Publications by authors named "Samuel Saporta"

32 Publications

Effects of isoflurane or propofol on postnatal hippocampal neurogenesis in young and aged rats.

Brain Res 2013 Sep 24;1530:1-12. Epub 2013 Jul 24.

Department of Pathology and Cell Biology/Neuroscience, School of Medicine, University Of South Florida, Tampa, FL 33612, USA.

An increasing number of in vitro and in vivo studies suggest that anesthesia and surgery could be risk factors for later cognitive impairment in the young and aged brain. General anesthesia has been shown to impair spatial memory in rats and this performance is dependent on hippocampal function and postnatal hippocampal neurogenesis. Anesthetic induced alteration of one or more stages of postnatal hippocampal neurogenesis may in part explain this cognitive impairment following anesthesia. Three different populations of proliferating cells in the dentate gyrus (DG) were labeled with different thymidine analogs (EdU, IdU, and CldU) at 4, 8, and 21 days, respectively, in young (3-month-old) and aged (20-month-old) rats prior to a 3h exposure to isoflurane, control, propofol, or 10% intralipid. 24h following general anesthesia, brains were collected for analysis. The number of cells co-localized with neuronal differentiation and maturation labels with each of the thymidine analogs was quantified. In addition, new cell proliferation 24hr following anesthesia was assessed with anti-Ki67. The effect of anesthesia on astrocytes was also assessed with anti-S100β. Isoflurane or propofol did not affect new cell proliferation, as assessed by Ki67, in the DG of young or aged rats. However, propofol significantly decreased the number of differentiating neurons and increased the number of astrocytes in the DG of young, but not aged, rats. Isoflurane significantly decreased the number of maturing neurons and increased the number of astrocytes in the DG of aged, but not young, rats. Isoflurane and propofol anesthesia altered postnatal hippocampal neurogenesis in an age and agent dependent matter.
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http://dx.doi.org/10.1016/j.brainres.2013.07.035DOI Listing
September 2013

Impaired blood-brain/spinal cord barrier in ALS patients.

Brain Res 2012 Aug 27;1469:114-28. Epub 2012 Jun 27.

Center of Excellence for Aging & Brain Repair, University of South Florida, Morsani College of Medicine, Tampa, FL 33612, USA.

Vascular pathology, including blood-brain/spinal cord barrier (BBB/BSCB) alterations, has recently been recognized as a key factor possibly aggravating motor neuron damage, identifying a neurovascular disease signature for ALS. However, BBB/BSCB competence in sporadic ALS (SALS) is still undetermined. In this study, BBB/BSCB integrity in postmortem gray and white matter of medulla and spinal cord tissue from SALS patients and controls was investigated. Major findings include (1) endothelial cell damage and pericyte degeneration, (2) severe intra- and extracellular edema, (3) reduced CD31 and CD105 expressions in endothelium, (4) significant accumulation of perivascular collagen IV, and fibrin deposits (5) significantly increased microvascular density in lumbar spinal cord, (6) IgG microvascular leakage, (7) reduced tight junction and adhesion protein expressions. Microvascular barrier abnormalities determined in gray and white matter of the medulla, cervical, and lumbar spinal cord of SALS patients are novel findings. Pervasive barrier damage discovered in ALS may have implications for disease pathogenesis and progression, as well as for uncovering novel therapeutic targets.
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http://dx.doi.org/10.1016/j.brainres.2012.05.056DOI Listing
August 2012

Quantitative assessment of new cell proliferation in the dentate gyrus and learning after isoflurane or propofol anesthesia in young and aged rats.

Brain Res 2012 Mar 13;1441:38-46. Epub 2011 Nov 13.

Department of Pathology and Cell Biology/ Neurosciences, School of Medicine, University Of South Florida, Tampa, FL, USA.

There is a growing body of evidence showing that a statistically significant number of people experience long-term changes in cognition after anesthesia. We hypothesize that this cognitive impairment may result from an anesthetic-induced alteration of postnatal hippocampal cell proliferation. To test this hypothesis, we investigated the effects of isoflurane and propofol on new cell proliferation and cognition of young (4 month-old) and aged (21 month-old). All rats were injected intraperitoneally (IP) with 50 mg/kg of 5-bromo-2-deoxyuridine (BrdU) immediately after anesthesia. A novel appetitive olfactory learning test was used to assess learning and memory two days after anesthesia. One week after anesthesia, rats were euthanized and the brains analyzed for new cell proliferation in the dentate gyrus, and proliferation and migration of newly formed cells in the subventricular zone to the olfactory bulb. We found that exposure to either isoflurane (p=0.017) or propofol (p=0.006) decreased hippocampal cell proliferation in young, but not in aged rats. This anesthetic-induced decrease was specific to new cell proliferation in the hippocampus, as new cell proliferation and migration to the olfactory bulb was unaffected. Isoflurane anesthesia produced learning impairment in aged rats (p=0.044), but not in young rats. Conversely, propofol anesthesia resulted in learning impairment in young (p=0.01), but not in aged rats. These results indicate that isoflurane and propofol anesthesia affect postnatal hippocampal cell proliferation and learning in an age dependent manner.
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http://dx.doi.org/10.1016/j.brainres.2011.11.025DOI Listing
March 2012

The effect of human umbilical cord blood cells on survival and cytokine production by post-ischemic astrocytes in vitro.

Stem Cell Rev Rep 2010 Dec;6(4):523-31

Center for Excellence in Aging and Brain Repair, University of South Florida, 12901 Bruce B. Downs Blvd, Tampa, FL 33612, USA.

