Striatal allografts in patients with Huntington's disease: impact of diminished astrocytes and vascularization on graft viability.

Neuronal transplantation has been proposed as a potential therapy to replace lost neurons in Huntington's disease. Transplant vascularization and trophic support are important for graft survival. However, very few studies have specifically addressed graft vascularization in patients with neurological disorders. In the present study, we analysed the vasculature of the host putamen and solid grafts of foetal striatal tissue transplanted into patients with Huntington's disease 9 and 12 years previously. Grafts were characterized by a significantly reduced number of large calibre blood vessels in comparison with the host brain. There were also significantly fewer astrocytes and gap junctions, suggesting a lack of functional blood-brain barrier components within the grafted tissue. Additionally, grafts demonstrated a nearly complete absence of pericytes (compared with the striatum) that are considered important for vascular stabilization and angiogenesis. Finally, the host striatum had a marked increase in atrophic astrocytes in comparison with controls and grafts. The extent to which the lower number of large calibre vessels and astrocytes within the transplants contributed to suboptimal graft survival is unknown. The marked increase in atrophic astrocytes in the host brain surrounding the grafts suggests that reduced host trophic support may also contribute to poor graft survival in Huntington's disease. A better understanding of the way in which these components support allografted tissue is critical to the future development of cell-based therapies for the treatment of Huntington's disease.

The role of embryonic motoneuron transplants to restore the lost motor function of the injured spinal cord.

Spinal cord injury or disease result in the loss of critical numbers of spinal motoneurons and consequentially, in severe functional impairment. The most successful way to replace missing motoneurons is the use of embryonic postmitotic motoneuron grafts. This method may also at least partially restore integrity of the injured spinal cord. It has been shown that grafted motoneurons survive, differentiate and integrate into the host cord and many of them are able to reinnervate the denervated muscles. If grafted motoneurons are provided with a conduit (e.g. reimplanted ventral root) the grafted cells are able to extend their axons along the entire length of the peripheral nerves and reach the hind or forelimb muscles and to restore limb locomotion patterns. Grafted motoneurons show excellent survival in motoneuron-depleted adult host cords, but the developing spinal cord appears to provide an unfavourable environment for these motoneurons as they do not survive in immature cords. The long term survival and maturation of the grafted neurons depend on the availability of a nerve conduit and one or more target muscles, independently of whether these are ectopic nerve-muscle implants or limb muscles in their original site. Thus, grafted and host motoneurons induce functional recovery in the denervated limb muscles when their axons can grow into an avulsed and reimplanted ventral root and then reach the limb muscles. Following segmental loss of motoneurons induced by partial spinal cord injury, motoneuron-enriched embryonic grafts can be placed into the gap-like hemisection cavity in the cervical spinal cord. Such transplants induce the regeneration of great numbers of host motoneurons possibly by the bridging effect of the grafts. In this case, the regenerating host motoneurons reinnervate their original target muscles while the small graft plays a minimal role in the reinnervation of muscles. These results suggest that reconstruction of the injured spinal cord using an embryonic motoneuron-enriched spinal cord graft is a feasible way to achieve improvement after severe functional motor deficits of the spinal cord.

Implanted neurosphere-derived precursors promote recovery after neonatal excitotoxic brain injury.

Brain damage through excitotoxic mechanisms is a major cause of cerebral palsy in infants. This phenomenon usually occurs during the fetal period in human, and often leads to lifelong neurological morbidity with cognitive and sensorimotor impairment. However, there is currently no effective therapy. Significant recovery of brain function through neural stem cell implantation has been shown in several animal models of brain damage, but remains to be investigated in detail in neonates. In the present study, we evaluated the effect of cell therapy in a well-established neonatal mouse model of cerebral palsy induced by excitotoxicity (ibotenate treatment on postnatal day 5). Neurosphere-derived precursors or control cells (fibroblasts) were implanted into injured and control brains contralateral to the site of injury, and the fate of implanted cells was monitored by immunohistochemistry. Behavioral tests were performed in animals that received early (4 h after injury) or late (72 h after injury) cell implants. We show that neurosphere-derived precursors implanted into the injured brains of 5-day-old pups migrated to the lesion site, remained undifferentiated at day 10, and differentiated into oligodendrocyte and neurons at day 42. Although grafted cells finally die there few weeks later, this procedure triggered a reduction in lesion size and an improvement in memory performance compared with untreated animals, both 2 and 5 weeks after treatment. Although further studies are warranted, cell therapy could be a future therapeutic strategy for neonates with acute excitotoxic brain injury.

