Search

Intracerebral transplantation of dopaminergic (DA) cells is currently further explored as a potential restorative therapy for Parkinson's disease (PD). However, before they can be considered for a more widespread clinical use a number of critical issues have to be resolved, including an optimized transplantation protocol. This study has been performed in a rat 6-hydroxydopamine model of PD and is based on the microtransplantation approach. The results demonstrate a reduced survival (threefold) for a single cell suspension of E14 rat ventral mesencephalon compared to a fragment suspension when a metal cannula is used for implantation. However, fragment suspensions result in a more variable graft survival and ectopically placed cells along the implantation tract. When a glass capillary is used for implantation, the survival of the single cell suspension (so-called “micrograft”) improved by fourfold (vs. single cells/metal cannula) and is superior to the combination of the metal cannula and fragment suspension (+40%). The micrografts show a reduced variability in DA neuron survival as well as fewer ectopically placed cells. Moreover, the implantation time can significantly be reduced from 19 to 7 min in micrografted animals without a compromise in DA graft survival and functional behavioral outcome. Using the microtransplantation approach graft size can be tailored effectively by varying the density of the final cell suspension at least between 11,000 and 320,000 cells/μl, resulting in comparable survival of tyrosine hydroxylase (TH)-positive neurons in the range of 2–4%. With this approach no more than 100 surviving TH-positive neurons are necessary to produce functional effects in the amphetamine-induced rotation test. Interestingly, we found that DA micrografts into lesion striatum present 20% higher survival rates of TH neurons in comparison to the intact striatum. In summary, these results provide further evidence for the usefulness of the microtransplantation approach and allow for a more precise and tailored adaptation of the implantation parameters for further studies on DA, and possibly also other neural-, glial-, and stem cell-derived grafts.

Restorative cell therapy concepts in neurodegenerative diseases are aimed at replacing lost neurons. Despite advances in research on pluripotent stem cells, fetal tissue from routine elective abortions is still regarded as the only safe cell source. Progenitor cells isolated from distinct first-trimester fetal CNS regions have already been used in clinical trials and will be used again in a new multicenter trial funded by the European Union (TRANSEURO). Bacterial contamination of human fetal tissue poses a potential risk of causing infections in the brain of the recipient. Thus, effective methods of microbial decontamination and validation of these methods are required prior to approval of a neurorestorative cell therapy trial. We have developed a protocol consisting of subsequent washing steps at different stages of tissue processing. Efficacy of microbial decontamination was assessed on rat embryonic tissue incubated with high concentrations of defined microbe solutions including representative bacterial and fungal species. Experimental microbial contamination was reduced by several log ranks. Subsequently, we have analyzed the spectrum of microbial contamination and the effect of subsequent washing steps on aborted human fetal tissue; 47.7% of the samples taken during human fetal tissue processing were positive for a microbial contamination, but after washing, no sample exhibited bacterial growth. Our data suggest that human fetal tissue for neural repair can carry microbes of various species, highlighting the need for decontamination procedures. The decontamination protocol described in this report has been shown to be effective as no microbes could be detected at the end of the procedure.

Cell replacement therapy by intracerebral transplantation of fetal dopaminergic neurons has become a promising therapeutic option for patients suffering from Parkinson's disease during the last decades. However, limited availability of human fetal tissue as well as ethical issues, lack of alternative nonfetal donor cells, and the absence of standardized transplantation protocols have prevented neurorestorative therapies from becoming a routine procedure in patients suffering from neurodegenerative diseases. Improvement of graft survival, surgery techniques, and identification of the optimal target area are imperative for further optimization of this novel treatment. In the present study, human primary fetal ventral mesencephalon-derived tissue from 7- to 9-week-old human fetuses was transplanted into 6-hydroxydopamine-lesioned adult Sprague–Dawley rats. Graft survival, fiber outgrowth, and drug-induced rotational behavior up to 14 weeks posttransplantation were compared between different intrastriatal transplantation techniques (full single cell suspension vs. partial tissue pieces suspension injected by glass capillary or metal cannula) and the intranigral glass capillary injection of a full (single cell) suspension. The results demonstrate a higher survival rate of dopamine neurons, a greater reduction in amphetamine-induced rotations (overcompensation), and more extensive fiber outgrowth for the intrastriatally transplanted partial (tissue pieces) suspension compared to all other groups. Apomorphine-induced rotational bias was significantly reduced in all groups including the intranigral group. The data confirm that human ventral mesencephalon-derived cells serve as a viable cell source, survive in a xenografting paradigm, and functionally integrate into the host tissue. In contrast to rat donor cells, keeping the original (fetal) neuronal network by preparing only a partial suspension containing tissue pieces seems to be beneficial for human cells, although a metal cannula that causes greater tissue trauma to the host is required for injection. In addition, homotopic intranigral grafts may represent a complimentary grafting approach to the “classical” ectopic intrastriatal target site in PD.