Cerebral ischemia induces death of all neural cell types within the region affected by the loss of blood flow. We have shown that administering human umbilical cord blood cells after a middle cerebral artery occlusion in rats significantly reduces infarct size, presumably by rescuing cells within the penumbra. In this study we examined whether the cord blood cells enhanced astrocyte survival in an in vitro model of hypoxia with reduced glucose availability. Primary astrocyte cultures were incubated for 2 h in no oxygen (95% N, 5% CO(2)) and low glucose (1% compared to 4.5%) media. Cord blood mononuclear cells were added to half the cultures at the beginning of hypoxia. Astrocyte viability was determined using fluorescein diacetate/propidium iodide (FDA/PI) labeling and cytokine production by the astrocytes measured using ELISA. In some studies, T cells, B cells or monocytes/macrophages isolated from the cord blood mononuclear fraction with magnetic antibody cell sorting (MACS) were used instead to determine which cellular component of the cord blood mononuclear fraction was responsible for the observed effects. Co-culturing mononuclear cord blood cells with astrocytes during hypoxia stimulated production of IL-6 and IL-10 during hypoxia. The cord blood T cells decreased survival of the astrocytes after hypoxia but had no effect on the examined cytokines. Our data demonstrate that the tested cord blood fractions do not enhance astrocyte survival when delivered individually, suggesting there is either another cellular component that is neuroprotective or an interaction of all the cells is essential for protection.
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http://dx.doi.org/10.1007/s12015-010-9174-xDOI Listing
December 2010

Human umbilical cord blood cells decrease microglial survival in vitro.

Stem Cells Dev 2010 Feb;19(2):221-8

Center of Excellence for Aging and Brain Repair, University of South Florida, Tampa, Florida 33612, USA.

When human umbilical cord blood (HUCB) cells are systemically administered following middle cerebral artery occlusion (MCAO) in rats, they produce a reduction in infarct size resulting in recovery of motor function. Rats receiving HUCB cells have a less severe inflammatory response compared to MCAO stroke rats. The purpose of this study was to determine the interaction between HUCB cells and the main resident immune cells of the brain (microglia) under normoxic and hypoxic conditions in vitro. Primary microglial cultures were incubated for 2 h in no oxygen (95% N, 5% CO(2)) and low glucose (1%) media. Mononuclear HUCB cells were added to half the cultures at the beginning of the hypoxia conditions. Microglial viability was determined using fluorescein diacetate/propidium iodide (FDA/PI) labeling and cytokine expression using ELISA. In some studies, CD11b+ or CD19+ cells isolated from the HUCB mononuclear fraction with magnetic antibody cell sorting (MACS) were used instead of the mononuclear fraction. Co-culturing mononuclear HUCB cells with microglia decreased viability of the microglia during hypoxia. In the microglial monocultures, hypoxia significantly increased release of IL-1beta compared to normoxia, while adding HUCB cells in the hypoxia condition decreased IL-1beta concentrations to the same level as in the normoxia monocultures. Both CD11b+ and CD19+ HUCB cells decreased microglial viability during normoxia and hypoxia. Our data suggest that HUCB cells may produce a soluble factor that decreases viability of microglia.
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http://dx.doi.org/10.1089/scd.2009.0170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2829348PMC
February 2010

Hyperglycemia not hypoglycemia alters neuronal dendrites and impairs spatial memory.

Pediatr Diabetes 2008 Dec;9(6):531-9

Department of Pediatrics, University of South Florida College of Medicine, Tampa, FL, USA.

Background/objective: We previously reported that chronic hyperglycemia, but not hypoglycemia, was associated with the reduction of neuronal size in the rat brain. We hypothesized that hyperglycemia-induced changes in neuronal structure would have negative consequences, such as impaired learning and memory. We therefore assessed the effects of hyperglycemia and hypoglycemia on neuronal dendritic structure and cognitive functioning in young rats.

Design/methods: Experimental manipulations were conducted on male Wistar rats for 8 wk, beginning at 4 wk of age. At the completion of the treatments, all rats were trained in the radial-arm water maze, a spatial (hippocampus-dependent) learning and memory task. Three groups of rats were tested: an untreated control group, a streptozotocin-induced diabetic (STZ-D) group, and an intermittent hypoglycemic group. Following behavioral training, the brains of all animals were examined with histologic and biochemical measurements.

Results: Peripheral hyperglycemia was associated with significant increases in brain sorbitol (7.5 +/- 1.6 vs. 5.84 +/- 1.0 microM/mg) and inositol (9.6 +/- 1.4 vs. 7.1 +/- 1.1 microM/mg) and reduced taurine (0.65 +/- 0.1 vs. 1.3 +/- 0.1 mg/mg). Histologic evaluation revealed neurons with reduced dendritic branching and spine density in STZ-D rats but not in control or hypoglycemic animals. In addition, the STZ-D group exhibited impaired performance on the water maze memory test.

Conclusions: Hyperglycemia, but not hypoglycemia, was associated with adverse effects on the brain polyol pathway activity, neuronal structural changes, and impaired long-term spatial memory. This finding suggests that the hyperglycemic component of diabetes mellitus has a greater adverse effect on brain functioning than does intermittent hypoglycemia.
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http://dx.doi.org/10.1111/j.1399-5448.2008.00431.xDOI Listing
December 2008

Implications of blood-brain barrier disruption in ALS.

Amyotroph Lateral Scler 2008 Dec;9(6):375-6

Our letter to the editor addresses important questions regarding the role of the blood-spinal cord barrier in amyotrophic lateral sclerosis. The novel finding of barrier dysfunction in ALS has implications for disease pathogenesis. This discussion should prove of widespread interest to researchers and may help in formulating various new therapeutic strategies to protect barrier function and thus extend functionality and lifespan in ALS patients.
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http://dx.doi.org/10.1080/17482960802160990DOI Listing
December 2008

Effects of Sertoli cell-conditioned medium on ventral midbrain neural stem cells: a preliminary report.

Neurotox Res 2008 May-Jun;13(3-4):241-6

Center of Excellence for Aging and Brain Repair, Department of Neurosurgery, University of South Florida College of Medicine, Tampa, FL 33612, USA.

The 796RMB cell line is a multipotent stem cell line isolated from human fetal midbrain tissues, a region from which dopamine neurons of the substantia nigra develop. It would be useful to increase the dopaminergic characteristics of this cell line to enhance its usefulness as a cell therapy for Parkinson's disease utilizing transplantation protocols. Sertoli cells and its conditioned media isolated from the testis have been previously shown to enhance tyrosine hydroxylase expression in ventral mesencephalon neurons both in vitro and in vivo. Therefore, the present preliminary study investigated the ability of Sertoli cell pre-conditioned medium to enhance differentiation of the 796MB cell line toward the domaminergic phenotype. Results showed that secretory products derived from Sertoli cell conditioned medium increased cell proliferation and enhanced dopaminergic neuronal differentiation of the 796RMB cell line. These findings may lead to alternative therapeutic cell transplantation protocols for the treatment of Parkinson's disease.
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http://dx.doi.org/10.1007/BF03033507DOI Listing
November 2008

MIP-1alpha and MCP-1 Induce Migration of Human Umbilical Cord Blood Cells in Models of Stroke.

