MiRNA-based mechanism for the commitment of multipotent progenitors to a single cellular fate.
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ABSTRACT:
SUBMITTER: Mann M
PROVIDER: S-EPMC2936650 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The mechanism of lineage commitment from hematopoietic stem cells (HSCs) is not well understood. Although commitment to either the lymphoid or the myeloid lineage is popularly viewed as the first step of lineage restriction from HSCs, this model of hematopoietic differentiation has recently been challenged. The previous identification of multipotent progenitors (MPPs) that can produce lymphocytes and granulocyte/macrophages (GMs) but lacks erythroid differentiation ability suggests the existence of an alternative HSC differentiation program. Contribution to different hematopoietic lineages by these MPPs under physiological conditions, however, has not been carefully examined. In this study, we performed a refined characterization of MPPs by subfractionating three distinct subsets based on Flt3 and vascular cell adhesion molecule 1 expression. These MPP subsets differ in their ability to give rise to erythroid and GM lineage cells but are equally potent in lymphoid lineage differentiation in vivo. The developmental hierarchy of these MPP subsets demonstrates the sequential loss of erythroid and then GM differentiation potential during early hematopoiesis. Our results suggest that the first step of lineage commitment from HSCs is not simply a selection between the lymphoid and the myeloid lineage.
| S-EPMC2118384 | biostudies-other
Project description:Stem cell fate is orchestrated by core transcription factors (TFs) and epigenetic modifications. Although regulatory genes that control cell type specification are identified, the transcriptional circuit and the cross-talk among regulatory factors during cell fate decisions remain poorly understood. To identify the "time-lapse" TF networks during B-lineage commitment, we used multipotent progenitors harboring a tamoxifen-inducible form of Id3, an in vitro system in which virtually all cells became B cells within 6 d by simply withdrawing 4-hydroxytamoxifen (4-OHT). Transcriptome and epigenome analysis at multiple time points revealed that ∼10%-30% of differentially expressed genes were virtually controlled by the core TFs, including E2A, EBF1, and PAX5. Strikingly, we found unexpected transcriptional priming before the onset of the key TF program. Inhibition of the immediate early genes such as Nr4a2, Klf4, and Egr1 severely impaired the generation of B cells. Integration of multiple data sets, including transcriptome, protein interactome, and epigenome profiles, identified three representative transcriptional circuits. Single-cell RNA sequencing (RNA-seq) analysis of lymphoid progenitors in bone marrow strongly supported the three-step TF network model during specification of multipotent progenitors toward B-cell lineage in vivo. Thus, our findings will provide a blueprint for studying the normal and neoplastic development of B lymphocytes.
| S-EPMC5830925 | biostudies-literature
Project description:Distinct families of multipotent heart progenitors play a central role in the generation of diverse cardiac, smooth muscle and endothelial cell lineages during mammalian cardiogenesis. The identification of precise paracrine signals that drive the cell-fate decision of these multipotent progenitors, and the development of novel approaches to deliver these signals in vivo, are critical steps towards unlocking their regenerative therapeutic potential. Herein, we have identified a family of human cardiac endothelial intermediates located in outflow tract of the early human fetal hearts (OFT-ECs), characterized by coexpression of Isl1 and CD144/vWF. By comparing angiocrine factors expressed by the human OFT-ECs and non-cardiac ECs, vascular endothelial growth factor (VEGF)-A was identified as the most abundantly expressed factor, and clonal assays documented its ability to drive endothelial specification of human embryonic stem cell (ESC)-derived Isl1+ progenitors in a VEGF receptor-dependent manner. Human Isl1-ECs (endothelial cells differentiated from hESC-derived ISL1+ progenitors) resemble OFT-ECs in terms of expression of the cardiac endothelial progenitor- and endocardial cell-specific genes, confirming their organ specificity. To determine whether VEGF-A might serve as an in vivo cell-fate switch for human ESC-derived Isl1-ECs, we established a novel approach using chemically modified mRNA as a platform for transient, yet highly efficient expression of paracrine factors in cardiovascular progenitors. Overexpression of VEGF-A promotes not only the endothelial specification but also engraftment, proliferation and survival (reduced apoptosis) of the human Isl1+ progenitors in vivo. The large-scale derivation of cardiac-specific human Isl1-ECs from human pluripotent stem cells, coupled with the ability to drive endothelial specification, engraftment, and survival following transplantation, suggest a novel strategy for vascular regeneration in the heart.
