Embryonic Donor Tissue Research Articles

Donor age dependent graft development and recovery in a rat model of Huntington's disease: Histological and behavioral analysis

Neural cell replacement therapy using fetal striatal cells has provided evidence of disease modification in clinical trials in Huntington's disease (HD) patients, although the results have been inconsistent. One of the contributing factors to the variable outcome could be the different capacity of transplanted cells derived from the primordial striatum to proliferate and maturate into striatal projection neurons. Based on the rodent lesion model of HD, the current study investigated how intrastriatal-striatal grafts from variable aged donors develop in vivo and how they influence functional recovery.Young adult female Sprague-Dawley rats were lesioned unilaterally in the dorso-striatum with quinolinic acid (0.12M) and transplanted 14 days later with single cell suspension grafts equivalent of one whole ganglionic eminence (WGE) from donors of embryonic developmental age E13, E14, or E15; animals with or without striatal lesion served as controls. All animals were tested on the Cylinder and the Corridor tests, as well as on apomorphine-induced rotation at baseline, post-lesion/pre-grafting, and at 6 and 10 weeks post-grafting. A week prior to perfusion, a sub-group in each grafted group received fluorogold injections into the ipsilateral globus pallidus to study graft efferent projections. In summary, the data demonstrates that the age of the embryonic donor tissue has an impact on both the graft mediated functional recovery, and on the in vivo cellular composition of the striatal transplant. E13 tissue grafts gave the best overall outcome indicating that WGE from different donor ages have different potential to promote functional recovery. Understanding the stages and process in rodent striatal development could improve tissue selection in clinical trials of cell therapy in HD.

Stem cell‐based therapy for Parkinson's disease

Motor dysfunctions in Parkinson's disease are considered to be primarily due to the degeneration of dopaminergic neurons in the substantia nigra pars compacta. Pharmacological therapies based on the principle of dopamine replacement are extremely valuable, but suffer from two main drawbacks: troubling side effects (e.g. dyskinesia) and loss of efficacy with disease progression. Transplantation of embryonic dopaminergic neurons has emerged as a therapeutic alternative. Enthusiasm following the success of the initial open‐label trials has been dampened by the negative outcome of double‐blind placebo controlled trials. Additionally, the emergence of graft‐related dyskinesia indicates that the experimental grafting procedure requires further refinement before it can be developed into a therapy. Shortage of embryonic donor tissue limits large‐scale clinical transplantation trials. We review three of the most attractive tissue sources of dopaminergic neurons for cell replacement therapy: human embryonic ventral mesencephalic tissue, embryonic and adult multipotent region‐specific stem cells and embryonic stem cells. Recent developments in embryonic stem cell research and on their implications for a future transplantation therapy in Parkinson's disease are described. Finally, we discuss how human embryonic stem cells can be differentiated into dopaminergic neurons, and issues such as the numbers of dopaminergic neurons required for success and the risk for teratoma formation after implantation.

Distinct Populations of Forebrain Neural Stem and Progenitor Cells Can Be Isolated Using Side-Population Analysis

The absence of stem cell-specific markers has posed challenges to the identification and isolation of stem cells. We report the isolation of a discrete and highly enriched population of neural stem cells from clonally derived colonies of neural stem cell and progenitor cells (neurospheres) after exposure to the fluorescent DNA binding dye Hoeschst 33342 and subsequent analysis via dual wavelength flow cytometry. The low fluorescent side population comprised only 3.6% of all live cells sorted yet contained >99% of all the neural stem cells as assayed by the formation of neurospheres in culture. Most neurosphere-derived cells are progenitor cells, and these are found within the higher fluorescence (non-side population) fraction. The isolation of a highly enriched population of self-renewing, multipotential neural stem cells was seen from both adult- and embryonic-derived neurospheres; however, the relative percentage of cells comprising the side-population and the mechanism of dye efflux varied between adult and embryonic donor tissue. Combining the side-population analysis with markers recently shown to enrich for neural stem cells afforded no further enrichment in the case of peanut agglutinin expression and size criteria; however, when the side-population analysis was combined with Lewis X (LeX) expression, a slight enrichment was seen over side-population analysis alone.

Improving the survival of grafted dopaminergic neurons: a review over current approaches.

