Understanding the Influence of Target Acquisition on Survival, Integration, and Phenotypic Maturation of Dopamine Neurons within Stem Cell-Derived Neural Grafts in a Parkinson's Disease Model.

Midbrain dopaminergic (DA) neurons include many subtypes characterized by their location, connectivity and function. Surprisingly, mechanisms underpinning the specification of A9 neurons [responsible for motor function, including within ventral midbrain (VM) grafts for treating Parkinson's disease (PD)] over adjacent A10, remains largely speculated. We assessed the impact of synaptic targeting on survival, integration, and phenotype acquisition of dopaminergic neurons within VM grafts generated from fetal tissue or human pluripotent stem cells (PSCs). VM progenitors were grafted into female mice with 6OHDA-lesions of host midbrain dopamine neurons, with some animals also receiving intrastriatal quinolinic acid (QA) injections to ablate medium spiny neurons (MSN), the A9 neuron primary target. While loss of MSNs variably affected graft survival, it significantly reduced striatal yet increased cortical innervation. Consequently, grafts showed reduced A9 and increased A10 specification, with more DA neurons failing to mature into either subtype. These findings highlight the importance of target acquisition on DA subtype specification during development and repair.SIGNIFICANCE STATEMENT Parish and colleagues highlight, in a rodent model of Parkinson's disease (PD), the importance of synaptic target acquisition in the survival, integration and phenotypic specification of grafted dopamine neurons derived from fetal tissue and human stem cells. Ablation of host striatal neurons resulted in reduced dopamine neuron survival within grafts, re-routing of dopamine fibers from striatal to alternate cortical targets and a consequential reduced specification of A9 dopamine neurons (the subpopulation critical for restoration of motor function) and increase in A10 DA neurons.

Isolation of LMX1a Ventral Midbrain Progenitors Improves the Safety and Predictability of Human Pluripotent Stem Cell-Derived Neural Transplants in Parkinsonian Disease.

Human pluripotent stem cells (hPSCs) are a promising resource for the replacement of degenerated ventral midbrain dopaminergic (vmDA) neurons in Parkinson's disease. Despite recent advances in protocols for the in vitro generation of vmDA neurons, the asynchronous and heterogeneous nature of the differentiations results in transplants of surprisingly low vmDA neuron purity. As the field advances toward the clinic, it will be optimal, if not essential, to remove poorly specified and potentially proliferative cells from donor preparations to ensure safety and predictable efficacy. Here, we use two novel hPSC knock-in reporter lines expressing GFP under the LMX1A and PITX3 promoters, to selectively isolate vm progenitors and DA precursors, respectively. For each cell line, unsorted, GFP+, and GFP- cells were transplanted into male or female Parkinsonian rodents. Only rats receiving unsorted cells, LMX1A-eGFP+, or PITX3-eGFP- cell grafts showed improved motor function over 6 months. Postmortem analysis revealed small grafts from PITX3-eGFP+ cells, suggesting that these DA precursors were not compatible with cell survival and integration. In contrast, LMX1A-eGFP+ grafts were highly enriched for vmDA neurons, and importantly excluded expansive proliferative populations and serotonergic neurons. These LMX1A-eGFP+ progenitor grafts accelerated behavioral recovery and innervated developmentally appropriate forebrain targets, whereas LMX1A-eGFP- cell grafts failed to restore motor deficits, supported by increased fiber growth into nondopaminergic target nuclei. This is the first study to use an hPSC-derived reporter line to purify vm progenitors, resulting in improved safety, predictability of the graft composition, and enhanced motor function.SIGNIFICANCE STATEMENT Clinical trials have shown functional integration of transplanted fetal-derived dopamine progenitors in Parkinson's disease. Human pluripotent stem cell (hPSC)-derived midbrain progenitors are now being tested as an alternative cell source; however, despite current differentiation protocols generating >80% correctly specified cells for implantation, resultant grafts contain a small fraction of dopamine neurons. Cell-sorting approaches, to select for correctly patterned cells before implantation, are being explored yet have been suboptimal to date. This study provides the first evidence of using 2 hPSC reporter lines (LMX1A-GFP and PITX3-GFP) to isolate correctly specified cells for transplantation. We show LMX1A-GFP+, but not PITX3-GFP+, cell grafts are more predictable, with smaller grafts, enriched in dopamine neurons, showing appropriate integration and accelerated functional recovery in Parkinsonian rats.

Lessons from inducible pluripotent stem cell models on neuronal senescence in aging and neurodegeneration.

Age remains the central risk factor for many neurodegenerative diseases including Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis. Although the mechanisms of aging are complex, the age-related accumulation of senescent cells in neurodegeneration is well documented and their clearance can alleviate disease-related features in preclinical models. Senescence-like characteristics are observed in both neuronal and glial lineages, but their relative contribution to aging and neurodegeneration remains unclear. Human pluripotent stem cell-derived neurons provide an experimental model system to induce neuronal senescence. However, the extensive heterogeneity in the profile of senescent neurons and the methods to assess senescence remain major challenges. Here, we review the evidence of cellular senescence in neuronal aging and disease, discuss human pluripotent stem cell-based model systems used to investigate neuronal senescence and propose a panel of cellular and molecular hallmarks to characterize senescent neurons. Understanding the role of neuronal senescence may yield novel therapeutic opportunities in neurodegenerative disease.