| S-EPMC3790234 | biostudies-literature
Project description:ObjectiveHuman multipotent stromal cells readily form single-cell-derived colonies when plated at clonal densities. However, the colonies are heterogeneous because cells from a colony form new colonies that vary in size and differentiation potential when replated at clonal densities. The experiments here tested the hypothesis that cells in the inner regions of colonies are partially differentiated, but the differentiation is reversible.Materials and methodsCells were separately isolated from the dense inner (IN) regions and less-dense outer regions (OUT) of single-cell-derived colonies. Cells were then compared by assays of their transcriptomes and proteins, and for clonogenicity and differentiation.ResultsIN cells expressed fewer cell-cycle genes and higher levels of genes for extracellular matrix than the OUT cells. When transferred to differentiation medium, differentiation of the colonies occurred primarily in the IN regions. However, the IN cells were indistinguishable from OUT cells when replated at clonal densities and assayed for rates of propagation and clonogenicity. Also, colonies formed by IN cells were similar to colonies formed by OUT cells because they had distinct IN and OUT regions. Cultures of IN and OUT cells remained indistinguishable through multiple passages (30 to 75 population doublings), and both cells formed colonies that were looser and less dense as they were expanded.ConclusionsThe results demonstrated that cells in the IN region of single-cell-derived colonies are partially differentiated, but the differentiation can be reversed by replating the cells at clonal densities.
| S-EPMC2628773 | biostudies-literature
Project description:BackgroundImproving the neuronal yield from in vitro cultivated neural progenitor cells (NPCs) is an essential challenge in transplantation therapy in neurological disorders. In this regard, Ascorbic acid (AA) is widely used to expand neurogenesis from NPCs in cultures although the mechanisms of its action remain unclear. Neurogenesis from NPCs is regulated by the redox-sensitive WNT/β-catenin signaling pathway. We therefore aimed to investigate how AA interacts with this pathway and potentiates neurogenesis.MethodsEffects of 200 μM AA were compared with the pro-neurogenic reagent and WNT/β-catenin signaling agonist lithium chloride (LiCl), and molecules with antioxidant activities i.e. N-acetyl-L-cysteine (NAC) and ruthenium red (RuR), in differentiating neural progenitor ReNcell VM cells. Cells were supplemented with reagents for two periods of treatment: a full period encompassing the whole differentiation process versus an early short period that is restricted to the cell fate commitment stage. Intracellular redox balance and reactive oxygen species (ROS) metabolism were examined by flow cytometry using redox and ROS sensors. Confocal microscopy was performed to assess cell viability, neuronal yield, and levels of two proteins: Nucleoredoxin (NXN) and the WNT/β-catenin signaling component Dishevelled 2 (DVL2). TUBB3 and MYC gene responses were evaluated by quantitative real-time PCR. DVL2-NXN complex dissociation was measured by fluorescence resonance energy transfer (FRET).ResultsIn contrast to NAC which predictably exhibited an antioxidant effect, AA treatment enhanced ROS metabolism with no cytotoxic induction. Both drugs altered ROS levels only at the early stage of the differentiation as no changes were held beyond the neuronal fate commitment stage. FRET studies showed that AA treatment accelerated the redox-dependent release of the initial pool of DVL2 from its sequestration by NXN, while RuR treatment hampered the dissociation of the two proteins. Accordingly, AA increased WNT/β-catenin signaling output i.e. MYC mRNA level, whereas RuR attenuated it. Moreover, AA improved neurogenesis as much as LiCl as both TUBB3-positive cell yield and TUBB3 mRNA level increased, while NAC or RuR attenuated neurogenesis. Markedly, the neurogenesis outputs between the short and the full treatment with either NAC or AA were found unchanged, supporting our model that neuronal yield is altered by events taking place at the early phase of differentiation.ConclusionsOur findings demonstrate that AA treatment elevates ROS metabolism in a non-lethal manner prior to the NPCs commitment to their neuronal fate. Such effect stimulates the redox-sensitive DVL2 activation and WNT/β-catenin signaling response that would enhance the ensuing neuronal cell differentiation.
| S-EPMC5641995 | biostudies-literature
Project description:Multipotent mesenchymal stem/stromal cells (MSC) are one of the crucial regulators of regeneration and tissue repair and possess an intrinsic program from self-organization mediated by condensation, migration and self-patterning. The ability to self-organize has been successfully exploited in tissue engineering approaches using cell sheets (CS) and their modifications. In this study, we used CS as a model of human MSC spontaneous self-organization to demonstrate its structural, transcriptomic impact and multipotent stromal cell commitment. We used CS formation to visualize MSC self-organization and evaluated the role of the Rho-GTPase pathway in spontaneous condensation, resulting in a significant anisotropy of the cell density within the construct. Differentiation assays were carried out using conventional protocols, and microdissection and RNA-sequencing were applied to establish putative targets behind the observed phenomena. The differentiation of MSC to bone and cartilage, but not to adipocytes in CS, occurred more effectively than in the monolayer. RNA-sequencing indicated transcriptional shifts involving the activation of the Rho-GTPase pathway and repression of SREBP, which was concordant with the lack of adipogenesis in CS. Eventually, we used an inhibitory analysis to validate our findings and suggested a model where the self-organization of MSC defined their commitment and cell fate via ROCK1/2 and SREBP as major effectors under the putative switching control of AMP kinase.