Trisomy 21 enhances human fetal erythro-megakaryocytic development.

Children with Down syndrome exhibit 2 related hematopoietic diseases: transient myeloproliferative disorder (TMD) and acute megakaryoblastic leukemia (AMKL). Both exhibit clonal expansion of blasts with biphenotypic erythroid and megakaryocytic features and contain somatic GATA1 mutations. While altered GATA1 inhibits erythro-megakaryocytic development, less is known about how trisomy 21 impacts blood formation, particularly in the human fetus where TMD and AMKL originate. We used in vitro and mouse transplantation assays to study hematopoiesis in trisomy 21 fetal livers with normal GATA1 alleles. Remarkably, trisomy 21 progenitors exhibited enhanced production of erythroid and megakaryocytic cells that proliferated excessively. Our findings indicate that trisomy 21 itself is associated with cell-autonomous expansion of erythro-megakaryocytic progenitors. This may predispose to TMD and AMKL by increasing the pool of cells susceptible to malignant transformation through acquired mutations in GATA1 and other cooperating genes.

Regeneration of kidney tissue using in vitro cultured fetal kidney cells.

Transplanting fetal kidney cells (FKCs) can regenerate kidney. This requires in vitro expansion in cell number to acquire enough cells for transplantation. However, FKCs may change their cellular characteristics during expansion and, thus, may not regenerate kidney tissue upon transplantation. We investigated how cell culture period affects cellular characteristics and in vivo regenerative potential of FKCs. As the passage number increased, cell growth rate and colony forming ability decreased while senescence and apoptosis increased. To examine in vivo regenerative potential, FKCs cultured through different numbers of passages were implanted into the parenchyma of kidneys of immunodeficient mice using fibrin gel for 4 wk. Histological analyses showed passage-dependent kidney tissue regeneration, and the regeneration was better when cells from lower number of passages were implanted. This result shows that in vitro culture of FKCs significantly affects the cell characteristics and in vivo tissue regenerative potential.

Long-term culture and neuronal survival after intraspinal transplantation of human spinal cord-derived neurospheres.

There is heterogeneity in neural stem and progenitor cell characteristics depending on their species and regional origin. In search for potent in vitro-expanded human neural precursor cells and cell therapy methods to repair the injured human spinal cord, the possible influence exerted by intrinsic cellular heterogeneity has to be considered. Data available on in vitro-expanded human spinal cord-derived cells are sparse and it has previously been difficult to establish long-term neurosphere cultures showing multipotentiality. In the present paper, human spinal cord-derived neurospheres were cultured in the presence of EGF, bFGF and CNTF for up to 25 passages (>350 days) in vitro. In contrast to the human first trimester subcortical forebrain, spinal cord tissue>9.5 weeks of gestation could not serve as a source for long-term neurosphere cultures under the present conditions. After withdrawal of mitogens, cultured neurospheres (at 18 passages) gave rise to cells with neuronal, astrocytic and oligodendrocytic phenotypes in vitro. After transplantation of human spinal cord-derived neurospheres to the lesioned spinal cord of immuno-deficient adult rats, large numbers of cells survived at least up to 6 weeks, expressing neuronal and astrocytic phenotypes. These results demonstrate that it is possible to expand and maintain multipotent human spinal cord-derived neurospheres in vitro for extended time-periods and that they have promising in vivo potential after engraftment to the injured spinal cord.

Amphetamine induced rotation in the assessment of lesions and grafts in the unilateral rat model of Parkinson's disease.

