FACS and immunomagnetic isolation of early erythroid progenitor cells from mouse fetal liver.
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ABSTRACT:
SUBMITTER: Braun TW
PROVIDER: S-EPMC8724924 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Prospective isolation and characterization of progenitor cells is a paradigmatic strategy for studies of organ development. However, extraction of viable cells, fractionation of lineages, and in vitro analysis of progenitors from the fetal pancreas in experimental organisms like mice has proved challenging and has not yet been reported for human fetal pancreas. Here, we report isolation of pancreatic islet progenitor cells from fetal mice by FACS. Monoclonal antibodies that recognize cell-surface proteins on candidate stem cells in brain, skin, and other organs enabled separation of major pancreatic cell lineages and isolation of native pancreatic cells expressing neurogenin 3, an established marker of islet progenitors. New in vitro cell culture methods permitted isolated mouse islet progenitors to develop into hormone-expressing endocrine cells. Insulin-producing cells derived in vitro required or expressed factors that regulate fetal beta cell differentiation; thus, the genetic programs normally controlling in vivo mouse islet development are similarly required in our system. Moreover, antibodies that recognize conserved orthologous cell-surface epitopes in human fetal pancreas allowed FACS-based enrichment of candidate islet progenitor cells expressing neurogenin 3. Our studies reveal previously undescribed strategies for prospective purification and analysis of pancreatic endocrine progenitor cells that should accelerate studies of islet development and replacement.
| S-EPMC1765430 | biostudies-literature
Project description:Little is known about the differentiation capabilities of nonhematopoietic cells of the human fetal liver. We report the isolation and characterization of a human fetal liver multipotent progenitor cell (hFLMPC) population capable of differentiating into liver and mesenchymal cell lineages. Human fetal livers (74-108 days of gestation) were dissociated and maintained in culture. We treated the colonies with geneticin and mechanically isolated hFLMPCs, which were kept in an undifferentiated state by culturing on feeder layers. We derived daughter colonies by serial dilution, verifying monoclonality using the Humara assay. hFLMPCs, which have been maintained in culture for up to 100 population doublings, have a high self-renewal capability with a doubling time of 46 h. The immunophenotype is: CD34+, CD90+, c-kit+, EPCAM+, c-met+, SSEA-4+, CK18+, CK19+, albumin-, alpha-fetoprotein-, CD44h+, and vimentin+. Passage 1 (P1) and P10 cells have identical morphology, immunophenotype, telomere length, and differentiation capacity. Placed in appropriate media, hFLMPCs differentiate into hepatocytes and bile duct cells, as well as into fat, bone, cartilage, and endothelial cells. Our results suggest that hFLMPCs are mesenchymal-epithelial transitional cells, probably derived from mesendoderm. hFLMPCs survive and differentiate into functional hepatocytes in vivo when transplanted into animal models of liver disease. hFLMPCs are a valuable tool for the study of human liver development, liver injury, and hepatic repopulation.
| S-EPMC1502553 | biostudies-literature
Project description:We used scRNAseq to profile CD71/CD24low fetal liver erythroid progenitor cells isolated by 2 distinct methods: FACS and immunomagnetic isolation. Cells from both isolation methods were hashtagged using Biolegend mouse hashtag antibodies and library prepped together on the 10X chromium platform with the 3'RNA v3 kit. We also performed CITE-seq to profile proteogenomic expression of CD117 and CD71 on lineage-depleted mouse fetal liver erythroid progenitor cells. CITE-seq was performed through a separate library prep on the 10X chromium platform with the 3'RNAv3 kit.
2021-11-12 | GSE188287 | GEO
Project description:BackgroundHematopoietic progenitors are generated in the yolk sac and aorta-gonad-mesonephros region during early mouse development. At embryonic day 10.5 the first hematopoietic stem cells emerge in the aorta-gonad-mesonephros. Subsequently, hematopoietic stem cells and progenitors are found in the fetal liver. The fetal liver is a potent hematopoietic site, playing an important role in the expansion and differentiation of hematopoietic progenitors and hematopoietic stem cells. However, little is known concerning the regulation of fetal liver hematopoietic stem cells. In particular, the role of cytokines such as interleukin-1 in the regulation of hematopoietic stem cells in the embryo has been largely unexplored. Recently, we observed that the adult pro-inflammatory cytokine interleukin-1 is involved in regulating aorta-gonad-mesonephros hematopoietic progenitor and hematopoietic stem cell activity. Therefore, we set out to investigate whether interleukin-1 also plays a role in regulating fetal liver progenitor cells and hematopoietic stem cells.Design and methodsWe examined the interleukin-1 ligand and receptor expression pattern in the fetal liver. The effects of interleukin-1 on hematopoietic progenitor cells and hematopoietic stem cells were studied by FACS and transplantation analyses of fetal liver explants, and in vivo effects on hematopoietic stem cell and progenitors were studied in Il1r1(-/-) embryos.ResultsWe show that fetal liver hematopoietic progenitor cells express the IL-1RI and that interleukin-1 increases fetal liver hematopoiesis, progenitor cell activity and promotes hematopoietic cell survival. Moreover, we show that in Il1r1(-/-) embryos, hematopoietic stem cell activity is impaired and myeloid progenitor activity is increased.ConclusionsThe IL-1 ligand and receptor are expressed in the midgestation liver and act in the physiological regulation of fetal liver hematopoietic progenitor cells and hematopoietic stem cells.
| S-EPMC2663609 | biostudies-literature
Project description:Exosc8 and Exosc9 are components of the exosome that establish a barricade to erythroid maturation. Here, we knocked down Exosc8 in fetal liver-derived erythroid progenitor cells to determine the cohort of Exosc8-regulated genes in erythroid cells.
