Striatal Grafts Research Articles

HESC-derived striatal progenitors grafted into a Huntington’s disease rat model support long-term functional motor recovery by differentiating, self-organizing and connecting into the lesioned striatum

BackgroundHuntington’s disease (HD) is a motor and cognitive neurodegenerative disorder due to prominent loss of striatal medium spiny neurons (MSNs). Cell replacement using human embryonic stem cells (hESCs) derivatives may offer new therapeutic opportunities to replace degenerated neurons and repair damaged circuits.MethodsWith the aim to develop effective cell replacement for HD, we assessed the long-term therapeutic value of hESC-derived striatal progenitors by grafting the cells into the striatum of a preclinical model of HD [i.e., adult immunodeficient rats in which the striatum was lesioned by monolateral injection of quinolinic acid (QA)]. We examined the survival, maturation, self-organization and integration of the graft as well as its impact on lesion-dependent motor alterations up to 6 months post-graft. Moreover, we tested whether exposing a cohort of QA-lesioned animals to environmental enrichment (EE) could improve graft integration and function.ResultsHuman striatal progenitors survived up to 6 months after transplantation and showed morphological and neurochemical features typical of human MSNs. Donor-derived interneurons were also detected. Grafts wired in both local and long-range striatal circuits, formed domains suggestive of distinct ganglionic eminence territories and displayed emerging striosome features. Moreover, over time grafts improved complex motor performances affected by QA. EE selectively increased cell differentiation into MSN phenotype and promoted host-to-graft connectivity. However, when combined to the graft, the EE paradigm used in this study was insufficient to produce an additive effect on task execution.ConclusionsThe data support the long-term therapeutic potential of ESC-derived human striatal progenitor grafts for the replacement of degenerated striatal neurons in HD and suggest that EE can effectively accelerate the maturation and promote the integration of human striatal cells.

Host-to-graft Propagation of α-synuclein in a Mouse Model of Parkinson's Disease: Intranigral Versus Intrastriatal Transplantation.

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and by the accumulation of misfolded α-synuclein (α-syn) in Lewy bodies. Ectopic transplantation of human fetal ventral mesencephalic DA neurons into the striatum of PD patients have provided proof-of-principle for the cell replacement strategy in this disorder. However, 10 to 22 y after transplantation, 1% to 27% of grafted neurons contained α-syn aggregates similar to those observed in the host brain. We hypothesized that intrastriatal grafts are more vulnerable to α-syn propagation because the striatum is not the ontogenic site of nigral DA neurons and represents an unfavorable environment for transplanted neurons. Here, we compared the long-term host-to-graft propagation of α-syn in 2 transplantation sites: the SNpc and the striatum. Two mouse models of PD were developed by injecting adeno-associated-virus2/9-human α-syn A53T into either the SNpc or the striatum of C57BL/6 mice. Mouse fetal ventral mesencephalic DA progenitors were grafted into the SNpc or into the striatum of SNpc or striatum of α-syn injected mice, respectively. First, we have shown a degeneration of the nigrostriatal pathway associated with motor deficits after nigral but not striatal adeno-associated-virus-hαsyn A53T injection. Second, human α-syn preferentially accumulates in striatal grafts compared to nigral grafts. However, no differences were observed for phosphorylated α-syn, a marker of pathological α-syn aggregates. Taken together, our results suggest that the ectopic site of the transplantation impacts the host-to-graft transmission of α-syn.

Panning for gold: Purifying mesencephalic dopaminergic progenitors differentiated from human pluripotent stem cells.

In a recently published study, Xu et al. used two surface markers, CLSTN2 and PTPRO, to generate highly purified donor dopaminergic neurons and achieved stable and predictable therapeutic outcomes by transplantation into the brain of PD animal models (Xu et al., 2022).

Is the Immunological Response a Bottleneck for Cell Therapy in Neurodegenerative Diseases?

