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**Leo** · 14 Feb 2005, 9:25 AM

Minnesota, eh

Transplanted Stem Cells Restore Function In Stroke (March 5, 2002) -- Researchers at the University of Minnesota department of neurosurgery and Stem Cell Institute (SCI) have demonstrated the ability of transplanted adult stem cells to restore function in laboratory ... > full story

Stroke Rats right?

"All you have to decide is what to do with the time that is given you."
Gandolf the Gray

**Wise Young** · 14 Feb 2005, 7:33 PM

Leo,

At the Henan's People Provincial Hospital in Zhengzhou, they havve transplanted autologous bone marrow in over 180 patients. Some had stroke but many had spinal cord injury. I get the impression that it is not a cure. Ira Black has been transplanting bone marrow stem cells into the spinal cords of rats with spinal cord injury for several years. A number of investigators have transplanted bone marrow stem cells into injured rat spinal cords. I attach some of the abstracts below.

Sasaki, et al. (2001) and Akiyama, et al. (2002) from the Kocsis lab at Yale showed that transplanted bone marrow stromal cells will remyelinate demyelinated spinal cords. Corti, et al. (2003) showed that green-fluorescent protein-expressing bone marrow cells transplanted to the spinal cord will show morphology and markers suggestive of a large range of cells. Mazzini, et al. (2003) transplanted bone marrow cells into the spinal cords of patients with ALS, reporting that it was safe but did not report improvements of function. Koshizuka, et al. (2004) transplanted bone marrow cells into mice with spinal cord injury and found cells showing markers of many cell types, including astrocytic and neuronal markers. In 2004, Corti, et al. reported that bone marrow cells transplanted into the spinal cords of SOD-1 mice (a mouse with the human gene for amyotrophic lateral sclerosis) will prolong life and improve function. Ankeny, et al., 2004 showed that bone marrow cells transplanted into contused spinal cords improved "air-walking" but not overground locomotion in rats. Bakshi, et al. (2004) reported that bone marrow and other stem cells "home" into injured spinal cords when given intravenously and intrathecally. Lu, et al. (2005) used BDNF-expressing bone marow stem cells and showed that it promoted regeneration but did not improve function.

The results are mixed. While a number of investigators have reported that bone marrow stem cells can produce a variety of cells expressing markers of neurons, astrocytes, microglia, and other cells but few have shown improved function in spinal cord injury, demyelination, and ALS models.

Wise.

Sasaki M, Honmou O, Akiyama Y, Uede T, Hashi K and Kocsis JD (2001). Transplantation of an acutely isolated bone marrow fraction repairs demyelinated adult rat spinal cord axons. Glia 35: 26-34. The potential of bone marrow cells to differentiate into myelin-forming cells and to repair the demyelinated rat spinal cord in vivo was studied using cell transplantation techniques. The dorsal funiculus of the spinal cord was demyelinated by x-irradiation treatment, followed by microinjection of ethidium bromide. Suspensions of a bone marrow cell fraction acutely isolated from femoral bones in LacZ transgenic mice were prepared by centrifugation on a density gradient (Ficoll-Paque) to remove erythrocytes, platelets, and debris. The isolated cell fraction contained hematopoietic and nonhematopoietic stem and precursor cells and lymphocytes. The cells were transplanted into the demyelinated dorsal column lesions of immunosuppressed rats. An intense blue beta-galactosidase reaction was observed in the transplantation zone. The genetically labeled bone marrow cells remyelinated the spinal cord with predominately a peripheral pattern of myelination reminiscent of Schwann cell myelination. Transplantation of CD34(+) hematopoietic stem cells survived in the lesion, but did not form myelin. These results indicate that bone marrow cells can differentiate in vivo into myelin-forming cells and repair demyelinated CNS. Department of Neurosurgery, Sapporo Medical University School of Medicine, Sapporo, Hokkaido, Japan.

