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1.
Development ; 147(20)2020 10 29.
Article in English | MEDLINE | ID: mdl-33028609

ABSTRACT

The genetic regulatory network controlling early fate choices during human blood cell development are not well understood. We used human pluripotent stem cell reporter lines to track the development of endothelial and haematopoietic populations in an in vitro model of human yolk-sac development. We identified SOX17-CD34+CD43- endothelial cells at day 2 of blast colony development, as a haemangioblast-like branch point from which SOX17-CD34+CD43+ blood cells and SOX17+CD34+CD43- endothelium subsequently arose. Most human blood cell development was dependent on RUNX1. Deletion of RUNX1 only permitted a single wave of yolk sac-like primitive erythropoiesis, but no yolk sac myelopoiesis or aorta-gonad-mesonephros (AGM)-like haematopoiesis. Blocking GFI1 and/or GFI1B activity with a small molecule inhibitor abrogated all blood cell development, even in cell lines with an intact RUNX1 gene. Together, our data define the hierarchical requirements for RUNX1, GFI1 and/or GFI1B during early human haematopoiesis arising from a yolk sac-like SOX17-negative haemogenic endothelial intermediate.


Subject(s)
Blood Cells/metabolism , Core Binding Factor Alpha 2 Subunit/metabolism , DNA-Binding Proteins/metabolism , Endothelium/metabolism , Hematopoiesis , Proto-Oncogene Proteins/metabolism , Repressor Proteins/metabolism , SOXF Transcription Factors/metabolism , Transcription Factors/metabolism , Yolk Sac/metabolism , Blood Cells/cytology , Cell Differentiation , Cell Lineage , Erythroid Cells/cytology , Erythroid Cells/metabolism , Histone Demethylases/antagonists & inhibitors , Histone Demethylases/metabolism , Humans , Models, Biological , Transcription, Genetic
2.
Stem Cell Res ; 34: 101380, 2019 01.
Article in English | MEDLINE | ID: mdl-30605840

ABSTRACT

We describe the generation and characterization of 5 human induced pluripotent stem cell (iPSC) lines derived from peripheral blood mononuclear cells (PBMCs) of healthy adult individuals. The PBMCs were reprogrammed using non-integrating Sendai viruses containing the reprogramming factors POU5F1 (OCT4), SOX2, KLF4 and MYC. The iPSC lines exhibited a normal karyotype, expressed pluripotency markers and differentiated into cells representative of the three embryonic germ layers. These iPSC lines can be used as controls in studying disease mechanisms.


Subject(s)
Cell Culture Techniques/methods , Induced Pluripotent Stem Cells/pathology , Leukocytes, Mononuclear/pathology , Adult , Cell Line , Female , Humans , Kruppel-Like Factor 4 , Male , Middle Aged , Young Adult
3.
PLoS One ; 6(6): e21136, 2011.
Article in English | MEDLINE | ID: mdl-21698266

ABSTRACT

BACKGROUND: Recent studies have identified stem/progenitor cells in human and mouse uterine epithelium, which are postulated to be responsible for tissue regeneration and proliferative disorders of human endometrium. These progenitor cells are thought to be derived from Müllerian duct (MD), the primordial female reproductive tract (FRT). METHODOLOGY/PRINCIPAL FINDINGS: We have developed a model of human reproductive tract development in which inductive neonatal mouse uterine mesenchyme (nMUM) is recombined with green fluorescent protein (GFP)-tagged human embryonic stem cells (hESCs); GFP-hESC (ENVY). We demonstrate for the first time that hESCs can be differentiated into cells with a human FRT epithelial cell phenotype. hESC derived FRT epithelial cells emerged from cultures containing MIXL1(+) mesendodermal precursors, paralleling events occurring during normal organogenesis. Following transplantation, nMUM treated embryoid bodies (EBs) generated epithelial structures with a typical MD phenotype that expressed the MD markers PAX2, HOXA10. Functionally, the hESCs derived FRT epithelium responded to exogenous estrogen by proliferating and secreting uterine-specific glycodelin A (GdA). CONCLUSIONS/SIGNIFICANCE: These data show nMUM can induce differentiation of hESC to form the FRT epithelium. This may provide a model to study early developmental events of the human FRT.


Subject(s)
Cell Differentiation , Embryonic Stem Cells/cytology , Epithelial Cells/cytology , Genitalia, Female/growth & development , Animals , Female , Genitalia, Female/cytology , Green Fluorescent Proteins/genetics , Humans , Immunohistochemistry , Polymerase Chain Reaction
4.
Int J Dev Biol ; 54(8-9): 1383-8, 2010.
Article in English | MEDLINE | ID: mdl-20563991

ABSTRACT

Slain1 was originally identified as a novel stem cell-associated gene in transcriptional profiling experiments comparing mouse and human embryonic stem cells (ESCs) and their immediate differentiated progeny. In order to obtain further insight into the potential function of Slain1, we examined the expression of beta-galactosidase in a gene-trap mouse line in which a beta-geo reporter gene was inserted into the second intron of Slain1. In early stage embryos (E7.5), the Slain1-betageo fusion protein was expressed within the entire epiblast, but by E9.5 became restricted to the developing nervous system and gastrointestinal tract. In later stage embryos (E11.5 - E13.5), expression was predominantly within the developing nervous system. Lower level expression was also observed in the developing limb buds, in the condensing mesenchyme, along the apical epidermal ridge and, at later stages, within the digital zones. These observations suggest that Slain1 may play a role in the development of the nervous system, as well as in the morphogenesis of several embryonic structures.


Subject(s)
Gene Expression Regulation, Developmental , Nervous System/metabolism , Proteins/genetics , beta-Galactosidase/genetics , Animals , Base Sequence , Embryo, Mammalian/embryology , Embryo, Mammalian/metabolism , Gastrointestinal Tract/embryology , Gastrointestinal Tract/metabolism , Gene Expression Profiling , Histocytochemistry , Humans , Limb Buds/embryology , Limb Buds/metabolism , Male , Mice , Molecular Sequence Data , Nervous System/embryology , Proteins/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Nucleic Acid , Time Factors , beta-Galactosidase/metabolism
5.
Stem Cell Res ; 4(2): 140-7, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20060373

ABSTRACT

Human ESCs (hESCs) are a valuable tool for the study of early human development and represent a source of normal differentiated cells for pharmaceutical and biotechnology applications and ultimately for cell replacement therapies. For all applications, it will be necessary to develop assays to validate the efficacy of hESC differentiation. We explored the capacity for FTIR spectroscopy, a technique that rapidly characterises cellular macromolecular composition, to discriminate mesendoderm or ectoderm committed cells from undifferentiated hESCs. Distinct infrared spectroscopic "signatures" readily distinguished hESCs from these early differentiated progeny, with bioinformatic models able to correctly classify over 97% of spectra. These data identify a role for FTIR spectroscopy as a new modality to complement conventional analyses of hESCs and their derivatives. FTIR spectroscopy has the potential to provide low-cost, automatable measurements for the quality control of stem and differentiated cells to be used in industry and regenerative medicine.


Subject(s)
Cell Differentiation/physiology , Embryonic Stem Cells/cytology , Spectroscopy, Fourier Transform Infrared , Computational Biology , Embryonic Stem Cells/metabolism , Least-Squares Analysis , Principal Component Analysis
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