Microrna-130a Regulates Hematopoietic Stem Cell Self-Renewal By Repressing Chromatin Modifiers and Shaping the Accessible Chromatin Landscape

Abstract Background: Residing at the apex of the blood system hierarchy, hematopoietic stem cells (HSCs) are endowed with multi-potency and self-renewal potential. Hematopoietic homeostasis is tightly regulated by controlling the balance between quiescence, self-renewal and lineage-commitment of HSCs. Although many studies have profiled gene expression patterns and epigenomes of HSC and downstream progenitors, post-transcriptional regulation of determinants that control these regulatory networks is largely unknown. MicroRNAs (miRNAs) represent a large class of post-transcriptional regulators that mediate repression of multiple target mRNAs by inhibiting their translation and/or inducing their degradation. A limited number of reports suggest that miRNAs are differentially expressed across the hematopoietic hierarchy and control lineage commitment and cell fate decisions by orchestrating gene regulatory networks, however the mechanisms remain unexplored. Methods: To identify miRNA(s) that play a functional role in human hematopoiesis, we performed an in vivo competitive repopulation screen in which candidate miRNAs were over-expressed (OE) in human CD34+CD38- umbilical cord blood (CB) cells and subsequently transplanted into immune-deficient mice for 24 weeks. miR-130a was shown to enhance long-term hematopoietic reconstitution and chosen for further investigation. Results: As miRNAs are negative regulators of gene expression, we studied the functional impact of miR-130a on long-term hematopoietic reconstitution by enforcing its expression in CB cells using lentiviral vector containing orange fluorescent protein (OFP) reporter. At 12 and 24 weeks after transplantation, increased miR-130a expression conferred a statistically significant, competitive advantage to transduced CB cells demonstrated by increased human chimerism and the proportion of OFP+/hCD45+ cells in the injected femur (IF), bone marrow (BM) and spleen of recipient mice. Xenografts produced by miR-130 O/E showed multi-lineage engraftment with myeloid skewing at the expense of B-lymphoid development and significantly enhanced erythroid output in RF, BM and spleen. In addition, ectopic expression of miR-130a caused splenomegaly in recipient mice. Flow cytometry analysis using several markers expressed during erythroid development revealed accumulation of immature GlyA+/CD71+/CD36+ erythroid progenitors, suggesting an erythroid differentiation block. Enforced expression of miR-130a also perturbed myeloid differentiation shown by the presence of abnormal CD14+/CD66b+ myeloid cells in the BM. At the primitive and progenitor cell stages, miR-130a O/E caused significant expansion of primitive CD34+/CD38- cells and increased the proportion of immuno-phenotypic HSC. Secondary transplantation involving limited dilution analysis revealed 10-fold increase in HSC frequency, consistent with a role of miR-130a in HSC self-renewal. Analysis of chromatin accessibility surrounding the miR-130a locus across the human hematopoietic hierarchy revealed peaks of accessible chromatin in HSC and downstream progenitors that were absent in mature cells. To ascertain the molecular mechanism of miR-130a function, label-free semi-quantitative proteomics was performed to determine differentially expressed proteins between miR-130a O/E and control-transduced CD34+ CB cells. Gene set enrichment analysis (GSEA) identified top miR-130a downregulated gene sets centered on chromatin remodelling. Components of SMRT/N-CoR co-repressor complex and polycomb repressive complex (PRC2) were identified to be among the top downregulated miR-130a targets. We assessed the impact of miR-130a O/E on the global chromatin accessibility landscape by performing ATAC-seq on CD34+ CB cells transduced with miR-130a or control lentivirus. Enforced expression of miR-130a resulted in a gain of approximately 450 accessible chromatin peaks. Transcription factor DNA recognition motif analysis revealed significant enrichment of GATA3 motif in accessible sites specific to miR-130a O/E cells. Conclusion: Together, our data suggests that miR-130a regulates HSC self-renewal and lineage specification. miR-130a mediates repression of several gene networks centered on chromatin remodelling and focally reshapes the accessible chromatin landscape of HSPC. Disclosures No relevant conflicts of interest to declare.