In the unilateral rat model of Parkinson's disease (PD), amphetamine induced rotation is widely used as an index of both lesion deficits and of graft-derived recovery. We have analysed the time course of the rotational response in lesioned rats, and in rats with lesions and dopamine grafts. In lesioned rats, the rotation exhibited a typical dose-dependent response, with low rates of rotation in the first 10 min after injection, rising gradually to a maximum after 20-30 min. Grafted rats exhibited a peak of rotation in the first 10 min after injection, which then fell to a minimum after 30 min. We demonstrate that the response seen in grafted rats is both drug and dose-dependent and show that the rotational profile results from interaction of the grafted and intact striata which exhibit differential temporal responses to the amphetamine.

Organogenesis of pancreatic anlagen allografted in rats.

AIMS: To study the possibility of revascularization, growth, and differentiation of embryonic pancreatic anlagen transplanted to adult hosts. While transplantations of pancreas and islets are the main methods to cure diabetes mellitus, the donor source is in shortage. So it's necessary to find a new source for transplantation. METHODS: The pancreas from embryonic day 14.5 (E14.5) and 15.5 (E15.5) Lewis rat embryos were implanted into either intraperitoneal or subrenal capsular site of healthy Lewis rats. at 3 weeks or 6 weeks after implantation, the pancreatic anlagen in the host rats were resected for size measurements, as well as histopathologic and immunohistochemical examinations. RESULTS: Three weeks after implantation into the renal-capsular site, the size of both E14.5 and E15.5 pancreatic anlagen had enlarged 10- to 15-fold with differentiation of acinar components upon histological examination. Moreover, increasing numbers of beta cells and islets stained positive for insulin, and newly generated vessels were observed around the tissues. Continued proliferation of the endocrine islets in E14.5 pancreatic anlagen grafts was observed after another 3 weeks, whereas further proliferation in the E15.5 pancreatic anlagen graft was not seen. Additionally fibrosis appeared in the exocrine component of both E14.5 and E15.5 pancreatic anlagen at this time point. When implanted into intraperitoneal site, enlarged E15.5 pancreatic anlagen with proliferatels beta cells were also observed after 3 weeks. However, both the size of the pancreatic anlagen and the proliferation of the beta cells were much less than that in the subrenal capsular site. CONCLUSIONS: The allografted E14.5 and E15.5 pancreatic anlagen revascularised and grew into tissues that were structurally similar to normal mature rats pancreatic tissue. Adequate embryonic age for the transplantation of pancreatic anlagen is 14.5 and 15.5 days old. Subrenal capsula is a more suitable site than the peritoneal cavity for implantation of pancreatic anlagen.

Transplantation of fetal kidney cells: neuroprotection and neuroregeneration.

Various trophic factors in the transforming growth factor-beta (TGF-beta) superfamily have been reported to have neuroprotective and neuroregenerative effects. Intracerebral administration of glial cell line-derived neurotrophic factor (GDNF) or bone morphogenetic proteins (BMPs), both members of the TGF-beta family, reduce ischemia- or 6-hydroxydopamine (6-OHDA)-induced injury in adult rat brain. Because BMPs and GDNF are highly expressed in fetal kidney cells, transplantation of fetal kidney tissue could serve as a cellular reservoir for such molecules and protect against neuronal injury induced by ischemia, neurotoxins, or reactive oxygen species. In this review, we discuss preclinical evidence for the efficacy of fetal kidney cell transplantation in neuroprotection and regeneration models.

Transforming growth factor beta-1 influence on fetal allografts during pregnancy.

