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Experimental Neurology 194 (2005) 230 – 242 www.elsevier.com/locate/yexnr Lineage-restricted neural precursors survive, migrate, and differentiate following transplantation into the injured adult spinal cord A.C. Lepore, I. FischerT Department of Neurobiology and Anatomy, 2900 Queen Lane, Drexel University College of Medicine, Philadelphia, PA 19129, USA Received 4 January 2005; revised 4 January 2005; accepted 15 February 2005 Available online 5 April 2005 Abstract Fetal spinal cord from embryonic day 14 (E14/FSC) has been used for numerous transplantation studies of injured spinal cord. E14/FSC consists primarily of neuronal (NRP)- and glial (GRP)-restricted precursors. Therefore, we reasoned that comparing the fate of E14/FSC with defined populations of lineage-restricted precursors will test the in vivo properties of these precursors in CNS and allow us to define the sequence of events following their grafting into the injured spinal cord. Using tissue derived from transgenic rats expressing the alkaline phosphatase (AP) marker, we found that E14/FSC exhibited early cell loss at 4 days following acute transplantation into a partial hemisection injury, but the surviving cells expanded to fill the entire injury cavity by 3 weeks. E14/FSC grafts integrated into host tissue, differentiated into neurons, astrocytes, and oligodendrocytes, and demonstrated variability in process extension and migration out of the transplant site. Under similar grafting conditions, defined NRP/GRP cells showed excellent survival, consistent migration out of the injury site and robust differentiation into mature CNS phenotypes, including many neurons. Few immature cells remained at 3 weeks in either grafts. These results suggest that by combining neuronal and glial restricted precursors, it is possible to generate a microenvironmental niche where emerging glial cells, derived from GRPs, support survival and neuronal differentiation of NRPs within the non-neurogenic and non-permissive injured adult spinal cord, even when grafted into acute injury. Furthermore, the NRP/GRP grafts have practical advantages over fetal transplants, making them attractive candidates for neural cell replacement. D 2005 Elsevier Inc. All rights reserved. Keywords: Fetal graft; Neural progenitor; Neural stem cell; Rat; Spinal cord injury Introduction Numerous studies over the last two decades have shown that grafting of embryonic day-14 fetal spinal cord tissue (E14/FSC) into the injured spinal cord rescues axotomized neurons from retrograde cell death and atrophy (Bregman and Reier, 1986; Mori et al., 1997), reduces scar formation (Houle, 1992), and promotes host axonal regeneration (Diener and Bregman, 1998b; Tessler et al., 1988). In the adult, host axons penetrate the graft, but do not transverse Abbreviations: AP, human placental alkaline phosphatase; E14/FSC, embryonic day-14 fetal spinal cord; GRP, glial-restricted precursor; NRP, neuronal-restricted precursor; NSC, multipotent neural stem cell. T Corresponding author. Fax: +1 215 843 9082. E-mail address: [email protected] (I. Fischer). 0014-4886/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2005.02.020 transplants to reenter host neuropil. Graft-derived fibers also project into host tissue (Jakeman and Reier, 1991; Reier et al., 1986). Although FSC grafts have been successful in improving functional recovery following complete spinal transection of neonates (Diener and Bregman, 1998a; Howland et al., 1995; Miya et al., 1997), little recovery occurs in adult models (Bregman et al., 1993; Stokes and Reier, 1992). Despite potential benefits, the use of fetal transplants has serious shortcoming including issues of supply, storage, cellular heterogeneity, and quality control. E14/FSC transplants consist primarily of lineagerestricted neural precursors, both neuronal (NRP)- and glial(GRP)-restricted precursors, as well as small populations of multipotent neural stem cells (NSCs) and differentiated cells (Kalyani and Rao, 1998). Previous studies A.C. Lepore, I. Fischer / Experimental Neurology 194 (2005) 230–242 231 have shown that NSCs grafted into non-neurogenic regions of intact and injured adult CNS survive poorly (Lepore et al., 2004) and differentiate mostly into glial cells (Cao et al., 2001). In contrast, lineage-restricted precursors survive for long periods of time and differentiate into neurons and glia, respectively (Han et al., 2002, 2004; Lepore et al., 2004). However, previous studies have raised concerns about the ability of grafted NSCs and NRPs to survive in the toxic environment of the injury site and to differentiate into mature neurons without the necessary instructive environment (Cao et al., 2002). We therefore reasoned that comparing the fate of E14/FSC transplants and transplants of a defined population of lineage-restricted precursors (NRPs and GRPs) in the injured adult spinal cord, using cells derived from alkaline phosphatase (AP) transgenic rats, will allow for elucidating the sequence of events that follows grafting of fetal cells in the injured adult CNS, including survival, migration, and differentiation. Using a lateral funiculus injury model, we found an early and extensive cell loss following acute transplants of