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Maximal STAT5-Induced Proliferation and Self-Renewal at Intermediate STAT5 Activity Levels

Molecular and Cellular Biology ◽

10.1128/mcb.01025-08 ◽

2008 ◽

Vol 28 (21) ◽

pp. 6668-6680 ◽

Cited By ~ 61

Author(s):

Albertus T. J. Wierenga ◽

Edo Vellenga ◽

Jan Jacob Schuringa

Keyword(s):

Gene Expression ◽

Cell Fate ◽

Target Genes ◽

Expression Patterns ◽

Activity Levels ◽

Dependent Manner ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cell Fate Decisions

ABSTRACT The level of transcription factor activity critically regulates cell fate decisions, such as hematopoietic stem cell (HSC) self-renewal and differentiation. We introduced STAT5A transcriptional activity into human HSCs/progenitor cells in a dose-dependent manner by overexpression of a tamoxifen-inducible STAT5A(1*6)-estrogen receptor fusion protein. Induction of STAT5A activity in CD34+ cells resulted in impaired myelopoiesis and induction of erythropoiesis, which was most pronounced at the highest STAT5A transactivation levels. In contrast, intermediate STAT5A activity levels resulted in the most pronounced proliferative advantage of CD34+ cells. This coincided with increased cobblestone area-forming cell and long-term-culture-initiating cell frequencies, which were predominantly elevated at intermediate STAT5A activity levels but not at high STAT5A levels. Self-renewal of progenitors was addressed by serial replating of CFU, and only progenitors containing intermediate STAT5A activity levels contained self-renewal capacity. By extensive gene expression profiling we could identify gene expression patterns of STAT5 target genes that predominantly associated with a self-renewal and long-term expansion phenotype versus those that identified a predominant differentiation phenotype.

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A Short Pulse of Prostaglandin E2 (PGE2) Induces Long Term Chromatin Changes in Hematopoietic Stem Cells Leading to Increased Self-Renewal and Engraftment

Blood ◽

10.1182/blood.v126.23.246.246 ◽

2015 ◽

Vol 126 (23) ◽

pp. 246-246

Author(s):

Eva M Fast ◽

Ellen M Durand ◽

Audrey Sporrij ◽

Leslie Ojeaburu ◽

Rebecca Maher ◽

...

Keyword(s):

Gene Expression ◽

Open Chromatin ◽

Advisory Committees ◽

Self Renewal ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cd34 Cells ◽

Equity Ownership ◽

Induced Genes

Abstract Hematopoietic stem cells (HSCs) offer promising treatment options for many blood diseases. We have previously identified Prostaglandin E2 (PGE2), a small molecule that increased HSC numbers in the zebrafish embryo. In an adult mammalian transplantation setting a two hour treatment significantly enhanced HSC engraftment. Currently PGE2 is being tested in a phase 2 clinical trial to improve cord blood transplants. To better understand PGE2 effect on HSCs mouse multipotent progenitors (MPP), short term (ST) HSCs, and long term (LT) HSCs were isolated via FACS and given a two hour pulse of PGE2 followed by a competitive transplantation assay. Surprisingly, PGE2 treatment mainly affected ST-HSCs by dramatically prolonging their ability to contribute to peripheral blood. The effect of the two hour treatment persisted through secondary competitive transplants in which robust peripheral blood chimerism of ST-HSCs was evident even 1.5 years after having been exposed to the drug. To elucidate underlying molecular changes gene expression right after PGE2 treatment as well as in ST-HSCs after transplantation was assessed. PGE2 target genes were divided into two categories; "transiently induced" and "permanently induced" genes. Most of the transcripts upregulated two hour after PGE2 treatment were "transiently induced" meaning that they did not continue to be differentially expressed after transplantation. In contrast, a few transcripts including chemokines such as Cxcl2, Cxcl3, members of the Fos gene family as well as Nr4a1, 2 and 3 were both upregulated right after PGE2 treatment as well as in ST-HSCs after transplantation. We classified these genes as "permanently induced". ATAC (Assay for Transposase-Accessible Chromatin)-seq analysis of the transplanted PGE2 treated cells indicated that these "permanently induced" genes maintained a distinctly open chromatin profile in both promotor and enhancer regions, whereas the "transiently induced" genes did not. Gene expression in human CD34+ cells included a signature implying CREB as the main transcription factor responsible for the acute PGE2 response. Phospho-FACS in mouse ST-HSCs and Western-blot analysis in human CD34+ cells confirmed a significant increase in CREB phosphorylation after PGE2 stimulation. Chromatin immunoprecipitation (ChIP)-seq analysis of pCREB was able to identify specific genomic regions where pCREB is recruited to after PGE2 treatment. Compared to unstimulated CD34+ cells an increased binding of pCREB could be detected in promotor regions near transcription start sites. In addition over 90% of de-novo pCREB binding occurred in intergenic and intronic regions. To determine the activation state of these putative enhancers changes in the histone mark H3K27ac and open chromatin state (via ATAC-seq) were assessed after PGE2 treatment. The data suggest that PGE2-induced pCREB binding correlates with remodeling of chromatin already after two hours of drug treatment. Furthermore chromatin sites opened by PGE2 were significantly enriched for the CREB motif both in human CD34+ cells acutely after treatment as well as in mouse ST-HSCs 1.5 years after transplant. In summary this work shows that a two hour treatment with PGE2 is sufficient to confer long-term engraftment properties to ST-HSCs. PGE triggers a chromatin remodeling event through CREB that can permanently alter epigenetic state and gene expression of ST-HSCs. Understanding the self-renewal network induced by PGE2 will not only enrich current clinical applications targeted at increasing engraftable HSC numbers but also further basic understanding of HSC self-renewal. Disclosures Zon: FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

