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Stem Cell Reports May 2024SOX2 is a transcription factor involved in the regulatory network maintaining the pluripotency of embryonic stem cells in culture as well as in early embryos. In...
SOX2 is a transcription factor involved in the regulatory network maintaining the pluripotency of embryonic stem cells in culture as well as in early embryos. In addition, SOX2 plays a pivotal role in neural stem cell formation and neurogenesis. How SOX2 can serve both processes has remained elusive. Here, we identified a set of SOX2-dependent neural-associated enhancers required for neural lineage priming. They form a distinct subgroup (1,898) among 8,531 OCT4/SOX2/NANOG-bound enhancers characterized by enhanced SOX2 binding and chromatin accessibility. Activation of these enhancers is triggered by neural induction of wild-type cells or by default in Smad4-ablated cells resistant to mesoderm induction and is antagonized by mesodermal transcription factors via Sox2 repression. Our data provide mechanistic insight into the transition from the pluripotency state to the early neural fate and into the regulation of early neural versus mesodermal specification in embryonic stem cells and embryos.
Topics: SOXB1 Transcription Factors; Animals; Enhancer Elements, Genetic; Mice; Neural Stem Cells; Mesoderm; Neurogenesis; Gene Expression Regulation, Developmental; Octamer Transcription Factor-3; Cell Differentiation; Nanog Homeobox Protein; Cell Lineage; Smad4 Protein; Embryonic Stem Cells; Mouse Embryonic Stem Cells; Chromatin; Protein Binding
PubMed: 38579708
DOI: 10.1016/j.stemcr.2024.03.003 -
Life Science Alliance Jun 2024Cardiovascular system develops from the lateral plate mesoderm. Its three primary cell lineages (hematopoietic, endothelial, and muscular) are specified by the...
Cardiovascular system develops from the lateral plate mesoderm. Its three primary cell lineages (hematopoietic, endothelial, and muscular) are specified by the sequential actions of conserved transcriptional factors. , a master regulator of mammalian hemangioblast development, however, is absent in the chicken genome and acts downstream of in zebrafish. Here, we investigated the epistatic relationship between NPAS4L and ETV2 in avian hemangioblast development. We showed that is deleted in all 363 avian genomes analyzed. Mouse ETV2 induced LMO2, but not NPAS4L or SCL, expression in chicken mesoderm. Squamate (lizards, geckos, and snakes) genomes contain both and In Madagascar ground gecko, both genes were expressed in developing hemangioblasts. Gecko ETV2 induced only LMO2 in chicken mesoderm. We propose that both and were present in ancestral amniote, with ETV2 acting downstream of NPAS4L in endothelial lineage specification. ETV2 may have acted as a pioneer factor by promoting chromatin accessibility of endothelial-specific genes and, in parallel with loss in ancestral mammals, has gained similar function in regulating blood-specific genes.
Topics: Animals; Mice; Cell Differentiation; Zebrafish; Hematopoietic Stem Cells; Transcription Factors; Birds; Mammals
PubMed: 38570190
DOI: 10.26508/lsa.202402694 -
Developmental Cell May 2024The developmental origin of blood-forming hematopoietic stem cells (HSCs) is a longstanding question. Here, our non-invasive genetic lineage tracing in mouse embryos...
The developmental origin of blood-forming hematopoietic stem cells (HSCs) is a longstanding question. Here, our non-invasive genetic lineage tracing in mouse embryos pinpoints that artery endothelial cells generate HSCs. Arteries are transiently competent to generate HSCs for 2.5 days (∼E8.5-E11) but subsequently cease, delimiting a narrow time frame for HSC formation in vivo. Guided by the arterial origins of blood, we efficiently and rapidly differentiate human pluripotent stem cells (hPSCs) into posterior primitive streak, lateral mesoderm, artery endothelium, hemogenic endothelium, and >90% pure hematopoietic progenitors within 10 days. hPSC-derived hematopoietic progenitors generate T, B, NK, erythroid, and myeloid cells in vitro and, critically, express hallmark HSC transcription factors HLF and HOXA5-HOXA10, which were previously challenging to upregulate. We differentiated hPSCs into highly enriched HLF+ HOXA+ hematopoietic progenitors with near-stoichiometric efficiency by blocking formation of unwanted lineages at each differentiation step. hPSC-derived HLF+ HOXA+ hematopoietic progenitors could avail both basic research and cellular therapies.
Topics: Animals; Humans; Mice; Cell Differentiation; Cell Lineage; Endothelial Cells; Hematopoiesis; Hematopoietic Stem Cells; Homeodomain Proteins; Pluripotent Stem Cells; Transcription Factors; Basic-Leucine Zipper Transcription Factors
PubMed: 38569552
DOI: 10.1016/j.devcel.2024.03.003 -
Circulation Research May 2024Pericytes are capillary-associated mural cells involved in the maintenance and stability of the vascular network. Although aging is one of the main risk factors for...