Neural transplantation is developing into a therapeutic alternative in Parkinson's disease. A major limiting factor is that only 3-20% of grafted dopamine neurons survive the procedure. Recent advances regarding how and when the neurons die indicate that events preceding actual tissue implantation and during the first week thereafter are crucial, and that apoptosis plays a pivotal role. Triggers that may initiate neuronal death in grafts include donor tissue hypoxia and hypoglycemia, mechanical trauma, free radicals, growth factor deprivation, and excessive extracellular concentrations of excitatory amino acids in the host brain. Four distinct phases during grafting that can involve cell death have been identified: retrieval of the embryo; dissection and preparation of the donor tissue; implantation procedure followed by the immediate period after graft injection; and later stages of graft maturation. During these phases, cell death processes involving free radicals and caspase activation (leading to apoptosis) may be triggered, possibly involving an increase in intracellular calcium. We review different approaches that reduce cell death and increase survival of grafted neurons, typically by a factor of 2-4. For example, changes in transplantation procedure such as improved media and implantation technique can be beneficial. Calcium channel antagonists such as nimodipine and flunarizine improve nigral graft survival. Agents that counteract oxidative stress and its consequences, such as superoxide dismutase overexpression, and lazaroids can significantly increase the survival of transplanted dopamine neurons. Also, the inhibition of apoptosis by a caspase inhibitor has marked positive effects. Finally, basic fibroblast growth factor and members of the transforming growth factor-beta superfamily, such as glial cell line-derived neurotrophic factor, significantly improve the outcome of nigral transplants. These recent advances provide hope for improved survival of transplanted neurons in patients with Parkinson's disease, reducing the need for human embryonic donor tissue and increasing the likelihood of a successful outcome.

Effects of anaesthetics and lazaroid U-83836E on survival of transplanted rat dopaminergic neurones

We investigated whether different methods of anaesthesia, used on the pregnant rat when collecting embryonic donor tissue, and a lipid peroxidation inhibitor (lazaroid U-83836E) affect the survival of grafted embryonic dopaminergic neurones in hemiparkinsonian rats. There was no difference in either functional recovery or survival of dopaminergic neurones between the different euthanasia groups: (a) isoflurane sedation followed by cervical dislocation, (b) equithesin- or (c) euthatal-anaesthesia. However, lazaroid-treatment enhanced both behavioural recovery and transplant survival.

Effects of hibernation or cryopreservation on the survival and integration of striatal grafts placed in the ibotenate-lesioned rat caudate-putamen

Tissue storage prior to intracerebral transplantation would represent a major advantage when conducting clinical transplantation trials in that the procurement of the embryonic donor tissue and the timing of neurosurgery could be planned more efficiently. In the present study, the effects of storing rat embryonic striatal tissue at either +4°C or below freezing temperature prior to grafting to the adult striatum, were assessed with regard to transplant survival, morphology and integration. Eleven days following a unilateral injection of ibotenic acid into the head of the caudate-putamen, a control group of rats received grafts of striatal primordium prepared immediately after dissection from rat embryos (embryonic day 16). A second group of rat embryonic striatal tissue was stored at 4°C (hibernation) for 5 days and then transplanted. A third group of the striatal donor tissue was cryopreserved in liquid nitrogen for 5 days before implantation surgery. Six to seven weeks following transplantation surgery, the grafts were analysed in brain sections processed for acetylcholinesterase histochemistry, DARPP-32 (dopamine and cyclic AMP regulated phosphoprotein with a molecular weight of 32 kDa) and tyrosine hydroxylase (TH) immunocytochemistry. The mean total graft volume and the relative size of the AChE-positive regions were not significantly different between the three groups. Striatal-specific graft regions, positively stained for AChE and DARPP-32, generally exhibited TH immunoreactivity, suggesting that they had received dopaminergic afferents from the host brain. We conclude that embryonic rat striatal tissue can be cryopreserved or hibernated over 5 days without significant impairment in the yield of striatal neurons following intrastriatal implantation and without markedly affecting transplant morphology.