| S-EPMC8470239 | biostudies-literature
Project description:The advent of single-cell transcriptomics has led to the proposal of a number of novel high-resolution models for the hematopoietic system. Testing the predictions generated by such models requires cell fate potential assays of matching, single-cell resolution. Here we detail the development of an in vitro high-throughput single-cell culture assay using flow cytometrically sorted single murine bone marrow progenitors, which measures their differentiation into any of five myeloid lineages. We identify critical parameters for single-cell culture outcome, including the choice of sorter nozzle size and pressure, culture media, and the coating of culture dishes with extracellular matrix proteins. Further, we find that accurate assay readout requires the titration of antibodies specifically for their use under low-cell-number conditions. Our approach may be used as a template for the development of single-cell fate potential assays for a variety of blood cell progenitors.
| S-EPMC5857247 | biostudies-literature
Project description:Adipose derived stem cells (ASCs) can be obtained from lipoaspirates and induced in vitro to differentiate into bone, cartilage, and fat. Using this powerful model system we show that after in vitro adipose differentiation a population of cells retain stem-like qualities including multipotency. They are lipid (-), retain the ability to propagate, express two known stem cell markers, and maintain the capacity for trilineage differentiation into chondrocytes, adipocytes, and osteoblasts. However, these cells are not traditional stem cells because gene expression analysis showed an overall expression profile similar to that of adipocytes. In addition to broadening our understanding of cellular multipotency, our work may be particularly relevant to obesity-associated metabolic disorders. The adipose expandability hypothesis proposes that inability to differentiate new adipocytes is a primary cause of metabolic syndrome in obesity, including diabetes and cardiovascular disease. Here we have defined a differentiation-resistant stem-like multipotent cell population that may be involved in regulation of adipose expandability in vivo and may therefore play key roles in the comorbidities of obesity.
| S-EPMC6013723 | biostudies-literature
Project description:BackgroundHematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) play a pivotal role in maintaining lifelong hematopoiesis. The distinction between stem cells and other progenitors, as well as the assessment of their functions, has long been a central focus in stem cell research. In recent years, deep learning has emerged as a powerful tool for cell image analysis and classification/prediction.MethodsIn this study, we explored the feasibility of employing deep learning techniques to differentiate murine HSCs and MPPs based solely on their morphology, as observed through light microscopy (DIC) images.ResultsAfter rigorous training and validation using extensive image datasets, we successfully developed a three-class classifier, referred to as the LSM model, capable of reliably distinguishing long-term HSCs, short-term HSCs, and MPPs. The LSM model extracts intrinsic morphological features unique to different cell types, irrespective of the methods used for cell identification and isolation, such as surface markers or intracellular GFP markers. Furthermore, employing the same deep learning framework, we created a two-class classifier that effectively discriminates between aged HSCs and young HSCs. This discovery is particularly significant as both cell types share identical surface markers yet serve distinct functions. This classifier holds the potential to offer a novel, rapid, and efficient means of assessing the functional states of HSCs, thus obviating the need for time-consuming transplantation experiments.ConclusionOur study represents the pioneering use of deep learning to differentiate HSCs and MPPs under steady-state conditions. This novel and robust deep learning-based platform will provide a basis for the future development of a new generation stem cell identification and separation system. It may also provide new insight into the molecular mechanisms underlying stem cell self-renewal.
| S-EPMC10935795 | biostudies-literature
Project description:Hematopoietic stem and progenitor cells (HSPCs) can self-renew and create committed progenitors, a process supposed to involve asymmetric cell divisions (ACDs). Previously, we had linked the kinetics of CD133 expression with ACDs but failed to detect asymmetric segregation of classical CD133 epitopes on fixed, mitotic HSPCs. Now, by using a novel anti-CD133 antibody (HC7), we confirmed the occurrence of asymmetric CD133 segregation on paraformaldehyde-fixed and living HSPCs. After showing that HC7 binding does not recognizably affect biological features of human HSPCs, we studied ACDs in different HSPC subtypes and determined the developmental potential of arising daughter cells at the single-cell level. Approximately 70% of the HSPCs of the multipotent progenitor (MPP) fraction studied performed ACDs, and about 25% generated lymphoid-primed multipotent progenitor (LMPP) as wells as erythromyeloid progenitor (EMP) daughter cells. Since MPPs hardly created daughter cells maintaining MPP characteristics, our data suggest that under conventional culture conditions, ACDs are lineage instructive rather than self-renewing.
| S-EPMC4263999 | biostudies-literature