An understanding of how the fetus escapes the maternal immune system may be relevant for the prevention of transplant rejection. There is evidence that the same immunosuppressive cytokines contribute to a successful pregnancy and transplant success. Transforming growth factor beta (TGF-beta) is a multifunctional cytokine that exhibits potent immunoregulatory and anti-inflammatory properties and may prolong graft survival. Recent reports suggest a role for TGF-beta in the generation of T-regulatory lymphocytes. Also, the role of TGF-beta in trophoblast differentiation and hypertension prompted us to evaluate maternal serum TGF-beta1 levels in normal allopregnant women and in pregnancies complicated by preeclampsia (PE), a disorder characterized by increased blood pressure, proteinuria, and end organ damage. Sixty-one pregnant preeclamptic women (32 cases with severe and 29 with mild PE), 22 normotensive healthy pregnant, and 20 nonpregnant controls formed the study groups. The active form of serum TGF-beta1 was investigated by an indirect ELISA technique. The results showed that TGF-beta1 was highly expressed in all three pregnant groups compared with the nonpregnant controls. No changes in TGF-beta1 serum levels was found in PE compared with a normal pregnancy. The results suggest that: (1) TGF-beta1 may function as a regulatory factor in fetal allograft survival during pregnancy and (2) TGF-beta1 does not have a pathophysiological role in PE.

Integration and differentiation of neural stem cells after transplantation into the dysmyelinated central nervous system of adult mice.

Mutant mice deficient in the myelin-associated glycoprotein (MAG) and the nonreceptor-type tyrosine kinase Fyn are characterized by a severely hypomyelinated central nervous system (CNS) and morphologically abnormal myelin sheaths. Despite this pronounced phenotype, MAG/Fyn-deficient mice have a normal longevity. In the present study, we took advantage of the normal life expectancy of this myelin mutant and grafted neural stem cells (NSCs) into the CNS of MAG/Fyn-deficient mice to study in short- and long-term experiments the fate of NSCs in adult dysmyelinated brains. Neural stem cells were isolated from spinal cords of transgenic mouse embryos ubiquitously expressing enhanced green fluorescent protein. Cells were expanded in vitro in the presence of mitogens for up to 5 weeks before they were grafted into the lateral ventricles or injected into white matter tracts. Analysis of mutant brains 3-15 weeks after intracerebroventricular transplantation of NSCs revealed only limited integration of donor cells into the host brains. However, injection of NSCs directly into white matter tracts resulted in widespread distribution of donor cells within the host tissue. Donor cells survived for at least 15 weeks in adult host brains. The majority of grafted cells populated white matter tracts and differentiated into oligodendrocytes that myelinated host axons. Results suggest that intraparenchymal transplantation of NSCs might be a strategy to reconstruct myelin in dysmyelinated adult brains.

Functional improvement with low-dose dopaminergic grafts in hemiparkinsonian rats.

OBJECTIVE: The beneficial functional effects of neural transplantation in Parkinson's disease are often directly attributed to the number of surviving dopaminergic cells within a graft. However, recent clinical trials of fetal neural transplantation suggest that a high number of dopaminergic cells may induce serious side effects. In this study, we explored the ability of low-dose dopaminergic grafts to produce functional benefits in the 6-hydroxydopamine rodent model of Parkinson's disease over a long period of observation. METHODS: Twelve rats received either 50,000 or 400,000 fetal ventral mesencephalic cells implanted into the striatum. Rotational behavior was assessed after the lesion and at 3, 6, 9, and 12 weeks after transplantation. Twelve weeks after transplantation, animals were perfused, and microtome sections were stained for tyrosine hydroxylase, glial fibrillary acidic protein, heat-shock protein 27, and vimentin. RESULTS: The low-dose group had a three-fold increase in tyrosine hydroxylase-positive cell survival rate compared with the high-dose group rate. The low-dose group also had a mean cell diameter significantly higher than the high-dose group. There was no significant difference between groups in fiber density; however, a higher percentage of longer fibers was encountered in the low-dose group. The low-dose group had a lower degree of trauma in the striatum, as assessed by optical density scores from glial fibrillary acidic protein, heat-shock protein 27, and vimentin staining. There was significant improvement in rotational behavior in the high-dose group at 3 weeks after transplantation, whereas the rotational behavior normalized in the low-dose group at 6 weeks after grafting. There was no significant difference in rotational behavior scores between groups at 6 weeks after grafting. CONCLUSION: This study demonstrates that over time, a low-dose dopaminergic graft has the capability of eliciting the same functional effect as a high-dose graft. Furthermore, low-dose grafts may increase graft survival, fiber outgrowth, and dopamine production and decrease trauma to the brain.