BACKGROUND
Pericytes are capillary-associated mural cells involved in the maintenance and stability of the vascular network. Although aging is one of the main risk factors for cardiovascular disease, the consequences of aging on cardiac pericytes are unknown.
METHODS
In this study, we have combined single-nucleus RNA sequencing and histological analysis to determine the effects of aging on cardiac pericytes. Furthermore, we have conducted in vivo and in vitro analysis of RGS5 (regulator of G-protein signaling 5) loss of function and finally have performed pericytes-fibroblasts coculture studies to understand the effect of RGS5 deletion in pericytes on the neighboring fibroblasts.
RESULTS
Aging reduced the pericyte area and capillary coverage in the murine heart. Single-nucleus RNA sequencing analysis further revealed that the expression of was reduced in cardiac pericytes from aged mice. In vivo and in vitro studies showed that the deletion of RGS5 impaired cardiac function, induced fibrosis, and morphological changes in pericytes characterized by a profibrotic gene expression signature and the expression of different ECM (extracellular matrix) components and growth factors, for example, and . Indeed, culturing fibroblasts with the supernatant of RGS5-deficient pericytes induced their activation as evidenced by the increased expression of αSMA (alpha smooth muscle actin) in a TGFβ (transforming growth factor beta)2-dependent mechanism.
CONCLUSIONS
Our results have identified RGS5 as a crucial regulator of pericyte function during cardiac aging. The deletion of RGS5 causes cardiac dysfunction and induces myocardial fibrosis, one of the hallmarks of cardiac aging.
Topics: Pericytes; Animals; RGS Proteins; Fibrosis; Fibroblasts; Mice; Cells, Cultured; Aging; Mice, Inbred C57BL; Mice, Knockout; Myocardium; Male; Coculture Techniques
PubMed: 38563133
DOI: 10.1161/CIRCRESAHA.123.324183 -
Stem Cell Research Jun 2024Phenylketonuria is a rare autosomal recessive metabolic disorder mainly due to a significant reduction in the enzyme phenylalanine hydroxylase, resulting in elevation of...
Phenylketonuria is a rare autosomal recessive metabolic disorder mainly due to a significant reduction in the enzyme phenylalanine hydroxylase, resulting in elevation of phenylalanine in the blood. Here, we have established two fibroblast-derived induced pluripotent stem cell lines using Sendai virus-based reprogramming. The established induced pluripotent stem cell lines exhibited a normal karyotype and expressed markers of pluripotency assessed through quantitative PCR, flow cytometry and immunocytochemistry. These cell lines also demonstrated the ability to differentiate into the three primary germ layers of the human body, including ectoderm, endoderm, and mesoderm.
Topics: Humans; Induced Pluripotent Stem Cells; Phenylketonurias; Fibroblasts; Cell Differentiation; Cell Line; Male; Child
PubMed: 38555716
DOI: 10.1016/j.scr.2024.103405 -
Stem Cell Research Jun 2024We employed a Sendai virus-based reprogramming method to transform human lymphoblastoid cell lines (LCL) derived from two individuals diagnosed with phenylketonuria...
We employed a Sendai virus-based reprogramming method to transform human lymphoblastoid cell lines (LCL) derived from two individuals diagnosed with phenylketonuria (PKU) into induced pluripotent stem cells (iPSC). This reprogramming process involved the expression of the four Yamanaka factors: KLF4, OCT4, SOX2, and C-MYC. The resulting patient-specific iPSCs exhibited a normal karyotype and expressed endogenous pluripotent markers NANOG and OCT-4. Notably, these iPSCs demonstrated strong differentiation capabilities, giving rise to cell populations representing the ectoderm, endoderm, and mesoderm germ layers.
Topics: Humans; Kruppel-Like Factor 4; Induced Pluripotent Stem Cells; Phenylketonurias; Cell Differentiation; Cell Line; Male; Lymphocytes; Cellular Reprogramming
PubMed: 38552357
DOI: 10.1016/j.scr.2024.103407 -
The Journal of Neuroscience : the... May 2024The neurovascular unit (NVU) includes multiple different cell types, including neurons, astrocytes, endothelial cells, and pericytes, which respond to insults on very...
The neurovascular unit (NVU) includes multiple different cell types, including neurons, astrocytes, endothelial cells, and pericytes, which respond to insults on very different time or dose scales. We defined differential vulnerability among these cell types, using response to two different insults: oxygen-glucose deprivation (OGD) and thrombin-mediated cytotoxicity. We found that neurons are most vulnerable, followed by endothelial cells and astrocytes. After temporary focal cerebral ischemia in male rats, we found significantly more injured neurons, compared with astrocytes in the ischemic area, consistent with differential vulnerability in vivo. We sought to illustrate different and shared mechanisms across all cell types during response to insult. We found that gene expression profiles in response to OGD differed among the cell types, with a paucity of gene responses shared by all types. All cell types activated genes relating to autophagy, apoptosis, and necroptosis, but the specific genes differed. Astrocytes and endothelial cells also activated pathways connected to DNA repair and antiapoptosis. Taken together, the data support the concept of differential vulnerability in the NVU and suggest that different elements of the unit will evolve from salvageable to irretrievable on different time scales while residing in the same brain region and receiving the same (ischemic) blood flow. Future work will focus on the mechanisms of these differences. These data suggest future stroke therapy development should target different elements of the NVU differently.
