Synthetic mRNA switches for detection and purification of cardiomyocytes and endothelial cells derived from human pluripotent stem cells [miRNA]
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE60631 | GEO | 2015/05/20
SECONDARY ACCESSION(S): PRJNA259166
REPOSITORIES: GEO
Similar Datasets
Project description:We designed microRNA-responsive, modified mRNA switches (miR-switches) that quantitatively control their own translation level by determining the miRNA activities. We found that the three miR-switches (i.e., miR-1-, miR-208-, and miR-499-switches) enable to purify cardiomyocytes differentiated from hPSCs with high efficiency, accuracy, and safety. The purified cardiomyocytes were engrafted in mouse heart and did not form tumor. Simultaneous purification of two different cell types, cardiomyocytes and endothelial cells, was also performed by transfecting the mRNA responding to the both miR-208 and miR-126, into heterogeneous cell population derived from hPSCs. In addition, selective induction of apoptosis in noncardiomyocytes triggered by miR-1/208-Bim switch enriched cardiomyocytes without cell sorting. The mRNA switch could purify desired cell types and control cell death pathways by monitoring the miRNA expression dynamics during cell fate conversion.
2015-05-20 | GSE60632 | GEO
Project description:We designed microRNA-responsive, modified mRNA switches (miR-switches) that quantitatively control their own translation level by determining the miRNA activities. We found that the three miR-switches (i.e., miR-1-, miR-208-, and miR-499-switches) enable to purify cardiomyocytes differentiated from hPSCs with high efficiency, accuracy, and safety. The purified cardiomyocytes were engrafted in mouse heart and did not form tumor. Simultaneous purification of two different cell types, cardiomyocytes and endothelial cells, was also performed by transfecting the mRNA responding to the both miR-208 and miR-126, into heterogeneous cell population derived from hPSCs. In addition, selective induction of apoptosis in noncardiomyocytes triggered by miR-1/208-Bim switch enriched cardiomyocytes without cell sorting. The mRNA switch could purify desired cell types and control cell death pathways by monitoring the miRNA expression dynamics during cell fate conversion. MYH6-EIP4 day8 differentiated cells (EGFP+) for miRNA, N=1 MYH6-EIP4 day8 differentiated cells (EGFP-) for miRNA, N=1 MYH6-EIP4 day20 differentiated cells (EGFP+) for miRNA, N=1 MYH6-EIP4 day20 differentiated cells (EGFP-) for miRNA, N=1 HUVEC for miRNA, N=2 AoSMC for miRNA, N=2 Hepatocytes for miRNA, N=2 NHDF for miRNA, N=2
2015-05-20 | E-GEOD-60631 | biostudies-arrayexpress
Project description:We designed microRNA-responsive, modified mRNA switches (miR-switches) that quantitatively control their own translation level by determining the miRNA activities. We found that the three miR-switches (i.e., miR-1-, miR-208-, and miR-499-switches) enable to purify cardiomyocytes differentiated from hPSCs with high efficiency, accuracy, and safety. The purified cardiomyocytes were engrafted in mouse heart and did not form tumor. Simultaneous purification of two different cell types, cardiomyocytes and endothelial cells, was also performed by transfecting the mRNA responding to the both miR-208 and miR-126, into heterogeneous cell population derived from hPSCs. In addition, selective induction of apoptosis in noncardiomyocytes triggered by miR-1/208-Bim switch enriched cardiomyocytes without cell sorting. The mRNA switch could purify desired cell types and control cell death pathways by