Neurodegenerative disorders such as Parkinson’s (PD) and Huntington’s disease (HD) are characterized by a selective detrimental impact on neurons in a specific brain area. Currently, these diseases have no cures, although some promising trials of therapies that may be able to slow the loss of brain cells are underway. Cell therapy is distinguished by its potential to replace cells to compensate for those lost to the degenerative process and has shown a great potential to replace degenerated neurons in animal models and in clinical trials in PD and HD patients. Fetal-derived neural progenitor cells, embryonic stem cells or induced pluripotent stem cells are the main cell sources that have been tested in cell therapy approaches. Furthermore, new strategies are emerging, such as the use of adult stem cells, encapsulated cell lines releasing trophic factors or cell-free products, containing an enriched secretome, which have shown beneficial preclinical outcomes. One of the major challenges for these potential new treatments is to overcome the host immune response to the transplanted cells. Immune rejection can cause significant alterations in transplanted and endogenous tissue and requires immunosuppressive drugs that may produce adverse effects. T-, B-lymphocytes and microglia have been recognized as the main effectors in striatal graft rejection. This review aims to summarize the preclinical and clinical studies of cell therapies in PD and HD. In addition, the precautions and strategies to ensure the highest quality of cell grafts, the lowest risk during transplantation and the reduction of a possible immune rejection will be outlined. Altogether, the wide-ranging possibilities of advanced therapy medicinal products (ATMPs) could make therapeutic treatment of these incurable diseases possible in the near future.

L-dopa response pattern in a rat model of mild striatonigral degeneration.

BackgroundUnresponsiveness to dopaminergic therapies is a key feature in the diagnosis of multiple system atrophy (MSA) and a major unmet need in the treatment of MSA patients caused by combined striatonigral degeneration (SND). Transgenic, alpha-synuclein animal models do not recapitulate this lack of levodopa responsiveness. In order to preclinically study interventions including striatal cell grafts, models that feature SND are required. Most of the previous studies focused on extensive nigral and striatal lesions corresponding to advanced MSA-P/SND. The aim of the current study was to replicate mild stage MSA-P/SND with L-dopa failure.Methods and resultsTwo different striatal quinolinic acid (QA) lesions following a striatal 6-OHDA lesion replicating mild and severe MSA-P/SND, respectively, were investigated and compared to 6-OHDA lesioned animals. After the initial 6-OHDA lesion there was a significant improvement of motor performance after dopaminergic stimulation in the cylinder and stepping test (p<0.001). Response to L-dopa treatment declined in both MSA-P/SND groups reflecting striatal damage of lateral motor areas in contrast to the 6-OHDA only lesioned animals (p<0.01). The remaining striatal volume correlated strongly with contralateral apomorphine induced rotation behaviour and contralateral paw use during L-dopa treatment in cylinder and stepping test (p<0.001).ConclusionOur novel L-dopa response data suggest that L-dopa failure can be induced by restricted lateral striatal lesions combined with dopaminergic denervation. We propose that this sequential striatal double-lesion model replicates a mild stage of MSA-P/SND and is suitable to address neuro-regenerative therapies aimed at restoring dopaminergic responsiveness.

Quantitative in vivo dual-color bioluminescence imaging in the mouse brain.

Bioluminescence imaging (BLI) is an optical imaging method that can be translated from the cell culture dish in vitro to cell tracking in small animal models in vivo. In contrast to the more widely used fluorescence imaging, which requires light excitation, in BLI the light is exclusively generated by the enzyme luciferase. The luciferase gene can be engineered to target and monitor almost every cell and biological process quantitatively in vitro and even from deep tissue in vivo. While initially used for tumor imaging, bioluminescence was recently optimized for mouse brain imaging of neural cells and monitoring of viability or differentiation of grafted stem cells. Here, we describe the use of bright color-shifted firefly luciferases (Flucs) based on the thermostable x5 Fluc that emit red and green for effective and quantitative unmixing of two human cell populations in vitro and after transplantation into the mouse brain in vivo. Spectral unmixing predicts the ratio of luciferases in vitro and a mixture of cells precisely for cortical grafts, however, with less accuracy for striatal grafts. This dual-color approach enables the simultaneous visualization and quantification of two cell populations on the whole brain scale, with particular relevance for translational studies of neurological disorders providing information on stem cell survival and differentiation in one imaging session in vivo.

Brain Region-Dependent Rejection of Neural Precursor Cell Transplants.

