Detailed analysis of the genetic and epigenetic signature of iPS cell-derived mesodiencephalic dopaminergic neurons
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE55475 | GEO | 2014/03/01
SECONDARY ACCESSION(S): PRJNA239709
REPOSITORIES: GEO
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Project description:Induced pluripotent stem cells (iPSCs) harbor great promise for in vitro generation of disease-relevant cell types, such as mesodiencephalic dopaminergic (mdDA) neurons involved in Parkinson’s disease. Although iPSC-derived midbrain DA neurons have been generated, detailed genetic and epigenetic characterization of such neurons is still lacking. The goal of this study is to examine the authenticity of iPSC-derived DA neurons obtained by established protocols. We FACS-purified mdDA (Pitx3gfp/+) neurons derived from mouse iPSCs and primary mdDA (Pitx3gfp/+) neurons to analyze and compare their genetic and epigenetic features. Although iPSC-derived DA neurons largely adopt characteristics of their in-vivo counterparts, relevant deviations in global gene expression and DNA methylation were found. Hypermethylated genes, mainly involved in neurodevelopment and basic neuronal functions, consequently showed reduced expression levels. Such abnormalities should be addressed as they might affect unambiguous long-term functionality and hamper the potential of iPSC-derived DA neurons for in-vitro disease modeling or cell-based therapy. RRBS methylation maps were generated for iPSCs cells, dopaminergic neurons derived from iPSCs and primary mesodiencephalic dopaminergic neurons
2014-03-01 | E-GEOD-55475 | biostudies-arrayexpress
Project description:Induced pluripotent stem cells (iPSCs) hold great promise for in vitro disease modeling and cell replacement therapy for Parkinson’s disease (PD). Both applications crucially require an in-depth profiling of the disease-relevant, iPSC-derived cell type. Midbrain dopaminergic (mDA) neurons derived from pluripotent stem cells are of substantial interest because of their instrumental value for PD therapy. IPSC-derived mDA neuron-like cells have been generated, however, detailed genetic and epigenetic characterization of strictly purified in vitro generated DA neurons has so far lagged behind. We generated mouse Pitx3gfp/+ iPSC-derived DA neurons that, after fluorescent activated cell sorting (FACS) allowed comprehensive comparison to mesodiencephalic dopaminergic (mdDA) neurons from Fac-sorted Pitx3gfp/+ ventral midbrains. We performed detailed analysis of global gene expression and genome-scale DNA methylation of CpG islands (CGIs) by reduced representation bisulfite sequencing. The reprogramming pathway from fibroblasts to iPSCs left parental cell footprints for both gene expression and DNA methylation. However, most gene expression patterns of iPSC-derived DA neurons closely resembled that of primary mdDA neurons with the strongest correlations for mdDA specific genes. Also, for DNA methylation patterns, high similarities were found for the vast majority of CGIs when comparing primary mdDA neurons with iPSC-derived DA neurons. Additionally, we found de novo DNA methylation during in vitro differentiation for hundreds of genes specifically in lineage-committed neural precursors that persisted in iPSC-derived DA neurons. Our study provides novel detailed characteristics of iPSC-derived DA neurons in comparison to the primary cell type. These findings add important information to our knowledge about these biomedically highly valuable, in vitro generated neurons. Microarray expression study comparing 3 samples of facs-sorted, Pitx3-gfp positive cells from each experimental group to a common reference consisting of adult mouse midbrain RNA. Each sample was analysed in normal and opposite dye orientation.
2014-05-13 | E-GEOD-47178 | biostudies-arrayexpress
Project description:Induced pluripotent stem cells (iPSCs) hold great promise for in vitro disease modeling and cell replacement therapy for Parkinson’s disease (PD). Both applications crucially require an in-depth profiling of the disease-relevant, iPSC-derived cell type. Midbrain dopaminergic (mDA) neurons derived from pluripotent stem cells are of substantial interest because of their instrumental value for PD therapy. IPSC-derived mDA neuron-like cells have been generated, however, detailed genetic and epigenetic characterization of strictly purified in vitro generated DA neurons has so far lagged behind. We generated mouse Pitx3gfp/+ iPSC-derived DA neurons that, after fluorescent activated cell sorting (FACS) allowed comprehensive comparison to mesodiencephalic dopaminergic (mdDA) neurons from Fac-sorted Pitx3gfp/+ ventral midbrains. We performed detailed analysis of global gene expression and genome-scale DNA methylation of CpG islands (CGIs) by reduced representation bisulfite sequencing. The reprogramming pathway from fibroblasts to iPSCs left parental cell footprints for both gene expression and DNA methylation. However, most gene expression patterns of iPSC-derived DA neurons closely resembled that of primary mdDA neurons with the strongest correlations for mdDA specific genes. Also, for DNA methylation patterns, high similarities were found for the vast majority of CGIs when comparing primary mdDA neurons with iPSC-derived DA neurons. Additionally, we found de novo DNA methylation during in vitro differentiation for hundreds of genes specifically in lineage-committed neural precursors that persisted in iPSC-derived DA neurons. Our study provides novel detailed characteristics of iPSC-derived DA neurons in comparison to the primary cell type. These findings add important information to our knowledge about these biomedically highly valuable, in vitro generated neurons.
