This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu)

Chiang, Yung-Hsiao

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This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu). CELL TRANSPLANTATION IN NEURODEGENERATIVE DISORDERS Yung-Hsiao Chiang Submitted to the faculty of the University Graduate School in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the School of Medicine, Indiana University October, 2000 Accepted by the graduate faculty, Indiana University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Shirley BayJr, Ph.D. Doctoral Committee 95avt~) David Stocum, Ph.D. September 28, 2000 ii ABSTRACT Neurodegenerative disorders, including Parkinson's disease and cerebral ischemia, are characterized by loss of neurons in the brain. We found that members of the transforming growth factor-~ family such as glial cell line-derived neurotrophic factor (GDNF) and osteogenic protein 1 (as known as bone morphogenic protein 7) were neuroprotective in models of ischemia-induced brain injury. Our studies also indicated that either stimulating endogenous cells to secrete trophic factors or directly injecting GDNF could effect regeneration of the nigrostriatal pathway in hemiparkinsonian rodents. These factors are also known to involve in the development of both the central nervous system and the kidney in mammals. Trophic factors which are critical for development of a structure would be synthesized and secreted within that structure. It is possible that implantation of the fetal kidney cells may have neuroprotective and neuroregenerative effects in the adult brain. A more exciting possibility involves the newly identified neural stem cell populations. These recently discovered multipotent stem cells may provide a source of replacement cells as well as trophic factors after implantation. The purpose of this dissertation is to test the hypotheses (1) that transplantation of fetal kidney cells may elicit neuroprotection during cerebral ischemia, (2) that endogenous GDNF, upregulated by pharmacological manipulation, may protect brain against ischemia-induced brain injury, (3) that the GDNF is the key factor in fetal kidney cell-induced neuroprotection, (4) that transplanted fetal kidney cells may be an endogenous source of GDNF and other trophic factors for regenerating the nigrostriatal pathway, and (5) that progenitor cells from embryonic rat brain are indeed multipotent VI neural stem cells. Transplantation of fetal kidney cells reduced the volume of cortical infarction and restored behavioral deficits in adult rats. This neuroprotection was not seen in animals which received adult kidney cell transplants, which contain much lower levels of trophic factors. Systemic administration of vitamin D3 for 8 days increased cortical GDNF levels and decreased ischemia-induced brain infarction. Since there are no GDNF receptor (GFRal) antagonists, the neuroprotective mechanism of GDNF and the efficacy of intracerebral kidney cell transplants were examined in heterozygous GFRalnull mutated (+/-) and wildtype (+/+) mice. GDNF administration and fetal kidney cell implantation decreased the motor deficits and cortical infarction induced by stroke in +/+ mice but not in+/- mice. Moreover, the extent of infarction in+/+ mice treated with fetal kidney transplants was significantly less than with GDNF, suggesting that fetal kidney transplants provide more neuroprotection than GDNF. Nigrostriatal transplantation of fetal kidney cells helped to regenerate dopaminergic fibers in the pathway of hemiparkinsonian rats. The neural stem cells from embryonic rat brain were undifferentiated, proliferative and capable of self-renewal over 2 years in vitro. After being transferred to an appropriate environment, they differentiate into neurons, astrocytes and oligodendrocytes. Their differentiation fates were affected by epigenetic factors. In conclusion, transplantation of fetal kidney cells provides neuroprotection as well as neuroregeneration in animal models of stroke and Parkinson's disease. The progenitor cells obtained from embryonic rat brains are indeed multipotent neural stem cells. vii TABLE OF CONTENTS Page I. Chapter I./Background----------------------------------------------------------------------1 A. Cerebral ischemia and trophic factors------------------------------------------------1 B. Parkinson's disease and trophic factors for dopamine neurons-------------------4 C. Fetal kidney cells and trophic factors------------------------------------------------6 D. Stem cells--------------------------------------------------------------------------------7 E. Delivery of trophic factors to the brain: stem cells and fetal cells--------------11 II. Chapter IL/Transplantation of Fetal Kidney Cells in Cerebral Ischemia-----------12 A. Introduction-----------------------------------------------------------------------------12 B. Materials and Methods----------------------------------------------------------------14 1. Animals-----------------------------------------------------------------------------14 2. Transplantation--------------------------------------------------------------------14 3. MCA ligation----------------------------------------------------------------------17 4. Behavioral measurements--------------------------------------------------------17 5. TTC staining-----------------------------------------------------------------------18 6. Pax-2 immunostaining and GDNF in situ hybridization---------------------18 7. Statistics----------------------------------------------------------------------------20 C. Results-----------------------------------------------------------------------------------20 1. Behavioral measurements--------------------------------------------------------20 2. Brain infarction--------------------------------------------------------------------21 3. Pax-2 immunoreactivity of fetal kidney transplants in the rat neocortex--24 4. GDNF expression in the kidney grafts-----------------------------------------24 D. Discussion------------------------------------------------------------------------------2 7 III. Chapter IIVManipulation of Endogenous Trophic Factors in Cerebral Ischemia-29 A. Introduction----------------------------------------------------------------------------29 B. Materials and methods----------------------------------------------------------------3 0 1. Animals and chemicals-----------------------------------------------------------30 2. MCA ligation and histology-----------------------------------------------------30 