Extracellular Matrix Biomimetic Hydrogels, Encapsulated with Stromal Cell-Derived Factor 1, Improve the Composition of Foetal Tissue Grafts in a Rodent Model of Parkinson's Disease.

Clinical studies have provided evidence for dopamine (DA) cell replacement therapy in Parkinson's Disease. However, grafts derived from foetal tissue or pluripotent stem cells (PSCs) remain heterogeneous, with a high proportion of non-dopaminergic cells, and display subthreshold reinnervation of target tissues, thereby highlighting the need to identify new strategies to improve graft outcomes. In recent work, Stromal Cell-Derived Factor-1 (SDF1), secreted from meninges, has been shown to exert many roles during ventral midbrain DA development and DA-directed differentiation of PSCs. Related, co-implantation of meningeal cells has been shown to improve neural graft outcomes, however, no direct evidence for the role of SDF1 in neural grafting has been shown. Due to the rapid degradation of SDF1 protein, here, we utilised a hydrogel to entrap the protein and sustain its delivery at the transplant site to assess the impact on DA progenitor differentiation, survival and plasticity. Hydrogels were fabricated from self-assembling peptides (SAP), presenting an epitope for laminin, the brain's main extracellular matrix protein, thereby providing cell adhesive support for the grafts and additional laminin-integrin signalling to influence cell fate. We show that SDF1 functionalised SAP hydrogels resulted in larger grafts, containing more DA neurons, increased A9 DA specification (the subpopulation of DA neurons responsible for motor function) and enhanced innervation. These findings demonstrate the capacity for functionalised, tissue-specific hydrogels to improve the composition of grafts targeted for neural repair.

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.

FGF-MAPK signaling regulates human deep-layer corticogenesis.

Despite heterogeneity across the six layers of the mammalian cortex, all excitatory neurons are generated from a single founder population of neuroepithelial stem cells. However, how these progenitors alter their layer competence over time remains unknown. Here, we used human embryonic stem cell-derived cortical progenitors to examine the role of fibroblast growth factor (FGF) and Notch signaling in influencing cell fate, assessing their impact on progenitor phenotype, cell-cycle kinetics, and layer specificity. Forced early cell-cycle exit, via Notch inhibition, caused rapid, near-exclusive generation of deep-layer VI neurons. In contrast, prolonged FGF2 promoted proliferation and maintained progenitor identity, delaying laminar progression via MAPK-dependent mechanisms. Inhibiting MAPK extended cell-cycle length and led to generation of layer-V CTIP2+ neurons by repressing alternative laminar fates. Taken together, FGF/MAPK regulates the proliferative/neurogenic balance in deep-layer corticogenesis and provides a resource for generating layer-specific neurons for studying development and disease.

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.

Effects of glucose and glutamine concentrations in human dental pulp stem cells viability. An approach for cell transplantation.

PURPOSE: To evaluate microscopic behavior and viability of dental pulp stem cells under glucose and glutamine deprivation. METHODS: Human tooth tissues were minced in isolated pieces and cultured until the desired cellular proliferation for experimental phases. Cells were cultured under variations of glucose and glutamine in both serum presence and absence, and then those cells were evaluated according to number and viability by MTT assay. The confocal microscopy analyzed cytoskeleton, nucleus, and mitochondria integrity. RESULTS: A low concentration of glucose favored cellular viability and microscopic behavior; the presence of glutamine in culture medium was favorable only when associated with glucose. The cellular biological potential in culture could be preserved in serum absence if nutritional requirements are adequate. CONCLUSION: Cell microscopic behavior and viability have demonstrated better patterns on serum-free low glucose culture medium with glutamine deprivation.

Effects of glucose and glutamine concentrations in human dental pulp stem cells viability. An approach for cell transplantation

PURPOSE: To evaluate microscopic behavior and viability of dental pulp stem cells under glucose and glutamine deprivation. METHODS: Human tooth tissues were minced in isolated pieces and cultured until the desired cellular proliferation for experimental phases. Cells were cultured under variations of glucose and glutamine in both serum presence and absence, and then those cells were evaluated according to number and viability by MTT assay. The confocal microscopy analyzed cytoskeleton, nucleus, and mitochondria integrity. RESULTS: A low concentration of glucose favored cellular viability and microscopic behavior; the presence of glutamine in culture medium was favorable only when associated with glucose. The cellular biological potential in culture could be preserved in serum absence if nutritional requirements are adequate. CONCLUSION: Cell microscopic behavior and viability have demonstrated better patterns on serum-free low glucose culture medium with glutamine deprivation. .