The concept of CNS as an immune-privileged site has been challenged by the occurrence of immune surveillance and allogeneic graft rejection in the brain. Here we examined whether the immune response to allogeneic neural grafts is determined by the site of implantation in the CNS. Dramatic regional differences were observed between immune responses to allogeneic neural precursor/stem cell (NPC) grafts in the striatum vs. the hippocampus. Striatal grafts were heavily infiltrated with IBA-1+ microglia/macrophages and CD3+ T cells and completely rejected. In contrast, hippocampal grafts exhibited milder IBA-1+ cell infiltration, were not penetrated efficiently by CD3+ cells, and survived efficiently for at least 2 months. To evaluate whether the hippocampal protective effect is universal, astrocytes were then transplanted. Allogeneic astrocyte grafts elicited a vigorous rejection process from the hippocampus. CD200, a major immune-inhibitory signal, plays an important role in protecting grafts from rejection. Indeed, CD200 knock out NPC grafts were rejected more efficiently than wild type NPCs from the striatum. However, lack of CD200 expression did not elicit NPC graft rejection from the hippocampus. In conclusion, the hippocampus has partial immune-privilege properties that are restricted to NPCs and are CD200-independent. The unique hippocampal milieu may be protective for allogeneic NPC grafts, through host-graft interactions enabling sustained immune-regulatory properties of transplanted NPCs. These findings have implications for providing adequate immunosuppression in clinical translation of cell therapy.

The Effect of Tissue Preparation and Donor Age on Striatal Graft Morphology in the Mouse

Huntington's disease (HD) is a progressive neurodegenerative disease in which striatal medium spiny neurons (MSNs) are lost. Neuronal replacement therapies aim to replace MSNs through striatal transplantation of donor MSN progenitors, which successfully improve HD-like deficits in rat HD models and have provided functional improvement in patients. Transplants in mouse models of HD are more variable and have lower cell survival than equivalent rat grafts, yet mice constitute the majority of transgenic HD models. Improving the quality and consistency of mouse transplants would open up access to this wider range of rodent models and facilitate research to increase understanding of graft mechanisms, which is essential to progress transplantation as a therapy for HD. Here we determined how donor age, cell preparation, and donor/host strain choice influenced the quality of primary embryonic grafts in quinolinic acid lesion mouse models of HD. Both a within-strain (W-S) and a between-strain (B-S) donor/host paradigm were used to compare transplants of donor tissues derived from mice at embryonic day E12 and E14 prepared either as dissociated suspensions or as minimally manipulated tissue pieces (TP). Good graft survival was observed, although graft volume and cellular composition were highly variable. The effect of cell preparation on grafts differed significantly depending on donor age, with E14 cell suspensions yielding larger grafts compared to TP. Conversely, TP were more effective when derived from E12 donor tissue. A W-S model produced larger grafts with greater MSN content, and while high levels of activated microglia were observed across all groups, a greater number was found in B-S transplants. In summary, we show that the effect of tissue preparation on graft morphology is contingent on the age of donor tissue used. The presence of microglial activation in all groups highlights the host immune response as an important consideration in mouse transplantation.

Striatal Transplantation of Human Dopaminergic Neurons Differentiated From Induced Pluripotent Stem Cells Derived From Umbilical Cord Blood Using Lentiviral Reprogramming.

Human induced pluripotent stem cells (hiPSCs) are promising sources for regenerative therapies like the replacement of dopaminergic neurons in Parkinson's disease. They offer an unlimited cell source that can be standardized and optimized to produce applicable cell populations to gain maximal functional recovery. In the present study, human cord blood-derived iPSCs (hCBiPSCs) were differentiated into dopaminergic neurons utilizing two different in vitro protocols for neural induction: (protocol I) by fibroblast growth factor (FGF-2) signaling, (protocol II) by bone morphogenetic protein (BMP)/transforming growth factor (TGF-β) inhibition. After maturation, in vitro increased numbers of tyrosine hydroxylase (TH)-positive neurons (7.4% of total cells) were observed by protocol II compared to 3.5% in protocol I. Furthermore, 3 weeks after transplantation in hemiparkinsonian rats in vivo, a reduced number of undifferentiated proliferating cells was achieved with protocol II. In contrast, proliferation still occurred in protocol I-derived grafts, resulting in tumor-like growth in two out of four animals 3 weeks after transplantation. Protocol II, however, did not increase the number of TH(+) cells in the striatal grafts of hemiparkinsonian rats. In conclusion, BMP/TGF-β inhibition was more effective than FGF-2 signaling with regard to dopaminergic induction of hCBiPSCs in vitro and prevented graft overgrowth in vivo.

