Lineage-restricted neural precursors survive, migrate, and differentiate following transplantation into the injured adult spinal cord

https://doi.org/10.1016/j.expneurol.2005.02.020Get rights and content

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.

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 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 lineage-restricted 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 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, Han et al., 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.

Section snippets

Cell isolation and culture

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

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

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

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

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