Akiyama Y, Radtke C and Kocsis JD (2002). Remyelination of the rat spinal cord by transplantation of identified bone marrow stromal cells. J Neurosci 22: 6623-30. Bone marrow contains a population of stem-like cells that can differentiate into neurons or glia. Stromal cells from green fluorescent protein (GFP)-expressing mice were isolated by initial separation on a density gradient and then cultured as adherent cells on plastic that proliferated in culture to confluency with a typical flattened elongative morphology. The large majority of the isolated stromal cells were GFP expressing and immunopositive for collagen type I, fibronectin, and CD44. Transplantation of these cells by direct microinjection into the demyelinated spinal cord of the immunosuppressed rat resulted in remyelination. The remyelinated axons showed characteristics of both central and peripheral myelination as observed by electron microscopy; conduction velocity of the axons was improved. GFP-positive cells and myelin profiles were observed in the remyelinated spinal cord region, indicating that the donor-isolated stromal cells were responsible for the formation of the new myelin. The GFP-positive cells were colocalized with myelin basic protein-positive and P0-positive cellular elements. These findings indicate that cells contained within the stromal cell fraction of the mononuclear cell layer of bone marrow can form functional myelin during transplantation into demyelinated spinal cord. Department of Neurology, Yale University School of Medicine, New Haven, Connecticut 06516, USA.

Corti S, Locatelli F, Donadoni C, Strazzer S, Salani S, Del Bo R, Caccialanza M, Bresolin N, Scarlato G and Comi GP (2002). Neuroectodermal and microglial differentiation of bone marrow cells in the mouse spinal cord and sensory ganglia. J Neurosci Res 70: 721-33. There is now evidence that bone marrow (BM) can generate cells expressing neuronal antigens in adult mouse brain. In the present study, we examined the spinal cord and dorsal root ganglia (DRG) of adult mice 3 months after BM cell transplantation from transgenic donor mice expressing the enhanced green fluorescent protein (GFP). To determine whether GFP(+) cells acquire neuroectodermal phenotypes, we tested, by immunocytochemistry followed by confocal analysis, the coexpression of the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal markers NeuN, neurofilament (NF), and class III beta-tubulin (TuJ1). Rare GFP(+) cells coexpressing TuJ1, NF, and NeuN were found both in spinal cord and in sensory ganglia. These cells have small dimensions and short cytoplasmic processes, probably reflecting an immature phenotype. Double GFP and GFAP positivity was found only in spinal cord. To determine whether cell fusion with endogenous cells occurred, we investigated the nuclear content of cells coexpressing GFP and neuronal or astrocytic markers, demonstrating that these cells have only one nucleus and a DNA ploidy that it is not different from that of surrounding neurons and astrocytes. Large numbers of GFP(+) cells are also positively stained for F4/80, a microglial-recognizing antibody, and present a characteristic microglial-like morphology both in spinal cord and, with a higher frequency, in sensory ganglia. These data support a potential role for BM-derived stem cells in spinal cord neuroneogenesis. They also confirm that the microglial compartment within the CNS and in DRG undergoes a relatively fast turnover, with the contribution of hematopoietic stem cells. Both these findings might prove useful for the development of treatments for spinal cord neurodegenerative and acquired disorders. Centro Dino Ferrari, Dipartimento di Scienze Neurologiche, Universita degli Studi di Milano, IRCCS Ospedale Maggiore Policlinico, Milano, Italy. stcorti@yahoo.it