2014-05-13 | GSE47178 | GEO
Project description:WNT1/beta-catenin signaling plays a crucial role in the generation of mesodiencephalic dopaminergic (mdDA) neurons including the Substantia nigra pars compacta (SNc) subpopulation, whose degeneration is a hallmark of Parkinson’s Disease (PD). However, the precise functions of WNT/beta-catenin signaling in this context remain unknown. Using mutant mice, primary ventral midbrain (VM) cells and pluripotent stem cells (mouse embryonic stem cells and induced pluripotent stem cells), we show that Dickkopf 3 (DKK3), a secreted glycoprotein that modulates WNT/beta-catenin signaling, is specifically required for the correct differentiation of a rostrolateral mdDA precursor subset into SNc DA neurons. Dkk3 transcription in the murine VM coincides with the onset of mdDA neurogenesis and is required for the maintenance of LMX1A and consequently PITX3 expression in rostrolateral mdDA precursors, without affecting the proliferation or specification of their progenitors. Treatment of primary VM cells or differentiating pluripotent stem cells with recombinant WNT1 and/or DKK3 proteins consistently increases the proportion of mdDA cells with SNc DA neuron identity and promotes their survival in vitro. The SNc DA pro-differentiation and pro-survival properties of DKK3, together with its known anti-tumorigenic effect, therefore make it an ideal candidate for the improvement of regenerative and neuroprotective strategies in the treatment of PD. We performed gene expression microarray analysis on iPSC-derived and FACS-sorted GFP-positive Pitx3GFP/+ mdDA neurons, differentiated in the presence or absence of recombinant human WNT1 and recombinant human DKK3. In addition, we analysed primary and FACS-sorted GFP-positive Pitx3+/GFP mdDA neurons isolated from the E13.5 and E14.5 ventral midbrain of Pitx3+/GFP embryos
2015-08-22 | E-GEOD-58813 | biostudies-arrayexpress
Project description:WNT1/beta-catenin signaling plays a crucial role in the generation of mesodiencephalic dopaminergic (mdDA) neurons including the Substantia nigra pars compacta (SNc) subpopulation, whose degeneration is a hallmark of Parkinson’s Disease (PD). However, the precise functions of WNT/beta-catenin signaling in this context remain unknown. Using mutant mice, primary ventral midbrain (VM) cells and pluripotent stem cells (mouse embryonic stem cells and induced pluripotent stem cells), we show that Dickkopf 3 (DKK3), a secreted glycoprotein that modulates WNT/beta-catenin signaling, is specifically required for the correct differentiation of a rostrolateral mdDA precursor subset into SNc DA neurons. Dkk3 transcription in the murine VM coincides with the onset of mdDA neurogenesis and is required for the maintenance of LMX1A and consequently PITX3 expression in rostrolateral mdDA precursors, without affecting the proliferation or specification of their progenitors. Treatment of primary VM cells or differentiating pluripotent stem cells with recombinant WNT1 and/or DKK3 proteins consistently increases the proportion of mdDA cells with SNc DA neuron identity and promotes their survival in vitro. The SNc DA pro-differentiation and pro-survival properties of DKK3, together with its known anti-tumorigenic effect, therefore make it an ideal candidate for the improvement of regenerative and neuroprotective strategies in the treatment of PD.
2015-08-22 | GSE58813 | GEO
Project description:Human induced pluripotent stem cells (iPSCs) can provide a promising source of midbrain dopaminergic (DA) neurons for cell replacement therapy for Parkinson’s disease. However, iPSC-derived donor cells may inevitably contain tumorigenic or inappropriate cells. Purification of neural progenitor cells or DA neurons as suitable donor cells has been attempted, but the isolation of DA progenitor cells derived from human pluripotent stem cells has so far been unsuccessful. Here we show human iPSC-derived DA progenitor cells can be efficiently isolated by cell sorting using a floor plate marker, Corin. we were able to develop a method for 1) scalable DA neuron induction on human laminin fragment and 2) sorting DA progenitor cells using an anti-Corin antibody. Furthermore, we determined the optimal timing for the cell sorting and transplantation. The grafted cells survived well and functioned as midbrain DA neurons in the 6-OHDA-lesioned rats, and showed minimal risk of tumor formation. The sorting of Corin-positive cells is favorable in terms of both safety and efficiency, and our protocol will contribute to the clinical application of human iPSCs for Parkinson’s disease. Differentiated human iPSC-derived neural progenitors just after sorting (day12 unsorted, day12 Corin+) and dopaminergic progenitors after an aggregation culture (day28 and day42, unsorted and day12-sorted, respectively), and human fetal ventral mesencephalon and dorsal mesencephalon (gestational age of 7.5 weeks) were subjected to RNA extraction and hybrdization on Affymetrix microarrays. Each sample except for human mesencephalon, undifferentiated iPSC, and day12-unsorted, day42-sample has 3 or 4 repeats.
