scholarly journals Distinct Contributions By Perivascular Niche Cells in Hematopoietic Stem Cell Maintenance

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 661-661
Author(s):  
Noboru Asada ◽  
Yuya Kunisaki ◽  
Takashi Nagasawa ◽  
Paul S. Frenette
Keyword(s):  
Stem Cell ◽  
3D Imaging ◽  
Leptin Receptor ◽  
Conflicts Of Interest ◽  
Cell Activity ◽  
Specific Cell ◽  
Hematopoietic Stem ◽  
Kolmogorov Smirnov ◽  
Ks Test ◽  
Smirnov Test

Abstract Hematopoietic stem cells (HSCs) self-renew and differentiate into all blood types in response to various demands through life. HSC functions are tightly and finely tuned by a specialized microenvironment called "niche" in the bone marrow (BM). Using Nestin-GFP transgenic mice, we have identified Nestin-GFP+ perivascular stromal cells exhibiting a mesenchymal stem/progenitor cell activity as niche cells. Furthermore, we found two types of Nestin-GFP+ cells expressing different surface markers, Nerve/glial antigen 2 (NG2) and Leptin receptor (Lepr) that are associated with arterioles and sinusoid, respectively, in the BM (Kunisaki et al. Nature, 2013). Both arteriolar and sinusoidal niche cells have been reported to show high gene expression of cytokines essential for HSC maintenance such as CXCL12 and stem cell factor (SCF), however, it remains unknown how the distinct niche cells differentially regulate HSC functions. To investigate the mechanisms, we utilized genetic mouse models, in which CXCL12 or SCF can be deleted in specific cell types. CXCL12 deletion in sinusoidal niche cells by using Lepr-cre/Cxcl12fl/− mice mobilized HSCs and lineage− Sca-1+ c-kit+ (LSK) progenitors into spleen (HSC, CT: Control/DL: Deleted: 760±165 / 2193±557 / spleen, n=6, p<0.05) and blood (LSK, CT/DL: 177±36 / 668±156 / mL blood, n=5, p<0.05), but had no effect on HSC numbers in the BM (CT/DL: 1435±101 / 1194±75 / femur, n=6, p=0.085), which is consistent with a previous report (Ding and Morrison, Nature, 2013). Furthermore, assessments of endogenous HSC localization using whole-mount 3D imaging technology revealed that the deletion of CXCL12 in Lepr+ niche cells had no impact on HSC location (KS-test: Two-sample Kolmogorov-Smirnov test, p=0.9981). By contrast, deletion of CXCL12 in NG2-cre derived cells, which recombines efficiently in the entire Nestin-GFP+ non-endothelial stromal fraction including both peri-arteriolar and peri-sinusoidal cells (96.9±1.3%), and overlapping with Lepr+ cells (88.5±1.6%) and CXCL12-abundant reticular cells (90.7±1.4%), led to a robust reduction of HSC numbers in the BM (CT/DL: 1487±87 / 179±40 / femur, n=10, p<0.0001) with HSC and progenitor mobilization into spleen (HSC, CT/DL: 705±262 / 3550±540 / spleen, n=6-8, p<0.01) and blood (LSK, CT/DL: 494±178 / 5357±896 / mL blood, n=5-7, p<0.01). In addition, deletion of CXCL12 in NG2-cre targeted cells led to HSC displacement away from arterioles (KS-test: Two-sample Kolmogorov-Smirnov test; p=0.001). To examine further a role of CXCL12 produced by NG2+ arteriolar niches on HSC maintenance, we generated tamoxifen-inducible NG2-creERTM/Cxcl12fl/− mice. Deletion of CXCL12 postnatally in NG2+ arteriolar niche cells significantly reduced the number of HSCs in the BM (CT/DL: 1617±160 / 960±95 / femur, n=10-13, p=0.0013), which was confirmed functionally by a competitive repopulation assay. Moreover, 3D imaging revealed that HSCs were located further away from arterioles in NG2-creERTM/Cxcl12fl/− marrow (KS-test: p<0.0001), suggesting a role for arteriolar niches in CXCL12-mediated HSC maintenance. As niche cells synthesize several factors, we evaluated the contribution of arteriolar niches in SCF synthesis, a cytokine shown to be critical for HSC maintenance. As expected, deletion of SCF in NG2-cre targeted cells led to a significant reduction of HSC numbers in the BM (CT/DL: 606±85 / 96±23 / femur, n=5-7, p<0.0001). To further evaluate functions of SCF produced by distinct vascular niches, we also compared these mice with deletions using Lepr-cre or tamoxifen-inducible NG2-creERTM mice. We found that deletion of SCF in Lepr-cre targeted cells showed a significant reduction of HSC numbers in the BM (CT/DL: 690±84 / 220±83 / femur, n=3-4, p<0.0118), consistent with previous studies (Ding et al., Nature, 2012), whereas there was no significant change observed in NG2-creERTM/SCFfl/− mice, suggesting that Lepr+ vascular niches rather than NG2+ arteriolar niches are the most important source of SCF in the BM. These results highlight distinct contributions of perivascular cells primarily located in separate vascular niches, arteriolar and sinusoidal, in HSC maintenance and mobilization. Disclosures No relevant conflicts of interest to declare.