Corti S, Locatelli F, Strazzer S, Guglieri M and Comi GP (2003). Neuronal generation from somatic stem cells: current knowledge and perspectives on the treatment of acquired and degenerative central nervous system disorders. Curr Gene Ther 3: 247-72. Stem cell transplantation through cell replacement or as vector for gene delivery is a potential strategy for the treatment of neurodegenerative diseases. Several studies have reported the transdifferentiation of different somatic stem cells into neurons in vitro or after transplantation into animal models. This observation has pointed out the perspective of using an ethical and accessible cell source to "replace" damaged neurons or provide support to brain tissue. However, recent findings such as the cell fusion phenomenon have raised some doubts about the real existence of somatic stem cell plasticity. In this review, we will discuss current evidence and controversial issues about the neuroneogenesis from various sources of somatic cells focusing on the techniques of isolation, expansion in vitro as well as the inductive factors that lead to transdifferentiation in order to identify the factors peculiar to this process. The morphological, immunochemical, and physiological criteria to correctly judge whether the neuronal transdifferentation occurred are critically presented. We will also discuss the transplantation experiments that were done in view of a possible clinical therapeutic application. Animal models of stroke, spinal cord and brain trauma have improved with Mesenchymal Stem Cells or Bone Marrow transplantation. This improvement does not seem to depend on the replacement of the lost neurons but may be due to increased expression levels of neurotrophic factors, thus suggesting a beneficial effect of somatic cells regardless of transdifferentiation. Critical understanding of available data on the mechanisms governing the cell fate reprogramming is a necessary achievement toward an effective cell therapy. Centro Dino Ferrari, Dipartimento di Scienze Neurologiche, Universita degli Studi di Milano, I.R.C.C.S. Ospedale Maggiore Policlinico, Centro di Eccellenza per lo Studio delle Malattie Neurodegenerative, Milano, Italy. stcorti@yahoo.it

Mazzini L, Fagioli F, Boccaletti R, Mareschi K, Oliveri G, Olivieri C, Pastore I, Marasso R and Madon E (2003). Stem cell therapy in amyotrophic lateral sclerosis: a methodological approach in humans. Amyotroph Lateral Scler Other Motor Neuron Disord 4: 158-61. INTRODUCTION: Recently it has been shown in animal models of amyotrophic lateral sclerosis (ALS) that stem cells significantly slow the progression of the disease and prolong survival. We have evaluated the feasibility and safety of a method of intraspinal cord implantation of autologous mesenchymal stem cells (MSCs) in a few well-monitored patients with ALS. METHOD: Bone marrow collection was performed according to the standard procedure by aspiration from the posterior iliac crest. Ex vivo expansion of mesenchymal stem cells was induced according to Pittenger's protocol. The cells were suspended in 2 ml of autologous cerebrospinal fluid and transplanted into the spinal cord by a micrometric pump injector. RESULTS: No patient manifested major adverse events such as respiratory failure or death. Minor adverse events were intercostal pain irradiation (4 patients) which was reversible after a mean period of three days after surgery, and leg sensory dysesthesia (5 patients) which was reversible after a mean period of six weeks after surgery. No modification of the spinal cord volume or other signs of abnormal cell proliferation were observed. CONCLUSIONS: Our results appear to demonstrate that the procedures of ex vivo expansion of autologous mesenchymal stem cells and of transplantation into the spinal cord of humans are safe and well tolerated by ALS patients. Department of Neurology, University of Torino, Italy. mazzini.l@libero.it

Koshizuka S, Okada S, Okawa A, Koda M, Murasawa M, Hashimoto M, Kamada T, Yoshinaga K, Murakami M, Moriya H and Yamazaki M (2004). Transplanted hematopoietic stem cells from bone marrow differentiate into neural lineage cells and promote functional recovery after spinal cord injury in mice. J Neuropathol Exp Neurol 63: 64-72. Recovery in central nervous system disorders is hindered by the limited ability of the vertebrate central nervous system to regenerate lost cells, replace damaged myelin, and re-establish functional neural connections. Cell transplantation to repair central nervous system disorders is an active area of research, with the goal of reducing functional deficits. Recent animal studies showed that cells of the hematopoietic stem cell (HSC) fraction of bone marrow transdifferentiated into various nonhematopoietic cell lineages. We employed a mouse model of spinal cord injury and directly transplanted HSCs into the spinal cord 1 week after injury. We evaluated functional recovery using the hindlimb motor function score weekly for 5 weeks after transplantation. The data demonstrated a significant improvement in the functional outcome of mice transplanted with hematopoietic stem cells compared with control mice in which only medium was injected. Fluorescent in situ hybridization for the Y chromosome and double immunohistochemistry showed that transplanted cells survived 5 weeks after transplantation and expressed specific markers for astrocytes, oligodendrocytes, and neural precursors, but not for neurons. These results suggest that transplantation of HSCs from bone marrow is an effective strategy for the treatment of spinal cord injury. Department of Orthopaedic Surgery, Chiba University, Graduate School of Medicine, Chiba, Japan. kossy-z@gb3.so-net.ne.jp