E14/ FSC. Surviving precursors expanded to fill the entire lesion by 3 weeks post-transplantation. E14/FSC grafts integrated and extended long processes into host spinal cord, differentiated into neurons, astrocytes, and oligodendrocytes and showed variability in migration out of the transplant site. Grafts of defined NRP/GRP cells filled the cavity without significant early cell loss, differentiated into mature CNS phenotypes and showed consistent cellular migration out of the injury site, even when grafted into the acute injury. This work suggests that mixed lineage-restricted precursor grafts generate a microenvironment that protects the cells from the detrimental effects of the injured CNS and provides them with a permissive niche for survival, differentiation, and migration. Furthermore, NRP/GRP grafts represent a practical alternative to fetal tissue transplants because of the ability to isolate, expand, store, and genetically manipulate these cells. medium [DMEM-F12, BSA (1 mg/mL; Sigma; St. Louis, MO), B27 (Invitrogen), bFGF (10 Ag/mL; Peprotech; Rocky Hill, NJ), Pen-Strep (100 IU/mL; Invitrogen), N2 (10 AL/ mL; Invitrogen), NT-3 (10 Ag/mL; Peprotech)] on poly-llysine (13.3 Ag/mL; Sigma)- and laminin (20 Ag/mL; Invitrogen)-coated dishes. Methods Adult spinal cord injury and transplants Cell isolation and culture Lateral funiculus injuries were created at the cervical 4 spinal cord level. Adult female Sprague–Dawley rats (approximately 250 g) received intraperitoneal injections of anesthetic cocktail [acepromazine maleate (0.7 mg/kg; Fermenta Animal Health, Kansas City, MO), ketamine (95 mg/kg; Fort Dodge Animal Health; Fort Dodge, IA), and xylazine (10 mg/kg; Bayer, Shawnee Mission, KS)]. The back musculature was excised, and a laminectomy was performed at the cervical 3/4 level. The dura was incised above the dorsal root entry zone. Microscissor cuts were created at the rostral and caudal extents of the injury. Aspiration was used to selectively ablate only the lateral white matter tracts, as well as a minimal portion of the dorsal and ventral gray matter. The dorsal columns and central canal were unaffected. Once hemostasis was NRPs and GRPs were isolated from embryonic day-13.5 transgenic Fischer 344 rats that express the marker gene, human placental alkaline phosphatase (AP). This transgenic animal has previously been characterized (Kisseberth et al., 1999; Mujtaba et al., 2002). Briefly, embryos were isolated in DMEM/F12 (Invitrogen; Carlsbad, CA). Trunk segments were incubated in collagenase Type I (10 mg/mL; Worthington Biochemicals; Lakewood, NJ)/dispase II (20 ng/ mL; Roche Diagnostics; Indianapolis, IN)/HBSS (Cellgro; Herndon, VA) solution for 8 min at room temperature to remove meninges from the cords. Cords were dissociated using a 0.05% trypsin/EDTA (Invitrogen) solution for 20 min at 378C. Cells were then plated in NRP complete Preparation of cells for grafting Following embryonic dissection, NRPs and GRPs were co-cultured for 3–10 days prior to transplantation. A mixed population of NRPs and GRPs was dissociated from culture flasks using 0.05% trypsin/EDTA, washed and resuspended at a concentration of 200,000 cells/AL [in Type 1 collagen matrix-Vitrogen (Cohesion; Palo Alto, CA)/ basal media mixture] for transplantation. Cells were placed on ice during surgery. After completion of surgery, cell viability was assessed using the trypan blue assay. Viability was always greater than 90%. The composition of the NRP/GRP cultures, with respect to the presence of undifferentiated neural precursors devoid of mature cells, was verified before grafting by staining for the immature neural marker nestin, for markers for NRPs (E-NCAM) and GRPs (A2B5), as well as for markers of mature cell types. Preparation of fetal spinal cord tissue (E14/FSC) for grafting Whole FSC tissue was isolated from embryonic day-14 rats as previously described (Bregman and McAtee, 1993). To obtain labeled tissue, we used transgenic Fischer 344 rats that express the AP marker gene. Briefly, embryos were isolated in DMEM/F12. Trunk segments were dissected, and the surrounding meninges were removed. Square pieces of tissue (1–3 mm) were kept in DMEM-F12 on ice until placed directly into the spinal cord lesion site (approximately 10 min). 232 A.C. Lepore, I. Fischer / Experimental Neurology 194 (2005) 230–242 achieved, NRP/GRP cells (mixed in Type I Collagen matrixVitrogen) or whole FSC tissue pieces were implanted into the injury cavity using either a 10-AL Hamilton Gastight syringe (Hamilton; Reno, NV) or forceps, respectively. The cell/matrix mixture and the tissue grafts both completely filled the cavities. Approximately 400,000 cells in the mixed NRP/GRP transplants were grafted into the injury site. Dura was closed with 9-0 suture; muscle was re-apposed; skin was closed with wound clips. Animals received Bupranorphin and methylprednisolone (Pharmacia and Upjohn; Kalamazoo, MI) postoperatively. Animals were immunosuppressed by subcutaneous administration of cyclosporine A (10 mg/kg; Sandoz Pharmaceuticals, East Hanover, NJ) daily beginning 3 days before grafting and continuously until sacrifice. The care and treatment of animals in all procedures was conducted in strict accordance with the guidelines set by the Drexel University