T. Zigova

Intravenous versus intrastriatal cord blood administration in a rodent model of stroke.

Human umbilical cord blood (hUCB) is a rich source of hematopoietic stem cells that have been used to reconstitute immune cells and blood lineages. Cells from another hematopoietic source, bone marrow, have been found to differentiate into neural cells and are effective in the treatment of stroke. In this study, we administered hUCB cells intravenously into the femoral vein or directly into the striatum and assessed which route of cell administration produced the greatest behavioral recovery in rats with permanent middle cerebral artery occlusion (MCAO). All animals were immunosuppressed with cyclosporine (CSA). When spontaneous activity was measured using the Digiscan automated system, it was found to be significantly less when hUCB was transplanted 24 hr after stroke compared with nontransplanted, stroked animals (P < 0.01). Furthermore, behavioral recovery was similar with both striatal and femoral hUCB delivery. This is in contrast to the step test, in which significant improvements were found only after femoral delivery of the hUCB cells. In the passive avoidance test, transplanted animals learned the task faster than nontransplanted animals (P < 0.05). Together, these results suggest that hUCB transplantation may be an effective treatment for brain injuries, such as stroke, or neurodegenerative disorders. In addition, intravenous delivery may be more effective than striatal delivery in producing long‐term functional benefits to the stroked animal.

Positive effect of transplantation of hNT neurons (NTera 2/D1 cell-line) in a model of familial amyotrophic lateral sclerosis.

Abstract Transplantation of hNT Neurons derived from the human teratocarcinoma cell-line (NTera2/D1) has been shown to ameliorate motor dysfunction in a number of injury or disease models in which the deficits are fairly localized. However, these cells have not been used before in a model with more extensive neurodegeneration. The aim of this study is to determine the effects of hNT Neuron transplants on motor neuron function in a mouse model of familial amyotrophic lateral sclerosis (FALS) in which there is a substitution of Alanine for Glycine at position 93 of the human SOD1 gene (G93A). Amyotrophic lateral sclerosis is a fatal degenerative motor neuron disease affecting the spinal cord, brainstem, and cortex. This disease clinically manifests as progressive muscular weakness and atrophy, leading to paralysis and death within 3–5 years of diagnosis. The FALS represents 10–13% of all cases. A range of behavioral tests was used to examine spontaneous locomotor activity, coordination, and muscle strength of mice. Long-term (10–11 weeks) transplantation of hNT Neurons into the L4–L5 segments of the ventral horn spinal cord of FALSG93A mice at 7 weeks of age (before onset of overt behavioral symptoms of disease) delayed the onset of motor dysfunction for at least 3 weeks. The average lifespan of the transplanted mice was 128 days compared to 106 days for media-injected group. The last mouse in the hNT Neuron transplanted group was euthanized at 135 days of age when it display partial paralysis of the hindlimbs. Immunohistochemical analysis of the implanted spinal cords demonstrated the survival of grafted hNT Neurons and showed many healthy-appearing motor neurons near the implant site. These results suggest that hNT Neuron transplantation may be a promising therapeutic strategy for ALS.

Expression of brain natriuretic peptide by human bone marrow stromal cells

Bone marrow stromal cells (BMSC) have been shown to generate neural cells under experimental conditions in vitro and following transplantation into animal models of stroke and traumatic CNS injury. Hastened recovery from the neurological deficit has not correlated with structural repair of the lesion in the stroke model. Secretory functions of BMSC, such as the elaboration of growth factors and cytokines, have been hypothesized to play a role in the enhanced recovery of neurological function. Using gene expression arrays, real time RT-PCR and radioimmunoassay, we have found that brain natriuretic peptide (BNP) is synthesized and released by BMSC at physiologically relevant levels in vitro. BNP, like its close homolog atrial natriuretic peptide (ANP), exerts powerful natriuretic, diuretic and vasodilatory effects. We speculate that transplanted BMSCs facilitate recovery from brain and spinal cord lesions by releasing BNP and other vasoactive factors that reduce edema, decrease intracranial pressure and improve cerebral perfusion.

Do hematopoietic cells exposed to a neurogenic environment mimic properties of endogenous neural precursors

Hematopoietic progenitors are cells, which under challenging experimental conditions can develop unusual phenotypic properties, rather distant from their original mesodermal origin. As previously reported, cells derived from human umbilical cord blood (HUCB) or human bone marrow (BM) under certain in vivo or in vitro conditions can manifest neural features that resemble features of neural‐derived cells, immunocytochemically and in some instances also morphologically. The present study explored how hematopoietic‐derived cells would respond to neurogenic signals from the subventricular zone (SVZ) of adult and aged (6 and 16 months old) rats. The mononuclear fraction of HUCB cells was transplanted into the SVZ of immunosuppressed (single cyclosporin or three‐drug treatment) animals. The triple‐suppression paradigm allowed us to protect transplanted human cells within the brain and to explore further their phenotypic and migratory properties. One week after implantation, many surviving HUCB cells were located within the SVZ and the vertical limb of the rostral migratory stream (RMS). The migration of HUCB cells was restricted exclusively to the pathway leading to the olfactory bulb. In younger animals, grafted cells navigated almost halfway through the vertical limb, whereas, in the older animals, the migration was less pronounced. The overall cell survival was greater in younger animals than in older ones. Immunocytochemistry for surface CD antigen expression showed that many HUCB cells, either cultured or within the brain parenchyma, retained their hematopoietic identity. A few cells, identified by using human‐specific antibodies (anti‐human nuclei, or mitochondria) expressed nestin and doublecortin, markers of endogenous neural progenitors. Therefore, it is believed that the environment of the neurogenic SVZ, even in aged animals, was able to support survival, “neuralization,” and migratory features of HUCB‐derived cells.