Effects of hibernation or cryopreservation on the survival and integration of striatal grafts placed in the ibotenate-lesioned rat caudate-putamen

Tissue storage prior to intracerebral transplantation would represent a major advantage when conducting clinical transplantation trials in that the procurement of the embryonic donor tissue and the timing of neurosurgery could be planned more efficiently. In the present study, the effects of storing rat embryonic striatal tissue at either +4°C or below freezing temperature prior to grafting to the adult striatum, were assessed with regard to transplant survival, morphology and integration. Eleven days following a unilateral injection of ibotenic acid into the head of the caudate-putamen, a control group of rats received grafts of striatal primordium prepared immediately after dissection from rat embryos (embryonic day 16). A second group of rat embryonic striatal tissue was stored at 4°C (hibernation) for 5 days and then transplanted. A third group of the striatal donor tissue was cryopreserved in liquid nitrogen for 5 days before implantation surgery. Six to seven weeks following transplantation surgery, the grafts were analysed in brain sections processed for acetylcholinesterase histochemistry, DARPP-32 (dopamine and cyclic AMP regulated phosphoprotein with a molecular weight of 32 kDa) and tyrosine hydroxylase (TH) immunocytochemistry. The mean total graft volume and the relative size of the AChE-positive regions were not significantly different between the three groups. Striatal-specific graft regions, positively stained for AChE and DARPP-32, generally exhibited TH immunoreactivity, suggesting that they had received dopaminergic afferents from the host brain. We conclude that embryonic rat striatal tissue can be cryopreserved or hibernated over 5 days without significant impairment in the yield of striatal neurons following intrastriatal implantation and without markedly affecting transplant morphology.

Effects of hibernation or cryopreservation on the survival and integration of striatal grafts placed in the ibotenate-lesioned rat caudate-putamen.

Tissue storage prior to intracerebral transplantation would represent a major advantage when conducting clinical transplantation trials in that the procurement of the embryonic donor tissue and the timing of neurosurgery could be planned more efficiently. In the present study, the effects of storing rat embryonic striatal tissue at either +4 degrees C or below freezing temperature prior to grafting to the adult striatum, were assessed with regard to transplant survival, morphology and integration. Eleven days following a unilateral injection of ibotenic acid into the head of the caudate-putamen, a control group of rats received grafts of striatal primordium prepared immediately after dissection from rat embryos (embryonic day 16). A second group of rat embryonic striatal tissue was stored at 4 degrees C (hibernation) for 5 days and then transplanted. A third group of the striatal donor tissue was cryopreserved in liquid nitrogen for 5 days before implantation surgery. Six to seven weeks following transplantation surgery, the grafts were analysed in brain sections processed for acetylcholinesterase histochemistry, DARPP-32 (dopamine and cyclic AMP regulated phosphoprotein with a molecular weight of 32 kDa) and tyrosine hydroxylase (TH) immunocytochemistry. The mean total graft volume and the relative size of the AChE-positive regions were not significantly different between the three groups. Striatal-specific graft regions, positively stained for AChE and DARPP-32, generally exhibited TH immunoreactivity, suggesting that they had received dopaminergic afferents from the host brain. We conclude that embryonic rat striatal tissue can be cryopreserved or hibernated over 5 days without significant impairment in the yield of striatal neurons following intrastriatal implantation and without markedly affecting transplant morphology.

A comparative study of preparation techniques for improving the viability of nigral grafts using vital stains, in vitro cultures, and in vivo grafts

The intracerebral transplantation of embryonic dopaminergic nigral neurons, although relatively successful, leads to a fairly low yield of surviving cells. Many factors may influence the viability of dopaminergic grafts and one of these is the preparation of the tissue prior to transplantation. We have investigated the effects of different steps during the preparation and storage of embryonic rat nigral cell suspensions on their subsequent survival at a variety of different time points using a combination of techniques and studies. For studies concerned with the first 24 h we employed vital stains, in the period covering the next 7 days we used in vitro cultures, and in the long term experiment we used in vivo grafts. The results suggest that nigral cell suspensions may remain sufficiently viable for grafting for much longer periods than previously reported. In addition a number of parameters which affect cell survival have been characterised, including the age of the embryonic donor tissue, the use of proteolytic enzymes and the trituration procedure used during the preparation of the suspension. The optimal preparation technique, therefore, uses E13–E14 embryos with the dissected ventral mesencephalon being incubated in purified 0.1% trypsin solutions for 60 min and triturated using a flame polished Pasteur pipette. This may have important implication in improving intracerebral transplantation for Parkinson's disease.

Expression of platelet-derived growth factor in and around intrastriatal embryonic mesencephalic grafts.