IACUC and the Institute of Laboratory Animal Resources, U.S. National Academy of Sciences. Tissue processing Animals were sacrificed at various time points (4 days, 3 and 5 weeks) following transplantation by transcardial perfusion with 0.9% saline, followed by ice-cold 4% paraformaldehyde (Fisher Scientific; Pittsburgh, PA). Spinal cords were removed from the animals, followed by cryprotection in 30% sucrose (Fisher Scientific)/0.1 M phosphate buffer at 48C for 3 days. The tissue was embedded in OCT (Fisher Scientific), fast frozen with dry ice, and stored at 808C until processed. Spinal cord tissue blocks were cut in the sagittal or transverse plane at 20 Am thickness. Sections were collected on gelatin- and poly-llysine-coated glass slides and stored at 808C until analyzed. Alkaline phosphatase histochemistry Serial sections were analyzed by AP histochemistry to assess the presence, location, migration, and morphology of graft-derived cells in the spinal cord. Sections were washed with PBS, heated at 608C for 1 h to inactivate endogenous enzyme activity, washed briefly in AP buffer (100 mM Tris, 100 mM NaCl, 50 mM MgCl2, pH 9.5; all Fisher Scientific) and finally incubated at room temperature in the dark with AP staining solution [(NBT; 1.0 mg/mL; Sigma), (BCIP; 0.1 mg/mL; Sigma), (levamisole; 5 mM; Sigma) in AP buffer] for 1.5–2.0 h. Slides were cover-slipped in hard-set Vectashield (Vector; Burlingame, CA) and visualized with light microscopy. Immunohistochemistry/phenotypic analysis Tissue sections and cultured cells were washed in PBS, blocked in 10% goat serum (Invitrogen) for 1 h at room temperature and incubated with primary antibody solution at 48C overnight. Both monoclonal and polyclonal (1:200; Accurate, Westbury, NY) antibodies against human placental alkaline phosphatase were used to identify graftderived cells. A number of primary antibodies were used to assess the phenotype of cells. Nestin (1:1000; monoclonal; Pharmingen; San Diego, CA) was used to identify undifferentiated neural precursor cells (NSCs, NRPs, and GRPs). Sox-2 (1:200; polyclonal; Chemicon; Temecula, CA) was used to identify NSCs. Dividing cells were identified using Proliferating Cell Nuclear Antigen (PCNA; 1:200; monoclonal; Chemicon). NRPs were identified specifically using E-NCAM (1:200; monoclonal; Chemicon), while GRPs were identified using A2B5 (1:500; monoclonal; Chemicon). Neurons were identified using the antibodies: MAP2 (1:100; monoclonal; Chemicon); NeuN (1:100; monoclonal; Chemicon). Astrocytes were identified using GFAP (1:100; monoclonal; Chemicon). Oligodendrocytes precursors were identified using the antibodies: NG2 (1:250; polyclonal; Chemicon), Sox10 (1:200; polyclonal; Chemicon). Mature oligodendrocytes were identified with RIP (1:1000; monoclonal; Chemicon). Samples were incubated for 2 h at room temperature with goat anti-mouse and goat anti-rabbit secondary antibodies (1:200; Jackson, West Grove, PA) conjugated to rhodamine or FITC. Samples were counterstained with DAPI (1:1000; Sigma) to identify nuclei, and cover-slipped with anti-fade mounting media (Fluorosave, CN Biosciences; La Jolla, CA). Slides were subsequently stored at 208C. Images were acquired on either a Leica DMRBE fluorescence microscope (Leica Microsystems; Bannockburn, IL) using a Photometric Sensys KAF-1400 CCD camera (Roper Scientific; Trenton, NJ) or on a Leica TCS SP2 laser confocal microscope (Leica). Images were analyzed using either IP Lab (Scanalytics; Fairfax, VA) or Leica confocal software version 2.0 (Leica). Adobe Photoshop CS (Adobe, San Jose, CA) was used to prepare figures. Results Graft composition E14 spinal cord grafts The phenotypic composition of E14/FSC was assessed to determine the specific cell types that make up the graft. DAPI staining shows the cellular pattern of E14/FSC (Fig. 1A). A large proportion of the cells expressed nestin (Fig. 1B), an early neural marker expressed by both NSCs and lineage-restricted precursors. Cells of the E14 FSC also expressed either E-NCAM (Fig. 1C) or A2B5 (Fig. 1D), markers of NRPs and GRPs, respectively. Only small numbers of cells expressed markers of NSCs (Sox-2) or neurons (NeuN) (data not shown). These results indicate that while E14/FSC grafts consisted mainly of lineage- A.C. Lepore, I. Fischer / Experimental Neurology 194 (2005) 230–242 233 Fig. 1. Graft characterization. Prior to transplantation, E14/FSC (A–D) and NRP/GRP cultures (E–F) were characterized to define their phenotypic profile. DAPI staining shows the cellular pattern of the tissue (A). Cells within the E14/FSC express the early neural marker nestin (B), as well as the cell surface antigens, E-NCAM (C) or A2B5 (D), markers of NRPs and GRPs, respectively. Cultures of NRPs and GRPs were harvested from E13.5 FSC. All cells in this population display the morphological characteristics of small phase-bright bipolar lineage-restricted cells (E). Similar to E14/FSC, NRP/GRP cells express nestin (F), as well as either E-NCAM (G) or A2B5 (H). Scale bars: 100 Am (A–D) or 50 Am (E–F). restricted precursors (NRPs and GRPs), they also included NSCs and differentiated neurons. NRP/GRP grafts NRPs and GRPs were isolated from the caudal neural tube of E13.5 AP rats and co-cultured for 3–10 days. During the culturing period, the