Evi1 Is a Stem Cell-Specific Regulator of Self-Renewal Capacity In the Definitive Hematopoietic System

Abstract Abstract 838 Self-renewal is a defining property of stem cells. Although a number of molecules have been implicated in the regulation of hematopoietic stem cell (HSC) self-renewal, loss of these genes is accompanied with other hematological abnormalities. Thus, it is unclear what will happen with a specific loss of self-renewal capacity of HSCs. Evi1 is an oncogenic transcription factor in myeloid malignancies. Evi1 expression is limited to hematopoietic stem/progenitor fraction, and Evi1 is essential for the maintenance of HSCs, but is dispensable for blood cell lineage commitment. Thus, we hypothesized that Evi1 expression could distinguish hematopoietic stem and progenitor cells, and reduction of Evi1 gene dosage might cause a specific loss of self-renewal activity. First, to elucidate Evi1 expression within the hematopoietic system, we have generated Evi1-IRES-green fluorescent protein (GFP) knock-in mice, in which GFP was expressed under the endogenous transcriptional regulatory elements of Evi1 gene. We found that Evi1 was predominantly expressed in the hematopoietic stem/progenitor fraction (Lin- Sca-1+ c-kit+ (LSK)), but its expression was rapidly extinguished during early stages of lineage commitment. Among the LSK compartment, Evi1 was expressed at the highest level in long-term HSCs (LT-HSCs; Flk2- CD34-, CD48- CD150+, or SP-tip fractions in LSK cells). Next, we hypothesized that Evi1 would have the potential to mark LT-HSCs effectively. To test this, we compared GFP+ and GFP- cells in the LSK fraction, and revealed that GFP+ LSK cells were more immature and quiescent with a higher colony-forming capacity than GFP- LSK cells. In addition, in vivo long-term multilineage repopulating cells were exclusively enriched in the GFP+ LSK fraction. In the embryo, Evi1 was highly expressed in the hematopoietic stem/progenitor fraction; that is, CD34+ c-kit+ cells in embryonic day 10.5 (E10.5) aorta-gonad-mesonephros, CD34+ c-kit+ CD48- cells in E12.5 placenta, and Mac-1+ Sca-1+ Lin- (MSL) CD48- cells in E14.5 fetal liver (FL). In vivo competitive repopulation assay showed that, in the MSL fraction of FL, GFP+ MSL cells exclusively had a long-term multilineage repopulating capacity. These results implied that Evi1 plays a more specific role in HSCs than in other hematopoietic cells. To clarify this, we analyzed heterozygous Evi1 knockout mice (Evi1 +/− mice), as it seems difficult to elucidate the function of a small population of HSCs in Evi1 conditional knockout mice due to the leaky expression of Cre recombinase. We have previously showed that haploinsufficiency of Evi1 leads to decreased numbers of LSK and CD34- LSK cells, and impaired long-term repopulating activity. Here we demonstrated the number of each fraction in Evi1 +/− LSK cells was reduced in proportion to their expression level of Evi1. But, there were no significant differences in the numbers of lymphoid and myeloid progenitors between Evi1 +/+ and Evi1 +/− mice. Evi1 +/− CD34+ LSK cells had an equivalent in vitro colony-forming capacity and day 11 colony-forming unit-spleen activity to Evi1 +/+ CD34+ LSK cells. However, in vivo short-term repopulation assay using CD34+ LSK cells showed that the percentage of donor-derived cells from Evi1 +/− mice was significantly declined at 4 weeks after transplantation. Moreover, Evi1 +/− CD34- LSK cells had a pronouncedly impaired in vivo repopulating capacity. These data suggested that the differentiation capacity of Evi1 +/− HSCs was maintained, but their self-renewal capacity was specifically reduced. Although flow cytometric analysis of cell-cycle status and apoptosis showed no differences in CD34- LSK cells between Evi1+/+ and Evi1 +/− mice, the G0 fraction of Evi1 +/− CD34+ LSK cells was significantly reduced, indicating that these cells might proliferate more rapidly to compensate for the impaired self-renewal capacity of HSCs. In conclusion, we showed that Evi1 is predominantly expressed in HSCs and its expression can mark long-term repopulating HSCs in the fetal and adult hematopoietic system. Moreover, functional loss caused by haploinsufficiency of Evi1 is limited to a defect of self-renewal capacity of HSCs, and the increased cell-cycle progression of CD34+ LSK cells in Evi1 +/− mice seems to be the consequence of the impaired self-renewal capacity. Our data may help to understand the unrevealed effects of loss of self-renewal activity of HSCs and compensative mechanism of their defects. Disclosures: No relevant conflicts of interest to declare.