Topics: Animals; Male; Rats; Astrocytes; Endothelial Cells; Neurons; Rats, Sprague-Dawley; Brain; Glucose; Brain Ischemia; Pericytes; Neurovascular Coupling
PubMed: 38548341
DOI: 10.1523/JNEUROSCI.1093-22.2024 -
Stem Cell Research Jun 2024We developed a well-characterized human induced pluripotent stem cell (iPSC) line obtained from healthy individuals' peripheral blood mononuclear cells (PBMC). The PBMCs...
We developed a well-characterized human induced pluripotent stem cell (iPSC) line obtained from healthy individuals' peripheral blood mononuclear cells (PBMC). The PBMCs were primed and reprogrammed using a non-integrating sendai viral vector, and the iPSC lines demonstrated complete differentiation capacity. This line, YBLi004-A, is available and registered in the human pluripotent stem cell registry. The line's legitimacy was validated using pluripotent marker expression, in vitro differentiation into three germ layers (ectoderm, mesoderm, and endoderm), karyotyping, and STR analysis. This iPSC line could be used as a healthy control for studies involving disease-specific-iPSCs, e.g. drug toxicity and efficacy testing.
Topics: Humans; Induced Pluripotent Stem Cells; Sendai virus; Leukocytes, Mononuclear; Cell Line; Cell Differentiation; Cellular Reprogramming
PubMed: 38547666
DOI: 10.1016/j.scr.2024.103402 -
Genes Feb 2024While animal model studies have extensively defined the mechanisms controlling cell diversity in the developing mammalian lung, there exists a significant knowledge gap...
While animal model studies have extensively defined the mechanisms controlling cell diversity in the developing mammalian lung, there exists a significant knowledge gap with regards to late-stage human lung development. The NHLBI Molecular Atlas of Lung Development Program (LungMAP) seeks to fill this gap by creating a structural, cellular and molecular atlas of the human and mouse lung. Transcriptomic profiling at the single-cell level created a cellular atlas of newborn human lungs. Frozen single-cell isolates obtained from two newborn human lungs from the LungMAP Human Tissue Core Biorepository, were captured, and library preparation was completed on the Chromium 10X system. Data was analyzed in Seurat, and cellular annotation was performed using the ToppGene functional analysis tool. Transcriptional interrogation of 5500 newborn human lung cells identified distinct clusters representing multiple populations of epithelial, endothelial, fibroblasts, pericytes, smooth muscle, immune cells and their gene signatures. Computational integration of data from newborn human cells and with 32,000 cells from postnatal days 1 through 10 mouse lungs generated by the LungMAP Cincinnati Research Center facilitated the identification of distinct cellular lineages among all the major cell types. Integration of the newborn human and mouse cellular transcriptomes also demonstrated cell type-specific differences in maturation states of newborn human lung cells. Specifically, newborn human lung matrix fibroblasts could be separated into those representative of younger cells ( = 393), or older cells ( = 158). Cells with each molecular profile were spatially resolved within newborn human lung tissue. This is the first comprehensive molecular map of the cellular landscape of neonatal human lung, including biomarkers for cells at distinct states of maturity.
Topics: Animals; Humans; Mice; Gene Expression Profiling; Lung; Mammals; Pericytes; Phenotype; Transcriptome; Infant, Newborn
PubMed: 38540357
DOI: 10.3390/genes15030298 -
Molecular Biology of the Cell May 2024The regulation of the cytoskeleton by multiple signaling pathways, sometimes in parallel, is a common principle of morphogenesis. A classic example of regulation by...
The regulation of the cytoskeleton by multiple signaling pathways, sometimes in parallel, is a common principle of morphogenesis. A classic example of regulation by parallel pathways is gastrulation, where the inputs from the Folded gastrulation (Fog)/Concertina (Cta) and the T48 pathways induce apical constriction and mesoderm invagination. Whether there are distinct roles for these separate pathways in regulating the complex spatial and temporal patterns of cytoskeletal activity that accompany early embryo development is still poorly understood. We investigated the roles of the Fog/Cta and T48 pathways and found that, by themselves, the Cta and T48 pathways both promote timely mesoderm invagination and apical myosin II accumulation, with Cta being required for timely cell shape change ahead of mitotic cell division. We also identified distinct functions of T48 and Cta in regulating cellularization and the uniformity of the apical myosin II network, respectively. Our results demonstrate that both redundant and distinct functions for the Fog/Cta and T48 pathways exist.
Topics: Animals; Drosophila; Gastrulation; Drosophila Proteins; Morphogenesis; Mesoderm; Myosin Type II; Drosophila melanogaster
PubMed: 38536475
DOI: 10.1091/mbc.E24-02-0050