Segregation of Human Neural Stem Cells in the Developing Primate Forebrain Vaclav Ourednik, Jitka Ourednik, Jonathan D. Flax, Michael Zawada, Cynthia Hutt, Chunhua Yang, Kook I. Park, Seung U. Kim, Richard L. Sidman, Curt R. Freed, Evan Y. Snyder

Supplementary Material

Supplemental Figure 1. Schematic of the experimental procedure. [A] BrdU-prelabelling of hNSCs prior to transplantation. Cells from a stable, self-maintaining clone of hNSCs (clone H6), originally isolated from the VZ of a 15-week human fetus and generated, grown, and characterized as previously described [5], were pre-incubated in culture (at a density of 5 x 10⁵ cells/ml) with BrdU (10µM, Sigma) for 48 hours prior to implantation in order to be subsequently identifiable in vivo by their darkly-stained BrdU-immunopositive nuclei. (The cells were derived from a human developmental stage more immature than that of the monkey recipients.) The BrdU-containing cells were then resuspended in PBS at 1.7x10⁷ cells/ml (as previously described [5]). [B] Implantation of hNSCs into the cerebral ventricle of fetal monkeys by transabdominal injection under ultrasonic guidance with the pregnant mother under general anesthesia, allowing the hNSCs access to the VZ. (The protocol was approved by the University of Colorado Animal Care and Use Committee, [#05004794(04)1F]). To the left, an actual ultrasonographic axial view that faces the top of the fetal head; black crosses define the bi-parietal distance (43.6 mm); arrows delimit the lateral cerebral ventricles into which hNSCs were implanted. Three pregnant bonnet monkeys were anaesthetised 30 min. prior to the procedure with an initial i.m. injection of acepromazine (0.5 mg/kg b.w.), ketamine (10 mg/kg b.w.), and atropine sulfate (0.4 mg/kg b.w.). Ketamine (0.02 mg/kg i.m.) was administered every 15 min. during the grafting procedure. The skin of the mother's abdomen was shaved, washed with betadine/alcohol, and locally anesthetized with 2% lidocaine HCl. The fetal head was palpated through the abdominal wall and visualized by ultrasound (Acuson 128XP, 5MHz transducer). Under ultrasound guidance, an 18 gauge spinal needle attached to a 10-ml syringe was inserted through the abdominal and uterine walls into the left lateral ventricle of the fetal brain. 1.53-2.21x10⁷BrdU-labelled hNSCs were injected in a volume of 0.9-1.3 ml over a period of 2 min. Implantation into the cerebral ventricle of fetal monkeys allows reliable and uniform access of the hNSCs to the VZ [ (5, 14); O. Brustle et al, Neuron 15, 1275-1285 (1995); K. Campbell et al, Neuron 15, 1259-1273 (1995); M. Olsson et al, Neuron 19, 761-772 (1997); H. Lacorazza et al, Nature Med. 4, 424-429 (1996); Snyder et al, Nature 374, 367-370 (1995); R. Fricker et al J. Neurosci. 19, 5990-6005 (1999); C.A. Walsh & C.L. Cepko. Science 255, 434-440, (1992); C.P. Austin & C.L. Cepko Development 110, 713-732, (1990) ]. The procedure for each animal took only ~30 min. Prophylactic antibiotics (ceftriaxon sodium) were given i.m. to the mothers at 30mg/kg b.w. daily for 3 days. Two of the pregnant monkeys also received an i.v. injection of cyclosporin (Sandoz) at 15mg/kg b.w. cyclosporin treatment continued at the same concentration on a daily basis p.o. (mixed in a banana mash) throughout the survival period. The 3^rd mother received no immunosuppression. After the procedure, the animals were returned to their cages for individual housing and were maintained on a regular feeding schedule. No pain, bruising, inflammation, or behavioral changes were observed in the injected animals and pregnancies continued normally without any obvious difficulties. [C] Evaluation of the engrafted monkey brain at the conclusion of neocortical development. One month following engraftment, each of the pregnant monkeys was anesthetized, intubated, and placed on an isoflurane ventilator (99.9% isoflurane mixed with oxygen, 2% flow rate). The abdomen was antiseptically prepared, and the fetus removed by Caesarean section and sacrificed by an overdose of pentobarbital (150 mg/fetus i.p.). All surgeries were successful and the mothers were returned to their breeding groups after 3 weeks of recovery in individual housing. The removed fetuses were perfused through the cardiac left ventricle with chilled heparinized saline (240 ml) followed by 4% paraformaldehyde (240 ml). Each brain was immersed in 250 ml of the fixative for an additional 5 hours at 4°C. Then, the tissue was cryoprotected in 30% sucrose in PBS and kept at 4°C until further processing. For histological analysis, the brains were cut at 3 coronal levels (I-III) and, at each level, 25-30 serial 35µm-sections were cut on a freezing microtome. Slides were kept at 4° C or, for longer storage, at -20° C until needed. For each individual immunostaining, 3 sections from each level were analysed. For histological methods see [27] in the text.