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Clonal-level lineage commitment pathways of hematopoietic stem cells in vivo

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1801480116 ◽

2019 ◽

Vol 116 (4) ◽

pp. 1447-1456 ◽

Cited By ~ 29

Author(s):

Rong Lu ◽

Agnieszka Czechowicz ◽

Jun Seita ◽

Du Jiang ◽

Irving L. Weissman

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem Cell ◽

Lineage Commitment ◽

Self Renewal ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Different Levels

While the aggregate differentiation of the hematopoietic stem cell (HSC) population has been extensively studied, little is known about the lineage commitment process of individual HSC clones. Here, we provide lineage commitment maps of HSC clones under homeostasis and after perturbations of the endogenous hematopoietic system. Under homeostasis, all donor-derived HSC clones regenerate blood homogeneously throughout all measured stages and lineages of hematopoiesis. In contrast, after the hematopoietic system has been perturbed by irradiation or by an antagonistic anti-ckit antibody, only a small fraction of donor-derived HSC clones differentiate. Some of these clones dominantly expand and exhibit lineage bias. We identified the cellular origins of clonal dominance and lineage bias and uncovered the lineage commitment pathways that lead HSC clones to different levels of self-renewal and blood production under various transplantation conditions. This study reveals surprising alterations in HSC fate decisions directed by conditioning and identifies the key hematopoiesis stages that may be manipulated to control blood production and balance.

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Pbx1 Regulates the Self-Renewal of Long-Term Hematopoietic Stem Cells by Maintaining Their Quiescence.

Blood ◽

10.1182/blood.v110.11.94.94 ◽

2007 ◽

Vol 110 (11) ◽

pp. 94-94 ◽

Cited By ~ 1

Author(s):

Francesca Ficara ◽

Mark J. Murphy ◽

Min Lin ◽

Michael L. Cleary

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Acute Leukemia ◽

B Cell ◽

Quiescent State ◽

Self Renewal ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Cell Fate Decisions

Abstract Pbx1 is a proto-oncogene that was originally discovered at the site of chromosomal translocations in pediatric acute leukemia. It codes for a homeodomain transcription factor, which is a component of hetero-oligomeric protein complexes that regulate developmental gene expression. Lack of Pbx1 is associated with multiple patterning malformations, defects in organogenesis, and severe fetal anemia, however embryonic lethality has prevented an assessment of its roles in the adult hematopoietic stem cell (HSC) compartment and in lymphoid differentiation. The objective of this study was to characterize the physiological roles for Pbx1 in the hematopoietic system, specifically in the regulation of cell fate decisions involved in the timing and/or extent of postnatal HSC and progenitor proliferation, self-renewal or differentiation capacity. A genetic approach was employed to conditionally inactivate Pbx1 in the hematopoietic compartment in vivo using Cre recombinase expressed under the control of the Tie2 or Mx1 promoters. A crucial role for Pbx1 in the development of the lympho-hematopoietic system was evidenced by reduced size, cell number, and altered architectures of the thymus and spleen in mutant mice. A marked reduction was observed in the bone marrow (BM) pro- and pre-B cell compartment, as well as a striking reduction (up to 10-fold) in common lymphoid progenitors (CLP), suggesting a role for Pbx1 at a critical stage of lymphoid development where acute leukemia likely originates. Accordingly, abnormal T cell development was observed in the thymus. Common myeloid progenitors (CMP) and Lin-cKit+Sca1+ (LKS, enriched in HSCs) cells were also reduced, as well as long-term stem cells (LT-HSCs, reduced 7-fold on average). Assessment of the proliferation status of LT- and ST (short-term)-HSCs, as well as multi-potent progenitors (MPP), revealed that the reduction of the HSC compartment was associated with a higher number of stem cells exiting the G0 phase, thus losing their quiescent state. Strikingly, Pbx1-deficient BM cells failed to engraft in competitive transplants, but were able to reconstitute congenic recipients in the absence of competition, indicating a profound defect of functional HSCs, which nevertheless retained reconstitution potential. Importantly, Pbx1 deficient HSCs progressively disappeared from primary transplant recipients, and were unable to engraft secondary recipients, demonstrating that Pbx1 is crucial for the maintenance of LT-HSC self-renewal. Microarray studies performed on mutant and wt LT- and ST-HSCs, followed by bioinformatics analysis, showed that in the absence of Pbx1 LT-HSCs are characterized by premature expression of a large subset of ST-HSC genes. The up-regulated differentially expressed transcripts are enriched for cell cycle regulatory genes, consistent with the observed increased cycling activity. Notably, more than 8% of the down-regulated genes are related to the Tgf-beta pathway, which serves a major role in maintaining HSC quiescence. Moreover, B-cell specific genes, which are expressed in the wt LT-HSC compartment, are down-regulated in the absence of Pbx1, suggesting that the observed reduction in CLP and B-cell numbers ultimately arose from a stem cell defect in lymphoid priming. We conclude that Pbx1 is at the apex of a transcriptional cascade that controls LT-HSC quiescence and differentiation, thus allowing the maintenance of their self-renewal potential, crucial for the homeostasis of the lympho-hematopoietic system.