Stem cell transplantation is a promising therapeutic approach in neurodegenerative diseases. Studying graft survival and development has important implications for the further development of experimental and clinical transplantation protocols. Cellular elements in neural transplants are sometimes difficult to identify. The existing labeling methods cannot reliably provide stably labeled cells that can be detected in long-term experiments. Transgenic (tg) Lewis rats ubiquitously expressing green fluorescent protein (GFP) provide an ideal donor source. The aim of this project was to investigate the potential of GFP-tg Lewis rats to serve as donor tissue for neural stem cell transplantation. Ventral mesencephalon (VM) GFP-tg E14.5-derived cells were compared to wild-type (wt) in vitro and in vivo. Firstly, cells from GFP and non-GFP VM tissue were compared with regard to their proliferation and response towards 6-OHDA-toxicity in culture. Secondly, 6-OHDA-lesioned hemiparkinsonian Sprague–Dawley/Crl:CD(SD) rats received intrastriatal grafts derived from VM of E14.5 GFP-tg rats. Due to the fact that donor and recipient belong to two different rat strains, we focused on graft survival in correlation with immunosuppression and graft GFP and tyrosine hydroxylase (TH) expression. In summary, in vitro tg cells exhibited 98% GFP expression and did not differ from wt cells in any of the measured parameters. In vivo, all experimental groups showed a significant compensation in rotation behavior after transplantation. Furthermore, there was no difference on rotation behavior or graft morphology and survival pattern as well as GFP expression between immunosuppressed and nonimmunosuppressed animals. The GFP-positive population of the graft was composed of 13.3% GFAP-positive, 56.1% NeuN-positive, and 1.9% TH-positive cells. Analysis of graft subpopulations manifested that 70.6% of GFAP-positive, 86.9% of NeuN-positive, and 80.1% of TH-positive cells coexpressed GFP. In conclusion, our data show that the Lewis GFP-tg rats serve as an excellent cell source for studying primary neural precursor cells in the transplantation paradigm.

Embryonic striatal graft-mediated functional recovery in the rodent lesion model of Huntington's disease (HD) has been shown to correlate with the proportion of dopamine- and adenosine 3′,5′-monophosphate-regulated phosphoprotein with a molecular weight of 32 kDa (DARPP-32)-positive neurons in the graft. The current study investigated the impact of graft distribution on the yield of DARPP-32-positive cells in the grafts following either single-tract or multitract cell delivery protocols using the microtransplantation approach. Cells derived from the whole ganglionic eminence of E15 rat embryos, ubiquitously expressing green fluorescent protein (GFP), were implanted into unilaterally QA-lesioned rat striatum either as 2 × 1.8 μl macrodeposits in a single tract, or as 18 × 0.2 μl microdeposits disseminated over six needle, multitract, penetrations. For both groups, an ultrathin glass capillary with an outer diameter of 50 μm was used. Histological assessment at 4 months after transplantation showed nearly twofold increase of DARRP-32-positive striatal-like neurons in the multitract compared to the single-tract group. However, the cellular make-up of the grafts did not translate into functional differences as tested in a basic spontaneous behavior test. Furthermore, the volumetric values for overall volume, DARPP-32-positive patches, and dopaminergic projection zones were similar between both groups. The results show that distribution of fetal striatal tissue in multiple submicroliter deposits provides for an increased yield of striatal-like neurons, potentially due to the enlargement of the graft–host border area intensifying the graft's exposure to host-derived factors. Furthermore, the use of embryonic tissue from GFP donors was validated in cell-based therapy studies in the HD model.