cells showed the characteristic morphology of small, phase-bright, lineage-restricted precursors (Fig. 1E). Before transplantation, the phenotypic composition of the grafted cells was again verified. We found that at the time of grafting, all cells in the NRP/GRP cultures expressed the early neural marker, nestin (Fig. 1F), showing that all cells were neural precursors. NRP/GRP cells did not express mature markers of neurons (NeuN), astrocytes (GFAP), or oligodendrocytes (RIP, NG2, and Sox-10) (data not shown), showing that cultures did not contain differentiated cell types. Cells in the NRP/GRP cultures expressed either E-NCAM (Fig. 1G) or A2B5 (Fig. 1H). Together, these results suggest that the cultures were composed almost exclusively of lineage-restricted precursors, both NRPs and GRPs. The mixed lineage-restricted cells used for grafting consisted of various ratios of NRPs and GRPs, dependent on the time spent in culture, and were devoid of NSCs and mature cells types. Furthermore, cultures of NRPs and GRPs maintained for multiple passages (up to 10 days) continued to be nestin-positive, without expressing mature markers. These results demonstrate that lineage-restricted precursors could be expanded in an undifferentiated state for extended periods of time. Mixed lineage-restricted cells used for grafting consisted of NRPs and GRPs at ratios that were dependent on the culturing time, but were always devoid of NSCs and mature cells types. Survival and migration E14 spinal cord grafts Small tissue segments of E14/FSC were grafted acutely into a lateral funiculus injury of the cervical spinal cord. Animals were sacrificed at 4 days, and 3 and 5 weeks postengraftment. Serial sections were assessed by AP histochemistry to detect the presence of AP-labeled cells. At the time of transplantation, the entire injury cavity was completely filled with graft tissue, but 4 days later only a small portion of the lesion site was filled with grafted cells (Figs. 2A–B). By 3 and 5 weeks post-transplantation, the entire lesion cavity was completely refilled with APlabeled cells, forming an even interface with the host (Figs. 2C–G; 3A, C). In addition, at the 3- and 5-week time points, long graft-derived fibers could be seen projecting from the transplant into the surrounding intact spinal cord (Figs. 2C–G). Graft-derived cells also migrated out of the transplant/injury site into the host parenchyma (Figs. 3A, B, D). The extent of migration of the transplanted cells and the extension of graft-derived fibers into the host spinal cord were variable and not observed in all animals (Fig. 3C). These observations show that after an initial cell loss, the grafted cells filled the injury site, most likely by proliferation, and appeared to have integrated with the host tissue. NRP/GRP grafts Mixed populations of NRPs and GRPs (400,000 cells/ transplant at various ratios) were grafted acutely into the lesion cavity via direct injection. These grafts were analyzed using the same protocols as with E14/FSC grafts. Despite the small number of precursor cells grafted relative to the 234 A.C. Lepore, I. Fischer / Experimental Neurology 194 (2005) 230–242 Fig. 2. Grafting of E14/FSC into the injured spinal cord: Survival and process extension. Undissociated E14/FSC was grafted acutely into a lateral funiculus injury of the cervical spinal cord. AP histochemistry was used to detect the presence of transplanted cells. Grafts initially filled the entire lesion cavity (data not shown), but suffered extensive cell loss by 4 days post-transplantation (A–B). By 3 weeks post-transplantation, E14/FSC grafts completely filled the lesion cavity and integrated with the adjacent host tissue (C–G). Graft-derived fibers project out of the transplant site into the host spinal cord in both the sagittal (C–E) and transverse (F–G) planes. These fibers project into both white (C–E) and gray (F–G) matter, and extend for distances greater than 5 mm in both the rostral (C) and caudal (D) direction. Panels are sagittal (A, C–E) or transverse (B, F–G) sections, with the dorsal aspect facing upwards. Scale bars: 1 mm (A–D, F) or 0.5 mm (E, G). number of cells contained in fetal tissue transplants, NRP/ GRP cells showed excellent survival in the injury site at 4 days (Figs. 4A–B). Grafts completely filled the injury cavity by 3 and 5 weeks, with no signs of a large cell mass suggestive of tumor formation (Fig. 4C). By 3 to 5 weeks post-engraftment, the NRP/GRP transplants robustly and consistently migrated out of the injury cavity along white matter tracts of the intact cord in both the rostral and caudal direction (Figs. 4C–E) to distances of up to 15 mm. This behavior has a general similarity to the fate of the E14/FSC grafts, but also underscores several differences between the NRP/GRP and E14/FSC transplants. These differences include characteristics of graft survival, migration, and process extension. Fig. 3. Grafting of E14/FSC into the injured spinal cord: Variability in migration and process extension. Undissociated E14/FSC was grafted acutely into a lateral funiculus injury of the cervical spinal cord. AP histochemistry was used to detect the presence of transplanted cells. By 3 weeks post-transplantation, E14/FSC-derived cells migrate out of the graft site