Dataset Information

Functional characterization and expression profiling of human induced pluripotent stem cell- and embryonic stem cell-derived endothelial cells.

ABSTRACT: With regard to human induced pluripotent stem cells (hiPSCs), in which adult cells are reprogrammed into embryonic-like cells using defined factors, their functional and transcriptional expression pattern during endothelial differentiation has yet to be characterized. In this study, hiPSCs and human embryonic stem cells (hESCs) were differentiated using the embryoid body method, and CD31(+) cells were sorted. Fluorescence activated cell sorting analysis of hiPSC-derived endothelial cells (hiPSC-ECs) and hESC-derived endothelial cells (hESC-ECs) demonstrated similar endothelial gene expression patterns. We showed functional vascular formation by hiPSC-ECs in a mouse Matrigel plug model. We compared the gene profiles of hiPSCs, hESCs, hiPSC-ECs, hESC-ECs, and human umbilical vein endothelial cells (HUVECs) using whole genome microarray. Our analysis demonstrates that gene expression variation of hiPSC-ECs and hESC-ECs contributes significantly to biological differences between hiPSC-ECs and hESC-ECs as well as to the "distances" among hiPSCs, hESCs, hiPSC-ECs, hESC-ECs, and HUVECs. We further conclude that hiPSCs can differentiate into functional endothelial cells, but with limited expansion potential compared with hESC-ECs; thus, extensive studies should be performed to explore the cause and extent of such differences before clinical application of hiPSC-ECs can begin.

SUBMITTER: Li Z

PROVIDER: S-EPMC3182033 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Functional characterization and expression profiling of human induced pluripotent stem cell- and embryonic stem cell-derived endothelial cells.

Li Zongjin Z Hu Shijun S Ghosh Zhumur Z Han Zhongchao Z Wu Joseph C JC

Stem cells and development 20110228 10

With regard to human induced pluripotent stem cells (hiPSCs), in which adult cells are reprogrammed into embryonic-like cells using defined factors, their functional and transcriptional expression pattern during endothelial differentiation has yet to be characterized. In this study, hiPSCs and human embryonic stem cells (hESCs) were differentiated using the embryoid body method, and CD31(+) cells were sorted. Fluorescence activated cell sorting analysis of hiPSC-derived endothelial cells (hiPSC- ...[more]

PMID: 21235328

Similar Datasets

Project description:Endothelial cells derived from human induced pluripotent stem cells (hiPSC-ECs) provide a new opportunity for mechanistic research on vascular regeneration and drug screening. However, functions of hiPSC-ECs still need to be characterized. The objective of this study was to investigate electrophysiological and functional properties of hiPSC-ECs compared with primary human cardiac microvascular endothelial cells (HCMECs), mainly focusing on ion channels and membrane receptor signaling, as well as specific cell functions. HiPSC-ECs were derived from hiPS cells that were generated from human skin fibroblasts of three independent healthy donors. Phenotypic and functional comparison to HCMECs was performed by flow cytometry, immunofluorescence staining, quantitative reverse-transcription polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), tube formation, LDL uptake, exosome release assays and, importantly, patch clamp techniques. HiPSC-ECs were successfully generated from hiPS cells and were identified by endothelial markers. The mRNA levels of KCNN2, KCNN4, KCNMA1, TRPV2, and SLC8A1 in hiPSC-ECs were significantly higher than HCMECs. AT1 receptor mRNA level in hiPSC-ECs was higher than in HCMECs. AT2 receptor mRNA level was the highest among all receptors. Adrenoceptor ADRA2 expression in hiPSC-ECs was lower than in HCMECs, while ADRA1, ADRB1, ADRB2, and G-protein GNA11 and Gai expression were similar in both cell types. The expression level of muscarinic and dopamine receptors CHRM3, DRD2, DRD3, and DRD4 in hiPSC-ECs were significantly lower than in HCMECs. The functional characteristics of endothelial cells, such as tube formation and LDL uptake assay, were not statistically different between hiPSC-ECs and HCMECs. Phenylephrine similarly increased the release of the vasoconstrictor endothelin-1 (ET-1) in hiPSC-ECs and HCMECs. Acetylcholine also similarly increased nitric oxide generation in hiPSC-ECs and HCMECs. The resting potentials (RPs), ISK1-3, ISK4 and IK1 were similar in hiPSC-ECs and HCMECs. IBK was larger and IKATP was smaller in hiPSC-ECs. In addition, we also noted a higher expression level of exosomes marker CD81 in hiPSC-ECs and a higher expression of CD9 and CD63 in HCMECs. However, the numbers of exosomes extracted from both types of cells did not differ significantly. The study demonstrates that hiPSC-ECs are similar to native endothelial cells in ion channel function and membrane receptor-coupled signaling and physiological cell functions, although some differences exist. This information may be helpful for research using hiPSC-ECs.