Donor-Specific Anti-HLA Antibodies in Huntington's Disease Recipients of Human Fetal Striatal Grafts

Fetal grafting in a human diseased brain was thought to be less immunogenic than other solid organ transplants, hence the minor impact on the efficacy of the transplant. How much prophylactic immune protection is required for neural allotransplantation is also debated. High-sensitive anti-HLA antibody screening in this field has never been reported. Sixteen patients with Huntington's disease underwent human fetal striatal transplantation in the frame of an open-label observational trial, which is being carried out at Florence University. All patients had both brain hemispheres grafted in two separate robotic-stereotactic procedures. The trial started in February 2006 with the first graft to the first patient (R1). R16 was given his second graft on March 2011. All patients received triple immunosuppressive treatment. Pre- and posttransplant sera were analyzed for the presence of anti-HLA antibodies using the multiplexed microsphere-based suspension array Luminex xMAP technology. Median follow-up was 38.5 months (range 13-85). Six patients developed anti-HLA antibodies, which turned out to be donor specific. Alloimmunization occurred in a time window of 0-49 months after the first neurosurgical procedure. The immunogenic determinants were non-self-epitopes from mismatched HLA antigens. These determinants were both public epitopes shared by two or more HLA molecules and private epitopes unique to individual HLA molecules. One patient had non-donor-specific anti-HLA antibodies in her pretransplant serum sample, possibly due to previous sensitization events. Although the clinical significance of donor-specific antibodies is far from being established, particularly in the setting of neuronal transplantation, these findings underline the need of careful pre- and posttransplant immunogenetic evaluation of patients with intracerebral grafts.

Differentiation of pluripotent stem cells into striatal projection neurons: a pure MSN fate may not be sufficient.

Huntington’s disease (HD) is an autosomal dominant inherited disorder leading to the loss inter alia of DARPP-32 positive medium spiny projection neurons (“MSNs”) in the striatum. There is no known cure for HD but the relative specificity of cell loss early in the disease has made cell replacement by neural transplantation an attractive therapeutic possibility. Transplantation of human fetal striatal precursor cells has shown “proof-of-principle” in clinical trials; however, the practical and ethical difficulties associated with sourcing fetal tissues have stimulated the need to identify alternative source(s) of donor cells that are more readily available and more suitable for standardization. We now have available the first generation of protocols to generate DARPP-32 positive MSN-like neurons from pluripotent stem cells and these have been successfully grafted into animal models of HD. However, whether these grafts can provide stable functional recovery to the level that can regularly be achieved with primary fetal striatal grafts remains to be demonstrated. Of particular concern, primary fetal striatal grafts are not homogenous; they contain not only the MSN subpopulation of striatal projection neurons but also include all the different cell types that make up the mature striatum, such as the multiple populations of striatal interneurons and striatal glia, and which certainly contribute to normal striatal function. By contrast, present protocols for pluripotent stem cell differentiation are almost entirely targeted at specifying just neurons of an MSN lineage. So far, evidence for the functionality and integration of stem-cell derived grafts is correspondingly limited. Indeed, consideration of the features of full striatal reconstruction that is achieved with primary fetal striatal grafts suggests that optimal success of the next generations of stem cell-derived replacement therapy in HD will require that graft protocols be developed to allow inclusion of multiple striatal cell types, such as interneurons and/or glia. Almost certainly, therefore, more sophisticated differentiation protocols will be necessary, over and above replacement of a specific population of MSNs. A rational solution to this technical challenge requires that we re-address the underlying question—what constitutes a functional striatal graft?