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Hematopoietic Stem Cells Have an Intrinsic Expansion Limit

Blood ◽

10.1182/blood.v120.21.4749.4749 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4749-4749

Author(s):

Shanti Rojas-Sutterlin ◽

André Haman ◽

Trang Hoang

Keyword(s):

Cell Cycle ◽

Stem Cell ◽

Chemotherapy Regimen ◽

Gene Dosage ◽

Hematopoietic Stem ◽

Cell Functions ◽

Cell Pool ◽

Stem Cell Pool

Abstract Abstract 4749 Hematopoietic stem cell (HSC) transplantation is the first successful cellular therapy and remains the only treatment providing long-term cure in acute myeloblastic leukemia. At the apex of the hematopoietic system, quiescent HSCs are spared by chemotherapeutic treatments that target proliferating cells and therefore can regenerate the entire blood system of a patient after drug exposure. Nevertheless, the consequence of repeated chemotherapy regimen on HSC function remains to be clarified. We previously showed that Scl/Tal1 gene dosage regulates HSC quiescence and functions when transplanted at limiting dilutions (Lacombe et al., 2010). In the present study, we investigate how massive expansion in vivo influences stem cell functions. To address this question, we optimized a protocol based on 5-fluorouracil (5-FU), an antimetabolite that has been used to treat colon, rectum, and head and neck cancers. In addition, we used Scl+/− mice to address the role of Scl in controlling HSCs expansion post-5-FU. We show that within 7 days following 5-FU treatment, HSCs exit quiescence and enter the cell cycle. To deplete cycling HSCs, we injected a second dose of 5-FU and showed that the stem cell pool was disseminated. Nonetheless, the remaining HSCs proliferated extensively to re-establish the HSC pool, which was twice larger than that of untreated mice. At this point, most HSCs have exited the cell cycle and were back to quiescence. Despite a near normal stem cell pool size and a quiescent status, HSCs from these 5-FU treated mice could not compete against untreated cells to regenerate the host in transplantation assays. Furthermore, we show that this extensive proliferation in vivo severely impaired the clonal expansion of individual HSC as measured by the mean activity of stem cell (MAS). Our results demonstrate that HSCs lose their competitive potential after two 5-FU treatments, suggesting that HSCs have an intrinsic expansion limit beyond which their regenerative potential is impaired. In addition, Scl is haplodeficient for cell cycle entry and cell division but Scl gene dosage does not affect this expansion limit. Therefore, our data dissociate the control of HSC expansion under extensive proliferative stress from cell cycle control during steady state. We surmise that chemotherapy regimen based on repeated administration of 5-FU or other antimetabolites are likely to severely impair long-term stem cell functions. Disclosures: No relevant conflicts of interest to declare.

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The Side Population Contains Functionally Distinct Hematopoietic Stem Cell Subpopulations

Blood ◽

10.1182/blood.v112.11.2464.2464 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2464-2464

Author(s):