Molecular Signature of Distinct CD34+ Hematopoietic Stem and Progenitor Cell Subsets of Patients with CML in Chronic Phase.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3170-3170
Author(s):  
Ingmar Bruns ◽  
Akos G. Czibere ◽  
Johannes C. Fischer ◽  
Ulrich Steidl ◽  
Sabrina Pechtel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cell ◽  
Healthy Volunteers ◽  
Leptin Receptor ◽  
Perfect Match ◽  
Chronic Phase ◽  
Hematopoietic Cell ◽  
Expression Level ◽  
Specific Cell ◽  
Hematopoietic Stem

Abstract During the last decade, chronic myeloid leukemia (CML) has been mainly characterized by the reciprocal translocation between chromosomes 9 and 22, resulting in the formation of the protooncogene BCR-ABL. This constitutively active tyrosine kinase is widely considered as the cause of the disease. Even though BCR-ABL transcripts are found in every dividing hematopoietic cell and thus, the disease is likely to originate from a primitive stem cell, the “cell of origin” is still a matter of debate. Despite the active “leukemia stem cell” discussion, very few characteristics of the “cancer stem cell” are established to date. In order to get further molecular insights into CML stem and progenitor cells, we examined CD34+ cell subsets obtained from bone marrow of 7 patients with CML in chronic phase in comparison with 5 healthy volunteers. CD34+ cells were immunomagnetically selected and high-speed cell sorting of lineage-negative, CD34+, CD38−, hematopoietic stem cells and myeloid progenitors was performed. Progenitors were further subdivided by anti-IL-3Ralpha and anti-CD45RA staining. Following RNA extraction, a two-cycle amplification procedure was used to generate cDNA for the hybridization with Affymetrix U133A2.0 arrays. After performing smoothening spline normalization, we applied the perfect match-mismatch difference model algorithm to calculate expression values (dChip). Hierarchical cluster analysis was performed using a correlation based centroid linkage algorithm. Hereby we could discriminate the HSCs, CMPs, and MEP subsets. Corroboration of RNA expression was performed by real-time RT-PCR for selected genes. Comparing the HSC subsets of CML patients with healthy controls we found 98 differentially expressed genes. 87 genes had a lower expression level in CML HSCs whereas 11 genes had a higher one. Among the downregulated genes in CML were transcriptions factors involved in myelogenesis and proliferation and several adhesion molecules associated with homing and migration of the HSCs. On the other hand, the Leptin receptor and BCR-ABL downstream targets were found to be upregulated. Within the common myeloid progenitor (CMP) compartment 37 genes were significantly differentially regulated. Twenty genes had a higher expression level in CML CMPs, 17 genes were downlegulated. Hematopoietic cell-specific cell cycle inhibitor MS4A3 was among the significantly downregulated genes whereas genes of the retinoblastoma and E2F families as well as inhibitors of the Wnt-signaling pathway were upregulated. Looking at megakaryocte-erythrocyte progenitors (MEP) in CML, key mediators of G2-M cell cycle transition were downregulated indicating a lower proliferative capacity of this subset. No transcriptional differences have been observed between granulocyte-macrophage progenitors from CML patients and healthy volunteers. Interestingly, among all other subsets myeloperoxidase (MPO) was downregulated in the CML samples and the Leptin receptor was upregulated. Our results provide novel insights into the biology of CML and potentially provide the basis for the characterization of a candidate CML stem cell.

