Biphasic activation of WNT signaling enhances the derivation of midbrain dopamine neurons for translational use [ChIP-seq]
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE162882 | GEO | 2020/12/18
REPOSITORIES: GEO
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Project description:We report a novel midbrain dopamine neuron derivation protocol from human pluripotent stem cells (hPSC)s based on a two-step WNT signaling activation strategy. By comparing next-generation sequencing (NGS) derived transcriptome profiling (RNA-seq) of dopamine neuron differentiated cells derived from hPSC with different concentration of Chir-boosting (day4 – day9) measured at day 11 including hPSC, we find that CHIR-boosting improves expression of midbrain markers such as EN1 while minimizing expression of contaminating posterior (hindbrain) and anterior (diencephalic) lineage markers. Furthermore, gene expression analysis from RNA-seq study confirmed the progressive differentiation and maturation of Boost CHIR treated precursors into mDA neurons by day 30 of differentiation. The protocol presented here is the basis for clinical grade dopamine neuron production and preclinical safety and efficacy studies.
2020-12-18 | GSE162883 | GEO
Project description:Human pluripotent stem cells (hPSCs) are a promising source of cells for applications in regenerative medicine. Directed differentiation of hPSCs into specialized cells such as spinal motoneurons or midbrain dopamine (DA) neurons has been achieved. However the effective use of hPSCs for cell therapy has lagged far behind. While mouse PSC-derived DA neurons have shown efficacy in models of Parkinson’s disease, DA neurons derived from human PSCs generally display poor in vivo performance. There are also considerable safety concerns for hPSCs related to their potential for teratoma formation or neural overgrowth. Here we present a novel floor plate-based strategy for the derivation of human DA neurons that efficiently engraft, suggesting that past failures were due to incomplete specification rather than a specific vulnerability of the cells. Midbrain floor plate precursors are derived from hPSCs in days following exposure to small molecule activators of sonic hedgehog (SHH) and canonical WNT signaling. Engraftable midbrain DA neurons are obtained by day 25 and can be maintained in vitro for several months. Extensive in vitro molecular profiling, biochemical and electrophysiological data define developmental progression and confirm identity of hPSC-derived midbrain DA neurons. In vivo survival and function is demonstrated in PD animal models in three host species. Long-term engraftment in 6-OHDA-lesioned mouse and rats demonstrates robust survival of midbrain DA neurons, complete restoration of amphetamine-induced rotation behavior and improvements in tests of forelimb use and akinesia. Finally, scalability is demonstrated by transplantation into Parkinsonian monkeys. Excellent DA neuron survival, function and lack of neural overgrowth in the three animal models tested indicate considerable promise for the development of cell based therapies in PD. Differentiated hESC with three conditions (LSB, LSB/S/F8, LSB/S/F8/CHIR) were subjected to RNA extraction in specific timepoint (day 0, 1, 3, 5, 7, 11, 13, 25) and hybridization on Illumina microarrays. Each sample has 3 or 4 biological repeats. Based on previous study* of dual SMAD inhibition neural induction, we developed new midbrain dopamine neuron protocol. It depends on time specific treatment of below factors (LSB/S/F8/CHIR): L (LDN193189 (BMP inhibitor) , day 0-11), SB (SB431542 (TGF-b signal inhibitor), day 0-5), S (SHH + Purmorphamine (Smo agonist), day 1-7), F8 (FGF8, day 1-7) and CHIR (CHIR99021 (GSK3b inhibitor), day 3-13) LSB and LSB/S/F8 are limited control conditions of dual SMAD only (LSB) or traditional patterning with Sonic and FGF (LSB/S/F8) *Chambers,S.M. et al. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat. Biotechnol. 27, 275-280 (2009).
2011-11-07 | E-GEOD-32658 | biostudies-arrayexpress
Project description:Here, we show that midbrain dopamine (DA) neurons derived from human pluripotent stem cells (hPSCs) are selectively permissive to SARS-CoV-2 infection both in vitro and upon transplantation in vivo, and that SARS-CoV-2 infection triggers a DA neuron inflammatory and cellular senescence response. A high-throughput screen in hPSC-derived DA neurons identified several FDA approved drugs, including riluzole, metformin, and imatinib, that can rescue the cellular senescence phenotype and prevent SARS-CoV-2 infection. RNA-seq analysis of human ventral midbrain tissue from COVID-19 patients, using formalin-fixed paraffin-embedded autopsy samples, confirmed the induction of an inflammatory and cellular senescence signature and identified low levels of SARS-CoV-2 transcripts. Our findings demonstrate that hPSC-derived DA neurons can serve as a disease model to study neuronal susceptibility to SARS-CoV-2 and to identify candidate neuroprotective drugs for COVID-19 patients. The susceptibility of hPSC-derived DA neurons to SARS-CoV-2 and the observed inflammatory and senescence transcriptional responses suggest the need for careful, long-term monitoring of neurological problems in COVID-19 patients.