Cell-based therapies for Parkinson's disease (PD) using neural stem cells to replace the lost dopamine neurons is currently an intense area of research. In this study we have evaluated the restorative potential of ectopic dopaminergic (DA) neurons derived from the rostral hindbrain (RH) of En1 +/Otx2lacZ transgenic mice. The genetic modification of the DA progenitor domain in the En1 +/Otx2lacZ mice is a gain of function, resulting in the enlargement of the area containing DA neurons, as well as an increase in their absolute number in the midbrain/hindbrain region. Amphetamine-induced rotation performed after cell transplantation into the unilaterally 6-hydroxydopamine-lesioned rat striatum revealed that animals with transgenic RH-derived DA grafts exhibited functional recovery similar to transgenic and wild-type ventral mesencephalon (VM)-derived DA grafts. Morphological analyses revealed equivalent numbers of surviving DA neurons from both homotopic VM- and ectopic RH-derived grafts from transgenic donors with low numbers of surviving serotonergic (5-HT) neurons. Conversely, grafts derived from wild-type donors contained predominantly surviving DA neurons or 5-HT neurons when they were prepared from the VM or RH, respectively. The study demonstrates the pattern of survival and functional potential of ectopic DA neurons derived from the RH of En1 +/Otx2lacZ transgenic mice and that cell transplantation is an important neurobiological tool to characterize newly generated DA neural stem cells in vivo.

Basic fibroblast growth factor (FGF-2) has been shown to enhance the survival and neurite extension of various types of neurons including spinal ganglion neurons. In addition, endogenous FGF-2 and FGF receptors are upregulated following peripheral nerve lesion in ganglia and at the lesion site. FGF-2 protein is expressed in different isoforms (18 kDa, 21 kDa, 23 kDa) and differentially regulated after nerve injury. In the rat we analyzed the regenerative capacity of the high molecular weight (HMW) FGF-2 isoforms (21/23 kDa) to support the regeneration of the axotomized adult sciatic nerve across long gaps. The nerve stumps were inserted into the opposite ends of a silicone chamber resulting in an interstump gap of 15 mm. Silicone tubes were filled with Matrigel or a mixture of Schwann cells (SC) and Matrigel. SC were prepared from newborn rats and transfected to overexpress HMW FGF-2. Four weeks after the operation procedure, channels were analyzed with regard to tissue cables bridging both nerve stumps and myelinated axons distal to the original proximal nerve stump. Peripheral nerves interposed with HMW Schwann cells displayed significantly enhanced nerve regeneration, with the greatest number of tissue cables containing myelinated axons and the highest number of myelinated axons. These results suggest that a cellular substrate together with a source of a trophic factor could be a promising tool to promote nerve regeneration and, therefore, become useful also for a clinical approach to repair long gaps.

In this study sensorimotor behavioral changes were monitored in rats following bilateral 6-hydroxydopamine (6-OHDA) axon terminal lesion and uni- or bilateral implantation of embryonic dopaminergic (DA) micrografts. A total of 28 μg of 6-OHDA was distributed over four injection tracts in the dorsolateral part of the caudate-putamen (CPU) bilaterally followed 4 months later by the implantation of DA micrografts spread over seven implantation tracts placed within the denervated area. Bilaterally 6-OHDA-lesioned animals exhibited significantly reduced behavioral performance scores in tests of explorational and stepping behavior as well as in skilled forelimb use. However, in contrast to the established medial forebrain bundle (MFB) lesion model of PD, these animals showed a spontaneous recovery in the side falling and skilled forelimb behavior and no deficits in overnight locomotor activity at 6 months after the lesion. Unilateral DA micrografts elicited a substantial amphetamine-induced rotational bias contralateral to the graft, but led to a significant impairment of contralateral skilled forelimb use and reduced scores in overnight locomotor activity. Bilateral DA micrografts caused a significant, though partial, increase in explorational and backhand stepping behavior, but resulted also in a significant decrease in performance levels in overnight locomotor activity and skilled forelimb use on both paws. In conclusion, DA grafts placed ectopically in the CPU in the partial lesion model of PD result in a double innervation of the GABAergic striatal neurons, arising from the residual nigrostriatal DA projections of the host and from the graft-derived DA efferent fibers. These two DA fiber systems may indeed function in a cooperative and competitive manner depending on their respective and different afferent and efferent connections, which, in turn, may lead to positive or negative influences on basal ganglia function and behavioral performances. The different patterns of 6-OHDA lesion and transplant-induced behavioral changes demonstrated in the present study compared to the “classical” MFB lesion model of PD may thus provide further insights in the complex functional organization of the basal ganglia and, thereby, may help to further optimize restorative strategies for neurodegenerative diseases, such as Parkinson's disease.