Mir-125a Confers Multi-Lineage Long-Term Repopulating Stem Cell Activity to Human Hematopoietic Committed Progenitors

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 900-900 ◽  
Author(s):  
Eric R. Lechman ◽  
Karin G. Hermans ◽  
Erwin M. Schoof ◽  
Aaron Trotman-Grant ◽  
Stephanie M Dobson ◽  
...  
Keyword(s):  
Stem Cell ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Cell Activity ◽  
Lymphoid Cells ◽  
Cell Populations ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Recent studies have shown that several miRNA are differentially expressed in hematopoietic stem cells (HSC) and involved in regulating self-renewal, pointing to a new axis of epigenetic control of HSC function. Murine studies have documented a role for miR-125a in regulating HSC as miR-125a enforced expression augments self-renewal. We examined whether these attributes are evolutionarily conserved within human hematopoiesis. Lentiviral vectors over-expressing miR-125a (miR-125OE) were developed and HSC function was investigated using xenotransplantation of CD34+ CD38- human umbilical cord blood (CB) hematopoietic stem and progenitor cells (HSPCs). miR-125OE resulted in significantly increased human bone marrow (BM) chimerism at 12 and 24 weeks post-transplantation and splenomegaly. Within enlarged spleens, there were significantly increased proportions of CD34+CD19+CD10+CD20-B lymphoid cells suggesting a partial B cell differentiation block at the pro-B cell stage. In the BM, CD41+ megakaryocytes, GlyA+ erythroid and CD3+ T cell populations were significantly expanded. Within the primitive compartment, multi-lymphoid progenitors (MLP) were massively expanded by 12 weeks, followed by a combined reduction of immuno-phenotypic HSC and multi-potent progenitors (MPP) by 24 weeks. Given this loss of immuno-phenotypic HSC, we wondered whether stem cell function was compromised in vivo. Secondary transplantation with limiting dilution (LDA) revealed that stem cell frequencies were increased by 4.5 fold in miR-125OE recipients. Using lentivirus sponge-mediated inhibition of miR-125 (miR-125KD) in CD34+CD38-human CB, we were able to directly link these effects to miR-125: B cells increased at the expense of T cells; immuno-phenotypic HSC increased with a concomitant loss of MLP; and functional HSC were decreased by 2.5 fold using secondary LDA assays. Together, these data strongly suggest that miR-125a expression levels regulate human HSC self-renewal and lineage commitment. Since HSC frequency increased so substantially upon miR-125OE, we asked whether more committed cell populations might also be endowed with enhanced self-renewal. Highly purified populations of HSC, MPP and MLP and CD34+CD38+ committed progenitors were transduced and transplanted cells into xenografts. Unexpectedly, miR-125OE transduced CD34+CD38+ progenitors produced a substantial graft after 12 weeks. Control transduced CD34+CD38+ cells did not engraft and only control transduced HSC generated a disseminating graft in recipient mice. miR-125OE transduced HSC and MPP generated robust engraftment, while MLP did not. In all cases, xenografts generated by CD34+CD38+ and MPP transduced with miR-125OE showed multi-lineage repopulation. Moreover, the miR-125OE grafts from CD34+CD38+ and MPP recipients were durable as secondary transplantation generated multi-lineage grafts for at least 20 weeks in 5/7 and 6/10 recipients, respectively; no control transduced groups generated secondary grafts. Thus, the enhancement of self-renewal by enforced expression of miR-125a occurs not only in HSC, but also in MPP and to an as yet unidentified subpopulation within the CD34+38+ committed progenitor compartment. Using protein mass spectrometry, we identified and validated a miR-125a target network in CD34+ CB that normally functions to restrain self-renewal in more committed progenitors. Together, our data suggest that increased miR-125a expression can endow an HSC-like program upon a selected set of non-self-renewing hematopoietic progenitors. Our findings offer the innovative potential to use MPP with enhanced self-renewal to augment limited sources of HSC to improve clinical outcomes. Disclosures No relevant conflicts of interest to declare.