2014-03-07 | E-GEOD-51214 | biostudies-arrayexpress
Project description:Induced pluripotent stem cells (iPSCs) are a promising source for cell-based therapy to treat Parkinson's disease (PD), in which midbrain dopaminegic (DA) neurons progressively degenerate. However, long-term analysis of human iPSC-derived DA neurons in primate PD models has never been performed. Here we show that DA progenitor cells derived from iPSCs of both healthy individuals and PD patients survived well in the brains of PD model primates and improved animal behavior. Magnetic resonance and positron emission tomography were useful to monitor the survival and function of the DA neurons. Score-based and video-recording analyses revealed an increase in spontaneous movement of the monkeys after transplantation. Histological studies showed that the mature DA neurons extended dense neurites into the host striatum. In addition, we never observed tumor formation for two years. Thus, this preclinical study using primate models indicates that human iPSC-derived DA progenitors are clinically applicable to treat PD patients.
2017-08-21 | GSE99253 | GEO
Project description:Human induced pluripotent stem cells (iPSCs) can provide a promising source of midbrain dopaminergic (DA) neurons for cell replacement therapy for Parkinson’s disease. However, iPSC-derived donor cells may inevitably contain tumorigenic or inappropriate cells. Purification of neural progenitor cells or DA neurons as suitable donor cells has been attempted, but the isolation of DA progenitor cells derived from human pluripotent stem cells has so far been unsuccessful. Here we show human iPSC-derived DA progenitor cells can be efficiently isolated by cell sorting using a floor plate marker, Corin. we were able to develop a method for 1) scalable DA neuron induction on human laminin fragment and 2) sorting DA progenitor cells using an anti-Corin antibody. Furthermore, we determined the optimal timing for the cell sorting and transplantation. The grafted cells survived well and functioned as midbrain DA neurons in the 6-OHDA-lesioned rats, and showed minimal risk of tumor formation. The sorting of Corin-positive cells is favorable in terms of both safety and efficiency, and our protocol will contribute to the clinical application of human iPSCs for Parkinson’s disease.
2014-03-07 | GSE51214 | GEO
Project description:The LIM homeodomain transcription factor Lmx1a is a very potential inducer of stem cells towards dopaminergic neurons. Despite several studies on the function of this gene, the exact in vivo role of Lmx1a in mesodiencephalic dopamine (mdDA) neuronal specification is still not understood. To analyze the genes functioning downstream of Lmx1a, we performed expression microarray analysis of LMX1A overexpressing MN9D dopaminergic cells. Several interesting regulated genes were identified, based on their regulation in other, previously generated expression arrays, and their expression pattern in the developing mdDA neuronal field. Post analysis through in vivo expression analysis in Lmx1a mouse mutant (drJ/drJ) embryos demonstrated a clear decrease in expression of the genes Grb10 and Rgs4, in and adjacent to the rostral and dorsal mdDA neuronal field and within the Lmx1a expression domain. Interestingly, the DA marker Vmat2 was significantly up-regulated as a consequence of increased LMX1A dose, and subsequent analysis on Lmx1a mutant E14.5 and adult tissue revealed a significant decrease in Vmat2 expression in mdDA neurons. Taken together, microarray analysis of an LMX1A overexpression cell system resulted in the identification of novel downstream targets of Lmx1A in mdDA neurons: Grb10, Rgs4 and Vmat2. RNA was isolated from MN9D cells. Each experimental sample consisted of a RNA pool derived from 3 separate 10-cm dishes containing Lmx1a overexpressing MN9D cells (transfected with pcDNA3.1(-)-Lmx1a). microarray analysis was performed in triplicate, each experimental sample was hybridized to the same reference pool of RNA derived from 9 10-cm dishes containing control MN9D cells (transfected with empty pcDNA3.1(-)). On each of three microarray samples, dye swap was performed to correct for dye effects.
2013-01-08 | E-GEOD-39174 | biostudies-arrayexpress
Project description:The LIM homeodomain transcription factor Lmx1a is a very potential inducer of stem cells towards dopaminergic neurons. Despite several studies on the function of this gene, the exact in vivo role of Lmx1a in mesodiencephalic dopamine (mdDA) neuronal specification is still not understood. To analyze the genes functioning downstream of Lmx1a, we performed expression microarray analysis of LMX1A overexpressing MN9D dopaminergic cells. Several interesting regulated genes were identified, based on their regulation in other, previously generated expression arrays, and their expression pattern in the developing mdDA neuronal field. Post analysis through in vivo expression analysis in Lmx1a mouse mutant (drJ/drJ) embryos demonstrated a clear decrease in expression of the genes Grb10 and Rgs4, in and adjacent to the rostral and dorsal mdDA neuronal field and within the Lmx1a expression domain. Interestingly, the DA marker Vmat2 was significantly up-regulated as a consequence of increased LMX1A dose, and subsequent analysis on Lmx1a mutant E14.5 and adult tissue revealed a significant decrease in Vmat2 expression in mdDA neurons. Taken together, microarray analysis of an LMX1A overexpression cell system resulted in the identification of novel downstream targets of Lmx1A in mdDA neurons: Grb10, Rgs4 and Vmat2.
2013-01-08 | GSE39174 | GEO