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Sphingolipid Perturbation Activates Proteostasis Programs to Govern Human Hematopoietic Stem Cell Self-Renewal

Blood ◽

10.1182/blood-2018-170 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 170-170

Author(s):

Stephanie Zhi-Juan Xie ◽

Laura Garcia Prat ◽

Veronique Voisin ◽

Alex Murison ◽

Olga I. Gan ◽

...

Keyword(s):

Stem Cells ◽

Ex Vivo ◽

Fold Increase ◽

Chromatin Accessibility ◽

Self Renewal ◽

Cellular Activation ◽

Hematopoietic Stem ◽

Risk Of Malignancy ◽

Ex Vivo Culture

Abstract The hematopoietic stem cells (HSC) field has long been perplexed by how the blood system d (~10e12 cells produced daily) - yet hematologic malignancies remain relatively rare. The risk of malignancy is mitigated in part by a complex hierarchy in which the quiescent long-term hematopoietic stem cells (LT-HSC) with high self-renewal capacity undergo a restricted number of cell divisions. Nonetheless, such a high production demand over a lifetime raises an inherent risk of malignancy due to DNA replication errors, misfolded proteins and metabolic stress that cause cellular damage in HSC. Previously, HSC dormancy, largely thought to be controlled by transcription factor networks, was held responsible for preventing mutation acquisition. However, recent studies suggest that LT-HSC contain critical cellular networks centered around the coordination of distinct HSC metabolic programs with proteostasis, which serve as crucial decision nodes to balance persistence or culling of HSC for lifelong blood production. While HSC culling mechanisms are known, the linkage between cellular stress programs and the self-renewal properties that underlie human HSC persistence remains unknown. Here, we ask how this HSC fate choice is influenced by lipid biosynthesis - an underexplored area of HSC metabolism. We observed a distinct sphingolipid transcriptional signature in human HSC and examined the consequences of sphingolipid perturbation in human cord blood (CB) stem cells during ex vivo activation. DEGS1 (Delta 4-Desaturase, Sphingolipid 1) is the final enzyme in de novo sphingolipid synthesis, converting dihydroceramide (dhCer) to ceramide (Cer); ablation of DEGS1 either genetically or by treatment with the synthetic retinoid fenretinide/N-(4-hydroxyphenyl) retinamide (4HPR) is sufficient to activate autophagy in mouse cells and human cell lines. DEGS1 gene expression was higher in HSC than in progenitors and was significantly increased in LT-HSC following 6 hours of cytokine stimulation, suggesting that it plays a role in cellular activation. Sphingolipid composition was altered in CB cultured with 4HPR for 8 days with an increase in dhCer levels and decrease in Cer levels shown by lipidomics. Remarkably, 4HPR treatment significantly increased in vitro colony forming efficiency from LT-HSC (50% over control), but not from short-term HSC or granulocyte-macrophage progenitors. Ex vivo 4HPR treatment of CB followed by serial xenotransplantation resulted in a 2.5-fold increase in long-term repopulation cell (LTRC) frequency over control-treated cells, suggesting that 4HPR treatment affects HSC self-renewal. RNA-seq analysis showed that 4HPR activates a set of proteostatic quality control (QC) programs that coalesce around the unfolded protein response (UPR) and autophagy, the latter confirmed by immunofluorescence and flow cytometry in CB stem cells. Ex vivo culture perturbs these programs and results in loss of chromatin accessibility at sites associated with uncultured LT-HSC as determined by ATAC-Seq. Addition of 4HPR to the culture activates these proteostatic programs to sustain immunophenotypic and functional HSC. These results suggest that ceramide, the central component to all sphingolipids, may act as a "lipid biostat" for measuring cellular stress and activating stress responses. We further asked if 4HPR could synergize with known compounds to enhance HSC self-renewal. Treatment of CB with a combination of 4HPR plus CD34+ agonists UM171 and StemRegenin-1 during ex vivo culture maintains a chromatin state more similar to uncultured LT-HSC as demonstrated by ATAC-seq, and led to a 4-fold increase in serial repopulating ability in xenotransplant assays over treatment with UM171 and SR1 alone. These results suggest that sphingolipid perturbation not only activates proteostatic mechanisms that protect HSC organelles from damage incurred during cellular activation, but also regulates the landscape of chromatin accessibility in cultured HSC when combined with CD34+ agonists. This work identifies a new linkage between sphingolipid metabolism, proteostatic QC systems and HSC self-renewal, and identifies novel strategies by which to expand HSC numbers and improve HSC quality for clinical applications. Disclosures Takayama: Megakaryon co. Ltd.: Research Funding.