3. Measurement ofGDNF by enzyme-linked immunosorbent assay (ELISA) analysis----------------------------------------------------------------31 4. Blood pressure, heart rate, glucose, calcium & blood gas measurements-32 viii 5. Body temperature-----------------------------------------------------------------3 3 C. Resul ts----------------------------------------------------------------------------------3 3 1. Cortical infarction----------------------------------------------------------------3 3 2. Effects of D3 on GDNF protein levels-----------------------------------------37 3. Physiological response to D3----------------------------------------------------3 8 D. Discussion------------------------------------------------------------------------------41 IV. Chapter IV/GDNF Receptor Mediates the Neuroprotective Effect of Fetal Kidney Cells---------------------------------------------------------------------------------44 A. Introduction-----------------------------------------------------------------------------44 B. Materials and methods----------------------------------------------------------------45 1. Animals-----------------------------------------------------------------------------45 2. GFRa 1 null (-/-) mutation-------------------------------------------------------46 3. Cerebral ischemia-----------------------------------------------------------------46 1. Animals--------------------------------------------------------------------46 11. Intracerebral injection of vehicle or GDNF--------------------------47 111. Kidney tissue implantation---------------------------------------------4 7 1v. MCA ligation-------------------------------------------------------------4 7 v. Behavioral measurements-----------------------------------------------48 v1. TTC staining--------------------------------------------------------------48 4. Enzyme-linked immunosorbent assay (ELISA) for GDNF-----------------49 5 . Statistics----------------------------------------------------------------------------5 0 C. Results-----------------------------------------------------------------------------------50 1. Cerebral ischemia-----------------------------------------------------------------50 1. Behavioral measurements-----------------------------------------------50 11. Brain infarction-----------------------------------------------------------51 2. GDNF levels in kidney-----------------------------------------------------------60 D. Discussion------------------------------------------------------------------------------60 V. Chapter V/Transplantation of Fetal Kidney Cells and Regeneration of the Nigrostriatal Pathway--------------------------------------------------------------------64 A. Introduction-----------------------------------------------------------------------------64 B. Materials and methods----------------------------------------------------------------66 1. S urgery-----------------------------------------------------------------------------66 2. Behavioral assays-----------------------------------------------------------------67 1. Rotation-------------------------------------------------------------------67 n. Postural imbalance------------------------------------------------------67 IX 3. TH immunocytochemistry ------------------------------------------------------6 7 4. Pax -2 immunostaining------------------------------------------------------------68 C. Resul ts-----------------------------------------------------------------------------------69 1. Rotational behavior --------------------------------------------------------------69 2. Postural asymmetry----------------------------------------------------------------7 0 3. TH immunocytochemistry--------------------------------------------------------72 4. Pax-2 immunocytochemistry-----------------------------------------------------75 D. Discussion-------------------------------------------------------------------------------7 5 VI. Chapter VI/Characterization of Long-Term Cultured EGF-Responsive Neural Stem Cells-----------------------------------------------------------------------------------79 A. Introduction-----------------------------------------------------------------------------79 B. Materials and methods-----------------------------------------------------------------80 1. Isolation of E-14 brain cells------------------------------------------------------80 2. Culture of neurospheres-----------------------------------------------------------81 3. Subculture of single neurospheres-----------------------------------------------81 4. Immunocytochemistry-------------------------------------------------------------81 5. BrdU labeling and immunodetection--------------------------------------------82 6. Measurement of cell proliferation------------------------------------------------83 7. Measurement of survival of cells-------------------------------------------------84 8. Measurement of process length---------------------------------------------------84 9. Statistics------------------------------------------------------------------------------84 C. Results------------------------------------------------------------------------------------85 1. Growing pattern of neuropsheres-------------------------------------------------85 2. Existence of neuroepithelial stem cells and precursor cells-------------------87 3. Proliferation characteristics of cells----------------------------------------------89 4. EGF-dependent cell proliferation-------------------------------------------------91 5. Stem cell characteristics------------------------------------------------------------96 6. Differentiation of neural stem cells----------------------------------------------100 7. Changes in differentiation by epigenetic factors-------------------------------106 1. Astroglial differentiation of neural stem cells in FCS----------------106 11. Astroglial differentiation of neural stem cells in bFGF--------------110 111. Astroglial differentiation of neural stem cells in retinol-------------110 D. Discussion-------------------------------------------------------------------------------113 VII. Chapter VII/General Conclusions-------------------------------------------------------121 VIII. References----------------------------------------------------------------------------------124 X IX. Curriculum Vita xi 

Cell transplantation in neurodegenerative disorders

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Cell transplantation in neurodegenerative disorders

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