Differentiation prevents assessment of neural stem cell pluripotency after blastocyst injection.

Earlier studies reported that neural stem (NS) cells injected into blastocysts appeared to be pluripotent, differentiating into cells of all three germ layers. In this study, we followed in vitro green fluorescent protein (GFP)-labeled NS and embryonic stem (ES) cells injected into blastocysts. Forty-eight hours after injection, significantly fewer blastocysts contained GFP-NS cells than GFP-ES cells. By 96 hours, very few GFP-NS cells remained in blastocysts compared with ES cells. Moreover, 48 hours after injection, GFP-NS cells in blastocysts extended long cellular processes, ceased expressing the NS cell marker nestin, and instead expressed the astrocytic marker glial fibrillary acidic protein. GFP-ES cells in blastocysts remained morphologically undifferentiated, continuing to express the pluripotent marker stage-specific embryonic antigen-1. Selecting cells from the NS cell population that preferentially formed neurospheres for injection into blastocysts resulted in identical results. Consistent with this in vitro behavior, none of almost 80 mice resulting from NS cell-injected blastocysts replaced into recipient mothers were chimeric. These results strongly support the idea that NS cells cannot participate in chimera formation because of their rapid differentiation into glia-like cells. Thus, these results raise doubts concerning the pluripotency properties of NS cells.

Fetal cortical allografts project massively through the adult cortex.

Allogeneic embryonic CNS tissue grafts placed in the mature brain are classically considered to lack significant long-range efferents. This problem was reexamined using 'green' cells from mice expressing ubiquitously an 'enhanced' green fluorescent protein as an alternative to classical tract tracing methods. The present study shows that fetal cortical neurons (E15; occipital origin) grafted in the occipitoparietal region of the adult cortex project massively throughout ipsilateral telencephalic structures. Two out of the nine grafted subjects had additional but sparse efferents in the visual thalamus, superior colliculus and pons.

Developmental changes in glutathione S-transferase isoforms expression and activity in intrasplenic fetal liver tissue transplants in rats.

The aim of the present study was to characterise developmental changes in glutathione S-transferase (GST) isoforms expression and in glutathione conjugation capacity in intrasplenic liver tissue transplants. For this purpose, syngenic fetal liver tissue suspensions were transplanted into the spleens of adult male Fischer 344 rats. Three days, 1, 2, 4 weeks, 2, 4, 6 months and 1 year later, transplant-recipients and control animals were sacrificed and class alpha, mu and pi GST isoforms expression and GST activities using the substrates o-dinitrobenzene and 1-chloro-2,4-dinitrobenzene were assessed in livers and spleens. In the hepatocytes of the adult livers no class pi, but a distinct class alpha and mu GST expression was seen. The bile duct epithelia were class pi GST positive. Fetal livers displayed almost no class alpha and mu, but a slight class pi GST expression. The same pattern was seen in 3-day-old intrasplenic liver tissue transplants. Up to 2 weeks after surgery the class alpha and mu GST expression increased in the hepatocytes of the transplants, whereas the immunostaining for class pi GST disappeared. No remarkable changes were seen thereafter. Normal conjugation capacities were observed with the livers of both groups of rats. Control spleens displayed only low GST activities. From 2 months after transplantation on activities were significantly higher in transplant-containing spleens than in respective control organs with a further increase up to one year after grafting. These results show that intrasplenically transplanted fetal liver cells proliferate and differentiate into mature cells displaying a GST expression pattern with respective enzyme activities similar to adult liver.

Triple immune suppression increases short-term survival of porcine fetal retinal pigment epithelium xenografts.