along white matter tracts of the intact host spinal cord (A–B, D). Cells migrate in both the rostral and caudal directions (A–B) for distances greater than 10 mm. However, FSC grafts demonstrate variability in migration and process extension. All FSC grafts survive and completely fill the lesion cavity, but some show only modest migration or process extension into the host spinal cord (C). All panels are sagittal sections, with the dorsal aspect facing upwards. Scale bars: 1 mm (A, C), 0.5 mm (B), or 0.1 mm (D). A.C. Lepore, I. Fischer / Experimental Neurology 194 (2005) 230–242 235 Fig. 4. Grafting of NRPs/GRPs into the injured spinal cord. Mixed populations of NRPs and GRPs were grafted acutely into a lateral funiculus injury of the cervical spinal cord. AP histochemistry was used to detect the presence of transplanted cells. NRP/GRP grafts filled the injury cavity only partially by 4 days post-transplantation (A–B), but expanded to fill the cavity by 3 weeks post-transplantation (C). NRP/GRP grafts demonstrated robust and consistent migration into the host spinal cord selectively along white matter tracts in both rostral and caudal directions (C–E). Cells were found at distances up to 15 mm by 3 weeks post-transplantation. All panels are sagittal sections, with the dorsal aspect facing upwards. Scale bars: 1 mm (A–C), 0.5 mm (D), or 0.1 mm (E). Phenotypic fate Fetal spinal cord grafts The presence of the transgenic AP marker allowed us to follow the phenotypic fate of transplant-derived cells at various time points after engraftment using double staining and confocal microscopy. We used co-localization of the AP transgene with markers of neural precursors (nestin), neurons (NeuN and MAP2), astrocytes (GFAP), and oligodendrocytes (RIP, Sox-10, and NG2). We found that at 4 days post-engraftment, a large proportion of graft-derived cells were still undifferentiated, expressing the early neural marker nestin (Figs. 5A– C). In addition, graft-derived neurons (Figs. 6A–C) could be found throughout the injury site, most likely originating from differentiated cells that were present in the E14/ FSC graft. In addition, astrocytes (Figs. 7A–C) were found in the injury site at 4 days post-engraftment. In contrast, no graft-derived oligodendrocytes could be detected at this early time point (Figs. 7G–I), consistent with the paucity of these cells in the E14 spinal cord. At 3 and 5 weeks post-engraftment, almost no graft-derived nestin-positive cells remained (Fig. 5D); whereas, numerous graft-derived neurons (Figs. 6D–F), astrocytes (Figs. 7D–F), and oligodendrocytes (Figs. 7J–L) were present throughout the transplant site. The grafted cells also expressed synaptophysin, a marker of synaptic vesicles, at 4 days (Figs. 6G–I) and at 3 and 5 weeks (Figs. 6J–L). Assessment of cell division of engrafted cells was conducted using the proliferation marker, PCNA. A large proportion of the grafted cells were proliferating at 4 days post-transplantation, while very few graft-derived cells continued to divide by 5 weeks (data not shown). Table 1 provides a summary of the fate of E14/FSC grafts. NRP/GRP grafts The vast majority of NRP/GRP graft-derived cells were nestin-positive at 4 days (Figs. 5E–G), confirming that the transplanted cells were undifferentiated prior to engraftment. No graft-derived cells had differentiated into neurons (Figs. 8A–C) or oligodendrocytes (Figs. 9G–I) by 4 days, but some cells differentiated into astrocytes (Figs. 9A–C). In contrast, very few graft-derived cells expressed nestin at 3 and 5 weeks post-transplantation (Fig. 5H), while most of the cells differentiated into neurons (Figs. 8D–F), astrocytes (Figs. 9D–F), and oligodendrocytes (Figs. 9J–L) throughout the injury site at these later time points. Grafted cells also expressed synaptophysin by 3 weeks post-transplantation (Figs. 8J–L). In addition to the injury/graft site, graft-derived cells that had migrated into the host spinal cord expressed markers of neurons, 236 A.C. Lepore, I. Fischer / Experimental Neurology 194 (2005) 230–242 Fig. 5. Persistence of grafted neural precursors. Both E14/FSC and NRP/GRP transplants were analyzed for nestin expression to determine if graft-derived precursors were still present. The majority of cells in both the E14/FSC (A–C) and NRP/GRP (E–G) grafts were positive for nestin at 4 days. Double immunofluorescence staining for AP (B and F, green) and nestin (C and G, red) shows the presence of graft-derived precursors (A and E, overlay). However, almost no graft-derived precursors were found at 5 weeks in both the E14/FSC (D) and NRP/GRP (H) grafts. Arrowheads denote double-labeled cells. All panels are sagittal sections. Scale bars: 30 Am. Fig. 6. E14/FSC grafts differentiate into neurons and express synaptic markers in the injured spinal cord. Graft-derived neurons are found at both 4 days (A–C) and 5 weeks (D–F) post-transplantation. Double immunofluorescence staining for AP (B and E, green) with NeuN (C and F, red) and MAP2 (data not shown) shows the presence of graft-derived neurons (A and D, overlay). E14/FSC grafts express synaptic markers at both 4 days (G–I) and 5 weeks (J–L) posttransplantation. Double immunofluorescence staining for AP (H and K, green) and synaptophysin (I and L, red) suggests the