Curr Neurovasc Res 2008 May;5(2):118-24

Center of Excellence for Aging & Brain Repair, MDC78, University of South Florida, 12901 Bruce B. Downs Blvd, Tampa FL 33612, USA.

Monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein (MIP-1alpha) are implicated in monocyte infiltration into the central nervous system (CNS) under pathological conditions. We previously showed that in vivo human umbilical cord blood cells (HUCB) migrate toward brain injury after middle cerebral artery occlusion (MCAO). We hypothesized that MCP-1 and MIP-1alpha may participate in the recruitment of HUCB towards the injury. Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO), and 24 hours later the production of MCP-1 and MIP-1alpha in the brain was examined with immunohistochemistry, ELISA, and western blotting. The chemotactic effect of MCP-1 and MIP-1alpha, and the expression of MCP-1 receptor CCR2 and MIP-1alpha receptor CCR1, CCR5 on the surface of HUCB were also examined. MCP-1 and MIP-1alpha expression were significantly increased in the ischemic hemisphere of brain, and significantly promoted HUCB cell migration compared to the contralateral side. This cell migration was neutralized with polyclonal antibodies against MCP-1 or MIP-1alpha. Also chemokine receptors were constitutively expressed on the surface of HUCB cells. The data suggested that the increased chemokines in the ischemic area can bind cell surface receptors on HUCB, and induce cell infiltration of systemically delivered HUCB cells into the CNS in vivo.
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http://dx.doi.org/10.2174/156720208784310259DOI Listing
May 2008

Routes of stem cell administration in the adult rodent.

Methods Mol Biol 2008 ;438:383-401

Center for Aging and Brain Repair Cell Biology, University of South Florida College of Medicine, Tampa, FL, USA.

Stem cell transplantation to replace damaged tissue or correct metabolic disease holds the promise of helping a myriad of human afflictions. Although a great deal of attention has focused on pluripotent stem cells derived from embryos, adult stem cells have been described in a variety of tissues, and they likely will prove to be as beneficial as embryonic stem cells in cell replacement therapy and control of inbred errors of metabolism. We describe methods by which stem cells can be introduced into the nervous system, although the techniques are applicable to any portion of the body to be targeted or any cell that may be used for cell therapy. The first and most straight-forward method is introduction of stem cells directly into the brain parenchyma. The second, which in our hands has proven to be superior in some instances, is introduction of the stem cells into the circulatory system.
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http://dx.doi.org/10.1007/978-1-59745-133-8_30DOI Listing
April 2008

Evidence of compromised blood-spinal cord barrier in early and late symptomatic SOD1 mice modeling ALS.

PLoS One 2007 Nov 21;2(11):e1205. Epub 2007 Nov 21.

Center of Excellence for Aging & Brain Repair, College of Medicine, University of South Florida, Tampa, Florida, United States of America.

Background: The blood-brain barrier (BBB), blood-spinal cord barrier (BSCB), and blood-cerebrospinal fluid barrier (BCSFB) control cerebral/spinal cord homeostasis by selective transport of molecules and cells from the systemic compartment. In the spinal cord and brain of both ALS patients and animal models, infiltration of T-cell lymphocytes, monocyte-derived macrophages and dendritic cells, and IgG deposits have been observed that may have a critical role in motor neuron damage. Additionally, increased levels of albumin and IgG have been found in the cerebrospinal fluid in ALS patients. These findings suggest altered barrier permeability in ALS. Recently, we showed disruption of the BBB and BSCB in areas of motor neuron degeneration in the brain and spinal cord in G93A SOD1 mice modeling ALS at both early and late stages of disease using electron microscopy. Examination of capillary ultrastructure revealed endothelial cell degeneration, which, along with astrocyte alteration, compromised the BBB and BSCB. However, the effect of these alterations upon barrier function in ALS is still unclear. The aim of this study was to determine the functional competence of the BSCB in G93A mice at different stages of disease.

Methodology/principal Findings: Evans Blue (EB) dye was intravenously injected into ALS mice at early or late stage disease. Vascular leakage and the condition of basement membranes, endothelial cells, and astrocytes were investigated in cervical and lumbar spinal cords using immunohistochemistry. Results showed EB leakage in spinal cord microvessels from all G93A mice, indicating dysfunction in endothelia and basement membranes and confirming our previous ultrastructural findings on BSCB disruption. Additionally, downregulation of Glut-1 and CD146 expressions in the endothelial cells of the BSCB were found which may relate to vascular leakage.

Conclusions/significance: Results suggest that the BSCB is compromised in areas of motor neuron degeneration in ALS mice at both early and late stages of the disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0001205PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2075163PMC
November 2007

Ultrastructure of blood-brain barrier and blood-spinal cord barrier in SOD1 mice modeling ALS.

Brain Res 2007 Jul 24;1157:126-37. Epub 2007 Apr 24.

Center of Excellence for Aging & Brain Repair, University of South Florida, College of Medicine, Tampa, FL 33612, USA.

The purpose of this study was to determine the ultrastructure of the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) in G93A SOD1 mice modeling ALS at different stages of disease. Electron microscope examination of brainstem, cervical and lumbar spinal cords was performed in ALS mice at early and late stages of disease. Our results show disorganized mitochondrial cristae and degenerating mitochondria in endothelial cells and neuropil, swollen astrocyte foot processes, swollen and degenerating capillary endothelial cells, astrocytes and motor neurons and extensive extracellular edema. In spite of progressive extracellular edema in neural tissue, capillary endothelial cell tight junctions appeared to remain intact in early and late symptomatic animals. Results show that disruption of BBB and BSCB was evident in areas of motor neuron degeneration in G93A mice at both early and late stages of disease. Capillary rupture was observed in brainstem in early symptomatic G93A mice. Capillary ultrastructure revealed that endothelial cell membrane and/or basement membrane damage occurred, followed by vascular leakage.
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http://dx.doi.org/10.1016/j.brainres.2007.04.044DOI Listing
July 2007

Multiple transplants of hNT cells into the spinal cord of SOD1 mouse model of familial amyotrophic lateral sclerosis.