PURPOSE: To determine the effect of triple drug immune suppression on RPE xenograft survival in the fetal pig after transplantation into the albino rabbit subretinal space. METHODS: Primary RPE microaggregates (approximately 40,000 RPE cells) were injected into the subretinal space of 24 albino rabbits, with half the rabbits maintained on triple systemic immune suppression. RPE survival was estimated with a DNA probe (porcine DNA repeat element; PRE) against a porcine-specific repetitive chromosomal marker or a RAM-11 antibody against rabbit macrophages. RESULTS: Numerous pigmented cells were visible in the subretinal space at all time points, but most pigment-containing cells 4 weeks or more after surgery were RAM-11 positive and PRE negative. The number of PRE-positive cells in the immune-suppressed group (4193 +/- 2461, 1184 +/- 1502, and 541 +/- 324 at 4, 8, and 12 weeks, respectively) was greater than in immune-competent control animals (292 +/- 506, 193 +/- 173, and 111 +/- 96), but the difference was only statistically significant at 4 weeks. The time-dependent decrease in PRE-positive cells was more pronounced in immune-suppressed animals. Image analysis performed on serial fundus photographs and fluorescein angiograms did not detect any difference in the appearance of the grafts in immune-suppressed versus immune-competent animals. CONCLUSIONS: Systemic immune suppression increased the 4-week survival of porcine RPE xenografts in the albino rabbit subretinal space, but there was poor survival in immune-suppressed and -competent animals 12 weeks after surgery. Many pigment-containing cells 4 or more weeks after surgery were PRE negative, indicating that they are of host origin.

Interference with anoikis-induced cell death of dopamine neurons: implications for augmenting embryonic graft survival in a rat model of Parkinson's disease.

One promising therapy for the treatment of Parkinson's disease is transplantation of embryonic ventral mesencephalic tissue. Unfortunately, up to 95% of grafted cells die, many via apoptosis. In this study we attempted to prevent anoikis-induced cell death, which is triggered during the preparation of cells for grafting, and examine the impact on graft viability and function. We utilized the extracellular matrix molecule tenascin-C (tenascin) and an antibody (Ab) to the cell adhesion molecule L1 to specifically mimic survival signals induced by cell-matrix and cell-cell interactions. In vitro, both tenascin- and L1 Ab-treated cultures doubled the number of tyrosine hydroxylase immunoreactive (THir) neurons compared to control. Additionally, cell survival assays determined that tenascin and L1 Ab-treated cell suspensions yielded more metabolically active and fewer dead cells than control suspensions. In contrast to the culture results, tenascin- and L1 Ab-treated mesencephalic grafts did not yield an increase in the number of THir neurons using our standard grafting paradigm (3 microl of 100,000 cells/microl). However, under low-density conditions (3 microl of 3,000 cells/microl), tenascin augmented grafted THir neuron survival. These findings are consistent with the view that cell density can dramatically influence the degree of stress placed on THir neurons and consequently affect the success of survival strategies in vivo. In conclusion, pretreatment with tenascin may prove beneficial to prevent anoikis in dilute cell suspension grafts, while long-term in vivo delivery methods need to be explored to determine if L1 can prevent anoikis in grafts of mesencephalic dopamine neurons after transplantation.

The future of cell-based transplantation therapies for neurodegenerative disorders.

Parkinson's disease is a common neurodegenerative disease with a lifetime incidence of 2.5% and a prevalence of at least 2% in individuals over 70 years old. Patients can be effectively treated with drugs that target the dopaminergic nigro-striatal pathway, but over time the efficacy of these medications is limited by the development of profound motor fluctuations and dyskinesias. This has prompted the search for alternative treatments, including the use of cell replacement therapies. Over the last decade, human fetal nigral transplants have demonstrated that dopaminergic neurons can survive and provide clinical benefit for patients with Parkinson's disease. However, there are clearly ethical concerns and a limit to the supply of this tissue as well as more recently anxieties over side effects. As a result, alternative sources of tissue have been investigated, and one such source are stem cells, which provide an attractive renewable tissue supply. In this review, we will discuss the current state-of-the-art and the characteristics of Parkinson's disease that increase its attraction as a target of stem cell therapy against results of current clinical trials using fetal neural grafts. Then we will discuss the various types and sources of stem cells, and some early transplantation results in animal models of Parkinson's disease. Finally we will discuss the prospect of using stem cells to deliver drugs and neurotrophic factors involved in neuroprotective and neuroreparative strategies in Parkinson's disease and other neurodegenerative conditions.