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Lack of α4 integrin expression in stem cells restricts competitive function and self-renewal activity

Blood ◽

10.1182/blood-2005-07-2670 ◽

2006 ◽

Vol 107 (7) ◽

pp. 2959-2967 ◽

Cited By ~ 63

Author(s):

Gregory V. Priestley ◽

Linda M. Scott ◽

Tatiana Ulyanova ◽

Thalia Papayannopoulou

Keyword(s):

Stem Cells ◽

Normal Function ◽

Short Term ◽

Self Renewal ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Progressive Decline ◽

Hematopoietic Reconstitution ◽

Competitive Disadvantage

AbstractAlpha4 integrin or VLA4 (CD49d/CD29) is a multitask molecule with wide expression within and outside the hematopoietic system. Because targeted ablation of α4 integrin leads to embryonic lethality, to study its effects on adult hematopoiesis, we used animals with conditional excision of α4 integrin (α4Δ/Δ) in hematopoietic cells. In such animals, we previously documented weakened bone marrow retention of progenitor cells during homeostasis and impaired homing and short-term engraftment after transplantation. In the present study we show that long-term repopulating cells lacking α4 integrins display a competitive disadvantage in hematopoietic reconstitution compared to normal competitors. Although initial dominance of α4+ competitors is due to their better homing and proliferative expansion early after transplantation, a progressive decline in contribution of α4Δ/Δ hematopoiesis is compatible with neither normal homing nor normal function of α4Δ/Δ hematopoietic stem cells (HSCs) in post-homing hematopoiesis. In the absence of α4+ competitor cells, α4Δ/Δ HSCs can establish long-term hematopoiesis in primary recipients, however, some resurgence of host hematopoiesis is evident, and it becomes dominant in secondary transplants, so that no survivors with exclusively α4Δ/Δ cells are seen in tertiary transplants. Collectively, our data provide compelling evidence that under regenerative stress α4 integrin assumes a greater importance than for maintenance of steady-state hematopoiesis.

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Distinct Regulatory Networks Govern Human Hematopoietic Stem Cell Across Development

Blood ◽

10.1182/blood.v126.23.2375.2375 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2375-2375

Author(s):

Sasan Zandi ◽

John E. Dick ◽

Faiyaz Notta ◽

Naoya Takayama

Keyword(s):

Stem Cell ◽

Cell Biology ◽

Regulatory Networks ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Single Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