Single-Cell Suspension Methodology Favors Survival and Vascularization of Fetal Striatal Grafts in the YAC128 Mouse Model of Huntington's Disease

Cell replacement therapies have yielded variable and short-lived benefits in Huntington's disease (HD) patients. This suboptimal outcome is likely due to the fact that graft survival is compromised long term because grafts are subjected to a host's microglial inflammatory response, to a lack of adequate trophic support, and possibly to cortical excitotoxicity. However, graft demise may also relate to more straightforward issues such as cell preparation methodology (solid grafts vs. cell suspension). Indeed, we recently reported that solid grafts are poorly revascularized in HD patients transplanted 9 and 12 years previously. To evaluate whether methodological issues relating to cell preparation may have an impact on graft viability, we implanted green fluorescent protein (GFP(+)) single-cell suspensions of fetal striatal neuronal cells into the striatum of YAC128 HD mice. Postmortem evaluation yielded comparable graft survival in YAC128 mice and their wild-type littermates (noncarrier) at 1 and 3 months posttransplantation. Additionally, the degrees of graft revascularization in the YAC128 and noncarrier mice were similar, with both capillaries and large-caliber vessels observable within the grafted tissue. Furthermore, GFP(+) cells interacted well with host blood vessels, indicating integration of the donor cells within the recipient brain. These observations, combined with our recent report of poor revascularization of solid grafts in the HD-transplanted patients, suggest that the success of cell transplantation can be improved by optimizing methodological aspects relating to cell preparation.

Multifaceted roles of BDNF and FGF2 in human striatal primordium development. An in vitro study

Grafting fetal striatal cells into the brain of Huntington's disease (HD) patients has raised certain expectations in the past decade as an effective cell-based-therapy for this devastating condition. We argue that the first requirement for successful transplantation is defining the window of plasticity for the striatum during development when the progenitor cells, isolated from their environment, are able to maintain regional-specific-identity and to respond appropriately to cues. The primary cell culture from human fetal striatal primordium described here consists of a mixed population of neural stem cells, neuronal-restricted progenitors and striatal neurons. These cells express trophic factors, such as BDNF and FGF2. We show that these neurotrophins maintain cell plasticity, inducing the expression of neuronal precursor markers and cell adhesion molecules, as well as promoting neurogenesis, migration and survival. We propose that BDNF and FGF2 play an important autocrine–paracrine role during early striatum development in vivo and that their release by fetal striatal grafts may be relevant in the setting of HD cell therapy.

Subthalamic nucleus lesion improves cell survival and functional recovery following dopaminergic cell transplantation in parkinsonian rats

Subthalamic nucleus (STN) modulation is currently the gold standard in the treatment of Parkinson's disease (PD) cases refractory to medication. Cell transplantation is a tissue-restorative approach and is a promising strategy in the treatment of PD. One of the obstacles to overcome in cell therapy is the poor dopaminergic cell survival. Our experiment investigates the impact of a partial subthalamotomy prior to ventral mesencephalic (VM) embryonic cell transplantation on dopaminergic cell survival and functional outcome. Unilateral dopamine depletion was carried out in rats, via medial forebrain bundle (MFB) injection of 6-hydroxydopamine, and half of the animals went on to receive unilateral excitotoxic lesions of the STN/Zone Incerta (ZI) causing partial lesion of these structures on the same side as the MFB lesion. All MFB-lesioned animals, with or without the STN/ZI lesion, received striatal ipsilateral embryonic VM cell grafts. The data suggest that the STN/ZI lesion could boost the dopamine cell survival in the grafts by 2.6-fold compared with the control grafted-only group. Moreover, performance on the drug-induced rotation and the spontaneous behavior tests were ameliorated on the STN/ZI-lesioned group to a significantly greater extent than the grafted-only group. These data suggest that the STN/ZI partial lesion optimized the striatal environment, promoting an improvement in cell survival. Further studies are needed to see whether the synergy between STN modulation via deep brain stimulation and cell therapy might have clinical applications in the management of PD.

