MAP2 expression in the developing human fetal spinal cord and following xenotransplantation

Cell Transplantation, Vol. 6, No. 3, pp. 339-346, 1997 Copyright 0 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0963.6897197 $17.00 + .OO

PII SO963-6897(97)00033-X

ELSEVIER

Original Contribution

MAP2 EXPRESSION

IN THE DEVELOPING HUMAN FETAL SPINAL CORD AND FOLLOWING XENOTRANSPLANTATION

Mark A. Giovanini,

Paul J. Reier, Thomas A. Eskin, and Douglas K. Anderson’

Department of Neurological Surgery, Department of Neuroscience, and Department of Neuropathology,Gainesville Veterans Affairs Medical Center, University of Florida College of Medicine, Gainesville, FL 32610 USA

in this process has been suggested by various lines of electrophysiological and behavioral evidence (30-32), showing that intraspinal grafts of fetal CNS tissue in rats and cats have the potential to influence functional recovery. As illustrated by recent trials for Parkinson’s disease, human fetal tissue is currently an available option for clinical application of intraspinal grafting techniques (11,12,19-21,26,29). Continued progress in our understanding of neural progenitor cell biology (13,27,33,35, 36,41&43) also could lead to an alternative source of human CNS tissue with advantages from a variety of biological and ethical perspectives. However, an important preclinical requisite in either case is to obtain a greater appreciation of the cellular dynamics of human embryonic CNS cells under different lesion conditions, especially in the case of SCI. In this context, xenotransplantation can serve as an important tool for studying development, plasticity, and regeneration of embryonic CNS tissue that could someday be employed to treat human conditions (4,6,14,16,44,45). The migration of donor cells and the detailed resolution of host-graft tissue and neuritic interactions, however, require reliable markers for distinguishing between transplanted and host cells. Ideally, this could emerge from the use of immunocytochemistry directed at specific donor or host antigens. Microtubule-associated proteins (MAPS) are among several mammalian CNS intermediate filament constituents that are differentially expressed during development (7,22,25,28,37,39,40). In most cases, MAP2 is highly detectable until shortly after birth, at which time its expression progressively diminishes as determined by immunohistochemical studies of adult rat, avian, and hu-

0 Abstract -Human fetal spinal cord (FSC) tissue was obtained from elective abortions at 6-14 wk gestational age (GA). The specimens were then either immediately processed for immunohistochemical analysis or xenotransplantation. In the latter case, donor tissue was prepared as a dissociated cell suspension and then introduced either subpially or intraspinally into contusion lesions of the adult rat midthoracic spinal cord. The xenografts were subsequently examined by conventional histological and immunohistochemical methods at 2-3 mo postgrafting. Immunostahdng showed that MAP2 was expressed heavily in cells residing in the mantle layer of the human fetal spinal cord in situ as early as 6 wk GA. Subpial and intraparenchymal xenografts also were intensely immunoreactive for MAPZ, but no staining of surrounding host neural tissue was detected. We conclude that the differential expression of MAP2 can be used to distinguish human graft tissue from the surrounding rat spinal cord in this xenograft paradigm. Under appropriate staining conditions, MAP2 can thus serve to facilitate analyses of host-graft integration, donor cell mi0 1997 Elsevier Science gration, and neuritic outgrowth. Inc. 0 Keywords - MAP2 expression; cord; Xenotransplantation.

Human

fetal spinal

INTRODUCTION New insights about neuronal responses to axotomy, neuroplasticity, and axonal regeneration have underscored exciting prospects for promoting functional improvement after spinal cord injury (SCI). A variety of experimental approaches are being considered as possible clinical interventions, and it is becoming increasingly likely that maximum benefits will ultimately evolve from a combination of therapeutic strategies (20,34,47). That some form of cellular replacement may play a vital role

1 l/25/96. ‘Correspondence should be addressed to Douglas K. Ander-

son, the University of Florida College of Medicine, Department of Neuroscience, Box 100244, Gainesville, FL 32610-0244.

ACCEPTED

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man spinal cords (28,39). The present investigation was carried out to determine whether this contrasting pattern of MAP2 expression in developing and adult neural tissue could be useful in characterizing developing human FSC xenografts and their interactions with the adjacent mature rat spinal cord. Results of this investigation have been previously summarized (15).

MATERIALS

AND METHODS

Human FSC Tissue Donor tissue at 6-14 wk gestational age (GA) was collected from elective therapeutic abortions according to federal guidelines for the procurement and use of human fetal tissue. Fetal spinal cord (FSC) specimens were identified and dissected free of surrounding mesodermal tissues and placed in a balanced salt medium on ice with as little delay as possible. The FSC material was then either immediately fixed by immersion in paraformaldehyde for histological/immunocytochemical analysis (n = 7) or transplanted within 4 h after procurement (n = 6). Gestational ages were estimated by footlength measurements (38).