Gpx3 Determines Competitiveness of Normal and Leukemic Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1587-1587 ◽  
Author(s):  
Olivier Herault ◽  
Kristin J Hope ◽  
Eric Deneault ◽  
Matthias Trost ◽  
Nadine Mayotte ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Insertional Mutagenesis ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Cell Activity ◽  
Relative Reduction ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Abstract 1587 Although important efforts have been invested in the discovery of genes that regulate normal or leukemic hematopoietic stem cells (HSC) self-renewal, the number of validated candidates remains low, due largely to the unavailability of functionally pure stem cell populations. Moreover, it is often difficult to identify the normal counterpart cell from which leukemia originated, further complicating studies based on comparative gene expression. In this study, we used a series of recently characterized Hoxa9 + Meis1 acute myeloid leukemias (AML) derived from fetal liver (FL) cells (Wilhelm BT et al., submitted). These leukemias are remarkably similar in several aspects including their L-HSC frequency (between ∼1 in 100 to 350) except for one leukemia (FLA2) in which 70% of the cells show repopulation ability (i.e., L-HSC). We reasoned that comparative mRNA profiling of FLA2 to the phenotypically similar FLB1 (0.3% L-HSC) might identify genes uniquely associated with L-HSC self-renewal. We observed a 2–3-fold upregulation of Gpx3 in FLA2, which was confirmed by qRT-PCR. In accordance with this, all 14 of the tested Gpx3 promoter region CpG sequences were methylated in FLB1 and hypomethylated in FLA2 cells. The higher expression of GPx3 in FLA2 was confirmed at the protein level and reflected in elevated glutathione peroxidase activity in comparison to FLB1. Importantly, we also observed in FLA2 a relative reduction in reactive oxygen species (ROS) level (DCFDA) and a concomitant decrease in p38 MAPK activation (western blot and mass spectrometry). The correlation of Gpx3 levels with L-HSC frequency could be reflective of their functional dependence on this enzyme. FLA2 cells being difficult to manipulate ex vivo, to address this we utilized retroviruses encoding shRNAs and a GFP reporter to explore the in vivo function of FLA2 cells with downregulated Gpx3. The decrease in percentage of GFP+ donor cells when leukemia became apparent (∼19 days) from that of populations initially transplanted, was 4-fold higher following Gpx3 knockdown in comparison to shLuciferase transduction. Moreover, those shGpx3 infected FLA2 remaining at day 19 displayed a 3-fold decrease in GFP mean fluorescence intensity relative to their control counterparts. These results show that GFPhigh cells were selectively depleted, and suggest that Gpx3 is critical for the competitiveness of L-HSCs. Because redox metabolism has been implicated in HSC self-renewal, we also analyzed its expression and function in normal HSC to gain further insight into the role of GPx3 in stem cell activity. Interestingly, compared to FL-HSCs, isolated 3 and 4 week bone marrow (BM), HSCs exhibited a 39- and 6-fold decrease in Gpx3 expression, respectively. A correlation of Gpx3 levels with enhanced self-renewal was also observed in vitro as overexpression of several nuclear determinants of HSC expansion such as Hoxb4, NA10HD, Klf10 and Prdm16 promoted Gpx3 expression by 3.2 to 19.2-fold. We next infected BM cells enriched for HSCs with retroviruses carrying shRNAs to Gpx3. shRNA targeting of Gpx3 dramatically inhibited hematopoietic reconstitution. Transplantations of sublethally irradiated recipients indicated that Gpx3 knockdown significantly impaired both early and late donor-derived hematopoiesis. These results suggest that GPx3 is critical for repopulation mediated by both short and long-term repopulating cells. In reciprocal gain-of-function experiments, Lin-CD150+CD48- cells engineered to overexpress Gpx3, showed a marked competitive advantage over controls when transplanted following a 7-day ex vivo culture step. Insertional mutagenesis was ruled out as proviral integration analyses of six recipients confirmed polyclonal hematopoiesis. Moreover, some mice were in part reconstituted by the same clones, indicating that self-renewal occurred in vitro prior to transplantation. Phenotypic analysis of late-transplant hematopoietic tissues showed that Gpx3-transduced cells contributed to lymphoid and myeloid repopulation, confirming their multipotentiality. Together, these results indicate that Gpx3 enhances HSC expansion ex vivo possibly through modulation of self-renewal activity. In conclusion, a unique model of primary L-HSC was exploited to identify Gpx3 as a critical determinant for the competitiveness of L-HSCs and complementary experiments demonstrated a key role for this gene in normal HSC self-renewal. Disclosures: No relevant conflicts of interest to declare.