The expression of platelet-derived growth factor (PDGF) was investigated in the embryonic donor tissue and surrounding host brain before and after intracerebral transplantation in a rat model of Parkinson's disease (PD). Ventral mesencephalic tissue from E13-E15 rat embryos was dissociated and implanted into adult rats with unilateral lesions of the mesostriatal dopamine system. Immunohistochemical studies showed that the majority of the grafted cells were PDGF-positive at early time points after grafting. However, the immunostaining gradually decreased, and had disappeared almost completely 3 wk after transplantation. These results were in agreement with in situ hybridization data demonstrating detectable levels of mRNA for PDGF chains in graft cells after 1, but not after 6 wk. In contrast, a large number of PDGF-immunoreactive cells was observed in the host brain adjacent to the grafts from 1 wk after transplantation, and increasing with time. Increased expression of PDGF was also observed in response to a sham-operation (injection of vehicle), although the number of PDGF-positive cells seemed lower than after grafting of embryonic tissue. Double immunofluorescence labeling of these cells with an anti-glial fibrillary acidic protein (GFAP) antiserum and a monoclonal antibody against PDGF B-chain, indicated that the PDGF-positive cells were astrocytes. The dynamic expression of PDGF in and around intrastriatal embryonic mesencephalic implants has several, potentially important, implications for graft survival and function. Glial cells could utilize the elevated levels of PDGF to proliferate in a reactive gliosis, and PDGF might also augment immune responses. It is also possible that PDGF increases the survival of, and promotes neurite outgrowth from, grafted dopaminergic neurons.

Integration of New Embryonic Nephrons Into the Kidney

The current report summarizes our experiments exploring the feasibility of creating a chimeric kidney, that is, an organ constituted by cells derived from more than one fertilized ovum. The overall strategy has been to obtain donor renal tissue from avian and murine embryos and to implant this into the host avian mesonephric mesoderm or into the cortex of murine neonatal kidney. In both models, donor cells were distinguished from the host by the presence of characteristic nuclear or cytoplasmic markers. Examination of quail to chick transplants showed the tandem development of mesonephric tissue in the form of bilobed organ. In the mouse chimeric kidney, examined 2 to 4 weeks postnatally, transplanted metanephric tissue grew and developed glomeruli, proximal tubules, and cords of cells, which extended into the medulla of the host kidney. Before death, intravenous FITC-dextran was administered to the host mouse. Some transplanted tubules were connected to filtering glomeruli, as judged by the presence of fluorescein within their lumens. These experimental models provide novel means with which to study nephrogenesis in vivo. Finally, if the embryonic donor tissue could be genetically engineered before implantation, the prospect of "nephron therapy" arises, in which altered implanted nephrons could deliver therapeutically useful molecules into the urine or kidney interstitium.

Embryonic entorhinal transplants project selectively to the deafferented entorhinal zone of adult mouse hippocampi, as demonstrated by the use of thy-1 allelic immunohistochemistry. Effect of timing of transplantation in relation to deafferentation

The mouse Thy-1. 1/Thy-1.2 allelic marking system is used to show that transplanted embryonic entorhinal cortex can reinnervate adult host hippocampi. The projection is limited to the appropriate terminal zones-viz. the outer two-thirds of the stratum moleculare of the dentate gyrus, and the stratum lacunosum-moleculare of the hippocampus-and extends for up to about 2 mm into the denervated host terminal field. The reconstruction of the entorhinal projections to the host requires direct contact between the embryonic donor tissue and the denervated adult host terminal field, and is dependent upon removal of the ipsilateral host entorhinal area. In the absence of an overall deafferenting host entorhinal lesion the transplanted entorhinal area forms only small local projections which are confined to areas which would have been locally deafferented as a result of direct damage to the host entorhinal afferents (i.e. during their intrahippocampal course) by the hippocampal lesion caused at the time when the transplant was inserted. The correct relative timing of deafferentation and transplantation is vital for the formation of the transplant-to-host projection. The host dendrites can be made receptive to entorhinal transplant projections by removal of the host entorhinal area at the time of transplantation. When deafferentation is performed first and transplantation is delayed, it is found that the deafferented host dendrites retain this receptivity even when deafferentation has been performed as much as two months before transplantation. Reversing the order of transplantation and deafferentation, however, shows that the transplants have only a transient ability to project to the deafferented host territory. Thus, transplants inserted and allowed to become established for one week before host deafierentation make very much reduced projections to the host, and from two weeks onwards are incapable of any detectable response to subsequent removal of the host entorhinal area. Coextensive with the formation of transplant-to-host entorhinodentate projections, the host entorhinal lesion also induces an intensification of the acetylcholinesterase staining of the host septodentate afferents in the denervated outer dentate stratum moleculare. The findings demonstrate the accurate reconstruction of a lost projection in adult brain by transplanting the appropriate type of embryonic tissue, but the results of altering the relative timing of deafferentation and transplantation raise currently unsolved questions about the nature of the competitive interactions between transplant and host axons. Surprisingly, a delay allowing time for the lesion-induced sprouting of intact host axons into the denervated area does not seem able to prevent formation of transplant-to-host projections by a subsequent entorhinal transplant. However, unlike the host's own entorhinal area (which retains the ability to sprout to the unilaterally deafierented dentate gyrus throughout adult life), the ability of an embryonic entorhinal area transplant to project to the deafferented host hippocampus and dentate gyrus appears to be transient, and to disappear completely between one and two weeks after transplantation.