monitoring the miRNA expression dynamics during cell fate conversion. MYH6-EIP4 iPSCs for mRNA, N=3 MYH6-EIP4 day18 cardiomyocytes (before transfection) for mRNA, N=3 MYH6-EIP4 day19 cardiomyocytes (miR-1-switch day1) for mRNA, N=3 MYH6-EIP4 day19 cardiomyocytes (without transfection) for mRNA, N=3 MYH6-EIP4 day25 cardiomyocytes (miR-1-switch day7) for mRNA, N=3 MYH6-EIP4 day25 cardiomyocytes (without transfection) for mRNA, N=3 201B7 d22 miR-208a-BFP sorted cells for mRNA, N=1 201B7 d22 miR-208a-BFP negative fraction sorted cells for mRNA, N=1 201B7 d22 SIRPA+LIN- sorted cells for mRNA, N=1 201B7 d22 SIRPA+LIN- negative fraction sorted cells for mRNA, N=1 201B7 d22 VCAM1+ sorted cells for mRNA, N=1 201B7 d22 VCAM1+negative fraction sorted cells for mRNA, N=1 MYH6-EIP4 day19 cardiomyocytes (miR-208a-Bim-switch day1) for mRNA, N=1 MYH6-EIP4 day19 cardiomyocytes (without transfection) for mRNA, N=1 MYH6-EIP4 day21 cardiomyocytes (miR-208a-Bim-switch day7) for mRNA, N=1 MYH6-EIP4 day21 cardiomyocytes (without transfection) for mRNA, N=1 MYH6-EIP4 day25 cardiomyocytes (miR-208a-Bim-switch day7) for mRNA, N=1 MYH6-EIP4 day25 cardiomyocytes (without transfection) for mRNA, N=1
2015-05-20 | E-GEOD-60632 | biostudies-arrayexpress
Project description:Isolation of specific cell types, including pluripotent stem cell (PSC)-derived populations, is frequently accomplished using cell surface antigens expressed by the cells of interest. However, specific antigens for many cell types have not been identified, making their isolation difficult. Here, we describe an efficient method for purifying cells based on endogenous miRNA activity. We designed synthetic mRNAs encoding a fluorescent protein tagged with sequences targeted by miRNAs expressed by the cells of interest. These miRNA switches control their own translation levels by sensing miRNA activities. Several miRNA switches (miR-1-, miR-208a-, and miR-499a-5p-switches) efficiently purified cardiomyocytes differentiated from human PSCs, and switches encoding the apoptosis inducer Bim enriched for cardiomyocytes without cell sorting. This approach is generally applicable, as miR-126-, miR-122-5p- and miR-375-switches purified endothelial cells, hepatocytes and INSULIN-producing cells differentiated from hPSCs, respectively. Thus, miRNA switches can purify cell populations for which other isolation strategies are unavailable. MYH6-EIP4 day8 differentiated cells (EGFP+) for miRNA, N=1 MYH6-EIP4 day8 differentiated cells (EGFP-) for miRNA, N=1 MYH6-EIP4 day20 differentiated cells (EGFP+) for miRNA, N=1 MYH6-EIP4 day20 differentiated cells (EGFP-) for miRNA, N=1 HUVEC for miRNA, N=2 AoSMC for miRNA, N=2 Hepatocytes for miRNA, N=2 NHDF for miRNA, N=2 MYH6-EIP4 iPSCs for mRNA, N=3 MYH6-EIP4 day18 cardiomyocytes (before transfection) for mRNA, N=3 MYH6-EIP4 day19 cardiomyocytes (miR-1-switch day1) for mRNA, N=3 MYH6-EIP4 day19 cardiomyocytes (without transfection) for mRNA, N=3 MYH6-EIP4 day25 cardiomyocytes (miR-1-switch day7) for mRNA, N=3 MYH6-EIP4 day25 cardiomyocytes (without transfection) for mRNA, N=3 201B7 d22 miR-208a-BFP sorted cells for mRNA, N=1 201B7 d22 miR-208a-BFP negative fraction sorted cells for mRNA, N=1 201B7 d22 SIRPA+LIN- sorted cells for mRNA, N=1 201B7 d22 SIRPA+LIN- negative fraction sorted cells for mRNA, N=1 201B7 d22 VCAM1+ sorted cells for mRNA, N=1 201B7 d22 VCAM1+negative fraction sorted cells for mRNA, N=1 MYH6-EIP4 day19 cardiomyocytes (miR-208a-Bim-switch day1) for mRNA, N=1 MYH6-EIP4 day19 cardiomyocytes (without transfection) for mRNA, N=1 MYH6-EIP4 day21 cardiomyocytes (miR-208a-Bim-switch day7) for mRNA, N=1 MYH6-EIP4 day21 cardiomyocytes (without transfection) for mRNA, N=1 MYH6-EIP4 day25 cardiomyocytes (miR-208a-Bim-switch day7) for mRNA, N=1 MYH6-EIP4 day25 cardiomyocytes (without transfection) for mRNA, N=1
