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Behavior of human neural progenitor cells transplanted to rat brain

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Abstract

Human neural stem/progenitor cells provide a useful tool for studies of neural development and differentiation, as well as a potential means for neuroreplacement therapeutic needs in the human CNS. Stem cells isolated from developing human central nervous system of 8–12-week fetuses were transplanted to the forebrain and cerebellum of young and adult rats after 14 days of in vitro expansion. Cells were labeled by bisbenzimide prior to transplantation without immunosuppression. Recipient brains were examined 10 and 20 days after transplantation. Labeled stem cells were found in the neocortex, lateral ventricle and caudate nucleus in the forebrain, and in the molecular layer, Purkinje cell layer, and granular layer of the cerebellum. Mitotically dividing stem cells were observed in graft core, confirming their proliferative potential in new microenvironment. Engrafted cells migrate through the parenchyme of striatum, along the ventricular ependymal layer and callosal fibers, some of them reaching the opposite hemisphere. Some cells migrating along the capillaries express glial acid fibrillary protein, demonstrating their differentiation into astrocytes. Grafted cells expressing calbindin were found in the Purkinje cell layer, suggesting their differentiation into the Purkinje cells. At the same time, some grafted cells were undifferentiated and expressed vimentin. Our results demonstrate that cultured human neural stem/progenitor cells migrate and differentiate into both neurons and astrocytes after transplantation to the rat forebrain or cerebellum of young and adult rats.

Introduction

During recent two decades a crucial revision of some cornerstone concepts has opened new horizons in neurosciences. Modern basic viewpoints include the idea of high CNS plasticity which means not only rearrangement of neurons and their interconnections, but also the formation of new neural cells in humans and animals during their whole life span [19]. This idea is bound with new knowledge regarding the neural stem cells.

Neural stem cells are multipotential progenitor cells that have self-renewal ability. A single neural stem cell is capable of generating various kinds of cells within the CNS, including neurons, astrocytes, and oligodendrocytes [2], [6], [9], [17], [18], [36].

Investigation of the biology of neural stem cells is driven not only by interest in fundamental problems of cell differentiation [1], [13], [16], but also by potential possibility of applied use of stem cells in clinic use for correction in different human brain diseases [3], [6], [7], [8], [29], [35], [39]. Recent investigations proved that stem cells, which play exceptional role in embryonal and early postnatal ontogenesis, exist in some distinct structures of adult human (and mammal in general) brain [2], [12], [21], [26], [28], [33]. The mechanisms driving functions of stem cells both in embryonal and in differentiated brain are not thoroughly understood, and nowadays main efforts of research are bound to the problems of their isolation, to their propagation and preservation as tissue culture, as well as to investigation of their potency to differentiate in vitro and after their transplantation into the brain. This explains the attention devoted to investigation of human neural stem cells which may serve as a source for neurotransplantation in cases of human neurodegenerative diseases.

Investigations of recent years demonstrated that human neural stem cells may be isolated from embryo brains [5], [10], [14], [16], [22], [31], [32], [33], [34], [37], [38], from brains of adult patients during surgical operation [4], [20], [21], [24] and even from cadaver brains [15]. Both fetal and adult human neural stem cells display similar proliferation kinetics, and are able to differentiate into neuronal and glia lineages and express the same regulatory genes [25]. Publications on development of human neural stem cells after their transplantation into adult brain are not numerous. Comparative analysis of the fate of stem cells in young and aged brains is absent.

Here we report our investigation of the development and differentiation of human neural stem/progenitor cells (NSC/NPC) that had been transplanted into different regions of young and adult rat brain after cultivation in vitro. Cultivated cells prior to transplantation had been characterized morphologically and phenotyped by means of flow cytometry according to a standard panel of antibodies, a testing procedure which permits us to evaluate important features of the cell culture sample. For further transplantation, the cell cultures having best viability and highest content of stem cells and neural progenitors, and the lowest proportion of cells expressing histocompatability antigens, were selected [27].

Section snippets

Material and methods

Brains from human 8–12-week fetuses were excised and suspended in NPBM (Clonetics) medium with addition of standard growth factor preparation (NPMM (Clonetics)), which includes hFGF, hEGF, NSF-1 and GA. In order to visualize human NSCs in the rat brain, they had been labeled by nuclear fluorescent dye, bisbenzimide (Hoechst 33342, Serva). Fifty Wistar rats served as NSC recipients. The cells were transplanted stereotaxically into the forebrain (including lateral ventricle, neocortex, and

In vitro properties of cells suggested for transplantation

Initial suspensions of human NPCs in selective medium formed typical neurospheres, which gradually grew in size during their cultivation. After 14 days of cultivation, part of the material had been used for transplantation in rat brains, and the remaining part was seeded on the serum-containing medium; 1 day later it was fixed and investigated immunocytochemically by means of antibodies to nestin, vimentin, GFAP and β-tubulin III.

Immunohistochemical investigation demonstrated that multipotent

Discussion

This investigation demonstrated that human neural/progenitor cells, propagated in cell culture survive successfully after transplantation into different structures of both young and aged rat brain, without immunosuppression, for no less than 20 days. Transplanted cells migrate widely from the place of injection as has been described in many publications [14], [16], [23], [30], [36], [38], [39]. Migration pattern is much like in young and adult rats. According to our observations, most distant

Acknowledgements

This work was supported by Russian Basic Science Foundation, ‘A-T Parents to Save Children’ Foundation, and MedBioComp Inc.

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