Ventral mesencephalic dopaminergic (vmDA) precursors derived from human embryonic stem cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE171769 | GEO | 2021/07/19
REPOSITORIES: GEO
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Project description:Ventral mesencephalic dopaminergic (vmDA) precursors derived from human embryonic stem cells
| PRJNA720859 | ENA
Project description:Successful cell therapy for Parkinson's disease (PD) requires large numbers of homogeneous ventral mesencephalic dopaminergic (vmDA) precursors. Enrichment of vmDA precursors via cell sorting is required to ensure high safety and efficacy of the cell therapy. Here, using LMX1A-eGFP knock-in reporter human embryonic stem cells, we discovered a novel surface antigen, trophoblast glycoprotein (TPBG), which was preferentially expressed in vmDA precursors. TPBG-targeted cell sorting enriched FOXA2+LMX1A+ vmDA precursors and helped attain efficient behavioral recovery of rodent PD models with increased numbers of TH+, NURR1+, and PITX3+ vmDA neurons in the grafts. Additionally, fewer proliferating cells were detected in TPBG+ cell-derived grafts than in TPBG- cell-derived grafts. Our approach is an efficient way to obtain enriched bona fide vmDA precursors, which could open a new avenue for effective PD treatment.
| S-EPMC8289854 | biostudies-literature
Project description:Despite the progress in safety and efficacy of cell therapy with pluripotent stem cells (PSCs), the presence of residual undifferentiated stem cells or proliferating neural progenitor cells (NPCs) with rostral identity has remained a major challenge. Here we reported the generation of an LMX1A knock-in GFP reporter human embryonic stem cell (hESC) line that marks the early dopaminergic progenitors during neural differentiation. Purified GFP positive cells in vitro exhibited expression of mRNA and proteins that characterized and matched the midbrain dopaminergic identity. Further proteomic analysis of enriched LMX1A+ cells identified several membrane associated proteins including CNTN2, enabling prospective isolation of LMX1A+ progenitor cells. Transplantation of hPSC-derived purified CNTN2+ progenitors enhanced dopamine release from transplanted cells in the host brain and alleviated Parkinson’s disease symptoms in animal models. Our study establishes an efficient approach for purification of large numbers of hPSC-derived dopaminergic progenitors for therapeutic applications.
2018-06-06 | PXD007837 | Pride
Project description:PurposePhotoreceptor precursor cells (PRPs) differentiated from human embryonic stem cells can serve as a source for cell replacement therapy aimed at vision restoration in patients suffering from degenerative diseases of the outer retina, such as retinitis pigmentosa and AMD. In this work, we studied the electrophysiologic maturation of PRPs throughout the differentiation process.MethodsHuman embryonic stem cells were differentiated into PRPs and whole-cell recordings were performed for electrophysiologic characterization at days 0, 30, 60, and 90 along with quantitative PCR analysis to characterize the expression level of various ion channels, which shape the electrophysiologic response. Finally, to characterize the electrically induced calcium currents, we employed calcium imaging (rhod4) to visualize intracellular calcium dynamics in response to electrical activation.ResultsOur results revealed an early and steady presence (approximately 100% of responsive cells) of the delayed potassium rectifier current. In contrast, the percentage of cells exhibiting voltage-gated sodium currents increased with maturation (from 0% to almost 90% of responsive cells at 90 days). Moreover, calcium imaging revealed the presence of voltage-gated calcium currents, which play a major role in vision formation. These results were further supported by quantitative PCR analysis, which revealed a significant and continuous (3- to 50-fold) increase in the expression of various voltage-gated channels concomitantly with the increase in the expression of the photoreceptor marker CRX.ConclusionsThese results can shed light on the electrophysiologic maturation of neurons in general and PRP in particular and can form the basis for devising and optimizing cell replacement-based vision restoration strategies.
| S-EPMC7533729 | biostudies-literature
Project description:Embryonic stem cells (ESCs) represent a promising source of midbrain dopaminergic (DA) neurons for applications in Parkinson disease. However, ESC-based transplantation paradigms carry a risk of introducing inappropriate or tumorigenic cells. Cell purification before transplantation may alleviate these concerns and enable identification of the specific DA neuron stage most suitable for cell therapy. Here, we used 3 transgenic mouse ESC reporter lines to mark DA neurons at 3 stages of differentiation (early, middle, and late) following induction of differentiation using Hes5::GFP, Nurr1::GFP, and Pitx3::YFP transgenes, respectively. Transplantation of FACS-purified cells from each line resulted in DA neuron engraftment, with the mid-stage and late-stage neuron grafts being composed almost exclusively of midbrain DA neurons. Mid-stage neuron cell grafts had the greatest amount of DA neuron survival and robustly induced recovery of motor deficits in hemiparkinsonian mice. Our data suggest that the Nurr1+ stage (middle stage) of neuronal differentiation is particularly suitable for grafting ESC-derived DA neurons. Moreover, global transcriptome analysis of progeny from each of the ESC reporter lines revealed expression of known midbrain DA neuron genes and also uncovered previously uncharacterized midbrain genes. These data demonstrate remarkable fate specificity of ESC-derived DA neurons and outline a sequential stage-specific ESC reporter line paradigm for in vivo gene discovery.