Loss of De Novo DNA Methylation Causes Expansion of the Mouse Hematopoietic Stem Cell Pool

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 835-835 ◽  
Author(s):  
Grant A. Challen ◽  
Jonathan S Berg ◽  
Margaret A. Goodell
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Stem Cell ◽  
Peripheral Blood ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Cell Activity ◽  
Aberrant Methylation ◽  
Wild Type ◽  
Hematopoietic Stem

Abstract Abstract 835 DNA methylation is one of the major epigenetic modifications in the vertebrate genome and is catalyzed by the DNA methyltransferase enzymes Dnmt1, Dnmt3a and Dnmt3b. We observed a dynamic expression profile of Dnmt3a and Dnmt3b in the hematopoietic system with both enzymes expressed at exponentially higher levels in hematopoietic stem cells (HSCs) compared to progenitors and differentiated cells and hypothesized that some of the unique characteristics of HSCs were epigenetically regulated by Dnmt3a and Dnmt3b. To study this, we crossed Dnmt3a and -3b conditional knock-out (KO) mice to Mx1-cre mice to generate inducible single- and double-KO (dKO) mice. We performed competitive transplantation of HSCs (side-population+c-Kit+Lineage-Sca-1+ = SPKLS) from these mice along with wild-type whole bone marrow competitor and induced deletion of the Dnmt3's in the donor cells by sequential pIpC injections in the wild-type recipients. No dramatic differences were observed in primary recipients in the absence of other hematopoietic perturbation, however when we re-transplanted Dnmt3a- and Dnmt3b-KO HSCs into secondary recipients, they exhibited surprisingly high peripheral blood reconstitution compared to control HSCs (>4-fold increase in engraftment). This was reflected in the bone marrow of these mice with a corresponding >4-fold expansion of the HSC pool (phenotypically defined by any of SPKLS; CD34-Flk2-KLS; CD150+CD48-KLS) with virtually all of these cells being derived from the Dnmt3a- and Dnmt3b-KO donor HSCs. Consistent with a previous study, we observed a decline in functional output of Dnmt3a/3b-dKO HSCs in secondary transplants in terms of peripheral blood chimerism, but surprisingly these mice also exhibited a modest expansion of the HSC pool (∼2-fold), the majority of which were derived from donor Dnmt3a/3b-dKO HSCs. In subsequent tertiary and quaternary transplantation, Dnmt3 single-KO HSCs remained highly superior in peripheral blood engraftment capacity relative to control HSCs and Dnmt3a/3b-dKO HSCs (Figure 1), although the expansion of the HSC pool in all Dnmt3-KOs continued to varying degrees. This enhanced HSC activity appears to be a cell autonomous mechanism as purified Dnmt3-KO SPKLS cells from transplanted mice have much greater hematopoietic colony forming potential in vitro compared to control HSCs on a per cell basis. However the observed HSC expansion does not appear attributable to either enhanced proliferation of Dnmt3-KO HSCs or more resistance to apoptosis. The serially-transplanted Dnmt3-KO HSCs are not overtly transformed, in that the levels of differentiated blood cells are still normal and the mice appear to be healthy. This may be akin to a pre-malignant state seen in human myelodysplastic syndrome. We have performed microarray expression profiling of serially-transplanted Dnmt3-KO HSCs and identified several candidate genes which are currently being investigated as the mechanism for HSC expansion. Our data suggest ablation of de novo DNA methylation in HSCs uncouples normal self-renewal and differentiation. These studies present further evidence for the contribution of epigenetic regulation to stem cell activity and provide a tantalizing link between potential aberrant methylation in HSCs contributing to leukemic transformation. Disclosures: No relevant conflicts of interest to declare.

