an underestimation of long-range projections in some studies. Mechanistically, axonal growth and pathfinding, as well as their inhibition, strongly depend on a precise interplay between endogenous signaling molecules and receptors on donor cells. Axonal outgrowth for example is, in the adult brain, inhibited by proteins associated with CNS myelin (e.g., Nogo) via signaling through respective receptors on growth cones . However, why axonal outgrowth from some donor populations escapes the inhibitory environment in the adult brain while it is blocked for others has not yet been determined. Species differences might contribute to the different results obtained in various xenograft models, potentially due to a mismatch of endogenous inhibitory molecules with xenogeneic receptors on human neurons. However, this notion is challenged by data from Gaillard et al., showing that long-range axonal outgrowth is possible following transplantation of murine cells into adult isogenic hosts . Moreover, the results of this and several other recent studies suggest that murine donor cells exhibiting the same regional identity as the implantation site can establish region-specific axonal projections in newborn [20, 21] and adult hosts . However, the potential of human neural grafts to establish axonal projections in the adult brain still deserves further investigation.
We have recently established a stable population of long-term self-renewing neuroepithelial stem cells (lt-NES cells) from pluripotent human ES cells , which give rise to neurons with a posteriorized regional phenotype in vitro. We used this highly uniform population to explore whether heterotopically grafted human neural stem cells with a highly restricted regional phenotype can give rise to region-specific axonal projections. To that end, lt-NES cells were transplanted into the cortex and hippocampus of adult rodents, i.e., locations exhibiting different and highly specific neuronal innervation patterns of clinical relevance. Our data show that lt-NES-derived neurons develop axonal projections highly specific for the implantation site and establish morphologically mature synapses.
Materials and methods
Human ES cell-derived long-term self-renewing neuroepithelial stem cells (lt-NES cells, derived from hES cell lines I3 and H9.2) were generated and transduced for GFP expression as described previously [2, 22] and in Supplementary Methods. For transplantation, donor cells passage 25–55 were trypsinized, washed in calcium- and magnesium-free PBS supplemented with 0.1% DNAse and concentrated to 7.5 × 104 cells/μl.
RNA was extracted using standard procedures from lt-NES cells and their differentiating progeny after 2, 4, and 8 weeks of in vitro differentiation and subsequently, cDNA was generated. Primer pairs (Supplementary Methods, table 1) were designed (Primer3) and PCRs performed using common cycling parameters.
Animals and transplantation
Severe combined immunodeficient-beige (SCID-bg) mice were used at an age of 8–10 weeks (n = 52, body weight 22–28 g). Alternatively, female 12-week-old Sprague–Dawley rats (n = 20, body weight 220–280 g) were used. Rats and mice were stereotactically transplanted according to coordinates adopted from Paxinos et al.  (Supplementary Methods). Sprague–Dawley rats were immunosuppressed with daily injections of cyclosporine (10 mg/kg i.p.). Animals were monitored for wound infections and neurological deficits on a daily basis during the first 2 weeks after transplantation and in weekly intervals thereafter. Care and use of the animals conformed to institutional policies and state legislation.
Immunohistochemistry and microscopy
Primary antibodies (Supplementary Methods, table 2) of the same species were never used together to avoid cross reactivity. Primary antibodies were visualized using corresponding FITC, Cy3 or Cy5 conjugated secondary antibodies. Sections were analyzed on a Fluoview 1000 confocal microscope (Olympus) or, if DAPI visualization was required, on a Zeiss Axioimager Z1 equipped with the Apotome technology (Zeiss) to reconstruct optical sections. Pre-embedding immunolabeling for electron microscopy was performed with the human specific anti-synaptophysin antibody. Ultrathin sections were examined under an electron microscope (CM-10, Philips).
In vivo analysis for the assessment of viability was performed in analogy to the Cavalieri method (Supplementary Methods). For determination of phenotypes in vivo at least 150 cells per animal (n = 3 per time point) were counted for every marker. Values represent % ± standard deviation. Statistical significance was calculated using paired Student’s t test [*p (two-sided) = 0.01−0.05].