Transplants Six Long-Evans adult female rats weighing 250 to 300 g were anesthetized with intraperitoneal Nembutal (50 mg/kg). A laminectomy was performed at T,$T,, and the spinal cords were each contused by dropping a 10 g weight from a height of 25 cm using the NYU spinal cord injury device (1). The rats were then allowed to recover for approximately 14-30 days after contusion injury, at which time they were reanesthetized with intraperitoneal Nembutal (50 mg/kg) and the contusion injury sites were reexposed. Fresh human FSC tissue was dissociated in DMEM by trituration with glass pipettes. A total of 20-50 pL of this cellular suspension was then injected into sites of cavitation at the contusion epicenter using a Hamilton syringe fitted with a 33 gauge needle in five animals and in the subpial plane above the contusion cavity in one rat (Fig 1). All rats were immunosuppressed with cyclosporine (15 mg/kg, subcutaneous) 24 h prior to transplantation and daily thereafter.

Tissue Preparation Human FSC specimens for immediate immunohistochemical analysis were postfixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 12 h and then embedded in paraffin. Rats with intraspinal transplants were anesthetized at 2 mo after transplantation with intraperitoneal Nembutal (50 mg/kg) and perfused intracardially with 100 mL of 0.9% saline followed by 500 mL of 4% paraformaldehyde in 0.1 M phosphate buffer. The result-

ing tissue specimens were then dissected from the spinal column and embedded in paraffin.

Immunohistochemistry Serial sections were then probed immunohistochemitally for several antigens: 1) “MAP2’‘-a monoclonal antibody directed against microtubule associated protein 2: dilution factor (1:4000; Zymed Laboratories, Inc., San Francisco, CA); 2) “GFAP’‘-a polyclonal antibody directed against glial fibrillary acidic protein (Dako Corporation, Carpinteria, CA); 3) ‘‘NFP’‘-a monoclonal antibody directed against middle and high molecular weight nonphosphorylated neurofilament (Zymed Laboratories, Inc., So. San Francisco, CA). Tissue sections at 6 pm were mounted on slides, deparaffinized, hydrated, and equilibrated in pH 7.4 phosphate buffer containing 0.3% Triton X-100. Endogenous peroxidase was quenched with either 0.3% hydrogen peroxide in methanol or 3% aqueous hydrogen peroxide. Various antigen retrieval techniques were applied to slides prior to addition of the primary antibody: for MAP2 and NFP, sections were exposed to microwaves for 15 min in 0.01 M citrate buffer, pH 6.0; for GFAP, tissue specimens were predigested with pepsin. Following primary antibody, all reactions were sequentially incubated in: biotinylated secondary antibody (Vector Laboratories, Inc., Burlingame, CA), and streptavidinhorseradish peroxidase complex (Zymed Laboratories, Inc., So. San Francisco, CA), with interspersed “washes” in buffer. Tissue-bound peroxidase complexes were visualized using 3’,3’diaminobenzidine as chromogen; the slides were then lightly counterstained with hematoxylin, dehydrated, and coverslipped.

RESULTS

Human donor FSCs FSC specimens that were obtained at 6-7 wk GA were generally well preserved and displayed considerable cytoarchitectural integrity by virtue of well-delineated embryonic cellular and fiber layers (Fig. 2a). The central germinal neuroepithelial layer showed only faint-tomoderate MAP2 immunoreactivity. The mantle layer contained immature cells medially. More differentiated cells containing prominent nucleoli with primitive processes, were present in the peripheral anterior and lateral zones. Intense MAP2 immunoreactivity was exhibited in the cell bodies of both the developing and more mature neurons and in the cytoplasmic extensions of the latter (Fig. 2b). Although fully differentiated intraspinal neurons could not be identified in 67 wk donor specimens, the prominent cells in the anterior and lateral mantle layer zones also were positive for NFP (Fig. 2~). The

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distinguish fetal donor tissue from the adjacent host spinal cord. The subpial graft grew as multiple nodules that were densely populated by small cells that were morphologically similar to those residing in the mantle layer of the intact 6-7 wk GA human spinal cord. The graft was intensely immunoreactive for MAP2, which contrasted sharply with the lack of MAP2 immunoreactivity in the underlying host spinal cord (Fig. 2d). Accordingly, a very unequivocal demarcation of graft and host tissues was obtained with the MAP2 antibody under the staining conditions employed. In contrast with MAP2 staining patterns, NFP immunoreactivity was observed in both the subpial graft, as well as the host spinal cord (Fig. 2e). Regions surrounding subpial grafts that failed to stain with anti-NFP, but which were faintly positive for MAP2, also showed GFAP immunoreactivity. This was consistent with some astroglial reactivity that was seen at the graft site. Host neurons generally failed to show any MAP2 immunoreactivity but were intensely stained with anti-NFP. Occasionally, some faint staining of axons with MAP2 could be found in the injured host spinal cord.

