Trophic and immunoregulatory properties of neural precursor cells: Benefit for intracerebral transplantation

https://doi.org/10.1016/j.expneurol.2010.04.021Get rights and content

Abstract

Intracerebral xenotransplantation of porcine fetal neuroblasts (pNB) is considered as an alternative to human neuroblasts for the treatment of neurodegenerative diseases. However, pNB are systematically rejected, even in an immunoprivileged site such as the brain. Within this context, neural stem/precursor cells (NSPC), which were suggested as exhibiting low immunogenicity, appeared as a useful source of xenogeneic cells. To determine the advantage of using porcine NSPC (pNSPC) in xenotransplantation, pNB and pNSPC were grafted into the striatum of rats without immunosuppression. At day 63, all the pNB were rejected while 40% of the rats transplanted with pNSPC exhibited large and healthy grafts with numerous pNF70-positive cells. The absence of inflammation at day 63 and the occasional presence of T cells in pNSPC grafts evoked a weak host immune response which might be partly due to the immunosuppressive properties of the transplanted cells. T cell proliferation assays confirmed such a hypothesis by revealing an inhibitory effect of pNSPC on T cells through a soluble factor. In addition to their immunosuppressive effect, in contrast to pNB, very few pNSPC differentiated into tyrosine hydroxylase-positive neurons but the cells triggered an intense innervation of the striatum by rat dopaminergic fibers coming from the substantia nigra. Further experiments will be required to optimize the use of pNSPC in regenerative medicine but here we show that their immunomodulatory and trophic activities might be of great interest for restorative strategies. This article is part of a Special Issue entitled "Interaction between repair, disease, & inflammation."

Introduction

Neural transplantation is a promising strategy to restore cell function in the human central nervous system (CNS). However, the limited access to human fetal neurons and the ethical concerns regarding their use have fuelled a search for alternative sources of transplantable cells. Among these, cells derived from pig embryos are of great interest (Isacson and Deacon, 1996, Isacson et al., 1995). Fetal pig neurons have the capacity to develop large axons (Deacon et al., 1994) and small-scale clinical trials indicate that neural cells isolated from porcine fetal brains integrate the host tissue after their transplantation into the brain of immunosuppressed patients (Deacon et al., 1997, Fink et al., 2000, Pakzaban and Isacson, 1994). For this reason, fetal porcine neurons have numerous advantages in addition to their wide availability. Their use as donor cells is however highly limited by the host immune response. Indeed, as previously shown for other intracerebral xenografts (Duan et al., 1995, Finsen et al., 1988, Lund et al., 1989, Pollack et al., 1990, Widner and Brundin, 1988), fetal porcine neurons implanted into the brain of immunocompetent rat are systematically rejected. This occurs within 5–7 weeks post-transplantation (Barker et al., 2000, Michel et al., 2006, Remy et al., 2001). The rejection process is accompanied by an infiltration of the graft by microglial and dendritic cells, and a sudden appearance of T lymphocytes (Barker et al., 2000, Michel et al., 2006, Remy et al., 2001). This coincides with a marked accumulation of transcripts encoding monocyte chemoattractants such as MCP-1 and RANTES, as well as proinflammatory lymphokines and Th1 cytokines (Barker et al., 2000, Melchior et al., 2002, Remy et al., 2001). Continuous exposure to high doses of cyclosporine A or treatments with several immunosuppressors prolong the survival of intracerebral xenografts (Cicchetti et al., 2003, Deacon et al., 1997, Fink et al., 2000, Jacoby et al., 1999, Larsson et al., 2000, Larsson et al., 2001, Pedersen et al., 1997) but few xenogeneic neurons survive and systemic treatments with immunosuppressors produce severe secondary effects (Rezzani, 2006). Progress has therefore to be made in order to control cell rejection even in a relatively immunoprivileged site such as the CNS (Barker and Widner, 2004).