The X-gal caution in neural transplantation studies.

Cell transplantation into host brain requires a reliable cell marker to trace lineage and location of grafted cells in host tissue. The lacZ gene encodes the bacterial (E. coli) enzyme β-galactosidase (β-gal) and is commonly visualized as a blue intracellular precipitate following its incubation with a substrate, “X-gal,” in an oxidation reaction. LacZ is the “reporter gene” most commonly employed to follow gene expression in neural tissue or to track the fate of transplanted exogenous cells. If the reaction is not performed carefully—with adequate optimization and individualization of various parameters (e.g., pH, concentration of reagents, addition of chelators, composition of fixatives) and the establishment of various controls—then misleading nonspecific background X-gal positivity can result, leading to the misidentification of cells. Some of this background results from endogenous nonbacterial β-gal activity in discrete populations of neurons in the mammalian brain; some results from an excessive oxidation reaction. Surprisingly, few articles have emphasized how to recognize and to eliminate these potential confounding artifacts in order to maximize the utility and credibility of this histochemical technique as a cell marker. We briefly review the phenomenon in general, discuss a specific case that illustrates how an insufficiently scrutinized X-gal positivity can be a pitfall in cell transplantation studies, and then provide recommendations for optimizing the specificity and reliability of this histochemical reaction for discerning E. coli β-gal activity.

Comparison of Calcium-Binding Proteins Expressed in Cultured hNT Neurons and hNT Neurons Transplanted into the Rat Striatum

An alternative source of cells for neural transplantation and brain repair that has many characteristics of immature neurons is the hNT neuron, derived from an embryonal human teratocarcinoma (NTera2) cell line that is terminally differentiated in vitro with retinoic acid. The majority of hNT neurons are GABAergic in cell culture. We have determined the calcium-binding protein (CBP) phenotypes of hNT neurons for three CBPs, calretinin (CR), calbindin D-28K (CB), and parvalbumin (PV), in cell culture and after transplantation into the rat striatum. In cell culture, 95% of all cell profiles were human nuclear matrix antigen (NuMA) positive. PV-positive hNT neurons constituted 50% of all neuron-like profiles, with CB+ and CR+ constituting 14 and 6% of cells, respectively. In contrast, when the striatal grafts were examined after 30 days survival using confocal microscopy, only 10% of hNT neurons immunopositive for NuMA were PV+; 19% were CB+/NuMA+, approximately the same percentage as was seen in vitro, and 82% of grafted hNT neurons were CR+. These results suggest that hNT neurons can be subdivided into at least three subpopulations based on the CBP phenotype that they express and that there is a CBP phenotypic shift following transplantation. Three related hypotheses are proposed to account for this phenotypic shift of hNT neurons after transplantation: (a) selective survival of the CR+ subpopulation of hNT neurons, (b) selective transitory quiescence of the transplanted PV+ cells due to transplantation stress, or (c) dedifferentiation of the hNT neurons following transplantation, which may allow them to respond to local environmental cues during the engraftment process.

Green fluorescent protein bone marrow cells express hematopoietic and neural antigens in culture and migrate within the neonatal rat brain.

Finding a reliable source of alternative neural stem cells for treatment of various diseases and injuries affecting the central nervous system is a challenge. Numerous studies have shown that hematopoietic and nonhematopoietic progenitors derived from bone marrow (BM) under specific conditions are able to differentiate into cells of all three germ layers. Recently, it was reported that cultured, unfractionated (whole) adult BM cells form nestin‐positive spheres that can later initiate neural differentiation (Kabos et al., 2002 ). The identity of the subpopulation of BM cells that contributes to neural differentiation remains unknown. We therefore analyzed the hematopoietic and neural features of cultured, unfractionated BM cells derived from a transgenic mouse that expresses green fluorescent protein (GFP) in all tissues. We also transplanted the BM cells into the subventricular zone (SVZ), a region known to support postnatal neurogenesis. After injection of BM cells into the neurogenic SVZ in neonatal rats, we found surviving GFP+ BM cells close to the injection site and in various brain regions, including corpus callosum and subcortical white matter. Many of the grafted cells were detected within the rostral migratory stream (RMS), moving toward the olfactory bulb (OB), and some cells reached the subependymal zone of the OB. Our in vitro experiments revealed that murine GFP+ BM cells retained their proliferation and differentiation potential and predominantly preserved their hematopoietic identity (CD45, CD90, CD133), although a few expressed neural antigens (nestin, glial fibrillary acdiic protein, TuJ1).

Neural Transplantation in Parkinson's Disease

In conclusion, proof of the principle exists that neural grafts can survive transplantation in PD and that this graft survival is related to preliminary evidence of clinical benefit and improvement on FD-PET. Two prospective, randomized, surgical placebo-controlled trials of fetal tissue transplantation for the treatment of PD will be published in the near future, as will results of a placebo-controlled xenograft trial. Lifelong survival of human fetal nigral grafts is likely. The striatum is comparatively simple to target surgically in comparison to other sites such as the subthalamic nucleus. Several new sources of dopamine cells are being developed for transplantation purposes. Long-term monitoring for toxicity, such as the development of dyskinesias, will be needed, and dose-escalation trials should be performed slowly due to the irreversible nature of transplants. There are numerous ways to improve current techniques of neural transplantation.