Amyotroph Lateral Scler 2006 Dec;7(4):221-6

Center of Excellence for Aging and Brain Repair, Department of Neurosurgery, University of South Florida, College of Medicine, Health Sciences Center, Tampa, FL 33612, USA.

hNT cells, derived from a human teratocarcinoma cell line, are versatile neuron-like cells that have been studied as possible treatment vehicles for neurodegenerative diseases. Previously, we showed the postponement of motor deficit symptoms in a G93A mouse model of amyotrophic lateral sclerosis (ALS) by transplanting hNT cells into the lumbar spinal cord. In this study, we examined the engraftment of hNT cells at multiple sites within the lumbar spinal cord by morphological analysis of neuritic process development. Results demonstrated that cells implanted at multiple sites established neuritic processes of different lengths independent of the number of cell implants. The hNT fiber outgrowth was a maximum of 0.15-0.3 mm from the transplants and mostly spread within the gray matter; interconnections between implants were not found. Therefore, we suggest that the observed postponement of motor deficit symptoms in G93A mice was not a result of neuritic outgrowth from the implanted hNT cells.
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http://dx.doi.org/10.1080/17482960600864470DOI Listing
December 2006

Novel cell therapy approaches for brain repair.

Prog Brain Res 2006 ;157:207-22

Center of Excellence for Aging and Brain Repair, Department of Neurosurgery, College of Medicine, University of South Florida, MDC 78, 12901 Bruce B. Downs Blvd., Tampa, FL 33612, USA.

Numerous reports elucidate that tissue-specific stem cells are phenotypically plastic and their differentiation pathways are not strictly delineated. Although the identity of all the epigenetic factors which may trigger stem cells to make a lineage selection are still unknown, the plasticity of adult stem cells opens new approaches for their application in the treatment of various disorders. There is increasing researcher interest in hematopoietic stem cells for treatment of not only blood-related diseases but also various unrelated disorders including neurodegenerative diseases. Human umbilical cord blood (hUCB) cells, due to their primitive nature and ability to develop into nonhematopoietic cells of various tissue lineages, including neural cells, may be useful as an alternative cell source for cell-based therapies requiring either the replacement of individual cell types and/or substitution of missing substances. Here we focus on recent findings showing the robustness of adult stem cells derived from hUCB and their potential as a source of transplant cells for the treatment of diseased or injured brains and spinal cords. Depending upon the pathological microenvironment in which the hUCB cells are introduced, neuroprotective and/or trophic effects of these cells, from release of various growth or anti-inflammatory factors to moderation of immune-inflammatory effectors, may be more likely than neural replacement. These protective effects may prove essential to maintaining restored tissue integrity over the course of various diseases or injuries.
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http://dx.doi.org/10.1016/S0079-6123(06)57014-1DOI Listing
January 2007

Cord blood mesenchymal stem cells: Potential use in neurological disorders.

Stem Cells Dev 2006 Aug;15(4):497-506

Department of Neurosurgery, Center of Excellence for Aging and Brain Repair, University of South Florida-College of Medicine, Tampa, FL 33612-4742, USA.

Our previous studies demonstrate enhanced neural protective effects of cord blood (CB) cells in comparison to stem cells from adult marrow. To determine further whether mesenchymal stem cells (MSCs) derived from human umbilical cord blood (hUCB) possess optimal characteristics for neural therapy, we isolated populations of plastic-adherent CB MSCs. These cells generated CD34-, CD45-, CD11b-, CD3-, CD19- cells in culture and failed to produce CFU-M, CFU-GEMM, or CFU-GM hematopoietic colonies in methylcellulose. However, cultured CB MSCs possessed a remarkable ability to support proliferation as well as differentiation of hematopoietic cells in vitro. In addition, supernatants from cultured CB MSCs promoted survival of NT2 N neural cells and peripheral blood mononuclear cells (MNCs) cultured under conditions designed to induce cell stress and limit protein synthesis. After incubation in neural differentiation medium, CB MSCs expressed the neural cell-surface antigen A2B5, the neurofilament polypeptide NF200, the oligodendrocyte precursor marker 04, intermediate filament proteins characteristic of neural differentiation (nestin and vimentin), as well as the astrocyte marker glial fibrillary acidic protein (GFAP) and the neural progenitor marker TUJ-1. We examined the immunomodulatory effects of the CB MSCs after co-culture with murine splenocytes. Whereas spleen cells from normal C57Bl/6 mice exhibited a prominent immunoglobulin M (IgM) response after immunization with the T cell-dependent antigen sheep red blood cells, this response was significantly decreased after incubation with CB MSCs. These data indicate that CB MSCs possess multiple utilities that may contribute to their therapeutic potency in the treatment of neurological disorders.
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http://dx.doi.org/10.1089/scd.2006.15.497DOI Listing
August 2006

Enhancing tyrosine hydroxylase expression and survival of fetal ventral mesencephalon neurons with rat or porcine Sertoli cells in vitro.

Brain Res 2006 Jun 15;1096(1):1-10. Epub 2006 Jun 15.

Department of Neurosurgery, Center of Excellence for Aging and Brain Repair, University of South Florida, Tampa, 33612, USA.

Sertoli cells (SCs) are testis-derived cells that secrete trophic factors important for the development of germ cells. Both porcine and rat SCs have been used as graft facilitators - neonatal porcine SCs to support islets in diabetes and 15-day-old rat SCs to enhance dopaminergic neuron transplants in Parkinson's disease models. However, there has never been a study examining the optimal SCs preparation to enhance tyrosine hydroxylase expression in the ventral mesencephalon (VM) neuron. The aim of this study was to compare the ability of both rat and porcine SCs to enhance tyrosine hydroxylase expression (TH) and neuronal survival at the same postnatal developmental ages. The SCs were isolated from 1-, 9-, or 15-day-old rat, or neonate (2-5 days), 2-month, or 4-month-old pig, and co-cultured with VM tissue from 13.5-day-old embryos. Our results showed that VM neurons co-cultured with SCs dispersed over the culture plate and had extensive neuritic outgrowth, while VM neurons cultured alone tended to cluster together forming a mass of cells with limited neurite outgrowth. TH expression was significantly increased when VM neurons were co-cultured with 15-day rat SCs or 2-month pig SCs but not when the cells were co-cultured with other ages of SCs. This suggests that secretion of trophic factors by SCs varies according to the developmental age, and it is critical for the success of graft facilitation that SCs from the appropriate age and species be used.
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http://dx.doi.org/10.1016/j.brainres.2006.04.058DOI Listing
June 2006

Hyperglycemic brain injury in the rat.