2021-10-20 | GSE174745 | GEO
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:FACS purified cells from differentiation day 14-15 cells from 3 BAC transgenic mESC lines: Hes::GFP (early), Nurr1::GFP (mid), and Pitx3::YFP (late) DA neuron development reporter lines All three lines were differentiated towards the midbrain dopamine phenotype, and FACS purification was performed at D14-15, and then subject to global transcriptome analysis
2012-07-11 | E-GEOD-37446 | biostudies-arrayexpress
Project description:COVID-19 patients commonly present with neurological signs of central nervous system (CNS) and/or peripheral nervous system dysfunction. However, which neural cells are permissive to infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been controversial. Here, we show that midbrain dopamine (DA) neurons derived from human pluripotent stem cells (hPSCs) are selectively susceptible and permissive to SARS-CoV-2 infection, and that SARS-CoV-2 infection triggers a DA neuron inflammatory and cellular senescence response. A high-throughput screen in hPSC-derived DA neurons identified several FDA approved drugs, including riluzole, metformin, and imatinib, that can rescue the cellular senescence phenotype by preventing SARS-CoV-2 infection. RNA-seq analysis of human ventral midbrain tissue from COVID-19 patients, using formalin-fixed paraffin-embedded autopsy samples, confirmed the induction of an inflammatory and cellular senescence signature and identified low levels of SARS-CoV-2 transcripts. Our findings demonstrate that hPSC-derived DA neurons can serve as a disease model to study neuronal susceptibility to SARS-CoV-2 and to identify candidate neuroprotective drugs for COVID-19 patients. The susceptibility of hPSC-derived DA neurons to SARS-CoV-2 and the observed inflammatory and senescence transcriptional responses suggest the need for careful, long-term monitoring of neurological problems in COVID-19 patients.
2023-12-04 | GSE248989 | GEO
Project description:Human pluripotent stem cell (hPSC)-based replacement therapy holds great promise in treating Parkinson’s disease (PD). However, the heterogeneity of hPSC-derived donor cells and the low yield of midbrain dopaminergic (mDA) neurons after transplantation hinder its broad clinical application. Here, we depicted the single-cell molecular landscape during mDA neuron differentiation. We found that this process recapitulated the development of multiple but adjacent fetal brain regions including ventral midbrain, isthmus, and ventral hindbrain, resulting in heterogenous donor cell population. We reconstructed the differentiation trajectory of mDA lineage and identified CLSTN2 and PTPRO as specific surface markers of mDA progenitors, which were predictive of mDA neuron differentiation and could facilitate highly enriched mDA neurons (up to 80%) following progenitor sorting and transplantation. Marker sorted progenitors exhibited higher therapeutic potency in correcting motor deficits of PD mice. Different marker sorted grafts had a strikingly consistent cellular composition, in which mDA neurons were enriched, while off-target neuron types were mostly depleted, suggesting stable graft outcomes. Our study provides a better understanding of cellular heterogeneity during mDA neuron differentiation, and establishes a strategy to generate highly purified donor cells to achieve stable and predictable therapeutic outcomes, raising the prospect of hPSC-based PD cell replacement therapies.
2022-06-14 | GSE204796 | GEO
Project description:Human pluripotent stem cell (hPSC)-based replacement therapy holds great promise in treating Parkinson’s disease (PD). However, the heterogeneity of hPSC-derived donor cells and the low yield of midbrain dopaminergic (mDA) neurons after transplantation hinder its broad clinical application. Here, we depicted the single-cell molecular landscape during mDA neuron differentiation. We found that this process recapitulated the development of multiple but adjacent fetal brain regions including ventral midbrain, isthmus, and ventral hindbrain, resulting in heterogenous donor cell population. We reconstructed the differentiation trajectory of mDA lineage and identified CLSTN2 and PTPRO as specific surface markers of mDA progenitors, which were predictive of mDA neuron differentiation and could facilitate highly enriched mDA neurons (up to 80%) following progenitor sorting and transplantation. Marker sorted progenitors exhibited higher therapeutic potency in correcting motor deficits of PD mice. Different marker sorted grafts had a strikingly consistent cellular composition, in which mDA neurons were enriched, while off-target neuron types were mostly depleted, suggesting stable graft outcomes. Our study provides a better understanding of cellular heterogeneity during mDA neuron differentiation, and establishes a strategy to generate highly purified donor cells to achieve stable and predictable therapeutic outcomes, raising the prospect of hPSC-based PD cell replacement therapies.
2022-06-14 | GSE204795 | GEO
Project description:Arabidopsis telomeric repeat binding factors (TRBs) can bind telomeric DNA sequences to protect telomeres from degradation. TRBs can also recruit Polycomb Repressive Complex 2 (PRC2) to deposit tri-methylation of H3 lysine 27 (H3K27me3) over certain target loci. Here, we demonstrate that TRBs also associate and colocalize with JUMONJI14 (JMJ14) and trigger H3K4me3 demethylation at some loci. The trb1/2/3 triple mutant and the jmj14-1 mutant show an increased level of H3K4me3 over TRB and JMJ14 binding sites, resulting in up-regulation of their target genes. Furthermore, tethering TRBs to the promoter region of genes with an artificial zinc finger (TRB-ZF) successfully triggers target gene silencing, as well as H3K27me3 deposition, and H3K4me3 removal. Interestingly, JMJ14 is predominantly recruited to ZF off-target sites with low levels of H3K4me3, which is accompanied with TRB-ZFs triggered H3K4me3 removal at these loci. These results suggest that TRB proteins coordinate PRC2 and JMJ14 activities to repress target genes via H3K27me3 deposition and H3K4me3 removal.
2023-02-22 | MSV000091349 | MassIVE
Project description:Stem cell treatments for neurodegenerative diseases are expected to reach clinical trials soon. Most of the approaches currently under development involve transplantation of immature progenitors that subsequently undergo phenotypic and functional maturation in vivo, and predicting the long term graft outcome already at the progenitor stage remains a challenge. Here, we took an unbiased approach to identify predictive markers expressed in dopamine neuron progenitors that correlate with graft outcome in an animal model of Parkinson's disease through gene expression analysis of >30 batches of grafted hESC-derived progenitors. We found that many of the commonly-used markers did not accurately predict in vivo subtype-specific maturation. Instead, we identified a specific set of markers associated with the caudal midbrain that correlate with high dopaminergic yield after transplantation in vivo. Using these markers, we developed a GMP differentiation protocol for highly efficient and reproducible production of transplantable dopamine progenitors from hESCs.
2017-01-01 | GSE86654 | GEO