Intraspinal Xenografts Fig. 1. Transplantation methods. The entire spinal cord is harvested from human fetuses 6 to 8 wk gestational age (a). The tissue is then homogenized gently into a dissociated tissue suspension (b). Contusion lesion made in the adult rat thoracic spinal cord 14 to 30 prior to transplant is then reexposed (c). Transplantation of the dissociated tissue suspension is then made either subpially above the injured rat spinal cord (d) or intraspinally into the contusion cavity (e).

majority of cells in the mantle layer, however, were NPP immunonegative. The marginal layer demonstrated a radial

pattern

emanating

of linear

MAP2

from the mantle

immunoreactive

profiles

layer.

Human FSCs obtained at 12-14 wk GA exhibited a histologically more mature ependymal layer and a distinct central gray with dorsal and ventral horns (not shown). These FSCs exhibited a heterogeneous population of both immature and mature neurons. As in the younger GA spinal cords, cells in the primitive gray matter were MAP2 immunoreactive with intense staining of both the cell bodies and cytoplasmic processes.

Subpial Xenograjl

In one animal, human FSC tissue was transplanted in a subpial location above the contused adult rat spinal cord. This ectopic site was chosen to minimize host-graft integration, thereby affording an initial opportunity to determine how well differences in MAP2 staining could

Intraspinal xenografts consistently grew in contusion cavities ultimately filling large areas of degenerating host gray and white matter (Fig. 2f). Graft tissue again demonstrated intense MAP2 immunoreactivity (Fig. 2g) similar to that seen in the subpial graft. Only faint MAP2 immunostaining was observed in the host white matter, and essentially no immunoreactivity was observed in gray matter. MAP2 staining in these grafts was heterogeneous with zones of moderate to intense MAP2 immunoreactivity being seen (Fig. 3a). Intense MAP2 immunoreactivity was found in areas that were composed of developing neurons identified by their strong NFP immunoreactivity (not shown). Graft regions exhibiting less intense MAP2 immunostaining were generally composed of glial elements as confirmed by their GFAP immunoreactivity (Fig. 3b). Differentiation of graft from host tissue with MAP2 permitted not only a general definition of the host-graft interface, but also of the degree to which transplantderived neuritic processes extended into the surrounding host white and gray matter. In some regions, a very intimate integration was observed (Fig. 2h and i). MAP2positive neuritic processes were observed extending from the grafts into the adjacent host neuropil (Fig. 2i) where they often formed calyxes around neuronal cell bodies. Some processes were also seen projecting into host white matter. By 2 mo postgrafting, neuritic outgrowth from the transplants was largely confined to the immediate host-graft interface region. In addition to

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Fig. 2. Seven week gestational age fetal spinal cord (a, x56), MAP2 stain. Three representative layers are present including: neuroepithelial (ne), mantle (m), and marginal (mg). There is intense staining of the cytoplasm of cells within the mantle layer (b, x192). Staining for NFP demonstrates immunoreactivity in the anterior/lateral portion of the mantle layer (c, x56). Longitudinal MAP2 section of a subpial transplant demonstrates marked immunoreactivity in the graft (g) without appreciable staining of the host (h) (d, x40). NFP staining of an adjacent section demonstrates robust immunoreactivity in both graft and host (e, x40). Longitudinal heamatoxylin and eosin section of an intraspinal transplant (f, x10). MAP2 staining of an adjacent section demonstrating demarcation of the graft with respect to host (g, x10). At closer inspection of the graft host interface, both immature neurons (arrow) and more mature neurons (arrow head) with cytoplasmic processes extending into the host can be appreciated (h, x96). Even closer inspection reveals islands of neuroblasts (arrow) and dendritic processes arborizing in the host neuropil (i, x192). Note the lack of immunoreactivity within host neurons (arrow head).

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Fig. 3. Longitudinal sections of an intraspinal xenograft 3 mo posttransplanation. This graft (g) is again well demarcated from host tissue (h) by MAP2 immunohistochemistry demonstrating faint (arrow head) and intense (arrow) regions of MAP2 staining (a, x28.8). The intense MAP2 immunopositive zones were composed of neuroblasts and neurons with elaborate processes demonstrated by NFP immunohistochemistry (not shown). Lighter staining regions within the graft (g) corresponded to glial zones demonstrated by GFAP (arrows) (b, x32). Note the indistinct graft/host interface (arrow heads).

these cytoplasmic vealed regions

the presence

of host neuropil

i). The overall however,

extensions, MAP2 immunostaining reof migrating donor cells in nearby

extent

appeared

and white

and amount

matter

(Figs.

of cellular

2h and

migration,

to be modest.

