Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Isolation of human iPSC-derived dopaminergic progenitors by cell sorting

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

SUBMITTER: Daisuke Doi

PROVIDER: E-GEOD-51214 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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.

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Isolation of human iPSC-derived dopaminergic progenitors by cell sorting

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