Brain Res 2006 Mar 17;1076(1):9-15. Epub 2006 Feb 17.

Department of Pediatrics, College of Medicine, University of South Florida, Tampa, FL 33612, USA.

Children with diabetes onset before 5 years of age have reduced neurocognitive function. This problem has been attributed to hypoglycemia, a complication of insulin therapy. The eye, kidney, and nerve complications of diabetes (hyperglycemia) have been reduced by intensified insulin therapy which is associated with a 3-fold increase in severe hypoglycemia and therefore is not recommended for children less than 13 years of age. Since hyperglycemia is much more common than intermittent hypoglycemia during early childhood diabetes, it is important to determine if hyperglycemia affects brain growth and development. Rats were exposed to 4 weeks of either continuous hyperglycemia (diabetes) or intermittent (3 h, 3 times/week) hypoglycemia from 4 to 8 weeks of age. The brains of these animals were compared to those of similarly aged normal control animals. The cell number was increased, and the cell size reduced in the cortex of diabetic animals as assessed by DNA/wet weight of brain and protein/DNA content. Reduced amounts of protein, fatty acids, and cholesterol/microgram DNA also indicate smaller cells with reduced myelin content in the cortex of the diabetic animals. Histologic evaluation of these brains confirmed the biochemical findings. These observations require further confirmation and evaluation but indicate that continuous hyperglycemia may be more damaging than intermittent hypoglycemia to the developing brain. This is an important consideration for the management of diabetes mellitus in young children.
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http://dx.doi.org/10.1016/j.brainres.2005.12.072DOI Listing
March 2006

Maternal transplantation of human umbilical cord blood cells provides prenatal therapy in Sanfilippo type B mouse model.

FASEB J 2006 Mar 9;20(3):485-7. Epub 2006 Jan 9.

Center of Excellence for Aging and Brain Repair, Department of Neurosurgery, University of South Florida, College of Medicine, Tampa, FL 33612, USA.

Numerous data support passage of maternal cells into the fetus during pregnancy in both human and animal models. However, functional benefits of maternal microchimerism in utero are unknown. The current study attempted to take advantage of this route for prenatal delivery of alpha-N-acetylglucosaminidase (Naglu) enzyme into the enzyme-deficient mouse model of Sanfilippo syndrome type B (MPS III B). Enzymatically sufficient mononuclear cells from human umbilical cord blood (MNC hUCB) were intravenously administered into heterozygote females modeling MPS III B on the 5th day of pregnancy during blastocyst implantation. The major findings were 1) administered MNC hUCB cells transmigrated and diffused into the embryos (E12.5); 2) some transmigrated cells expressed CD34 and CD117 antigens; 3) transmigrated cells were found in both the maternal and embryonic parts of placentas; 4) transmigrated cells corrected Naglu enzyme activity in all embryos; 5) administered MNC hUCB cells were extensively distributed in the organs and the blood of heterozygote mothers at one week after transplantation. Results indicate that prenatal delivery of Naglu enzyme by MNC hUCB cell administration into mothers of enzyme-deficient embryos is possible and may present a significant opportunity for new biotechnologies to treat many inherited disorders.
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http://dx.doi.org/10.1096/fj.05-4684fjeDOI Listing
March 2006

Umbilical cord blood-derived stem cells and brain repair.

Ann N Y Acad Sci 2005 May;1049:67-83

Center of Excellence for Aging and Brain Repair, Department of Neurosurgery, College of Medicine, University of South Florida, 12901 Bruce B. Downs Blvd., MDC 78, Tampa, FL 33612, USA.

Human umbilical cord blood (HUCB) is now considered a valuable source for stem cell-based therapies. HUCB cells are enriched for stem cells that have the potential to initiate and maintain tissue repair. This potential is especially attractive in neural diseases for which no current cure is available. Furthermore, HUCB cells are easily available and less immunogenic compared to other sources for stem cell therapy such as bone marrow. Accordingly, the number of cord blood transplants has doubled in the last year alone, especially in the pediatric population. The therapeutic potential of HUCB cells may be attributed to inherent ability of stem cell populations to replace damaged tissues. Alternatively, various cell types within the graft may promote neural repair by delivering neural protection and secretion of neurotrophic factors. In this review, we evaluate the preclinical studies in which HUCB was applied for treatment of neurodegenerative diseases and for traumatic and ischemic brain damage. We discuss how transplantation of HUCB cells affects these disorders and we present recent clinical studies with promising outcome.
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http://dx.doi.org/10.1196/annals.1334.008DOI Listing
May 2005

Methylprednisolone inhibits production of interleukin-1beta and interleukin-6 in the spinal cord following compression injury in rats.

J Neurosurg Anesthesiol 2005 Apr;17(2):82-5

Department of Anesthesiology, University of Miami School of Medicine, Miami, Florida 33136, USA.

Interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) are major inflammatory cytokines produced after spinal cord injury (SCI). This study sought to evaluate the effects of methylprednisolone (MP) on IL-1beta and IL-6 protein in spinal cord tissue following SCI. Halothane-anesthetized, female Sprague-Dawley rats weighing (280-320 g) underwent laminectomy at T7-T8. No lesions were produced in animals in the saline control and MP control groups. SCI was induced by temporary placement of an aneurysm clip at T7-T8, with a closing pressure of 55 g at the spinal level of T7-T8, resulting in spinal cord compression for one minute. Animals with SCI were treated with MP (30 mg/kg sc) or an equal volume of saline. IL-1beta and IL-6 spinal cord protein were measured by enzyme-linked immunosorbent assays (ELISA). Data were summarized as mean +/- SD and compared by two-way analysis of variance (ANOVA). IL-1beta and IL-6 levels were elevated in the SCI + Saline animals (P < 0.01) compared with saline control, MP control, and SCI + MP-treated animals. The rise in IL-1beta and IL-6 levels after SCI was blunted after administration of MP, suggesting an interaction between glucocorticosteroids and the cytokine cascade after spinal cord trauma. Further evaluation of the effects of MP on the cytokine cascade may be important in assessing whether or not the anti-inflammatory effects of glucocorticosteroids confer neuroprotection after SCI.
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http://dx.doi.org/10.1097/01.ana.0000163199.10365.38DOI Listing
April 2005

Formation and structure of transplantable tissue constructs generated in simulated microgravity from Sertoli cells and neuron precursors.