Genetic modifications of porcine neurons are currently performed to evaluate the advantage of a local production of immunosuppressive molecules such as CTLA4-Ig (Martin et al., 2005). Transplantation of multipotent stem cells is another alternative. Xenogeneic mesenchymal stem cells or expanded neural precursors show long-term survival in the brain of immunocompetent animals (Armstrong et al., 2001, Rossignol et al., 2009). Such lengthy survival has been partially attributed to the low immunogenicity of multipotent cells (Armstrong et al., 2001, Odeberg et al., 2005), but recent evidence points to the sizeable immunosuppressive effect of mesenchymal stem cells (for review, see Di Nicola et al., 2002, Krampera et al., 2003, Rasmusson, 2006, Uccelli et al., 2007). Neural precursor cells (NSPC) may also display such immunoregulatory properties. In 2005, Pluchino et al., showed that syngeneic NSPC systemically injected in a mouse model of multiple sclerosis promoted neuroprotection by immunomodulatory mechanisms (Pluchino et al., 2003, Pluchino et al., 2005). If porcine NSPC (pNSPC) exhibit such immunoregulatory properties, their use would be of great interest for restorative strategies. In fact, NSPC derived from fetal or adult pig brains could be easily expanded upon treatment with bFGF, providing an indefinite source of transplantable cells. Like human NSPC, porcine NSPC are able to generate the three major neural lineages–oligodendrocytes, astrocytes and neurons—both in vitro and following transplantation in vivo (Harrower et al., 2006, Smith and Blakemore, 2000). In addition, experimental work on immunosuppressed rats has shown a good differentiation of pNSPC into neurons with the formation of synapses and the extension of fibers (Harrower et al., 2006).

In the present paper, we show that pNSPC are a valuable source of donor cells, displaying immunosuppressive properties while also producing a trophic effect upon the host dopaminergic system.

Section snippets

Cell preparation

Porcine embryos were obtained from Large White domestic pigs, 28 days after artificial insemination. Animals were obtained from the Institut National de la Recherche Agronomique (INRA, Nouzilly, France) and killed in the institute's accredited slaughterhouse. After hysterectomy, the embryos were collected and washed in Hank's balanced salt solution (HBSS; Life Technologies Ltd, CergyPontoise, F) before dissection of the brain tissue.

Porcine neuroblasts (pNB) were prepared from G28 ventral

Survival of pNSPC in the rat brain

pNSPC were isolated from the forebrain of G28 porcine embryos and expanded as neurospheres for 10 days in the presence of bFGF (Fig. 1). After 7 days in differentiation conditions, 98.4% of the neurospheres contained both GFAP+ and NF70+ cells (Fig. 1b, c). Very few pNSPC were undergoing a dopaminergic differentiation. Indeed, immunocytochemistry analyses revealed an amount of one tyrosine hydroxylase+ cells for 100 neurospheres (Fig. 1d).

To analyze the survival of pNSPC in the rat brain, the

Discussion

The intensity and rapidity of the rejection in the brain depend on the phylogenetic distance between donor and host (Dymecki et al., 1990, Mason et al., 1986, Pakzaban and Isacson, 1994) and the status of the host immune system (Marion et al., 1990) but our present data emphasize the fact that the host immune response is also influenced by the nature of the transplanted cells. Porcine aortic endothelial cells (PAEC) implanted into the rat striatum are eradicated within 1–2 weeks (Remy et al.,

Acknowledgments

The authors are grateful to Pr. Jean-Paul Soulillou for his support. We also thank Joanna Ashton-Chess for critical reading the manuscript. The nestin antibody obtained from the Developmental Studies Hybridoma Bank (DSHB) was developed by Susan Hockfield developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. The DP5 antibody was a gift of Dr Denise Paulin, Université Pierre et Marie Curie, Paris, F. The work

References (71)

  • T.P. Harrower et al.

    Long-term survival and integration of porcine expanded neural precursor cell grafts in a rat model of Parkinson's disease

    Exp. Neurol.

    (2006)
  • O. Isacson et al.