possible association of grafted cells with synapse formation (G and J, overlay). Arrowheads denote double-labeled cells. All panels are sagittal sections. Scale bars: 30 Am. A.C. Lepore, I. Fischer / Experimental Neurology 194 (2005) 230–242 237 Fig. 7. E14/FSC grafts differentiate into astrocytes and oligodendrocytes in the injured spinal cord. Graft-derived astrocytes are found at both 4 days (A–C) and 5 weeks (D–F) post-transplantation. Double immunofluorescence staining for AP (B and E, green) and GFAP (C and F, red) shows the presence of graftderived astrocytes (A and D, overlay). Graft-derived oligodendrocytes are found at 5 weeks (J–L) post-transplantation, but not at 4 days (G–I). Double immunofluorescence staining for AP (K, green) with RIP (L, red), Sox-10 (data not shown) and NG2 (data not shown) shows the presence of graft-derived oligodendrocytes (J, overlay). Arrowheads denote double-labeled cells. All panels are sagittal sections. Scale bars: 30 Am. astrocytes, and oligodendrocytes (data not shown). However, the neurons were found predominantly close to the injury site, while their glial counterparts were also found at greater distances. This finding is in agreement with previous reports that demonstrated the limited migratory capacity of NRPs compared to GRPs (Han et al., 2002, 2004). Similar differentiation results were obtained from cells maintained in culture for 3–10 days and were in agreement with the grafting results into the intact spinal cord (Lepore et al., 2004). Similar to E14/FSC transplants, it appears that a significant proportion of the grafted cells expressed PCNA at 4 days post-transplantation, while very few cells continued to divide by 5 weeks (data not shown). Table 1 provides a summary of the fate of NRP/GRP grafts. Discussion Fetal spinal cord tissue has been used as a transplant source for two decades, but the events that follow grafting into the injured spinal cord were not well understood because of difficulties in cell detection and the limited knowledge of neural precursor biology at the time. Our studies were designed to elucidate the sequence of events that follow FSC transplantation into the injured spinal cord and at the same time address important issues concerning the fate of neural precursor cells in the injured CNS. The close relationship between these topics stems from the finding that E14/FSC, a transplant source for many of the grafting studies, consists primarily of neuronal- and glialrestricted precursors (Rao, 1999). In this study, we Table 1 Summary of graft fate Neural precursors Cell division Neurons Astro’s Oligo’s 4 days Synaptic proteins 4 days 5 weeks 4 days 5 weeks 4 days 5 weeks 4 days 5 weeks E14/FSC NRP/GRP ++ ++ + + ++ ++ + + + ++ ++ + + ++ ++ 5 weeks 4 days 5 weeks + + + ++ ++ Long-term survival Initial loss Fill cavity (5 w) Migration Tumors Process extension E14/FSC NRP/GRP Yes Yes Large Small Yes Yes Variable Consistent None None Variable Minimal This table provides a summary of the fate of both E14/FSC and NRP/GRP grafts at 4 days and 5 weeks following transplantation into the injured spinal cord. (+) Denotes few cells; (++) denotes many cells; ( ) denotes no cells. 238 A.C. Lepore, I. Fischer / Experimental Neurology 194 (2005) 230–242 Fig. 8. NRP/GRP grafts differentiate into neurons and express synaptic markers in the injured spinal cord. Graft-derived neurons are found at 5 weeks (D–F) post-transplantation, but not at 4 days (A–C). Double immunofluorescence staining for AP (E, green) with NeuN (F, red) and MAP2 (data not shown) shows the presence of graft-derived neurons (D, overlay). NRP/GRP grafts express synaptic markers at 5 weeks (J–L) post-transplantation, but not at 4 days (G–I). Double immunofluorescence staining for AP (K, green) and synaptophysin (L, red) suggests the possible association of grafted cells with synapse formation (J, overlay). Arrowheads denote double-labeled cells. All panels are sagittal sections. Scale bars: 30 Am. Fig. 9. NRP/GRP grafts differentiate into astrocytes and oligodendrocytes in the injured spinal cord. Graft-derived astrocytes are found at both 4 days (A–C) and 5 weeks (D–F) post-transplantation. Double immunofluorescence staining for AP (B and E, green) and GFAP (C and F, red) shows the presence of graftderived astrocytes (A and D, overlay). Graft-derived oligodendrocytes are found at 5 weeks (J–L) post-transplantation, but not at 4 days (G–I). Double immunofluorescence staining for AP (K, green) with RIP (L, red), Sox-10 (data not shown) and NG2 (data not shown) shows the presence of graft-derived oligodendrocytes (J, overlay). Arrowheads denote double-labeled cells. All panels are sagittal sections. Scale bars: 30 Am. A.C. Lepore, I. Fischer / Experimental Neurology 194 (2005) 230–242 employed a well-defined population of lineage-restricted neural precursors, cells whose properties have been previously characterized in vitro (Mayer-Proschel et al., 1997; Rao and Mayer-Proschel, 1997) and in the normal uninjured CNS (Han et al., 2002, 2004; Lepore et al., 2004). Furthermore, we were able to clearly interpret the grafting experiments because the tissue and the cells were obtained from alkaline phosphatase transgenic rats, allowing us unambiguously to track the grafts (Kisseberth et al., 1999; Mujtaba