Prolonged differentiation into mature, non-tumorigenic grafts
Human ES cell-derived long-term self-renewing neuroepithelial stem cells (lt-NES cells) were propagated as described previously . In the presence of FGF2 and EGF, these cells exhibit uniform expression of the neural stem cell-associated genes nestin and sox2, a rosette-like growth pattern, high neurogenic differentiation potential, and a regional phenotype corresponding to an anterior hindbrain location, with all these properties remaining stable for at least 80 passages . For transplantation, 7.5 × 104 lt-NES cells (passage 25–55; derived from lines I3 and H9.2 [24, 25]) expressing EGFP from the PGK promoter , were stereotactically injected into the dentate gyrus or motor cortex of adult immunodeficient SCID-bg mice or immunosuppressed Sprague–Dawley rats. Recipient mouse brains were analyzed 3, 6, 12, 24, and 48 weeks after transplantation, rat brains at 3, 6, and 12 weeks after transplantation.
Donor cells could not only be identified by virtue of their GFP expression, but also using human-specific antibodies to human nuclei, Ki67, nestin, synaptophysin and NF-M . Furthermore, human nuclei show a remarkably homogenous heterochromatin pattern in DAPI stains (Fig. 1D, H, asterisks), which can be distinguished from the coarse heterochromatin of mouse cells (e.g., Figs. 1H, 3H). Nonetheless, fusion of donor cells with host cells remains a concern in the interpretation of transplant studies, in particular when donor cells appear to acquire traits of resident cells. It is thus essential to distinguish whether regional differentiation patterns are truly due to donor cell plasticity rather than mimicry through cell fusion. We addressed this issue and stained grafted brain slices 6 and 9 months after transplantation into SCID-bg mice with an antibody against human nuclei and combined this staining with an in situ hybridization for the detection of mouse satellite DNA. More than 500 human nuclei were analyzed in both transplantation sites, but not a single nucleus co-stained positive for the murine DNA in situ probe (Fig. 1I), suggesting that cell fusion is not a relevant event after transplantation of human lt-NES cells into the adult mouse brain.
lt-NES-derived neurons generate axons in vitro and in vivo
After initiation of in vitro differentiation by growth factor withdrawal, lt-NES cells formed a complex axonal network within several weeks (Fig. S2A). RT-PCR analyses showed that differentiated lt-NES cells express a number of factors and receptors known to be involved in axon outgrowth and guidance  (Fig. S2B). Members of the netrin, ephrin, semaphorin and robo/slit families of guidance molecules and receptors were expressed in proliferating lt-NES cells, but some of them decreased during in vitro differentiation, suggesting the presence of a cell-autonomous time window for the direction of axonal outgrowth after transplantation. On the contrary, within the adult mammalian brain, axonal growth is limited to an absolute minimum . Molecular stop signals like Nogo, Myelin-associated glycoprotein (MAG), Myelin-oligodendrocyte glycoprotein (MOG) and the repulsive guidance molecule (RGM-A)  play an important role. These inhibitors signal through a receptor complex composed of the Nogo receptor (NgR1), LINGO and p75. RT-PCR analysis of proliferating and differentiating human lt-NES cells revealed a continuous expression of Nogo and RGM-A, whereas MAG and MOG were not expressed (Fig. S2C). NgRs and Lingo were expressed at a constant level, whereas p75 was upregulated upon neuronal maturation (also reflected at the immunohistochemical level in Fig. S3).
Evidence for the formation of xenogenic synapses
The clear identification of hSyn-positive terminals and co-localization with markers for specific neurotransmitters allowed the quantification of inhibitory and excitatory donor-derived synaptic terminals in different projection fields. This analysis revealed that within the ipsilateral pyramidal cell layer, 36.7 ± 5.9% of human terminals co-stained positive for GAD67 whereas 12.8 ± 3.2% of the human terminals co-stained positive for vGlut2 24 weeks after transplantation. Within the contralateral pyramidal cell layer 32.9 ± 5.9% of the human terminals stained positive for vGlut2 and no clear co-localization with GAD67 could be detected.
The most important finding of this study is the remarkable specificity with which human ES cell-derived long-term self-renewing neuroepithelial stem cells (lt-NES) recapitulate endogenous axonal projections within the adult brain. Some previous studies with primary and in vitro propagated human cells had already hinted at their capacity for extensive axonal innervation [9, 10, 15, 31], which was mainly found to follow white matter tracts close to the site of transplantation. Our study revealed that upon transplantation into the motor cortex lt-NES establish ipsi- and contralateral projections as well as trajectories into the pyramidal and extrapyramidal motor system, including the cerebral peduncles and the cervical spinal cord. Interestingly, the same cells adopted a hippocampus-specific projection profile with laminar specificity when transplanted into the dentate gyrus. The contralateral fiber projection and termination pattern via the fimbria-fornix closely resembles that of endogenous commissural fibers . Importantly, only glutamatergic human terminals were detectable along these long-range fibers, despite the fact that the majority of donor cells show a GABAergic phenotype after transplantation. To exclude fusion with host cells as a possible explanation for this phenomenon, we used all available genetic, immunological, and morphological markers to unambiguously identify human cells. We also demonstrate that human axon terminals establish contacts displaying all morphological characteristics of normal active synapses.