Cell Transplant 2004 ;13(7-8):755-63

Department of Anatomy, University of South Florida College of Medicine, Tampa, FL 33612, USA.

Cell transplantation therapy for Parkinson's disease (PD) has received much attention as a potential treatment protocol for this neurodegenerative condition. Although there have been promising successes with this approach, it remains problematic, especially regarding the inability to provide immediate trophic support to the newly grafted cells and the inability to prevent acute and/or long-term graft rejection by the host. To address these issues of cell graftability, we have created a novel tissue construct from isolated rat Sertoli cells (SC) and the NTerra-2 immortalized human neuron precursor cell line (NT2) utilizing NASA-developed simulated microgravity technology. The two cell types were cocultured at a 1:4 (SC/NT2) ratio in the High Aspect Rotating Vessel (HARV) biochamber for 3 days, after which a disc-shaped aggregate (1-4 mm diameter) was formed. Sertoli neuron aggregated cells (SNAC) were collected by gravity sedimentation and processed either for light and electron microscopy or for fluorescent immunocytochemistry. Intra-SNAC clusters of SC and NT2 cells were identified by anti-human mitochondrial protein (huMT--specific for NT2 cells) and cholera toxin subunit B (CTb--specific for SC). There was little evidence of cell death throughout the aggregate and the absence of central necrosis, as might be expected in such a large aggregate in vitro. Ultrastructurally, SC did not express junctional modifications with NT2 cells nor with adjacent SC as is typical of SC in vivo and, in some protocols, in vitro. NT2 cells, however, showed distinct intercellular junction-like densities with adjacent NT2 cells, often defining canaliculi-like channels between the microvillus borders of the cells. The results show that the use of simulated microgravity coculture provides a culture environment suitable for the formation of a unique and viable Sertoli-NT2 (i.e., SNAC) tissue construct displaying intra-aggregate cellular organization. The structural integration of SC with NT2 cells provides a novel transplantable tissue source, which can be tested to determine if SC will suppress rejection of the grafted NT2 cells and provide for their short- and long-term trophic support in situ in the treatment of experimental PD.
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http://dx.doi.org/10.3727/000000004783983431DOI Listing
May 2005

Rapid differentiation of NT2 cells in Sertoli-NT2 cell tissue constructs grown in the rotating wall bioreactor.

Brain Res Bull 2004 Dec;64(4):347-56

Department of Anatomy, College of Medicine, Health Sciences Center, University of South Florida, Tampa, FL 33612, USA.

Cell replacement therapy is of great interest as a long-term treatment of neurodegenerative diseases such as Parkinson's disease (PD). We have previously shown that Sertoli cells (SC) provide neurotrophic support to transplants of dopaminergic fetal neurons and NT2N neurons, derived from the human clonal precursors cell line NTera2/D1 (NT2), which differentiate into dopaminergic NT2N neurons when exposed to retinoic acid. We have created SC-NT2 cell tissue constructs cultured in the high aspect ratio vessel (HARV) rotating wall bioreactor. Sertoli cells, NT2, and SC plus NT2 cells combined in starting ratios of 1:1, 1:2, 1:4 and 1:8 were cultured in the HARV in DMEM with 10% fetal bovine serum and 1% growth factor reduced Matrigel for 3 days, without retinoic acid. Conventional, non-HARV, cultures grown in the same culture medium were used as controls. The presence of tyrosine hydroxylase (TH) was assessed in all culture conditions. Sertoli-neuron-aggregated-cell (SNAC) tissue constructs grown at starting ratios of 1:1 to 1:4 contained a significant amount of TH after 3 days of culture in the HARV. No TH was detected in SC HARV cultures, or SC, NT2 or SC-NT2 conventional co-cultures. Quantitative stereology of immunolabled 1:4 SNAC revealed that approximately 9% of NT2 cells differentiate into TH-positive (TH+) NT2N neurons after 3 days of culture in the HARV, without retinoic acid. SNAC tissue constructs also released dopamine (DA) when stimulated with KCl, suggesting that TH-positive NT2N neurons in the SNAC adopted a functional dopaminergic phenotype. SNAC tissue constructs may be an important source of dopaminergic neurons for neuronal transplantation.
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http://dx.doi.org/10.1016/j.brainresbull.2004.09.003DOI Listing
December 2004

Effects of Sertoli cell transplants in a 3-nitropropionic acid model of early Huntington's disease: a preliminary study.

Neurotox Res 2003 ;5(6):443-50

Center of Excellence for Aging and Brain Repair, Department of Neurosurgery, The Neuroscience Program, College of Medicine, University of South Florida, Tampa, FL 33612, USA.

Problems with immunosuppression and graft survival limit clinical applications of neurotransplantation protocols for neurodegenerative disease. Sertoli cells, testes-derived cells with immunosuppressive and trophic properties, may serve as an alternative cell source for transplantation. Sertoli cells were transplanted into the striatum of rats following two injections of 3-nitropropionic acid (3-NP) to determine whether they could ameliorate abnormalities in a model of early stage Huntington's disease. 3-NP-induced locomotor hyperactivity was significantly reduced in rats receiving Sertoli transplants compared to controls, with some behaviors returning to baseline. Sertoli cells survived in the striatum without systemic immunosuppression and some formed tubule-like structures. These results show that Sertoli transplants are able to ameliorate locomotor abnormalities in a 3-NP model of early HD. Thus, Sertoli cells should be further evaluated as a possible treatment strategy for the early stages of Huntington's disease.
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http://dx.doi.org/10.1007/BF03033174DOI Listing
February 2004

Human umbilical cord blood stem cells infusion in spinal cord injury: engraftment and beneficial influence on behavior.