et al., 2002). Previous studies have shown that E14/FSC, grafted into adult models of traumatic spinal cord injury, rescue axotomized neurons (Mori et al., 1997), induce regeneration into but not through the transplant (Tessler et al., 1988), and promote limited recovery of function (Bregman et al., 1993; Stokes and Reier, 1992). The efficacy of these grafts can be enhanced with delayed transplantation, together with the administration of exogenous growth factors (Bregman et al., 2002; Coumans et al., 2001). Despite their limited success, the analysis of fetal grafts derived from various developmental ages has yielded important information with respect to the survival and fate of grafted cells. For example, studies examining FSC derived from different embryonic ages have shown excellent survival and differentiation at the E12–15 range, while the survival rate decreases dramatically in tissue taken from older fetuses (Bernstein et al., 1985; Reier et al., 1983). We now recognize that at the optimal embryonic age (e.g., E13–14), FSC contains primarily lineage-restricted precursor cells (Rao, 1999). At later times (e.g., NE15), there are increasing numbers of mature cell types such as post-mitotic neurons, which survive poorly following transplantation into the adult CNS (Sieradzan and Vrbova, 1991). Furthermore, our previous grafting experiments into the adult CNS demonstrated excellent survival and robust differentiation of lineage-restricted precursors derived from E13.5, compared with poor survival of NSCs derived from E10.5 (Lepore et al., 2004). We have therefore argued that lineage-restricted cells, rather than NSCs, represent the cell population critical for successful grafting into the spinal cord (Han et al., 2002, 2004; Lepore et al., 2004). Nevertheless, it is important to note that NSCs can produce neurons when grafted into a neurogenic environment such as the SVZ-rostral migratory stream (Fricker et al., 1999), hippocampus (Shihabuddin et al., 2000), or the developing CNS (Campbell et al., 1995). While these grafting studies indicate that lineagerestricted precursors derived from the developing spinal cord are potentially an excellent source for transplantation into the adult CNS, it has remained unclear whether these properties will stand the challenges of the injured spinal cord. The main concern raised by previous work has been the ability of the grafted cells to survive the toxic site of the injury and subsequently differentiate into mature neurons in a non-neurogenic environment. For example, various classes of isolated glial-restricted precursors robustly survive and differentiate in vivo (Han et al., 2004; Roy et 239 al., 1999; Windrem et al., 2004; Zhang et al., 1999), but neuronal-restricted precursors survive poorly and fail to differentiate into neurons in the injured adult spinal cord (Cao et al., 2002). The failure to obtain neurons in the injury site suggests that the microenvironment of the graft needs to be modified to improve survival and to include instructive cues for neuronal differentiation that are missing. Examples of such strategies include in vitro manipulation of grafted cells (Le Belle et al., 2004; Wu et al., 2002), delayed engraftment (Ogawa et al., 2002), co-grafting with other cells (Schumm et al., 2004; Wilby et al., 1999), immune modulation (Zhang et al., 2003), genetic modification of transplanted cells (Castellanos et al., 2002; Ostenfeld et al., 2002), and exogenous administration of mitogenic (Martens et al., 2002), neuroprotective (Cicchetti et al., 2002; Helt et al., 2001), or differentiation factors (Chmielnicki et al., 2004). The present results represent an effective example of overcoming the non-neurogenic and non-permissive environment of injured spinal cord and the lack of instructive cues in the adult CNS. We show that mixed grafts of neuronal- and glial-restricted precursors support survival, neuronal, and glial differentiation, and migration by creating a microenvironmental niche that is both protective and instructive, resembling a neurogenic CNS environment. Potential explanations for the survival and neuronal differentiation abilities of mixed NRP/GRP grafts include the use of lineage-restricted precursors committed to a neuronal fate and the presence of glial cells in the form of noninflammatory astrocytes, via differentiation of transplanted GRPs. The fetal-derived astrocytes provide factors supporting precursor survival and neuronal maturation. Astrocytes are known to secrete a wide variety of trophic molecules that can enhance survival (Banker, 1980; Lindsay, 1979; Muller et al., 1995) and regeneration (Kliot et al., 1990) such as GDNF (Schaar et al., 1993), CNTF (Dallner et al., 2002; Sendtner et al., 1994), NT-3 (Dreyfus et al., 1999; Rudge et al., 1995), and NGF (Houlgatte et al., 1989; Rudge et al., 1992). Interestingly, hippocampal but not spinal cord astrocytes have been shown to play an important role in neurogenesis by instructing NSCs to adopt a neuronal fate (Song et al., 2002; Ueki et al., 2003). Our studies suggest that while astrocytes derived from spinal cord may not be able to instruct NSCs toward a neuronal lineage choice, they can promote or at least support neurogenesis of lineagerestricted neuronal precursors. It is also possible