Corti S, Locatelli F, Donadoni C, Guglieri M, Papadimitriou D, Strazzer S, Del Bo R and Comi GP (2004). Wild-type bone marrow cells ameliorate the phenotype of SOD1-G93A ALS mice and contribute to CNS, heart and skeletal muscle tissues. Brain 127: 2518-32. Amyotrophic lateral sclerosis (ALS) is a progressive, lethal neurodegenerative disease without any effective therapy. To evaluate the potential of wild-type bone marrow (BM)-derived stem cells to modify the ALS phenotype, we generated BM chimeric Cu/Zn superoxide dismutase (SOD1) mice by transplantation of BM cells derived from mice expressing green fluorescent protein (GFP) in all tissues and from Thy1-YFP mice that express a spectral variant of GFP (yellow fluorescent protein) in neurons only. In the recipient cerebral cortex, we observed rare GFP+ and YFP+ neurons, which were probably generated by cell fusion, as demonstrated by fluorescence in situ hybridization (FISH) analysis, suggesting that this phenomenon is not limited to Purkinje cells. GFP-positive microglial cells were extensively present in both the brain and spinal cord of the affected animals. Completely differentiated and immature GFP+ myofibres were also present in the heart and skeletal muscles of SOD1 mice, confirming that BM cells can participate in striated muscle tissue regeneration. Moreover, wild-type BM chimeric SOD1 mice showed a significantly delayed disease onset and an increased life span, probably due to a positive 'non-neuronal environmental' effect rather than to neuronogenesis. This improvement in SOD1-G93A mouse survival is comparable with that previously obtained using some safer pharmacological agents. BM transplantation-related complications in humans preclude its clinical application for ALS treatment. However, our data suggest that further studies aimed at improving the degree of tissue chimerism by BM-derived cells may provide valuable insights into strategies to slow ALS progression. Centro Dino Ferrari, Dipartimento di Scienze Neurologiche, Universita degli Studi di Milano, IRCCS Ospedale Maggiore Policlinico, Milano, Italy.

Ankeny DP, McTigue DM and Jakeman LB (2004). Bone marrow transplants provide tissue protection and directional guidance for axons after contusive spinal cord injury in rats. Exp Neurol 190: 17-31. Contusive spinal cord injury (SCI) produces large fluid-, debris- and inflammatory cell-filled cystic cavities that lack structure to support significant axonal regeneration. The recent discovery of stem cells capable of generating central nervous system (CNS) tissues, coupled with success in neurotransplantation strategies, has renewed hope that repair and recovery from CNS trauma is possible. Based on results from several studies using bone marrow stromal cells (MSCs) to promote CNS repair, we transplanted MSCs into the rat SCI lesion cavity to further investigate their effects on functional recovery, lesion morphology, and axonal growth. We found that transplanted MSCs induced hindlimb airstepping--a spontaneous locomotor movement associated with activation of the stepping control circuitry--but did not alter the time course or extent of overground locomotor recovery. Using stereological techniques to describe spinal cord anatomy, we show that MSC transplants occupied the lesion cavity and were associated with preservation of host tissue and white matter (myelin), demonstrating that these cells exert neuroprotective effects. The tissue matrix formed by MSC grafts supported greater axonal growth than that found in specimens without grafts. Moreover, uniform random sampling of axon profiles revealed that the majority of neurites in MSC grafts were oriented with their long axis parallel to that of the spinal cord, suggesting longitudinally directed growth. Together, these studies support further investigation of marrow stromal cells as a potential SCI repair strategy. Department of Physiology and Cell Biology, The Ohio State University, 333 West 10th Avenue, Columbus, OH 43210, USA. Ankeny.3@osu.edu