2014-08-09 | GSE60136 | GEO
Project description:Primitive erythroid cells (EryP) are the earliest differentiated cell type of the mammalian embryo. They appear in the yolk sac by embryonic day 7.5, begin to enter the embryonic circulation 2 days later and continue to mature in a stepwise and synchronous fashion. Like their adult counterparts, EryP enucleate. However, EryP circulate throughout the embryo for several days before the first enucleated forms can be identified in the blood. We have used transgenic mouse lines in which GFP marks EryP to investigate this seemingly long lag and have identified a previously unrecognized developmental niche for EryP maturation. After exiting the yolk sac, EryP begin to express cell adhesion proteins, including alpha4, alpha5, and beta1 integrins, on their surface and migrate into the fetal liver (FL), where they interact with macrophages within erythroblastic islands. Binding of EryP to FL macrophages in vitro is stage-specific and partly depends on VCAM-1. The ability to tag and track EryP nuclei using a transgenic mouse line expressing an H2B-EGFP fusion allowed us to identify and characterize extruded EryP nuclei and to demonstrate that molecules such as alpha4, alpha5, and beta1 integrins are redistributed onto the plasma membrane surrounding the extruding nucleus. FL macrophages engulf extruded EryP nuclei in cocultures and in the native FL in vivo. We conclude that EryP home to, complete their maturation, and enucleate within the FL, a tissue that is just developing as EryP begin to circulate. Our observations suggest a simple solution for a puzzling aspect of the development of the primitive erythroid lineage.
| S-EPMC2373346 | biostudies-literature
Project description:Exosc8 and Exosc9 are components of the exosome that establish a barricade to erythroid maturation. Here, we knocked down Exosc8 in fetal liver-derived erythroid progenitor cells to determine the cohort of Exosc8-regulated genes in erythroid cells. Freshly isolated fetal liver progenitor cells were infected with retrovirus expressing shRNA targeting either luciferase or Exosc8. Total RNA was isolated from these cells after 3 days ex-vivo culture, during which the cells underwent erythroid maturation.
2014-08-09 | E-GEOD-60136 | biostudies-arrayexpress
Project description:Liver epithelioid progenitor cells (LEPCs) have important roles in liver therapy because of their hepatic differentiation potency in vitro and in vivo. Despite many researches on humans, mice, and rats, equivalent progenitor cells derived from bovine are relatively rare. The purpose of our current study is to characterize bovine LEPCs, and research on the cure potency of this heteroplastic progenitor cells on mice liver fibrosis. We have used collagenase IV digesting and differential adhesion method to isolate slabstone shape, EpCAM, LGR5, NCAM1 and SOX9 positive progenitor cells from fetal Luxi bovine liver. When cultured in hepatic differentiation media containing 20 ng/ml Oncostatin M, LEPCs can differentiate into hepatocytes in vitro. After 4 weeks of intravenous tail vein injection into CCl4-injured mouse liver, LEPCs engrafted into liver parenchyma, differentiated into ALB positive hepatocytes, and could alleviate liver fibrosis through down regulating fibrosis genes-Tgfb1 and ?-SMA as well as decreasing expression of collagen gene Col1a1, Col3a1, and Col4a1, and regain liver function by recovering ALT and AST. Our findings provided a useful tool for studying liver development in vitro, new cell resource for heterograft on mouse liver diseases, and a new platform for researches on immune rejection of heterogeneous cell transplantation.
| S-EPMC5809641 | biostudies-literature
Project description:The Krüppel-like factor (KLF) family dominates highly conserved three zinc finger DNA binding domains at the C-terminus and variable transactivation domains at the N-terminus. Humans possess 18 KLF genes that are differentially expressed in various tissues. Several KLFs recognize a specific CACCC DNA motif that is commonly found within hematopoietic-specific promoters. To investigate those KLFs that are involved in human hemoglobin (Hb) switching, the present study analyzed a previous microarray data set from fetal and adult erythroid cells and validated the mRNA expression levels of 18 KLFs by reverse transcription-quantitative PCR (RT-qPCR). KLF with a decreased expression level in the fetuses was selected for a functional study in human erythroid progenitor cells using lentiviral-based short hairpin RNA knockdown. The fetuses demonstrated a lower level of KLF4 mRNA expression when compared with the adults. Downregulation of KLF4 in erythroid progenitor cells from healthy individuals and individuals with β0-thalassemia/HbE evidenced the increasing embryonic and fetal globin mRNA expression with neither significant cytotoxicity nor gene expression alteration of the examined globin regulators, KLF1, B-cell lymphoma/leukemia 11A and lymphoma/leukemia-related factor. These findings demonstrate that the downregulation of KLF4 is associated with increased embryonic and fetal globin gene expression in human erythroid progenitor cells. Moreover, identifying putative compounds or molecular approaches that effectively downregulate KLF4 and further induce embryonic globin expression may provide an alternative therapeutic strategy for α-globin substitution in severe α-thalassemia.
| S-EPMC8383760 | biostudies-literature
Project description:Human embryonic and induced pluripotent stem cells are a promising cell source for the future treatment of Parkinson´s disease. It has been shown that mesencephalic dopaminergic (mesDA) neurons arise from the midbrain floor plate in which FOXA2 acts as a key transcription factor. We identified IAP which is specifically co-expressed with FOXA2 positive cells and is suitable to isolate mesDA progenitors utilizing MACS Technolog.The sorted iPSCs were viable, enriched for midbrain specific markers, lacked expression of pluripotency markers, and differentiated into mature dopaminergic neurons in vitro. IAP might be used as a tool to purify mesDA progenitor cells for regenerative therapy in PD patients
2017-09-25 | GSE74991 | GEO