2015-05-20 | E-GEOD-60633 | biostudies-arrayexpress
Project description:Isolation of specific cell types, including pluripotent stem cell (PSC)-derived populations, is frequently accomplished using cell surface antigens expressed by the cells of interest. However, specific antigens for many cell types have not been identified, making their isolation difficult. Here, we describe an efficient method for purifying cells based on endogenous miRNA activity. We designed synthetic mRNAs encoding a fluorescent protein tagged with sequences targeted by miRNAs expressed by the cells of interest. These miRNA switches control their own translation levels by sensing miRNA activities. Several miRNA switches (miR-1-, miR-208a-, and miR-499a-5p-switches) efficiently purified cardiomyocytes differentiated from human PSCs, and switches encoding the apoptosis inducer Bim enriched for cardiomyocytes without cell sorting. This approach is generally applicable, as miR-126-, miR-122-5p- and miR-375-switches purified endothelial cells, hepatocytes and INSULIN-producing cells differentiated from hPSCs, respectively. Thus, miRNA switches can purify cell populations for which other isolation strategies are unavailable.
2015-05-20 | GSE60633 | GEO
Project description:We showed novel synthetic microRNA (miRNA) switch system, which can purify large quantities of iPSC-CMs using magnetic-activated cell sorting (MACS). We used miR-208a, which is specifically expressed in cardiomyocytes, as a specific miRNA of CMs, and CD4 as a selection marker for MACS. We synthesized a miRNA switch encoding CD4 tagged with complementary sequences against miR-208a (miR-208a CD4 switch). We transfected this switch into differentiated cells, and easily got efficiently purified CMs in a large scale with MACS. In addition, we demonstrated that purified cells were shown to be engrafted as CMs in mouse hearts.
2022-05-30 | GSE153714 | GEO
Project description:A hallmark of cancer cells is the metabolic switch from oxidative phosphorylation (OXPHOS) to glycolysis, a phenomenon referred to as the “Warburg effect”, which is also observed in primed human pluripotent stem cells (hPSCs). Here, we report that downregulation of SIRT2 and upregulation of SIRT1 is a molecular signature of primed hPSCs and that SIRT2 critically regulates metabolic reprogramming during induced pluripotency by targeting glycolytic enzymes including aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and enolase. Remarkably, knockdown of SIRT2 in human fibroblasts resulted in significantly decreased OXPHOS and increased glycolysis. In addition, we found that miR-200c-5p specifically targets SIRT2, downregulating its expression. Furthermore, SIRT2 overexpression in hPSCs significantly affected energy metabolism, altering stem cell functions such as pluripotent differentiation properties. Taken together, our results identify the miR-200c-SIRT2 axis as a key regulator of metabolic reprogramming (Warburg-like effect), via regulation of glycolytic enzymes, during human induced pluripotency and pluripotent stem cell function. To address our hypothesis that acetylation affects the metabolic switch, we compared protein acetylation in hESCs and hDFs by liquid chromatography-tandem mass spectrometry (LCMS/ MS) analyses following immunoprecipitation with acetyl-Lys antibody.