Primary cell culture from human striatal primordium. Contribution to research on neuronal plasticity

Throughout fetal life, striatal neurons originated in the ganglionic eminence from a population of dividing stem cells. Little is known about the molecular mechanisms that regulate the activation, self-renewal and differentiation of striatal neuronal precursors. In order to identify the regulatory mechanisms controlling striatal cell neurogenesis and differentiation, we have recently isolated and propagated in vitro primary cell cultures from the human fetal striatal primordium (1). These cells express both neuronal and striatal properties, and are responsive to BDNF and FGF2. In this study, we found that human striatal precursor (HSP) cells are a mixed population mainly constituted of neuronal-restricted progenitors and striatal neurons (DARPP32-, GAD1-expressing cells) and neural/stem cells, which under specific in vitro differentiating conditions not only generate neurons, astrocytes and oligodendrocytes, but also possess the ability of osteogenic differentiation. We also observed that BDNF and FGF2 exert different effects on HSP depending on the differentiation state of these cells. In fact, both neurotrophins promote cell proliferation, migration and the expression of neural stem/progenitor markers in undifferentiated HSP cells, while they stimulate neuritogenesis in the neuronal differentiated component as demonstrated after specific neuronal induction. We have previously reported that striatal primordium from human fetus was able to grow into the brain of Huntington’s disease (HD) patients and that this process was associated with metabolic change and some clinical benefit (2,3). Our results add new insight into the developmental processes of human fetal striatal grafts in HD and, in addition, have implications for cell based transplantation approaches in the CNS.

Large-sized Fetal Striatal Grafts in Huntington's Disease Do Stop Growing: Long-term Monitoring in the Florence Experience.

Development of six large nodules of solid tissue after bilateral human fetal striatal transplantation in four Huntington’s disease patients has raised concern about the safety of this experimental therapy in our setting. We investigated by serial MRI-based volumetric analysis the growth behaviour of such grafts. After 33-73 months from transplantation the size of five grafts was stable and one graft showed a mild decrease in size. Signs neither of intracranial hypertension nor of adjuctive focal neurological deficit have ever been observed. This supports long-term safety of the grafting procedure at our Institution.

Fetal striatal grafting slows motor and cognitive decline of Huntington's disease

ObjectiveTo assess the clinical effect of caudate-putaminal transplantation of fetal striatal tissue in Huntington's disease (HD).MethodsWe carried out a follow-up study on 10 HD transplanted patients and 16 HD not-transplanted...

Engraftment of Mouse Embryonic Stem Cells Differentiated by Default Leads to Neuroprotection, Behaviour Revival and Astrogliosis in Parkinsonian Rats

We report here protection against rotenone-induced behavioural dysfunction, striatal dopamine depletion and nigral neuronal loss, following intra-striatal transplantation of neurons differentiated from murine embryonic stem cells (mES). mES maintained in serum free medium exhibited increase in neuronal, and decrease in stem cell markers by 7th and 10th days as revealed by RT-PCR and immunoblot analyses. Tyrosine hydroxylase, NURR1, PITX3, LMX1b and c-RET mRNA showed a significant higher expression in differentiated cells than in mES. Dopamine level was increased by 3-fold on 10th day as compared to 7 days differentiated cells. Severity of rotenone-induced striatal dopamine loss was attenuated, and amphetamine-induced unilateral rotations were significantly reduced in animals transplanted with 7 days differentiated cells, but not in animals that received undifferentiated ES transplant. However, the ratio of contralateral to ipsilateral swings in elevated body swing test was significantly reduced in both the transplanted groups, as compared to control. Striatal grafts exhibited the presence of tyrosine hydroxylase positive cells, and the percentage of dopaminergic neurons in the substantia nigra was also found to be higher in the ipsilateral side of 7 days and mES grafted animals. Increased expression of CD11b and IBA-1, suggested a significant contribution of these microglia-derived factors in controlling the limited survival of the grafted cells. Astrocytosis in the grafted striatum, and significant increase in the levels of glial cell line derived neurotrophic factor may have contributed to the recovery observed in the hemiparkinsonian rats following transplantation.

The long-term safety and efficacy of bilateral transplantation of human fetal striatal tissue in patients with mild to moderate Huntington's disease

Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disease involving progressive motor, cognitive and behavioural decline, leading to death approximately 20 years after motor onset. The disease is characterised...

Q11 Huntington's disease Italian trial in neuro-transplantation: update on 17 patients group

IntroductionEmbryonic striatal cells grafts transplanted into rat striatum have been shown to be effective in reversing behavioural deficits, including motor and cognitive impairments in Huntington's disease (HD) rat models. For...