    Specific axon guidance factors persist in the adult brain as demonstrated by pig neuroblasts transplanted to the rat

    Neuroscience

    (1996)
  • M. Krampera et al.

    Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen-specific T cells to their cognate peptide

    Blood

    (2003)
  • L.C. Larsson et al.

    Porcine neural xenografts in rats and mice: donor tissue development and characteristics of rejection

    Exp. Neurol.

    (2001)
  • Z.D. Ling et al.

    Differentiation of mesencephalic progenitor cells into dopaminergic neurons by cytokines

    Exp. Neurol.

    (1998)
  • J. Liu et al.

    Suppression of human peripheral blood lymphocyte proliferation by immortalized mesenchymal stem cells derived from bone marrow of Banna Minipig inbred-line

    Transplant. Proc.

    (2004)
  • P. Lu et al.

    Neural stem cells constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury

    Exp. Neurol.

    (2003)
  • D.W. Marion et al.

    Patterns of immune rejection of mouse neocortex transplanted into neonatal rat brain, and effects of host immunosuppression

    Brain Res.

    (1990)
  • D.W. Mason et al.

    The fate of allogeneic and xenogeneic neuronal tissue transplanted into the third ventricle of rodents

    Neuroscience

    (1986)
  • D.C. Michel et al.

    Dendritic cell recruitment following xenografting of pig fetal mesencephalic cells into the rat brain

    Exp. Neurol.

    (2006)
  • J. Odeberg et al.

    Low immunogenicity of in vitro-expanded human neural cells despite high MHC expression

    J. Neuroimmunol.

    (2005)
  • P. Pakzaban et al.

    Neural xenotransplantation: reconstruction of neuronal circuitry across species barriers

    Neuroscience

    (1994)
  • E.B. Pedersen et al.

    Triple immunosuppression protects murine intracerebral, hippocampal xenografts in adult rat hosts: effects on cellular infiltration, major histocompatibility complex antigen induction and blood-brain barrier leakage

    Neuroscience

    (1997)
  • I.F. Pollack et al.

    MHC antigen expression in spontaneous and induced rejection of neural xenografts

    Prog. Brain Res.

    (1990)
  • I. Rasmusson

    Immune modulation by mesenchymal stem cells

    Exp. Cell Res.

    (2006)
  • I. Rasmusson et al.

    Mesenchymal stem cells inhibit lymphocyte proliferation by mitogens and alloantigens by different mechanisms

    Exp. Cell Res.

    (2005)
  • S. Sergent-Tanguy et al.

    Cell surface antigens on rat neural progenitors and characterization of the CD3 (+)/CD3 (−) cell populations

    Differentiation

    (2006)
  • M. Shinoda et al.

    Microglial cell responses to fetal ventral mesencephalic tissue grafting and to active and adoptive immunizations

    Exp. Neurol.

    (1996)
  • T. Subramanian et al.

    Rejection of mesencephalic retinal xenografts in the rat induced by systemic administration of recombinant interferon-gamma

    Exp. Neurol.

    (1995)
  • C.N. Svendsen et al.

    Survival and differentiation of rat and human epidermal growth factor-responsive precursor cells following grafting into the lesioned adult central nervous system

    Exp. Neurol.

    (1996)
  • A. Uccelli et al.

    Mesenchymal stem cells: a new strategy for immunosuppression?

    Trends Immunol.

    (2007)
  • L. Wang et al.

    Neural stem/progenitor cells modulate immune responses by suppressing T lymphocytes with nitric oxide and prostaglandin E2

    Exp. Neurol.

    (2009)
  • H. Widner et al.

    Immunological aspects of grafting in the mammalian central nervous system. A review and speculative synthesis

    Brain Res.

    (1988)
  • R.A. Barker et al.

    A role for complement in the rejection of porcine ventral mesencephalic xenografts in a rat model of Parkinson's disease

    J. Neurosci.

    (2000)
  • C. Beauvillain et al.

    Neonatal and adult microglia cross-present exogenous antigens

    Glia

    (2008)
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