Grant Anthony Challen ◽

Margaret A Goodell

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Side Population ◽

Clonal Diversity ◽

Stem Cell Markers ◽

Self Renewal ◽

Hematopoietic Stem ◽

Hematopoietic System

Abstract Over the decades since hematopoietic stem cells (HSCs) were first identified, the traditional view has been that the hematopoietic system is regenerated by a single pool of multipotent, quiescent HSCs that are sequentially recruited into cell cycle and which then progressively divide and differentiate until they are exhausted and ultimately replaced by the next cohort of stem cells. However, recent evidence has challenged this classical clonal succession model of HSC hierarchy by suggesting that the hematopoietic system is maintained by a pool of different HSC subtypes, with distinct self-renewal and differentiation potentials (the clonal diversity model, Figure 1). The side population (SP), characterized by Hoechst dye efflux, has been used as a method for isolating HSCs for over a decade and the SP has been shown to be highly enriched for HSC activity. While the entire SP is strikingly homogeneous with respect to expression of canonical stem cell markers such as Sca-1 and c-Kit, we recently observed heterogeneous expression for the SLAM family molecule CD150 within the SP, with CD150+ cells more prevalent in the lower SP and CD150− cell more prevalent in the upper SP. We decided to examine this observation further by investigating the properties of cells from different regions of the SP. Functional capacity was assessed by competitive bone marrow transplantation of upper SP cells, lower SP cells, and a combination of the two populations. Lower SP cells showed better engraftment than upper SP cells in recipient mice, a trend that continued when donor HSCs were isolated from primary recipients and re-transplanted into secondary hosts. Lower SP cells showed 3-fold better engraftment than upper SP cells in secondary transplants, suggesting better self-renewal capacity. However, analysis of the hematopoietic lineages formed by donor cells in recipient mice demonstrated that while both upper and lower SP cells were capable of forming all mature lineages, lower SP cells were biased towards myeloid differentiation while upper SP cells were biased towards lymphoid differentiation. The lineage biases observed from transplantation of one cell population alone were exacerbated when both upper and lower SP cells were co-transplanted into the same recipient mouse, suggesting that while both populations are capable of forming all hematopoietic lineages, in the presence of the other stem cell type (as would be the case in normal homeostasis) that the majority of the output from each HSC subtype is almost exclusively lymphoid or myeloid. The lineage contribution trends observed in the peripheral blood were also reproduced when bone marrow of transplanted mice was analyzed, including at the level of progenitors with lower SP cells showing greater ability to make myeloid progenitors (megakaryocyte-erythrocyte progenitors and granulocyte-macrophage progenitors) and upper SP cells producing proportionately more common lymphoid progenitors. Microarray analysis of upper and lower SP cells to determine the molecular signatures underlying these functional differences found many genes critical for long-term HSC self-renewal to be highly expressed in lower SP cells including Rb1, Meis1, Pbx1 and TGFbr2 while upper SP cells showed higher expression of cell cycle and activation genes. Cell cycle analysis showed upper SP cells to be approximately 2-fold more proliferative than lower SP cells (18.9% to 8.3% Ki-67+, 39.4% to 20.1% BrdU+ 3-days post-BrdU administration). The clonal diversity model which proposes the adult HSC compartment consists of a fixed number of different HSC subtypes each with pre-programmed behavior has important implications for using HSCs in experimental and clinical settings. While other studies have provided functional evidence for the clonal diversity model, this is the first study to prospectively isolate the functionally distinct HSC subtypes prior to transplantation. Figure Figure

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Pleiotrophin Signaling Is Necessary and Sufficient for Hematopoietic Stem Cell Self-Renewal In Vivo

Blood ◽

10.1182/blood.v116.21.404.404 ◽

2010 ◽

Vol 116 (21) ◽

pp. 404-404 ◽

Cited By ~ 1

Author(s):

Heather A Himburg ◽

Pamela Daher ◽

J. Lauren Russell ◽

Phuong Doan ◽

Mamle Quarmyne ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Signaling Pathways ◽