M Phase

Abstract Introduction: Much of our fundamental understanding of stem cell biology comes from studies of hematopoiesis where single cells produce differentiated progeny while still retaining the ability to produce daughter stem cells (self-renewal). The cardinal property of a stem cell, whether normal or malignant, is self-renewal; the key biological process that ensures the ability of the stem cell to maintain long-term clonal growth. However, our understanding of the molecular basis of self-renewal in human hematopoiesis is limited. At the embryonic stage fetal liver is the main source of hematopoiesis; from week 6 of gestation until before birth. At this stage HSCs are in a different microenvironment but capable of self-renewing and differentiation to the full spectrum of blood lineages. While murine studies uncovered several intrinsic differences between fetal and adult HSCs, a comprehensive analysis of human HSC compartment across development is lacking. In this study we have combined HSC purification methods and xenograft quantitative assay in conjunction with low input RNA sequencing and Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) to provide a comprehensive functional and molecular outlook of human stem cell compartment across development. Results: We followed the dynamics of four sub-fractions of CD34+CD38- divided by CD90 and CD49f expression across human blood development: fetal liver (hFL) and adult bone marrow (hBM). Using xenograft model, we identified human long, intermediate and short term HSCs in hFL and hBM. 5 single CD90+CD49f+ hFL cells were capable of sustaining the multilineage graft for over 52 weeks up to tertiary recipient, while BM cells only last for 20 weeks in the primary recipient. The frequency of LT-HSC in the CD90+CD49f+ compartment goes from 1/8 in hFL to 1/50 in hBM. hFL CD90-CD49f+ cells showed an intermediate repopulation capacity up to 44 weeks in secondary recipient. On average 10% of hFL long term HSC (LT-HSC) were in S/G2/M phase, in contrast only 0.4% of BM LT-HSC were in S/G2/M phase indicating that hFL HSCs are 20 times more in cycle compare to BM. We found that 320 genes were expressed differentially between LT-HSC and multipotent progenitors (MPP) in hBM as oppose to only 32 genes found to be differentially expressed in hFL (FDR<0.1). Interestingly, we found only 2 genes in common between these two groups. ERRBS showed an overall increase in methylation of HSC compartment in hBM compare to hFL and gradual demethylation of lineage associated genes in MPP. Conclusion: Our data indicate that there are distinct regulatory networks that govern hFL and hBM HSC self-renewal. We found very little differences in gene expression between all hFL HCS compartments (average 20 genes) compare to hBM (average 224), indicating that by adulthood self-renewal is becoming more restricted to the LT-HSC compartment. Disclosures No relevant conflicts of interest to declare.

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Integrating RNA-seq and assay for transposase-accessible chromatin by sequencing (ATAC-seq) predicts functionally-relevant chromatin regions

10.1101/2021.05.06.443010 ◽

2021 ◽

Author(s):

Collin B Merrill ◽

Austin B Montgomery ◽

Miguel A Pabon ◽

Aylin R Rodan ◽

Adrian Rothenfluh

Keyword(s):

Gene Expression ◽

Regulatory Networks ◽

Transcriptional Response ◽

Chromatin Accessibility ◽

Rna Seq ◽

Data Types ◽

Promoter Regions ◽

Next Generation Sequencing Technology ◽

Distal Promoter ◽

Accessible Chromatin

Gene regulation is critical for proper cellular function. Next-generation sequencing technology has revealed the presence of regulatory networks that regulate gene expression and essential cellular functions. Studies investigating the epigenome have begun to uncover the complex mechanisms regulating transcription. Assay for transposase-accessible chromatin by sequencing (ATAC-seq) is quickly becoming the assay of choice for epigenomic investigations. Integrating epigenomic and transcriptomic data has the potential to reveal the chromatin-mediated mechanisms regulating transcription. However, integrating these two data types remains challenging. We used the insulin signaling pathway as a model to investigate chromatin regions and gene expression changes using ATAC- and RNA-seq in insulin-treated Drosophila S2 cells. We show that insulin causes widespread changes in chromatin accessibility and gene expression. Then, we attempted to integrate ATAC- and RNA-seq data to predict functionally-relevant chromatin regions that control the transcriptional response to insulin. We show that using differential chromatin accessibility can predict functionally-relevant genome regions, but that stratifying differentially-accessible chromatin regions by annotated feature type provides a better prediction of whether a chromatin region regulates gene expression. In particular, our data demonstrate a strong correlation between chromatin regions annotated to distal promoters (1-2 kb from the transcription start site). To test this prediction, we cloned candidate distal promoter regions upstream of luciferase and validated the functional relevance of these chromatin regions. Our data show that distal promoter regions selected by correlations with RNA-seq are more likely to control gene expression. Thus, correlating ATAC- and RNA-seq data can home in on functionally-relevant chromatin regions.