| S-EPMC3408729 | biostudies-literature
Project description:Retinal degeneration is a leading cause of irreversible blindness in the developed world. Differentiation of retinal cells, including photoreceptors, from both mouse and human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), potentially provide a renewable source of cells for retinal transplantation. Previously, we have shown both the functional integration of transplanted rod photoreceptor precursors, isolated from the postnatal retina, in the adult murine retina, and photoreceptor cell generation by stepwise treatment of ESCs with defined factors. In this study, we assessed the extent to which this protocol recapitulates retinal development and also evaluated differentiation and integration of ESC-derived retinal cells following transplantation using our established procedures. Optimized retinal differentiation via isolation of Rax.GFP retinal progenitors recreated a retinal niche and increased the yield of Crx(+) and Rhodopsin(+) photoreceptors. Rod birth peaked at day 20 of culture and expression of the early photoreceptor markers Crx and Nrl increased until day 28. Nrl levels were low in ESC-derived populations compared with developing retinae. Transplantation of early stage retinal cultures produced large tumors, which were avoided by prolonged retinal differentiation (up to day 28) prior to transplantation. Integrated mature photoreceptors were not observed in the adult retina, even when more than 60% of transplanted ESC-derived cells expressed Crx. We conclude that exclusion of proliferative cells from ESC-derived cultures is essential for effective transplantation. Despite showing expression profiles characteristic of immature photoreceptors, the ESC-derived precursors generated using this protocol did not display transplantation competence equivalent to precursors from the postnatal retina.
| S-EPMC3580313 | biostudies-literature
Project description:We show that human embryonic stem cell-derived dopaminergic neurons survived transplantation to the neurotoxin 6-hydroxydopamine-lesioned rat striatum and, in combination with the cells newly differentiated from their progenitors, contributed to locomotive function recovery at 5 months. The animal behavioral improvement was correlated with the dopamine neurons present in the graft. Although the donor cells contained forebrain and midbrain dopamine neurons, the dopamine neurons present in the graft mainly exhibited a midbrain, or nigra, phenotype, suggesting the importance of midbrain dopamine neurons in functional repair. Furthermore, progenies of grafted cells were neurons and glia with greatly diminished mitotic activity by 5 months. Thus, the in vitro-produced human dopamine neurons can functionally engraft in the brain.
| S-EPMC2707927 | biostudies-literature
Project description:Evidence from carefully conducted open label clinical trials suggested that therapeutic benefit can be achieved by grafting fetal dopaminergic (DAergic) neurons derived from ventral mesencephalon (VM) into the denervated striatum of Parkinson's disease (PD) patients. However, two double-blind trials generated negative results reporting deleterious side effects such as prominent dyskinesias. Heterogeneous composition of VM grafts is likely to account for suboptimal clinical efficacy.We consider that gene expression patterns of the VM tissue needs to be better understood by comparing the genetic signature of the surviving and functioning grafts with the cell suspensions used for transplantation. In addition, it is crucial to assess whether the grafted cells exhibit the DAergic phenotype of adult substantia nigra pars compacta (SNpc). To investigate this further, we used a GFP reporter mouse as source of VM tissue that enabled the detection and dissection of the grafts 6 weeks post implantation. A comparative gene expression analysis of the VM cell suspension and grafts revealed that VM grafts continue to differentiate post-implantation. In addition, implanted grafts showed a mature SNpc-like molecular DAergic phenotype with similar expression levels of TH, Vmat2 and Dat. However, by comparing gene expression of the adult SNpc with dissected grafts we detected a higher expression of progenitor markers in the grafts. Finally, when compared to the VM cell suspension, post-grafting there was a higher expression of markers inherent to glia and other neuronal populations.In summary, our data highlight the dynamic development of distinctive DAergic and non-DAergic gene expression markers associated with the maturation of VM grafts in vivo. The molecular signature of VM grafts and its functional relevance should be further explored in future studies aimed at the optimization of DAergic cell therapy approaches in PD.
| S-EPMC3510255 | biostudies-literature
Project description:Current differentiation protocols for generating mesencephalic dopaminergic (mesDA) neurons from human pluripotent stem cells result in grafts containing only a small proportion of mesDA neurons when transplanted in vivo. In this study, we develop lineage-restricted undifferentiated stem cells (LR-USCs) from pluripotent stem cells, which enhances their potential for differentiating into caudal midbrain floor plate progenitors and mesDA neurons. Using a ventral midbrain protocol, 69% of LR-USCs become bona fide caudal midbrain floor plate progenitors, compared to only 25% of human embryonic stem cells (hESCs). Importantly, LR-USCs generate significantly more mesDA neurons under midbrain and hindbrain conditions in vitro and in vivo. We demonstrate that midbrain-patterned LR-USC progenitors transplanted into 6-hydroxydopamine-lesioned rats restore function in a clinically relevant non-pharmacological behavioral test, whereas midbrain-patterned hESC-derived progenitors do not. This strategy demonstrates how lineage restriction can prevent the development of undesirable lineages and enhance the conditions necessary for mesDA neuron generation.
| S-EPMC10698156 | biostudies-literature
Project description:Human embryonic stem cells have been a great interest due to its potential to provide a renewable source of surrogate dopamine neurons for transplantation into ParkinsonM-bM-^@M-^Ys disease patients. We have differentiated hESCs with a monolayer of PA6 cells under 2D and 3D systems for 28 days. Genome-wide microarray analysis showed that our 3D samples increased expression of genes that are involved in neuronal developments such as Wnt, hedgehog and mitogen-activated protein kinase (MAPK) signalling pathways. The results suggest that the 3D differentiation system may have affected the regulatory or signalling mechanisms which enhanced the rate of differentiation towards ectoderm. 3 biological replicates for 2D samples and 2 biological replicates for 3D samples.
2013-01-03 | E-GEOD-43235 | biostudies-arrayexpress