IP3 3-Kinase B Controls Hematopoietic Stem Cell Homeostasis and Prevents Lethal Hematopoietic Failure in Mice

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 250-250
Author(s):  
Karsten Sauer
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Conflicts Of Interest ◽  
Cell Activity ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Phosphoinositide 3 Kinase ◽  
And Function

Tight regulation of hematopoietic stem cell (HSC) homeostasis ensures life-long hematopoiesis and prevents blood cancers. The mechanisms balancing HSC quiescence with expansion and differentiation into hematopoietic progenitors are incompletely understood. Here, we identify inositoltrisphosphate (IP3) 3-kinase B (Itpkb) as a novel essential regulator of HSC quiescence and function. Young Itpkb-/- mice accumulated phenotypic HSC which were less quiescent and proliferated more than wildtype controls. Itpkb-/- HSC downregulated quiescence and stemness associated mRNAs, but upregulated activation, oxidative metabolism, protein synthesis and lineage associated transcripts. Although they showed no significant homing defects and had normal to elevated viability, Itpkb-/- HSC had a severely reduced competitive long-term repopulating potential. Aging Itpkb-/- mice lost hematopoietic stem and progenitor cells and died with severe anemia. Wildtype HSC normally repopulated Itpkb-/- hosts, indicating a HSC-intrinsic Itpkb requirement. In vitro, Itpkb-/- HSC had reduced cobblestone-area forming cell activity and showed increased stem cell factor activation of the phosphoinositide 3-kinase (PI3K) effector Akt. This was reversed by exogenous provision of the Itpkb product IP4, a known PI3K/Akt antagonist. Itpkb-/- HSC also showed transcriptome changes consistent with hyperactive Akt/mTOR signaling. Thus, we propose that Itpkb ensures HSC quiescence and function in part by limiting cytokine-induced PI3K signaling in HSC. Disclosures No relevant conflicts of interest to declare.

scholarly journals Histone Acetyltransferases and Stem Cell Identity

Cancers ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 2407
Author(s):  
Ruicen He ◽  
Arthur Dantas ◽  
Karl Riabowol
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Fate ◽  
Epigenetic Modification ◽  
Cell Types ◽  
Histone Acetyltransferases ◽  
Specific Cell ◽  
Cell Fates ◽  
Cell Identity ◽  
Hematopoietic Stem

Acetylation of histones is a key epigenetic modification involved in transcriptional regulation. The addition of acetyl groups to histone tails generally reduces histone-DNA interactions in the nucleosome leading to increased accessibility for transcription factors and core transcriptional machinery to bind their target sequences. There are approximately 30 histone acetyltransferases and their corresponding complexes, each of which affect the expression of a subset of genes. Because cell identity is determined by gene expression profile, it is unsurprising that the HATs responsible for inducing expression of these genes play a crucial role in determining cell fate. Here, we explore the role of HATs in the maintenance and differentiation of various stem cell types. Several HAT complexes have been characterized to play an important role in activating genes that allow stem cells to self-renew. Knockdown or loss of their activity leads to reduced expression and or differentiation while particular HATs drive differentiation towards specific cell fates. In this study we review functions of the HAT complexes active in pluripotent stem cells, hematopoietic stem cells, muscle satellite cells, mesenchymal stem cells, neural stem cells, and cancer stem cells.

HLF Expression Defines the Human Hematopoietic Stem Cell State.