J Hematother Stem Cell Res 2003 Jun;12(3):271-8

Center for Aging and Brain Repair and Department of Anatomy, University of South Florida College of Medicine, Tampa, FL 33612, USA.

The use of human umbilical cord blood (hUCB)--a rich source of nonembryonic or adult stem cells--has recently been reported to ameliorate behavioral consequences of stroke. In this study, we tested whether human cord blood leukocytes also ameliorate behavioral impairments of spinal cord injury. Rats were divided into five groups: (1) laminectomy (without spinal cord injury) only; (2) laminectomy + cord blood infusion; (3) spinal cord injury + cord blood infused 1 day post injury; (4) spinal cord injury + cord blood infused 5 days post injury; and (5) spinal cord injury only. Spinal cord injury was induced by compressing the spinal cord for 1 min with an aneurysm clip calibrated to a closing pressure of 55 g. Open-field behavior was assessed 1, 2, and 3 weeks after intravenous injection of prelabeled human cord blood cells. Open-field test scores of spinal cord injured rats treated with human cord blood at 5 days were significantly improved as compared to scores of rats similarly injured but treated at day 1 as well as the otherwise untreated injured group. The results suggest that cord blood stem cells are beneficial in reversing the behavioral effects of spinal cord injury, even when infused 5 days after injury. Human cord blood-derived cells were observed in injured areas, but not in noninjured areas, of rat spinal cords, and were never seen in corresponding areas of spinal cord of noninjured animals. The results are consistent with the hypothesis that cord blood-derived stem cells migrate to and participate in the healing of neurological defects caused by traumatic assault.
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http://dx.doi.org/10.1089/152581603322023007DOI Listing
June 2003

Intravenous administration of human umbilical cord blood cells in a mouse model of amyotrophic lateral sclerosis: distribution, migration, and differentiation.

J Hematother Stem Cell Res 2003 Jun;12(3):255-70

Center of Excellence for Aging and Brain Repair and Department of Neurosurgery, University of South Florida, College of Medicine, Tampa, FL 33612, USA.

Amyotrophic lateral sclerosis (ALS), a multifactorial disease characterized by diffuse motor neuron degeneration, has proven to be a difficult target for stem cell therapy. The primary aim of this study was to determine the long-term effects of intravenous mononuclear human umbilical cord blood cells on disease progression in a well-defined mouse model of ALS. In addition, we rigorously examined the distribution of transplanted cells inside and outside the central nervous system (CNS), migration of transplanted cells to degenerating areas in the brain and spinal cord, and their immunophenotype. Human umbilical cord blood (hUCB) cells (10(6)) were delivered intravenously into presymptomatic G93A mice. The major findings in our study were that cord blood transfusion into the systemic circulation of G93A mice delayed disease progression at least 2-3 weeks and increased lifespan of diseased mice. In addition, transplanted cells survived 10-12 weeks after infusion while they entered regions of motor neuron degeneration in the brain and spinal cord. There, the cells migrated into the parenchyma of the brain and spinal cord and expressed neural markers [Nestin, III Beta-Tubulin (TuJ1), and glial fibrillary acidic protein (GFAP)]. Infused cord blood cells were also widely distributed in peripheral organs, mainly the spleen. Transplanted cells also were recovered in the peripheral circulation, possibly providing an additional cell supply. Our results indicate that cord blood may have therapeutic potential in this noninvasive cell-based treatment of ALS by providing cell replacement and protection of motor neurons. Replacement of damaged neurons by progeny of cord blood stem cells is probably not the only mechanism by which hUCB exert their effect, since low numbers of cells expressed neural antigens. Most likely, cord blood efficacy is partially due to neuroprotection by modulation of the autoimmune process.
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http://dx.doi.org/10.1089/152581603322022990DOI Listing
June 2003

Preliminary study of the behavioral effects of LBS-neuron implantation on seizure susceptibility following middle cerebral artery occlusion in the rats.

Neurotox Res 2002 Mar;4(2):111-8

Department of Neurosurgery, Center for Aging and Brain Repair, University of South Florida, MDC 78, 12901 Bruce B. Downs Blvd., Tampa, FL 33612, USA.

Neural transplantation is a promising treatment strategy that can restore the motor, sensory and cognitive functions in the rat middle cerebral artery occlusion (MCAO) model of stroke. In particular, neuronal cells derived from a human teratocarcinoma cell line, called hNT neurons or LBS neurons (clinical grade preparation), are effective in improving behavioral recovery after stroke. In the elderly, epilepsy is a common sequela of stroke, especially if the infarction involves cerebral cortex. However, the effect of implanting neural cells on seizure susceptibility in the MCAO model has not yet been determined. The purpose of this study was to determine the susceptibility to pentylenetetrazol (PTZ)-induced seizures in normal, MCAO-lesioned and MCAO-lesioned rats in which the LBS neurons were injected. Adult, male Sprague-Dawley rats were subjected to 60 min of MCAO using the intraluminal filament technique followed 3-4 weeks later by transplantation of 80,000 LBS-neurons into the ipsilateral cortex. Susceptibility to PTZ-induced seizures was tested 4-6 weeks post-transplant at doses of 35, 50 and 70 mg/kg, administered subcutaneously. Latency to the first lethal response, latency to first generalized seizure, duration of the first generalized seizure, and the number of generalized seizures in an hour post-PTZ treatment observation period was determined. Even thought there was a tendency for groups that underwent MCAO to be more susceptible to seizures, there were no statistically significant differences between the groups and no differences between MCAO alone and MCAO animals in which cells had been implanted. While grafted cells were identified in all but one injected animal, the results suggest that the grafts may not have been healthy either from immunological rejection or PTZ-induced injury. These results suggest that while placing cells within the cortex does not reduce seizure susceptibility, it also does not increase the incidence of seizures. Further investigations are warranted.
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http://dx.doi.org/10.1080/10298420290015908DOI Listing
March 2002

Neurobehavioral assessment of transplanted porcine Sertoli cells into the intact rat striatum.

Neurotox Res 2002 Mar;4(2):103-9

Department of Neurosurgery and The Neurosciences Program, Center for Aging and Brain Repair, College of Medicine, University of South Florida, Tampa, FL 33612, USA.