DISCUSSION

In this study, we have observed that MAP2 antibody, when used at the appropriate dilution, can effectively distinguish human fetal tissue from the surrounding adult rat spinal cord for at least up to 2 mo after transplantation. In the host spinal cord, only faint MAP2 staining was detected in surrounding white matter and only rarely was MAP2 immunoreactivity seen in mature host neurons. Human donor tissue, however, exhibited robust immunoreactivity to anti-MAP2. This sharp contrast in MAP2 affinity provides a straightforward way to demonstrate host-graft tissue integration and neuritic projections in a human-to-rat xenograft paradigm.

MAP2 Expression in Rat and Human Neural Tissue The fact that developing human fetal tissue exhibited intense MAP2 immunostaining is consistent with previous descriptions of strong MAP2 immunoreactivity in immature neurons of various species (3,7,8,10,22,25, 28,37,39,40), with MAP2 showing a progressive decline in expression as a function of neuronal maturation (28). In the developing rat spinal cord, MAP2 first appears in the mantle layer at E12. Increased MAP2 immunostaining intensity is seen in both the ventral and dorsal horns, as well as the intermediate gray neuropil until El6 (28). MAP2 is then highly expressed from El6 until P5, at which time staining intensity starts to gradually diminish.

In the adult rat spinal cord, MAP2 staining is usually faint in the gray matter with the exception of a subpopulation of neurons in the dorsal horn in Rexed laminae II and III. The human-to-rat xenografts examined in this study were largely composed of immature cells corresponding to those found in the developing spinal cord in situ at 6-14 wk GA. As our results and a previous description (37) show, MAP2 is extensively expressed in all cells of the developing human FSC that reside in the mantle layer at 6 wk GA. Conversely, MAP2 is only marginally detectable in cells that occupy the germinal neuroepithelial zone at this time. MAP2 was clearly identified in the grafts as islands of neuroblasts that were not NFP immunoreactive, as well as in more mature-appearing cells that did exhibit NFP expression. Intense MAP2 immunoreactivity thus seems to precede detectable NFP expression in the majority of cells in xenografts of human FSC tissue. Similar patterns of MAP2 immunoreactivity also have been noted in the in situ human FSC in this and other studies (28,37). Others have considered MAP2 immunoreactive cells to represent presumptive neuroblasts in both the developing human and rat spinal cord (28,37). Our observations showing that 1) anti-MAP2 staining was in register with NFP-positive cellular elements and 2) no overlap in MAP2 and GFAP immunoreactivity lead to the same interpretation. Initial Observations of Host-Graft Interactions Two previous studies of human fetal neural xenografts to the adult rat CNS have identified donor cells and axonal processes with species-specific antibody directed against a human neurofilament protein constituent (44,45). In the present study, differential immunostaining of host and graft tissue with anti-MAP2, at a prescribed

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dilution, has afforded another approach whereby hostgraft interactions can be readily visualized. Relative to what has already been described based on antineurofilament staining, MAP2 also provides a useful complementary demonstration of graft-derived neuritic projections and neuronal distributions at the host-graft interface by virtue of its strong expression in dendritic processes and cell bodies of young neurons (2,5,9,23,24). From these initial results, it does not appear that donor human neurons migrate extensively into adjacent regions of the host CNS, though MAP-2 immunostained cell bodies were often seen at the host-graft interface. Dendritic processes, on the other hand, did extend into the surrounding host spinal cord, particularly in regions of spinal gray matter close to the transplants, Together with previous findings based on neurofilament immunostaining of intraspinal transplants (44,45), human FSC tissue is capable of extensive neuronal integration with the rat CNS. This also is consistent with previous results derived from experiments in which human fetal dopaminergic neurons were introduced into the rat striatum (l&46). The degree of neural integration suggested in the spinal cord supports the possibility of neuronal relays being established across separated segments of the host spinal cord (30,31). This potential mechanism of functional recovery has been largely examined with allografts into the rat spinal cord (17); however, human xenografts may represent an even more useful setting in which to test this mode of graft-mediated repair. More detailed analyses of host-graft neuronal interactions are currently in progress to determine how various lesion conditions may affect human xenograft maturation and patterns of connectivity in the injured adult spinal cord (Giovannini et al., in preparation). Acknowledgments - This work was supported in part by the Department of Veterans Affairs (D.K.A.), the University of Florida Brain Institute; C.M., K.E. Overstreet (D.K.A.) and Mark F. Overstreet (P.J.R.) Endowments for Spinal Cord Injury Research; The University of Florida Brain Institute andThe State of Florida Brain and Spinal Cord Injury Research Trust Fund. We would also like to thank Judy Olmsted MS. for her technical assistance in preparing the immunohistochemical sections shown in this manuscript.

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