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Evidence for the Involvement of CXCR4 Signaling in In Vivo Self-Renewal of Transplanted Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v118.21.1888.1888 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1888-1888 ◽

Cited By ~ 1

Author(s):

Chen-YI LAI ◽

Makoto Otsu ◽

Motohito Okabe ◽

Sachie Suzuki ◽

Satoshi Yamazaki ◽

...

Keyword(s):

Post Transplantation ◽

Gain Of Function ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cxcr4 Signaling ◽

Hematopoietic Reconstitution

Abstract Abstract 1888 Hematopoietic stem cells (HSCs) represent the unique cell population capable of self-renewal and multi-lineage differentiation, thereby lifelong sustainment of the hematopoiesis. HSC transplantation has proven beneficial for various diseases, it is therefore important to elucidate the molecular determinants for successful HSC engraftment. Signaling through the chemokine receptor CXCR4 has been implicated in HSC engraftment by the observation that transplantation of HSCs lacking this molecule results in poor hematopoietic reconstitution. Because this impairment, however, can be attributed to the defects in any of the post-transplantation processes that include bone marrow (BM)-homing, -repopulation, or –retention, it is still unclear whether CXCR4 plays an essential role in HSC self-renewal upon transplantation. To elucidate the role of CXCR4 signaling in HSC self-renewal in conjunction with transplantation, we used a purified CD34neg/low c-Kit+ Sca-1+ Lineage-markerneg population as the defined stem cell source. As a loss-of-function study, CXCR4 was conditionally deleted in HSCs before transplantation. As a gain-of-function study, we generated the HSC populations overexpressing either wild-type (wt)- or C-terminal truncated (δC)-CXCR4 (OE-HSCs), the latter of which is known to exhibit enhancement in the SDF-1 signaling, by gene transfer and subsequent cell sorting. We compared these cells with control HSCs in in vitro assays with regard to the biological characteristics including chemotaxis, proliferation, colony formation, and cobblestone-area (CA) forming ability. To dissect in vivo post-transplantation processes, we investigated hematopoietic repopulation kinetics in the recipient BM at the homing/lodging phase (within 1 wk) and the early repopulation phase (2–3 wks) for the above test HSCs. The self-renewal potential of each HSC population was estimated by competitive repopulation assay. In vitro studies: OE-HSCs with wt- or δC-CXCR4 exhibited enhanced chemotaxis and proliferation in response to SDF1, confirming the gain-of-function effects of these modifications. CA forming ability was greater in OE-HSCs with δC-CXCR4 than control counterparts and absent in CXCR4-KO HSCs, suggesting the critical role of CXCR4-signaling in HSC proliferation in the presence of stromal support. In vivo studies: 1) the homing/lodging phase. Unexpectedly, we did not find significant alteration in the numbers of early progenies detectable in recipient BM 3 days after transplantation of HSCs receiving either loss- or gain-of-function modification to CXCR4, indicating that this signaling is indispensable in HSC homing. 2) the early repopulation phase. Impairment of hematopoietic repopulation in BM became evident for CXCR4-KO HSCs through 2–3 wks. On the other hand, OE-HSCs with CXCR4, more remarkably of ΔC-mutation, showed enhanced BM repopulation kinetics at ∼3 wks post transplantation, suggesting the importance of CXCR4 signaling in HSC amplification at this post-transplantation phase. 3) long-term hematopoiesis. CXCR4-KO-HSCs showed poor hematopoietic reconstitution potentials, consistent with previous observations. Interestingly, impaired peripheral repopulation was also observed with OE-HSCs with wt- or ΔC-CXCR4. Further characterization revealed that the recipients of CXCR4-overexpressing HSCs did retain their progenies, which showed multilineage differentiation, but exhibited impaired release of mature leukocytes from the BM to the peripheral blood. Most importantly, however, test-cell chimerism in the long-term HSC fraction was significantly higher in the mice receiving OE-HSCs with CXCR4, especially of ΔC-type, than those transplanted with control HSCs, indicating that the augmentation of CXCR4 signaling enhanced competitive repopulation ability of HSCs. These modified HSCs demonstrated repopulation abilities also in secondary recipients. We demonstrated that CXCR4 signaling is indispensible for HSC homing and that continuous overexpression of CXCR4 cannot benefit the peripheral reconstitution in contrary to the expectation. More importantly, our studies showed that augmentation of CXCR4 signaling leads to HSC expansion in vivo upon transplantation. We thus conclude that CXCR4 signaling has a role in HSC self-renewal and that its regulation may find the approach that will improve HSC transplantation outcomes. Disclosures: No relevant conflicts of interest to declare.

