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.
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.
Project description:Analysis of the effect of isolation methods (fluorescence activated cell sorting (FACS), positive and negative immunomagnetic selection) on gene expression in human primary CD4+, CD8+ T cells, B cells and monocytes. FACS incurs the least short-term changes in gene expression signature.
Project description:Overexpression of transcription factor Sox17 in human ES cells-derived endothelial cells and hematopoietic cells enhances expansion of hemogenic endothelium-like cells. Human ES cells were differentiated for 6 days, 8 days or 12 days in EBs, then CD34+CD43-CD45- endothelial cells, CD34+CD43+CD45- pre-hematopoietic progenitor cells (HPCs) or CD34+CD43+CD45+ HPCs were isolated by fluorescence activated cell sorting (FACS) and subjected to a microarray analysis.M-cM-^@M-^@Some samples were plated onto OP9 cells after the isolation by FACS, and transduced with the 4OH-tamoxifen-inducible 1M-CM-^WFLAG-tagged Sox17-ERT retrovirus. The cells were cultured with 4OH-tamoxifen. CD34+CD43+CD45low hemogenic endothelium-like cells expanded by Sox17-ERT were collected by magnetic-activated cell sorting (MACS) and subjected to a ChIP-chip analysis.
Project description:Analysis of the surfaceome of a blood cell subset requires cell sorting, followed by surface protein enrichment. Here, we present a protocol combining magnetically activated cell sorting (MACS) and surface biotinylation of the target cell subset from human peripheral blood mononuclear cells (PBMCs). We describe the steps for isolating target cells and their in-column surface biotinylation, followed by isolation and mass spectrometry analysis of biotinylated proteins. The protocol enables in-column surface biotinylation of specific cell subsets with minimal membrane disruption.
Project description:We identified APLNR as a surface marker for in vitro cardiac progenitors derived from human induced pluripotent stem cells (hiPSC). To gain further insight on the differentiation trajectory and its relevance with in vivo cardiac development, we performed polyA mRNA-sequencing on APLNR+ in vitro cardiac progenitors derived from 3 hiPSC lines at 0, 24, 48 and 72 hours post-immunomagnetic isolation. Our study revealed APLNR+ in vitro cardiac progenitors differentiate via a transient progenitor stage before further differentiation into cardiomyocytes and cardiac mesenchyme.
Project description:Identification of 187AA unique peptides by mass spectrometry and detection of 187AA by mass spectrometry in human cells (GZF2-iPSC, GZF2-hep, 293T, SK-hep1, ESC-derived cardiomyocytes, Hep-G2 and Hela).
Project description:Identification of 187AA unique peptides by mass spectrometry and detection of 187AA by mass spectrometry in human cells (GZF2-iPSC, GZF2-hep, 293T, SK-hep1, ESC-derived cardiomyocytes, Hep-G2 and Hela).
Project description:Analysis of induced nephron progenitor cells from female/male urine cells (iNPC-F/INPC-M) by defined transcription factors vs. ESC derived nephron progenitor cell (ESC-NPC_H9/ESC-NPC_BG01) and female/male urine cells (UC-F/UC-M). Results provide insight into molecular similarities between induced nephron progenitor cells and human ESC derived nephron progenitor cell