Sertoli cells, a testes-derived cell with immunosuppressive and trophic properties, may serve as an alternative cell source for transplantation in a number of neurodegenerative diseases. However, before Sertoli cells can be considered for clinical use, safety studies must be conducted to ensure that the cells themselves produce no adverse effects when transplanted into the central nervous system. The present study assessed the behavioral effects of transplanting porcine Sertoli cells into the striatum of normal rats and provided a histological examination of the graft site and host striatum. Activity monitors revealed significant increases in nocturnal locomotor activity over time following both sham and Sertoli transplants. Ambulation and rearing, but not stereotypic measures, were increased compared to pre-transplant levels. Sertoli animals exhibited less behavioral alteration than sham controls. Histological examination of the striatum demonstrated surviving Sertoli cell transplants in an intact striatum. These results indicated that Sertoli cell xenografts might be a safe alternative cell source for neurotransplantation procedures requiring immune or trophic support.
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http://dx.doi.org/10.1080/10298420290015935DOI Listing
March 2002

Lithium exposure enhances survival of NT2N cells (hNT neurons) in the hemiparkinsonian rat.

Eur J Neurosci 2002 Dec;16(12):2271-8

Department of Neurosurgery, Center for Aging and Brain Repair, University of South Florida College of Medicine, MDC 78, 12901 Bruce B. Downs Blvd, Tampa 33612, USA.

Lithium (Li +) treatment of NTera2/D1 (or hNT Neurons) in culture increases tyrosine hydroxylase (TH) expression in this cell-line [Zigova et al., (1999) Exp. Neurol., 157, 251-258]. It is not known if these Li + treated cells maintain TH expression once transplanted into the striatum of the hemiparkinsonian rats. hNT neurons were either treated with 1 mm LiCl or left untreated and then transplanted into the striatum of Sprague-Dawley rats. Some cells were exposed to the lithium for 24 h in culture while others were exposed only briefly (2-3 h) just prior to transplantation. We also examined whether Li + treatment of the animal after transplantation (0.24% w/w lithium carbonate in chow) was effective in increasing neuronal survival. One week after transplantation, the animals were perfused with 4% paraformaldehyde and immunocytochemistry was performed on 30 micro m sections through the transplant. Human nuclear matrix antigen immunostaining demonstrated that there was significantly better survival of cells in the group treated briefly with lithium compared to all other groups. Brief exposure to lithium resulted in a greater expression of TH in situ as well. Neuron specific enolase immunohistochemistry showed that there was extensive fibre outgrowth in all groups. These results suggest that brief Li + exposure may enhance survival to over 60% and increase TH expression of hNT Neurons transplanted in the hemiparkinsonian rat nearly three-fold.
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http://dx.doi.org/10.1046/j.1460-9568.2002.02300.xDOI Listing
December 2002

Functional recovery after complete contusion injury to the spinal cord and transplantation of human neuroteratocarcinoma neurons in rats.

J Neurosurg 2002 Jul;97(1 Suppl):63-8

Center for Aging and Brain Repair, Department of Anatomy, College of Medicine, University of South Florida, Tampa 33612, USA.

Object: Human neuroteratocarcinoma (hNT)-derived neurons are differentiated postmitotic neurons derived from a human teratocarcinoma cell line following treatment with retinoic acid. In preclinical transplantation studies investigators have demonstrated both their safety as a source of neurons for transplantation and efficacy in treating stroke-related behavioral deficits. The objective of this study was to examine whether hNT neurons transplanted in an area of complete spinal cord contusion would improve electrophysiological measures of spinal cord function.

Methods: Complete spinal cord contusion injury, defined as the complete loss of motor evoked potentials (MEPs), was produced in 30 rats at T-8. Ten rats with contused spinal cords underwent transplantation with hNT neurons within the site of contusion immediately after injury (immediate transplant group). Ten rats underwent hNT neuron transplantation following a 2-week evaluation for loss of MEPs (delayed transplant group). Ten other rats with contusion injury served as a spinal cord injury control group, and 10 rats underwent only a T-8 laminectomy and served as non-injured controls. All rats survived 8 weeks after transplantation. In the delayed transplant group significant functional recovery was observed, as demonstrated by return of MEPs and a modest improvement of motor function. Immunohistochemical analysis showed the survival, integration, and long fiber outgrowth of the grafted hNT neurons.

Conclusions: These findings suggest that the transplantation of the hNT neurons may be an effective means of reestablishing electrical connectivity of the injured spinal cord.
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http://dx.doi.org/10.3171/spi.2002.97.1.0063DOI Listing
July 2002

Human umbilical cord blood cells express neural antigens after transplantation into the developing rat brain.

Cell Transplant 2002 ;11(3):265-74

Center for Aging and Brain Repair, Department of Neurosurgery, University of South Florida College of Medicine, Tampa 33612, USA.

Recently, our laboratory began to characterize the mononuclear cells from human umbilical cord blood (HUCB) both in vitro and in vivo. These cryopreserved human cells are available in unlimited quantities and it is believed that they may represent a source of cells with possible therapeutic and practical value. Our previous molecular and immunocytochemical studies on cultured HUCB cells revealed their ability to respond to nerve growth factor (NGF) by increased expression of neural markers typical for nervous system-derived stem cells. In addition, the DNA microarray detected downregulation of several genes associated with development of blood cell lines. To further explore the survival and phenotypic properties of HUCB cells we transplanted them into the developing rat brain, which is known to provide a conducive environment for development of neural phenotypes. Prior to transplantation, HUCB cells were either cultured with DMEM and fetal bovine serum or were exposed to retinoic acid (RA) and nerve growth factor (NGF). Neonatal pups (1 day old) received unilateral injection of cell suspension into the anterior part of subventricular zone. One month after transplantation animals were perfused, their brains cryosectioned, and immunocytochemistry was performed for identification of neural phenotypes. Our results clearly demonstrated that approximately 20% of transplanted HUCB survived (without immunosuppression) within the neonatal brain. Additionally, double-labeling with cell-type-specific markers revealed that some HUCB-derived cells (recognized by anti-human nuclei labeling) were immunopositive for glial fibrillary acidic protein (GFAP) and few donor cells expressed the neuronal marker TuJ1 (class III beta-tubulin). These findings suggest that at least some of the transplanted HUCB cells differentiated into cells with distinct glial or neuronal phenotypes after being exposed to instructive signals from the developing brain.
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February 2003