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Stag2 regulates Hematopoietic Differentiation and Self-Renewal

Blood ◽

10.1182/blood.v130.suppl_1.790.790 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 790-790

Author(s):

Yotaro Ochi ◽

Ayana Kon ◽

Kenichi Yoshida ◽

Keisuke Kataoka ◽

Masahiro Marshall Nakagawa ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Bone Marrow ◽

Peripheral Blood ◽

Chromatin Accessibility ◽

The Self ◽

Cohesin Complex ◽

Hematopoietic Differentiation ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Cohesin is a multimeric protein complex, which has been initially implicated in the cohesion of replicated sister chromatids but more recently shown to be involved in the long-range regulation of gene expression by stabilizing 3-dememsional structure of the genomic DNA. Recently, multiple components of the cohesin complex has been shown to be a recurrent target of somatic mutations in various myeloid malignancies, in which STAG2 is most frequently mutated and undergoes loss-of-function. However, the leukemogenic mechanism of mutated- STAG2 has not been fully understood, although recent studies have reported deregulated differentiation and enhanced self-renewal of STAG2 -mutated stem cells. To investigate the functional role of STAG2 in leukemogenesis as well as normal hematopoiesis, we generated Stag2 conditional knockout (cKO) mice with an Mx1 -cre allele, in which Stag2 deletion was induced by polyIC injection. When assessed in the peripheral blood of 12 to 20 week-old mice, the complete blood count showed no significant changes between Stag2 cKO mice and controls. No morphological alterations were observed in the peripheral blood as well as in the bone marrow. However, in the spleen, the extramedullary hematopoiesis was evident, exhibiting increased myeloid progenitors and erythroblasts. In repeated methylcellulose cultures, Stag2 deficient BM cells showed an enhanced serial replating capacity, suggesting an increased self-renewal potential in Stag2 deficient hematopoietic stem cells (HSCs) in vitro. Flow cytometry of bone marrow cells revealed increased numbers of hematopoietic stem and progenitor cells (HSPCs) defined as Lineage−Sca-1+Kit+(LSK) cells in Stag2 cKO mice compared with controls. Within the myeloid progenitor (MP) compartment, we observed increased common myeloid progenitors (CMPs), while decreased megakaryocyte/erythroid (MEPs) and common lymphoid progenitors (CLPs), compared to controls. Moreover, CD11b+Gr-1+ mature myeloid cells were significantly increased in the bone marrow of Stag2 cKO mice. These results suggest that Stag2 deficiency causes myeloid skewing. In competitive bone marrow transplantation assays assessing the reconstitution potential, Stag2 cKO-derived cells showed reduced chimerism in the peripheral blood compared to wild-type mice-derived cells. However, the reduced chimerism of Stag2 cKO-derived cells was largely confined to B-lymphocytes showing a severe reduction, while the chimerism of Stag2 cKO-derived myeloid cells was not affected compared to controls. In the bone marrow, by contrast, the chimerism of Stag2 cKO cells was not significantly changed, but rather tended to show increased numbers in the LSK, CMP and granulocyte/macrophage progenitor (GMP) fractions, while the MEP and CLP fractions were reduced. These results suggest that Stag2 deficiency could enhance the self-renewal capacity of HSCs in vivo, but Stag2 -deleted cells may not uniformly contribute to all hematopoietic cell fractions probably due to the impaired differentiation of these HSCs. Next, we assessed the effect of Stag2 -deficiency on gene expression, where RNA sequencing of Stag2 -deleted HSPCs revealed a subset of genes differentially expressed between Stag2 WT and cKO cells. Including key myeloid-specific regulators, these genes were considered to be potential gene targets of Stag2, deregulation of which is implicated in the abnormal hematopoiesis of Stag2 cKO mice. Given that the cohesin complex is known to be involved in the establishing and maintaining DNA accessibility, we also assessed the global chromatin accessibility by assays for transposase accessible chromatin with sequencing (ATAC-seq) of Stag2 -deleted HSPCs. Of interest, chromatin accessibility in Stag2 -deleted cells was increased for genes implicated in myeloid differentiation, whereas reduced for those in lymphoid differentiation. Motif analysis of more accessible regions in Stag2 -deleted cells revealed an enrichment of the binding site for the transcription factor Runx1, which is known to regulate HSC differentiation. Our results demonstrate that Stag2 loss leads to the impaired hematopoietic differentiation and enhances the self-renewal potential of HSCs through the modulation of chromatin accessibility and consequent abnormal gene expression, possibly contributing to leukemogenesis. Disclosures Takaori-Kondo: Novartis: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Research Funding; Janssen Pharma: Honoraria; Pfizer: Honoraria.