Tyrosine hydroxylase is expressed by neocortical neurons after transplantation.

Tyrosine hydroxylase [TyrOHase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2], an essential enzyme in the synthesis of catecholamines, is expressed normally by neurons in the brainstem but not by those in mature neocortex. When embryonic neocortex is transplanted into adult neocortex, TyrOHase-immunoreactive cells develop and continue to be present in the transplants for the life of the host animal. The percentage of transplant neurons that express TyrOHase is highly correlated with the age of the embryonic donor tissue at the time of transplantation. Many TyrOHase-immunoreactive cells are present in transplants from embryonic day 12 (E12) embryos. The labeled cells are frequently arrayed in striking clusters of cell bodies and their processes, which ramify densely within the transplants. Moderate numbers of cells are found scattered throughout transplants from E14 donors, while E17 donors consistently develop small numbers of TyrOHase-containing cells. Tissue removed for transplantation on the day before birth (E19) never contains cells that express TyrOHase. The TyrOHase-positive cells are mostly bipolar and stellate in shape and show neither immunoreactivity for other catecholamine-synthesizing enzymes nor catecholamine fluorescence. These results provide a demonstration of continued TyrOHase synthesis in central nervous system cells that normally do not express this enzyme. Because of these and similar results with other neurotransmitter enzymes, the transplantation paradigm is particularly useful as a technique for studying the factors that regulate enzyme induction and activity during development of the nervous system.

Acetylcholine-rich neuronal grafts in the forebrain of rats: Effects of environmental enrichment, neonatal noradrenaline depletion, host transplantation site and regional source of embryonic donor cells on graft size and acetylcholinesterase-positive fibre outgrowth

The importance of several factors influencing the survival of cholinergic-rich embryonic tissue transplanted to the adult rat forebrain and the extent of acetylcholinesterase-positive fibre innervation of the host brain was investigated in 3 experiments. In the first two experiments, embryonic ventral forebrain tissue was grafted to the neocortex of rats in which the intrinsic cortical cholinergic innervation had been removed by nucleus basalis lesions. Housing the host rats in an enriched environment produced a temporary enhancement of fibre outgrowth 4 weeks after transplantation, but this was not maintained after 10 weeks. Fibre outgrowth was greater when the grafts were transplanted to the noradrenaline-depleted neocortex than to the intact neocortex. Neither environmental enrichment nor noradrenaline depletion influenced graft survival or size. In the third experiment, the embryonic donor tissue was dissected to separate regions containing precursors of the nucleus basalis cholinergic cells from regions containing precursors of the septal cholinergic cells, and transplanted to either the neocortex following nucleus basalis lesions or to the hippocampus following fimbria-fornix lesions. Nucleus basalis grafts showed greater growth in size than septal grafts, and grafts placed into the hippocampus showed greater growth in size than grafts placed into the neocortex. More interestingly, the extent of fibre outgrowth depended on the appropriateness of the donor tissue to the host transplantation site: nucleus basalis tissue showed greater acetylcholinesterase-positive outgrowth than septal tissue in the neocortex, whereas septal tissue showed greater outgrowth than nucleus basalis tissue in the hippocampus.