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Supplemental Figure 2. Clonal hNSCs migrate from the ventricular germinal zone (VZ) into the developing neocortex of fetal Old World monkeys after implantation at 12-13 wks post-conception (pc).

In the left part of this figure are 2 schematics depicting stratification of the developing monkey neopallium at the time of transplantation [I.] (12-13 wks pc) and at the time of sacrifice near term [II.] (16-17 wks pc). At the time of transplantation, neurogenesis is complete in subcortical zones, and the cerebral cortex itself is represented by a cortical plate (CP) and subplate (SP). The 6-layer stratification typical of the mature neocortex is not yet detectable. Young postmitotic neurons already in the CP will differentiate further to form the relatively sparse neuronal population of layer I (marginal zone [MZ]) and the numerous neurons of the deeper cortical layers IV-VI. On the other hand, neurons destined for superficial layers II/III at the time engraftment are just being generated and are destined to migrate out from the VZ mainly during the post-grafting survival period of the subsequent 4 weeks. Considerable migration of postmitotic precursor cells along the leading processes of radial glia extending centrifugally from the VZ to the cortical surface can be observed in the growing cerebral wall [24-26]. Also generated during the 1 month survival period are glial cells destined to reside in the subcortex and deeper cortical layers; most glial cells destined for the superficial cortical layers will not be generated until after the time of sacrifice. (The subsequent gain in cortical volume after 17 wks pc is attributable mainly to increases in the amount of neuropil and to the production of glia) (For review, see [24-26,35].)

[A-C] Photomicrographs from selected locations spanning the thickness of the neopallium. (Their location relative to the schematic is indicated by brackets.) [A] Injected into the left lateral ventricle and having integrated throughout the VZ, the hNSC-derived cells ["d"], identified by their BrdU immunoreactivity (black nuclei), migrated along the monkey's radial glial processes (visualized with an antibody to vimentin [brown]), through the neopallial wall to reach their temporally-appropriate destination in the nascent superficial layers II/III [A], where they detached from the radial glia and took up residence as neurons (see Fig. 3 for close-ups and characterization). Arrows indicate climbing cells (both donor- and host-derived) positioned along the processes of the vimentin+ radial glia. Some cells (inset) are pictured still attached to these fibers and in the process of migration. The photomicrographs in [B,C] show examples of immature, donor hNSC-derived (BrdU+ black nuclei, "d") astrocytes (brown vimentin+ immunostain) intermixed with host-derived astrocytes in deeper cortical lamina, having differentiated in the expected manner given that gliogenesis is the prevalent developmental program ongoing in those layers at that time. These observations suggest that hNSCs respond to the molecular signals of the developing primate brain directing them to recognize the centrifugal migratory pathways defined by radial glia and to participate in formation of the CNS architecture. Additional Abbreviations: WM, white matter; II-VI, cortical layers. Bar: 35 µm.

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Defining Neural Stem Cells: Neural stem cells (NSCs) are primordial, uncommitted cells that can give rise to the array of more specialized cells of the CNS throughout the neuraxis. They are operationally defined by their ability (a) to differentiate into cells of all neural lineages (neurons -- ideally of multiple subtypes, oligodendroglia, astroglia) in multiple regional and developmental contexts (i.e., to be multipotent); (b) to self-renew (i.e., to give rise also to new NSCs with similar potential); (c) to populate multiple developing and/or degenerating CNS regions. An unambiguous demonstration of monoclonal derivation of progeny is obligatory to the definition -- i.e., a single cell must possess these attributes. The clone used in these studies (clone H6) has been shown to fulfill these characteristics in rodents.