Blood ◽  
2021 ◽  
Author(s):  
Bernhard Lehnertz ◽  
Jalila Chagraoui ◽  
Tara MacRae ◽  
Elisa Tomellini ◽  
Sophie Corneau ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Fetal Liver ◽  
Cell Activity ◽  
Mouse Strains ◽  
Marker Genes ◽  
Hematopoietic Stem ◽  
Cell State ◽  
Human Hscs ◽  
Stem Cell State

Hematopoietic stem cells (HSCs) sustain blood cell homeostasis throughout life and can regenerate all blood lineages following transplantation. Despite this clear functional definition, highly enriched isolation of human HSCs can currently only be achieved through combinatorial assessment of multiple surface antigens. While several transgenic HSC reporter mouse strains have been described, no analogous approach to prospectively isolate human HSCs has been reported. To identify genes with the most selective expression in human HSCs, we profiled population- and single-cell transcriptomes of un-expanded and ex vivo cultured cord blood-derived HSPCs as well as peripheral blood, adult bone marrow, and fetal liver. Based on these analyses, we propose the master transcription factor HLF (Hepatic Leukemia Factor) as one of the most specific HSC marker genes. To directly track its expression in human hematopoietic cells, we developed a genomic HLF reporter strategy, capable of selectively labeling the most immature blood cells based on a single engineered parameter. Most importantly, HLF-expressing cells comprise all of the stem cell activity in culture and in vivo during serial transplantation. Taken together, these results experimentally establish HLF as a defining gene of the human hematopoietic stem cell state and outline a new approach to continuously mark these cells with high fidelity.

scholarly journals Cytokine-Mediated Natural Killer Cells Effects Impair Hematopoietic Stem Cell Function

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2641-2641
Author(s):  
Lorena Lobo Figueiredo-Pontes ◽  
Robert S. Welner ◽  
Miroslava Kardosova ◽  
Hong Zhang ◽  
Meritxell Alberich-Jorda ◽  
...  
Keyword(s):  
Stem Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Cytokine Production ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Cytokine Signaling ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Nk Cytotoxicity

Abstract Natural killer (NK) cells participate in innate and adaptive immune responses, and upon activation rapidly produce cytokines, chemokines, and growth factors, including IFNγ, TNFα, TGFβ, GM-CSF, MIP1α, MIP1β, IL-10, and others, which can affect the function of other hematopoietic cells. Considering the recent evidences that hematopoietic stem cells (HSCs) respond to cytokine signaling, we hypothesized that NK cell-mediated cytokine production could mediate HSC function. By the use of co-cultures of purified Ly5.1 murine NK cells and congenic Ly5.2 HSCs, we concluded that NK activity affects HSC frequency in vitro as well as hematopoietic reconstitution in vivo. Sorted NK cells (CD3- NK1.1+) and HSCs (Lin-, Sca1+, ckithi, CD48-, CD150+) were co-cultured in the presence or absence of IL2 over an OP9 stromal cells layer for 14 to 28 days. After 14 days, the addition of NK cells to HSC cultures resulted in an approximate 2-fold reduction of lineage negative cells (Lin-) recovered cells, as compared to control HSC cultures, as determined by flow cytometry analysis. Lin- counts were even lower in HSC+NK long-term cultures when compared to HSC only cultures. Ly5.1 HSCs and/or Ly5.2 NK cells were injected into sublethally irradiated Ly5.1/2 chimeric mice in a ratio of 105 NK to 103 HSCs per mouse. The addition of IL2-stimulated NK to injected HSCs reduced engraftment from 15.7% to 1.82% when the 16 weeks bone marrow (BM) chimerism was analyzed. In agreement, donor CD45.1 cells contribution to the LSK and HSC subpopulations was reduced in the HSC+NK transplanted mice. To test whether NK depletion from BM grafts would affect HSC function, we performed limiting dilution transplantation assays where whole BM from Ly5.2 mice was submitted to immunonagnetic NK1.1 or IgG depletion and injected into lethally irradiated Ly5.1 animals. Donor chimerism after 8 and 16 weeks of transplant showed that depleting NK cells improves the engraftment ability of HSC in a cell dose-dependent manner. When 25 x104 BM cells were injected, chimerism increased from 40 to more than 90% in NK depleted group. Of note, HSC frequency was 1 in 1595 in the control and 1 in 95 in the NK depleted group. In order to understand the mechanisms by which NK cells could regulate HSCs, we took advantage of a CCAAT/enhancer-binding protein gamma (C/ebpg) knockout (KO) conditional mouse model generated in our laboratory, considering that C/ebpg had been previously shown to regulate NK cytotoxicity. Using similar culture conditions, HSCs and NK cells isolated from control (CT) or Cebpg KO mice were injected into congenic sublethally irradiated recipients. Results showed that Cebpg-deficient NK cells do not harm HSC engraftment as CT NK cells do. For instance, after 8 weeks, the addition of CT non-stimulated and IL-2-stimulated NK cells to normal transplanted HSCs reduced the engraftment from 40% to 20% and 10%, respectively. In contrast, chimerism was not different when HSCs only or HSCs + stimulated KO NK cells were transplanted. Gene expression and cytokine profiles of deficient and normal NK cells revealed the potential players of this HSC-NK regulation. Of these, interferon gamma (IFNg), was lower produced by the C/ebpg deficient NK cells. Therefore, besides controlling NK cytotoxicity, we showed here that C/ebpg also plays a role in the regulation of HSCs by NK-mediated cytokine production. Next, we investigated whether depletion of NK cells from human BM samples would improve transplantation efficiency. NK cells were removed using CD56 antibody and transplanted into sublethally irradiated NSG mice. Sixteen weeks after transplantation, animals were sacrificed and the percentage of human CD45 cells in blood, BM, and spleen demonstrated that NK depletion from human BM favors engraftment. Altogether, these findings provide new insights to the knowledge of HSC regulation by NK cells, which are present in BM transplantation (BMT) grafts. Although the alloreactive effect of NK cells against non-identical tumor cells from BMT recipients is well known, its cytokine-mediated effects over identical progenitor cells from the graft were not previously explored. We show that NK-secreted cytokines harm stem cell function, thus suggesting that depletion of NK cells from BM donor cells preparations can improve stem cell engraftment, particularly in the setting of alternative transplants with limiting cell numbers or non-myeloablative conditioning regimens. Disclosures No relevant conflicts of interest to declare.