So far, only few studies employing murine cells have reported such a highly region-specific projection pattern of grafted neurons. Gaillard et al.  showed that motor cortex grafts can reestablish appropriate long-range projections within the adult murine motor system. They found the homotopic nature of their explants to be important for successful reconstruction. This notion is further supported by two recent publications employing transplantation of murine ES cell-derived cortical precursors into newborn mice [20, 21]. In contrast, the human ES cell-derived lt-NES cells used in our study do not exhibit a telencephalic phenotype. They are posteriorized and show a marker profile compatible with an anterior hindbrain identity, a bias acquired during long-term in vitro expansion in the presence of growth factors . After transplantation into the adult hippocampus and motor cortex, they do not acquire a region-specific phenotype as indicated by the absence of BF1, a transcription factor broadly expressed in both telencephalic regions. Thus, for both target regions, our donor cell population can be regarded as heterotopic. Yet, the cells exhibit highly specific patterns of axonal outgrowth.
The precise mechanisms for this remarkable specificity remain to be elucidated. As in the study by Gaspard et al., we left the surrounding endogenous motor cortex or hippocampal tissue intact in order to maintain potential local guidance cues . However, it seems unlikely that complex non-linear trajectories such as innervation of target regions in the contralateral hemisphere via the fimbria-fornix pathway are merely guided by chemoattractants and repellents. A more likely explanation could be that the newly formed axons grow alongside host fiber tracts, which, by nature, represent region-specific trajectories. Considering that many of the newly formed axonal projections pass myelinated fiber tracts such as the corpus callosum and the fimbria-fornix, it is remarkable that this process appears not to be inhibited by myelin-associated inhibitors of axonal growth. In this respect, the slow maturation of human neurons might provide a substantial advantage. We found that p75 is only upregulated after several weeks of in vitro differentiation. This delayed expression of an important member of the receptor complex inhibiting axonal outgrowth [32, 33] might determine a permissive time window for axonal outgrowth from human lt-NES cells in the adult brain.
Prospects for experimental brain repair
Although the mechanisms enabling long-range axonal projections in the adult mammalian nervous system require further investigation, the results of our and other studies in related systems [9, 10, 15, 31] indicate that human neural grafts may, under appropriate conditions, eventually be used for the innervation of remote targets in the host brain. This prospect could be particularly relevant for diseases affecting a specific group of neurons with defined projections. Examples include central motoneurons, affected in ALS, but also nigral dopamine neurons projecting into the striatum, the main target of Parkinson’s disease. In this regard, it is important to realize that substantial functional benefits might already result from incomplete structural repair . However, functional data going beyond the morphological detection of axons and synapses will be essential to validate the efficacy of graft-derived projections.
Safety considerations play an essential role with respect to the source of the donor cells [35, 36]. The potential to proliferate human ES cell-derived lt-NES cells over many passages without compromising their neuronal differentiation potential enables the generation of large numbers of pure neural donor cells. In our preclinical model, and with the cell numbers used, this population did not result in tumor/teratoma formation in any of the transplant recipients, which were followed up to the limit of the recipient animal’s lifespan.
Taken together, human neuroepithelial stem cells derived from pluripotent cells show promising results in terms of transplant survival, safety, neuronal differentiation, axonal pathfinding, and synaptogenesis in vivo. In combination with efficient strategies to direct the donor cells towards fates of therapeutic value [37–41] and iPS technology [36, 42] for the generation of immunocompatible grafts, they should provide a versatile tool for experimental nervous system repair.
We are grateful to Joseph Itskovitz-Eldor (Technion, Israel Institute of Technology, Haifa, Israel) for providing the hESC lines I3 and H9.2. We thank Michaela Segschneider, Anke Leinhaas, Christina Leufgen, and Justus Ritter for outstanding technical support. We would further like to thank Jörg Bedorf and Reinhard Büttner for their support regarding the EM studies. This work was supported by Deutsche Forschungsgemeinschaft Grants SFB-TR3 (TP D2 and TP C1), European Union Grant FP7-HEALTH-2007-B-22943-NeuroStemcell and the Hertie Foundation.
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