that astrocytes derived from GRPs of embryonic origin are more permissive than adult spinal cord astrocytes with respect to supporting neurogenesis. The latter interpretation is less likely given that neurons are generated before astrocytes during development (Lee et al., 2000). Interestingly, E14/FSC and NRP/GRP grafts both show selective migration out of the injury site into white matter of the intact host CNS. Previous work has demonstrated that both endogenous (Arvidsson et al., 2002; Nakatomi et al., 2002; Parent and Lowenstein, 2002; Parent et al., 1997) and 240 A.C. Lepore, I. Fischer / Experimental Neurology 194 (2005) 230–242 grafted precursors (Aboody et al., 2000; Snyder et al., 1997; Svendsen et al., 1997) can migrate to sites of CNS pathology. Presumably, factors produced by dying/dysfunctional cells, invading immune cells, and other potential players stimulate this precursor response. In vitro studies have demonstrated that microglia (Aarum et al., 2003), chemokines, and chemokine receptors such as SDF-1a and CXCR4, respectively (Ehtesham et al., 2004; Ji et al., 2004; Peng et al., 2004; Tran et al., 2004), and inflammatory cytokines such as TNFa and IFNg (Ben-Hur et al., 2003) and PDGF (Armstrong et al., 1990; Forsberg-Nilsson et al., 1998) can stimulate migration of neural precursor. Many of these pro-migratory elements are found at spinal cord injury sites (Bartholdi and Schwab, 1997; Wang et al., 1996), and thus may be playing a role in instructing grafted NRPs and GRPs to migrate, possibly along degenerating white matter tracts. However, our data indicate that while the E14/FSC grafts show variable migration out of the transplant site, the NRP/GRP grafts show consistent and robust migration. We propose that these differences may be the result of the extensive cell loss and the presence of adherent cellular matrix in the E14/FSC graft, as well as the variable graft– host interface associated with E14/FSC grafts, altogether generating conditions that compete and interfere with signals directing migration out of the grafting site. The migration of NRPs and GRPs in the injured spinal cord is particularly attractive for CNS repair. NRPs, which by and large remain at the grafting site, have the potential of becoming relays for reconstructing circuits and creating a permissive environment at the injury site. The migration stream, consisting mostly of grafted GRPs and subsequently glial cells, can potentially guide and myelinate growing axons. Despite the relatively small number of NRP/GRP cells injected, the mixed precursor grafts show excellent survival that is equivalent, or greater, than E14/FSC transplants. In this case, we propose that the increased cell death observed with fetal tissue transplants may be a product of poor survival of multipotent and/or mature cell types present in the heterogeneous fetal tissue, as well as the poor access of nutrients to the grafted tissue. In contrast, the survival of NRP/GRP grafts reflects the stable microenvironment generated by the mixed population of neuronal and glial precursors and good nutrient supply accessible to cultured cells. In both cases, we found that the grafted cells continued to divide for at least 4 days after grafting, a process that can explain how these grafts were able to fill the lesion cavity. In both graft types, we found neither significant proliferation at 5 weeks post-transplantation nor any signs of a large cell mass suggestive of tumor formation. Regardless of their potential efficacy, FSC grafts face daunting ethical and practical problems with respect to their clinical use in CNS repair, including tissue availability, cellular heterogeneity and logistics of harvesting and storing tissue. We therefore argue that because mixed NRP/GRP grafts are able to recapitulate the utility of the FSC grafts, along with added benefits of increased survival and migration, they should be considered as preferred candidates for therapy. In addition, lineage-restricted precursor cell transplantation offers practical benefits such as the capacity to appropriately select specific classes of precursors according to the specific pathological needs and the ability to derive these cells from alternative sources such as embryonic stem cells. Acknowledgments We acknowledge all the members of our group for their assistance, particularly Carla Tyler-Poltz for excellent tissue culture, Dr. Birgit Neuhuber and Dr. Steve Han for critical discussions, Dr. Timothy Himes and Theresa Connors for their technical expertise, and Andrea Ketschek for help in early experiments. A.C.L. thanks the Neuroscience graduate program for support and Stephen, Leopold, and Molly for a much needed diversion. I.F. is supported by NIH grants NS24707 and NS 37515. This work has been inspired and encouraged by the continued collaboration with Dr. Mahendra Rao. References Aarum, J., Sandberg, K., Haeberlein, S.L., Persson, M.A., 2003. Migration and differentiation of neural precursor cells can be directed by microglia. Proc. Natl. Acad. Sci. U. S. A. 100, 15983 – 15988. Aboody, K.S., Brown, A., Rainov, N.G., Bower, K.A., Liu, S., Yang, W., Small, J.E., Herrlinger, U., Ourednik, V., Black, P.M., Breakefield, X.O., Snyder, E.Y., 2000. 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