Human stem cells harboring a suicide gene improve the safety and standardisation of neural transplants in Parkinsonian rats.

Despite advancements in human pluripotent stem cells (hPSCs) differentiation protocols to generate appropriate neuronal progenitors suitable for transplantation in Parkinson's disease, resultant grafts contain low proportions of dopamine neurons. Added to this is the tumorigenic risk associated with the potential presence of incompletely patterned, proliferative cells within grafts. Here, we utilised a hPSC line carrying a FailSafeTM suicide gene (thymidine kinase linked to cyclinD1) to selectively ablate proliferative cells in order to improve safety and purity of neural transplantation in a Parkinsonian model. The engineered FailSafeTM hPSCs demonstrated robust ventral midbrain specification in vitro, capable of forming neural grafts upon transplantation. Activation of the suicide gene within weeks after transplantation, by ganciclovir administration, resulted in significantly smaller grafts without affecting the total yield of dopamine neurons, their capacity to innervate the host brain or reverse motor deficits at six months in a rat Parkinsonian model. Within ganciclovir-treated grafts, other neuronal, glial and non-neural populations (including proliferative cells), were significantly reduced-cell types that may pose adverse or unknown influences on graft and host function. These findings demonstrate the capacity of a suicide gene-based system to improve both the standardisation and safety of hPSC-derived grafts in a rat model of Parkinsonism.

Viral Delivery of GDNF Promotes Functional Integration of Human Stem Cell Grafts in Parkinson's Disease.

Dopaminergic neurons (DAns), generated from human pluripotent stem cells (hPSCs), are capable of functionally integrating following transplantation and have recently advanced to clinical trials for Parkinson's disease (PD). However, pre-clinical studies have highlighted the low proportion of DAns within hPSC-derived grafts and their inferior plasticity compared to fetal tissue. Here, we examined whether delivery of a developmentally critical protein, glial cell line-derived neurotrophic factor (GDNF), could improve graft outcomes. We tracked the response of DAns implanted into either a GDNF-rich environment or after a delay in exposure. Early GDNF promoted survival and plasticity of non-DAns, leading to enhanced motor recovery in PD rats. Delayed exposure to GDNF promoted functional recovery through increases in DAn specification, DAn plasticity, and DA metabolism. Transcriptional profiling revealed a role for mitogen-activated protein kinase (MAPK)-signaling downstream of GDNF. Collectively, these results demonstrate the potential of neurotrophic gene therapy strategies to improve hPSC graft outcomes.

Transcriptional Profiling of Xenogeneic Transplants: Examining Human Pluripotent Stem Cell-Derived Grafts in the Rodent Brain.

Human pluripotent stem cells are a valuable resource for transplantation, yet our ability to profile xenografts is largely limited to low-throughput immunohistochemical analysis by difficulties in readily isolating grafts for transcriptomic and/or proteomic profiling. Here, we present a simple methodology utilizing differences in the RNA sequence between species to discriminate xenograft from host gene expression (using qPCR or RNA sequencing [RNA-seq]). To demonstrate the approach, we assessed grafts of undifferentiated human stem cells and neural progenitors in the rodent brain. Xenograft-specific qPCR provided sensitive detection of proliferative cells, and identified germ layer markers and appropriate neural maturation genes across the graft types. Xenograft-specific RNA-seq enabled profiling of the complete transcriptome and an unbiased characterization of graft composition. Such xenograft-specific profiling will be crucial for pre-clinical characterization of grafts and batch-testing of therapeutic cell preparations to ensure safety and functional predictability prior to translation.

Specification of murine ground state pluripotent stem cells to regional neuronal populations.

Pluripotent stem cells (PSCs) are a valuable tool for interrogating development, disease modelling, drug discovery and transplantation. Despite the burgeoned capability to fate restrict human PSCs to specific neural lineages, comparative protocols for mouse PSCs have not similarly advanced. Mouse protocols fail to recapitulate neural development, consequently yielding highly heterogeneous populations, yet mouse PSCs remain a valuable scientific tool as differentiation is rapid, cost effective and an extensive repertoire of transgenic lines provides an invaluable resource for understanding biology. Here we developed protocols for neural fate restriction of mouse PSCs, using knowledge of embryonic development and recent progress with human equivalents. These methodologies rely upon naïve ground-state PSCs temporarily transitioning through LIF-responsive stage prior to neural induction and rapid exposure to regional morphogens. Neural subtypes generated included those of the dorsal forebrain, ventral forebrain, ventral midbrain and hindbrain. This rapid specification, without feeder layers or embryoid-body formation, resulted in high proportions of correctly specified progenitors and neurons with robust reproducibility. These generated neural progenitors/neurons will provide a valuable resource to further understand development, as well disorders affecting specific neuronal subpopulations.