scholarly journals TWIST1 Preserves Hematopoietic Stem Cell Function Via Repression of Cacna1b Expression

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 12-12
Author(s):  
Nan Wang ◽  
Jing Yin ◽  
Na You ◽  
Dan Guo ◽  
Yangyang Zhao ◽  
...  
Keyword(s):  
Stem Cell ◽  
Steady State ◽  
Calcium Channel ◽  
Cell Fate ◽  
Hematopoietic Stem Cell ◽  
Cell Function ◽  
Conflicts Of Interest ◽  
Hematopoietic Stem ◽  
Voltage Gated ◽  
Voltage Gated Calcium Channel

The mitochondria of hematopoietic stem cell (HSC) play crucial roles in regulating cell fate and in preserving HSC functionality and survival. However, the mechanism underlying its regulation remain poorly understood. Here, we identify transcription factor TWIST1 as a novel regulator of HSC maintenance through modulating mitochondrial function. We demonstrate that Twist1 deletion results in a significantly decreased long-term HSC (LT-HSC) frequency, markedly reduced dormancy and self-renewal capacities and skewed myeloid differentiation in steady-state hematopoiesis. Twist1-deficient LT-HSC are more compromised in tolerance of irradiation and 5 fluorouracil-induced stresses, and exhibit typical phenotypes of senescence and higher levels of DNA damage and apoptosis. Mechanistically, Twist1 deficiency upregulates the expression of voltage-gated calcium channel Cacna1b in HSC, leading to noticeable increases in mitochondrial calcium levels, biogenesis, metabolic activity and reactive oxygen species production. Suppression of voltage-gated calcium channel by a calcium channel blocker largely rescues the phenotypic and functional defects in Twist1-deleted HSCs under both steady-state and stress conditions. Collectively, our data, for the first time, characterize TWIST1 as a critical regulator of HSC function acting through CACNA1B/Ca2+/mitochondria axis, and highlight the importance of Ca2+ in HSC maintenance. These observations provide new insights into the mechanisms for the control of HSC fate. Disclosures No relevant conflicts of interest to declare.

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