Bakshi A, Hunter C, Swanger S, Lepore A and Fischer I (2004). Minimally invasive delivery of stem cells for spinal cord injury: advantages of the lumbar puncture technique. J Neurosurg Spine 1: 330-7. OBJECT: Stem cell therapy has been shown to have considerable therapeutic potential for spinal cord injuries (SCIs); however, most experiments in animals have been performed by injecting cells directly into the injured parenchyma. This invasive technique compromises the injured spinal cord, although it delivers cells into the hostile environment of the acutely injured cord. In this study, the authors tested the possibility of delivering stem cells to injured spinal cord by using three different minimally invasive techniques. METHODS: Bone marrow stromal cells (BMSCs) are clinically attractive because they have shown therapeutic potential in SCI and can be obtained in patients at the bedside, raising the possibility of autologous transplantation. In this study transgenically labeled cells were used for transplantation, facilitating posttransplantation tracking. Inbred Fisher-344 rats received partial cervical hemisection injury, and 2 x 10(6) BMSCs were intravenously, intraventricularly, or intrathecally transplanted 24 hours later via lumbar puncture (LP). The animals were killed 3, 10, or 14 days posttransplantation, and tissue samples were submitted to histochemical and immunofluorescence analyses. For additional comparison and validation, lineage restricted neural precursor (LRNP) cells obtained from E13.5 rat embryos were transplanted via LP, and these findings were also analyzed. CONCLUSIONS: Both BMSCs and LRNP cells home toward injured spinal cord tissues. The use of LP and intraventricular routes allows more efficient delivery of cells to the injured cord compared with the intravenous route. Stem cells delivered via LP for treatment of SCI may potentially be applicable in humans after optimal protocols and safety profiles are established in further studies. Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, Pennsylvania 19129, USA. ab99@drexel.edu

Lu P, Jones LL and Tuszynski MH (2005). BDNF-expressing marrow stromal cells support extensive axonal growth at sites of spinal cord injury. Exp Neurol 191: 344-60. Bone marrow stromal cells (MSCs) constitute a heterogeneous cell layer in the bone marrow, supporting the growth and differentiation of hematopoietic stem cells. Recently, it has been reported that MSCs harbor pluripotent stem cells capable of neural differentiation and that simple treatment of MSCs with chemical inducing agents leads to their rapid transdifferentiation into neural cells. We examined whether native or neurally induced MSCs would reconstitute an axonal growth-promoting milieu after cervical spinal cord injury (SCI), and whether such cells could act as vehicles of growth factor gene delivery to further augment axonal growth. One month after grafting to cystic sites of SCI, native MSCs supported modest growth of host sensory and motor axons. Cells "neurally" induced in vitro did not sustain a neural phenotype in vivo and supported host axonal growth to a degree equal to native MSCs. Transduction of MSCs to overexpress brain-derived neurotrophic factor (BDNF) resulted in a significant increase in the extent and diversity of host axonal growth, enhancing the growth of host serotonergic, coerulospinal, and dorsal column sensory axons. Measurement of neurotrophin production from implanted cells in the lesion site revealed that the grafts naturally contain nerve growth factor (NGF) and neurotrophin-3 (NT-3), and that transduction with BDNF markedly raises levels of BDNF production. Despite the extensive nature of host axonal penetration into the lesion site, functional recovery was not observed on a tape removal or rope-walking task. Thus, MSCs can support host axonal growth after spinal cord injury and are suitable cell types for ex vivo gene delivery. Combination therapy with other experimental approaches will likely be required to achieve axonal growth beyond the lesion site and functional recovery. Department of Neurosciences and Center for Neural Repair, University of California at San Diego, La Jolla, CA 92093-0626, USA.

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Circles Of DNA Might Help Predict Success Of Stem Cell Transplantation

Circles Of DNA Might Help Predict Success Of Stem Cell Transplantation

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