Project description:BackgroundEndothelial cells (ECs) are essential regulators of the vasculature, lining arteries, veins, and capillary beds. While all ECs share a number of structural and molecular features, heterogeneity exists depending on their resident tissue. ECs lining the choriocapillaris in the human eye are lost early in the pathogenesis of age-related macular degeneration (AMD), a common and devastating form of vision loss. In order to study the mechanisms leading to choroidal endothelial cell (CEC) loss and to develop reagents for repairing the choroid, a reproducible in vitro model, which closely mimic CECs, is needed. While a number of protocols have been published to direct induced pluripotent stem cells (iPSCs) into ECs, the goal of this study was to develop methods to differentiate iPSCs into ECs resembling those found in the human choriocapillaris specifically.MethodsWe transduced human iPSCs with a CDH5p-GFP-ZEO lentiviral vector and selected for transduced iPSCs using blasticidin. We generated embryoid bodies (EBs) from expanded iPSC colonies and transitioned from mTESR™1 to EC media. One day post-EB formation, we induced mesoderm fate commitment via addition of BMP-4, activin A, and FGF-2. On day 5, EBs were adhered to Matrigel-coated plates in EC media containing vascular endothelial cell growth factor (VEGF) and connective tissue growth factor (CTGF) to promote CEC differentiation. On day 14, we selected for CECs using either zeocin resistance or anti-CD31 MACS beads. We expanded CECs post-selection and performed immunocytochemical analysis of CD31, carbonic anhydrase IV (CA4), and RGCC; tube formation assays; and transmission electron microscopy to access vascular function.ResultsWe report a detailed protocol whereby we direct iPSC differentiation toward mesoderm and utilize CTGF to specify CECs. The CDH5p-GFP-ZEO lentiviral vector facilitated the selection of iPSC-derived ECs that label with antibodies directed against CD31, CA4, and RGCC; form vascular tubes in vitro; and migrate into empty choroidal vessels. CECs selected using either antibiotic selection or CD31 MACS beads showed similar characteristics, thereby making this protocol easily reproducible with or without lentiviral vectors.ConclusionECs generated following this protocol exhibit functional and biochemical characteristics of CECs. This protocol will be useful for developing in vitro models toward understanding the mechanisms of CEC loss early in AMD.

Project description:ImportanceThree-dimensional cerebral organoids generated from patient-derived induced pluripotent stem cells (iPSCs) may be used to interrogate cellular-molecular underpinnings of schizophrenia.ObjectiveTo determine transcriptomic profiles and functional characteristics of cerebral organoids from patients with schizophrenia using gene expression studies, complemented with investigations of mitochondrial function through measurement of real-time oxygen consumption rate, and functional studies of neuronal firing with microelectrode arrays.Design, setting, and participantsThis case-control study was conducted at Massachusetts General Hospital between 2017 and 2019. Transcriptomic profiling of iPSC-derived cerebral organoids from 8 patients with schizophrenia and 8 healthy control individuals was undertaken to identify cellular pathways that are aberrant in schizophrenia. Induced pluripotent stem cells and cerebral organoids were generated from patients who had been diagnosed as having schizophrenia and from heathy control individuals.Main outcomes and measuresTranscriptomic analysis of iPSC-derived cerebral organoids from patients with schizophrenia show differences in expression of genes involved in synaptic biology and neurodevelopment and are enriched for genes implicated in schizophrenia genome-wide association studies (GWAS).ResultsThe study included iPSC lines generated from 11 male and 5 female white participants, with a mean age of 38.8 years. RNA sequencing data from iPSC-derived cerebral organoids in schizophrenia showed differential expression of genes involved in synapses, in nervous system development, and in antigen processing. The differentially expressed genes were enriched for genes implicated in schizophrenia, with 23% of GWAS genes showing differential expression in schizophrenia and control organoids: 10 GWAS genes were upregulated in schizophrenia organoids while 15 GWAS genes were downregulated. Analysis of the gene expression profiles suggested dysregulation of genes involved in mitochondrial function and those involved in modulation of excitatory and inhibitory pathways. Studies of mitochondrial respiration showed lower basal consumption rate, adenosine triphosphate production, proton leak, and nonmitochondrial oxygen consumption in schizophrenia cerebral organoids, without any differences in the extracellular acidification rate. Microelectrode array studies of cerebral organoids showed no differences in baseline electrical activity in schizophrenia but revealed a diminished response to stimulation and depolarization.Conclusions and relevanceInvestigations of patient-derived cerebral organoids in schizophrenia revealed gene expression patterns suggesting dysregulation of a number of pathways in schizophrenia, delineated differences in mitochondrial function, and showed deficits in response to stimulation and depolarization in schizophrenia.

Dataset Information

Functional characterization and expression profiling of human induced pluripotent stem cell- and embryonic stem cell-derived endothelial cells.

Publications

Functional characterization and expression profiling of human induced pluripotent stem cell- and embryonic stem cell-derived endothelial cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets