Human neural precursor cells in spinal cord repair
Author: Piao, Junghua
Date: 2007-12-07
Location: Hörsal 9Q, Alfred Nobels Allé 8, Karolinska Institutet, Huddinge
Time: 09.30
Department: Institutionen för neurobiologi, vårdvetenskap och samhälle / Department of Neurobiology, Care Sciences and Society
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thesis.pdf (1.270Mb)
Abstract
Traumatic spinal cord injury (SCI) often results in substantial neural
cell death, axonal degeneration and demyelination, accompanied by loss of
sensory and motor functions. Cell therapy may have neuroprotective
effects, substitute for lost neural cells, promote regeneration and
enhance remyelination in the lesioned spinal cord. The availability of
neural precursor cells (NPCs) suitable for both experimental and clinical
purposes may be significantly improved by expansion of these cells in
vitro. The primary objectives of the present thesis have been to evaluate
conditions for culturing and in vitro expansion of human NPCs and to
characterize the suitability of these cells for therapeutic treatment of
SCI.
NPCs derived from the human first trimester forebrain and spinal cord
were cultured as neurospheres in the presence of epidermal growth factor
(EGF) and basic fibroblast growth factor (bFGF), with or without ciliary
neurotrophic factor (CNTF). First trimester forebrain up to 12 weeks of
gestation can serve as source for neurospheres, while spinal cord NPCs
only expand successfully when the originating tissue is ≤ 9.5 weeks of
gestation. Within these developmental time frames, gestational age of the
donor tissue did not affect the proportion of cells with different
phenotypes in culture. In vitro propagation enhanced the size of the cell
population immunoreactive (IR) for CD133 without altering the numbers of
cells expressing Tra-1-60-, Tra-1-81-, stage-specific embryonic antigen-4
(SSEA-4)-, nestin- and proliferating cell nuclear antigen (PCNA). The
fraction of glial fibrillary acidic protein (GFAP)-IR cells increased,
while the proportion of β-tubulin III-IR cells was reduced following five
or more passages in the case of cultures originating from the spinal
cord, but not from forebrain. Thus, even under the same culture
conditions, the cellular composition of human neurospheres was dependent
on the length of propagation in vitro and the regional heterogeneity of
the tissue from which they were derived.
CNTF enhanced the rate of cell proliferation and expansion of human NPCs
derived from the embryonic spinal cord without significantly altering the
proportions of cells that expressed nestin, GFAP,β-tubulin III or O4. In
contrast, this factor exerted no such influence on human NPCs derived
from agematched forebrain. This difference might reflect, at least in
part, differences in the expression of the CNTF receptor-α protein in
these two regions of the central nervous system during development.
In vitro cultures of human NPCs expressed human leukocyte antigen (HLA)
class I and II, but almost no co-stimulatory proteins (CD40, CD80 and
CD86). The level of HLA increased during expansion of both spinal cord
and forebrain NPCs. This phenomenon could, at least in theory, enhance
the immunogenicity of NPCs and the risk for rejection after
transplantation, but human peripheral lymphocytes exhibited no response
when co-cultured with NPCs expanded in vitro. Together, these
observations indicated that expanded NPCs demonstrate a low degree of
immunogenicity, despite their pronounced expression of HLA, and the
incompatibility of this antigen with that of a potential recipient.
In order to study the patterns of migration of human NPCs implanted into
the injured spinal cord, these cells were transplanted close to traumatic
spinal cord lesions performed in rats by clip compression or partial
transection. A small fraction of the grafted NPCs had migrated, always
confined to the white matter, several millimeters away from the site of
implantation. There was no evidence of directed migration of grafted NPCs
towards any of the two spinal cord lesions at six weeks after
transplantation. The proportion of cells expressing nestin, GFAP or
β-tubulin III, was similar at the graft center and among the cells that
had migrated furthest, suggesting that their motility was not influenced
by the degree of differentiation. In agreement, pre-differentiation of
NPCs in vitro, achieved by inhibiting γ-secretase, did not affect
migration of the grafted cells. Reduction of the extracellular
chondroitin sulfate proteoglycans by chondroitinase ABC promoted
migration of NPCs, demonstrating that although no evidence for homing of
grafted NPCs was detected, extrinsic factors can affect their migration.
In conclusion, implanted human NPCs mainly remained at the site of
transplantation, a feature that may be beneficial for purposes of local
neuroprotection and cell replacement following focal SCI.
List of papers:
I. Piao JH, Odeberg J, Samuelsson EB, Kjaeldgaard A, Falci S, Seiger A, Sundström E, Akesson E. (2006). "Cellular composition of long-term human spinal cord- and forebrain-derived neurosphere cultures." J Neurosci Res 84(3): 471-82
Pubmed
II. Piao JH, Samuelsson EB, Kjældgaard A, Falci S, Seiger Å, Sundström E, Åkesson E. (1970). "Regional-dependent proliferative response to ciliary neurotrophic factor (CNTF) by human neural stem and progenitor cells." (Submitted)
III. Odeberg J, Piao JH, Samuelsson EB, Falci S, Akesson E. (2005). "Low immunogenicity of in vitro-expanded human neural cells despite high MHC expression." J Neuroimmunol 161(1-2): 1-11
Pubmed
IV. Piao JH, Holmberg L, Delfani K, Seiger Å, Kjældgaard A, Falci S, Åkesson E, Sundström E. (1970). "Migration of human neural precursor cells grafted to the injured spinal cord." (Submitted)
I. Piao JH, Odeberg J, Samuelsson EB, Kjaeldgaard A, Falci S, Seiger A, Sundström E, Akesson E. (2006). "Cellular composition of long-term human spinal cord- and forebrain-derived neurosphere cultures." J Neurosci Res 84(3): 471-82
Pubmed
II. Piao JH, Samuelsson EB, Kjældgaard A, Falci S, Seiger Å, Sundström E, Åkesson E. (1970). "Regional-dependent proliferative response to ciliary neurotrophic factor (CNTF) by human neural stem and progenitor cells." (Submitted)
III. Odeberg J, Piao JH, Samuelsson EB, Falci S, Akesson E. (2005). "Low immunogenicity of in vitro-expanded human neural cells despite high MHC expression." J Neuroimmunol 161(1-2): 1-11
Pubmed
IV. Piao JH, Holmberg L, Delfani K, Seiger Å, Kjældgaard A, Falci S, Åkesson E, Sundström E. (1970). "Migration of human neural precursor cells grafted to the injured spinal cord." (Submitted)
Issue date: 2007-11-16
Rights:
Publication year: 2007
ISBN: 978-91-7357-288-0
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