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Maintenance of Hematopoietic Stem Cells Through Regulation of Wnt and mTOR Pathways.

Blood ◽

10.1182/blood.v120.21.2309.2309 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2309-2309

Author(s):

Jian Huang ◽

Peter S. Klein

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Signaling Pathways ◽

Glycogen Synthase ◽

Dependent Manner ◽

Mtor Inhibition ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Abstract 2309 Hematopoietic stem cells (HSCs) maintain the ability to self-renew and to differentiate into all lineages of the blood. The signaling pathways regulating hematopoietic stem cell (HSCs) self-renewal and differentiation are not well understood. We are very interested in understanding the roles of glycogen synthase kinase-3 (Gsk3) and the signaling pathways regulated by Gsk3 in HSCs. In our previous study (Journal of Clinical Investigation, December 2009) using loss of function approaches (inhibitors, RNAi, and knockout) in mice, we found that Gsk3 plays a pivotal role in controlling the decision between self-renewal and differentiation of HSCs. Disruption of Gsk3 in bone marrow transiently expands HSCs in a b-catenin dependent manner, consistent with a role for Wnt signaling. However, in long-term repopulation assays, disruption of Gsk3 progressively depletes HSCs through activation of mTOR. This long-term HSC depletion is prevented by mTOR inhibition and exacerbated by b-catenin knockout. Thus GSK3 regulates both Wnt and mTOR signaling in HSCs, with opposing effects on HSC self-renewal such that inhibition of Gsk3 in the presence of rapamycin expands the HSC pool in vivo. In the current study, we found that suppression of the mammalian target of rapamycin (mTOR) pathway, an established nutrient sensor, combined with activation of canonical Wnt/ß-catenin signaling, allows the ex vivo maintenance of human and mouse long-term HSCs under cytokine-free conditions. We also show that combining two clinically approved medications that activate Wnt/ß-catenin signaling and inhibit mTOR increases the number of long-term HSCs in vivo. Disclosures: No relevant conflicts of interest to declare.

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Hematopoietic recovery after transplantation is primarily derived from the stochastic contribution of hematopoietic stem cells

10.1101/2021.09.21.461235 ◽

2021 ◽

Author(s):

Stefan Radtke ◽

Mark Enstrom ◽

Dnyanada P. Pande ◽

Margaret L. Cui ◽

Ravishankar Madhu ◽

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Keyword(s):

Clonal Diversity ◽

Enhanced Sampling ◽

Primate Model ◽

Hematopoietic Stem ◽

Neutrophil Recovery ◽

Hematopoietic Recovery ◽

Hematopoietic Reconstitution ◽

Initial Recovery ◽

Two Phases

Reconstitution after hematopoietic stem cell (HSC) transplantation is assumed to occur in two distinct phases: initial recovery mediated by short-term progenitors and long-term repopulation by multipotent HSCs which do not contribute to hematopoietic reconstitution during the first 6-9 months. We have previously reported the transplantation and exclusive engraftment of the HSC-enriched CD34+CD45RA-CD90+ phenotype in a nonhuman primate model. Here, we closely followed the clonal diversity and kinetics in these animals. Enhanced sampling and high density clonal tracking within the first 3 month revealed that multipotent HSCs actively contributed to the early phases of neutrophil recovery and became the dominant source for blood cells as early as 50 days after transplant. Longitudinal changes in clonal diversity supported a stochastic engraftment of HSCs with the majority of HSCs clones vanishing early during neutrophil recovery and a smaller fraction of HSC clones expanding into bigger pools to support long-term hematopoiesis. In contrast to the bi-phasic model, we propose that hematopoietic recovery after myeloablation and transplantation is primarily derived from HSCs in a stochastic manner rather than in two phases by independent cell populations.

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