2018-10-26 | PXD006036 | Pride
Project description:This experiment was conducted to identify target genes of the microRNA-499 in skeletal muscle of transgenic mice that overexpressed miR-499. The following abstract from the submitted manuscript describes the major findings of this work. Coupling of mitochondrial function and skeletal muscle fiber type by a miR-499/Fnip1/AMPK circuit. Jing Liu, Xijun Liang, Danxia Zhou, Ling Lai, Tingting Fu, Yan Kong, Qian Zhou, Rick B. Vega, Min-Sheng Zhu, Daniel P. Kelly, Xiang Gao, and Zhenji Gan. Upon adaption of skeletal muscle to physiological and pathophysiological stimuli, muscle fiber type and mitochondrial function are coordinately regulated. Recent studies have identified pathways involved in control of contractile proteins of oxidative type fibers. However, the mechanism for coupling of mitochondrial function to muscle contractile machinery during fiber type transition remains unknown. Here, we show that the expression of the genes encoding type I myosins, Myh7/Myh7b and their intronic miR-208b/miR-499 parallels mitochondrial function during fiber type transitions. Using in vivo approaches in mice, we found that miR-499 drives a PGC-1a-dependent mitochondrial oxidative metabolism program to match shifts in slow-twitch muscle fiber composition. Mechanistically, miR-499 directly targets Fnip1, a known AMP-activated protein kinase (AMPK)-interacting protein that negatively regulates AMPK, a known activator of PGC-1a. Inhibition of Fnip1 reactivated AMPK/PGC-1a signaling and mitochondrial function in myocytes. Restoration of the expression of miR-499 in the mdx mouse model of Duchenne muscular dystrophy (DMD) reduced the severity of DMD. Thus, we have identified a miR-499/Fnip1/AMPK circuit that can serve as a mechanism to couple muscle fiber type and mitochondrial function. Keywords: muscle, contractile fiber type, mitochondrial function, microRNA, gene regulation RNA from three wild-type (non-transgenic (NTG)) and three miR-499 overexpressing (MCK-miR-499) mice was analyzed. three replicates of each are provided.
2016-09-03 | E-GEOD-72581 | biostudies-arrayexpress
Project description:This experiment was conducted to identify target genes of the microRNA-499 in skeletal muscle of transgenic mice that overexpressed miR-499. The following abstract from the submitted manuscript describes the major findings of this work. Coupling of mitochondrial function and skeletal muscle fiber type by a miR-499/Fnip1/AMPK circuit. Jing Liu, Xijun Liang, Danxia Zhou, Ling Lai, Tingting Fu, Yan Kong, Qian Zhou, Rick B. Vega, Min-Sheng Zhu, Daniel P. Kelly, Xiang Gao, and Zhenji Gan. Upon adaption of skeletal muscle to physiological and pathophysiological stimuli, muscle fiber type and mitochondrial function are coordinately regulated. Recent studies have identified pathways involved in control of contractile proteins of oxidative type fibers. However, the mechanism for coupling of mitochondrial function to muscle contractile machinery during fiber type transition remains unknown. Here, we show that the expression of the genes encoding type I myosins, Myh7/Myh7b and their intronic miR-208b/miR-499 parallels mitochondrial function during fiber type transitions. Using in vivo approaches in mice, we found that miR-499 drives a PGC-1a-dependent mitochondrial oxidative metabolism program to match shifts in slow-twitch muscle fiber composition. Mechanistically, miR-499 directly targets Fnip1, a known AMP-activated protein kinase (AMPK)-interacting protein that negatively regulates AMPK, a known activator of PGC-1a. Inhibition of Fnip1 reactivated AMPK/PGC-1a signaling and mitochondrial function in myocytes. Restoration of the expression of miR-499 in the mdx mouse model of Duchenne muscular dystrophy (DMD) reduced the severity of DMD. Thus, we have identified a miR-499/Fnip1/AMPK circuit that can serve as a mechanism to couple muscle fiber type and mitochondrial function. Keywords: muscle, contractile fiber type, mitochondrial function, microRNA, gene regulation
2016-09-03 | GSE72581 | GEO
Project description:Human pluripotent stem cells (hPSCs) such as embryonic stem cells and induced pluripotent stem cells are promising materials for cell-based regenerative therapies to heart diseases. However, until realization there are many hurdles such as high efficiency of cardiac differentiation of hPSCs and production of clinical-grade cardiac cells derived from hPSCs. Here, we show that a novel small molecule KY02111 robustly enhances differentiation to functional cardiomyocytes from hPSCs. To reveal how KY02111 function on promoting cardiac differentiation of hPSCs, we analyzed the gene expression profiles in KY02111-treated IMR90-1 hiPSCs using the microarray technique. At Day3 of cardiac differentiation from hiPSCs, KY02111 or DMSO was added in the culture and then the cell population was harvested after 12 or 24 hours for RNA extraction.
2012-10-30 | E-GEOD-33622 | biostudies-arrayexpress