Fold Increase ◽

Self Renewal ◽

Hematopoietic Stem ◽

Human Hscs ◽

Necessary And Sufficient

Abstract Abstract 404 Several signaling pathways have been elucidated which regulate hematopoietic stem cell self-renewal, including the Notch, Wnt, HOX and BMP signaling pathways. However, several of these pathways (e.g. Notch, Wnt) may not be necessary for maintenance of HSCs in vivo. We recently demonstrated that treatment of murine and human HSCs with the heparin binding growth factor, pleiotrophin (PTN), was sufficient to induce self-renewal of murine and human HSCs in culture (Himburg, Nat Med, 2010). In order to determine if PTN signaling is necessary for HSC self renewal and normal hematopoiesis in vivo, we examined the bone marrow HSC content and hematopoietic profile of mice bearing a constitutive deletion of PTN (PTN−/− mice) as well as mice bearing constitutive deletion of the PTN receptor, receptor protein tyrosine phosphatase β/ζ (RPTPβ/ζ) (courtesy of Dr. Gonzalo Herradon, Spain and Dr. Sheila Harroch, L'Institut Pasteur, Paris, FR). PTN−/− mice demonstrated no significant differences in total bone marrow (BM) cells or BM colony forming cells (CFCs) but had significantly decreased bone marrow CD34(-)c-kit(+)sca-1(+)lin(-) (34-KSL) cells compared to littermate controls which retained PTN (PTN+/+) mice (0.007% vs. 0.02%, p=0.03). Consistent with this phenotype, PTN−/− mice also contained 2–fold decreased CFU-S12 compared to control PTN+/+ mice (p= 0.003). PTN−/− mice also demonstrated an 11-fold reduction in long-term repopulating HSC content compared to PTN+/+ mice as measured via competitive repopulating assay (12 week CRU frequency: 1 in 6 cells vs. 1 in 66 cells). Taken together, these data demonstrate that PTN signaling is necessary for maintenance of the BM HSC pool in vivo. Since PTN is known to antagonize the phosphatase activity of RPTPβ/ζ, we hypothesized that deletion of RPTPβ/ζ would increase BM HSC self-renewal and result in expansion of the BM HSC pool in vivo. Consistent with this hypothesis, RPTPβ/ζ−/− mice displayed a 1.3-fold increase in total BM cells (p= 0.04), 1.8-fold increase in BM 34-KSL cells (p=0.03), 1.6-fold increase in BM CFCs (p= 0.002) and 1.6–fold increase in BM CFU-S (p< 0.0001). RPTPβ/ζ−/− mice also demonstrated 1.4–fold higher long-term repopulating capacity (12 weeks) following competitive repopulating assay compared to RPTPβ/ζ+/+ mice (Donor CD45.1+ cell engraftment: 4.2% vs. 1.5%). Interestingly, RPTPβ/ζ −/− mice had significantly increased PB white blood cell counts, hemoglobin and platelet counts compared to RPTPβ/ζ+/+ mice coupled with splenomegaly. The RPTPβ/ζ−/− mice also had significantly increased BM vascular density (via quantitative mouse endothelial cell antigen staining) compared to RPTPβ/ζ+/+ mice, suggesting that PTN/RPTPβ/ζ signaling may augment the HSC pool size directly and also indirectly via activation of the BM vascular niche. These results demonstrate that PTN signaling is necessary and sufficient for induction of HSC self-renewal in vivo. Disclosures: No relevant conflicts of interest to declare.

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The F-Box Protein NIPA Limits Hematopoietic Stem Cell Survival and Transplantation Efficiency

Blood ◽

10.1182/blood.v122.21.1175.1175 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1175-1175

Author(s):

Stefanie Kreutmair ◽

Anna Lena Illert ◽

Rouzanna Istvanffy ◽

Christina Eckl ◽

Christian Peschel ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cell ◽

Spleen Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Mitotic Entry ◽

Stem Cell Maintenance ◽

F Box Protein

Abstract Hematopoietic stem cells (HSCs) are characterized by their ability to self-renewal and multilineage differentiation. Since mostly HSCs exist in a quiescent state re-entry into cell cycle is essential for their regeneration and differentiation. We previously characterized NIPA (Nuclear Interaction Partner of ALK) as a F-Box protein that defines an oscillating ubiquitin E3 ligase and contributes to the timing of mitotic entry. To examine the function of NIPA in vivo, we generated Nipa deficient animals, which are viable but sterile due to a defect in testis stem cell maintenance. To further characterize the role of NIPA in stem cell maintenance and self-renewal we investigated hematopoiesis in Nipa deficient animals. FACS analyses of spleen cells and bone marrow (BM) showed differences in Leucocyte subpopulations. Measuring the CD4 and CD8 positivity within all Thy1.2+ cells, the balance in NIPA-/- T-lymphocytes is destabilised in favour of CD4 positive cells. Besides CD43/CD19 positive as well as CD43/B220 positive cells within all leukocytes are increased in NIPA deficient spleen cells. Analysing more primitive cells, FACS data of bone marrow showed significantly decreased numbers of Lin-Sca1+cKit+ (LSK) cells in NIPA-/- mice (age > 20 month), where LSKs were reduced to 40% of wildtype (wt) littermates (p=0,0171). Additionally, in such older NIPA-/- mice, only half the number of multipotent myeloid progenitors were detected in comparison to wt mice. To examine efficient response of stem cells to myeloid depression, mice were treated with 5-FU four days before BM harvest. We found that in NIPA-/- mice, both the number of myeloid progenitors as well as the number of LSKs were severely reduced compared to those in wt levels after 5-FU treatment (p<0.001). Interestingly, the reduction of progenitors and LSK cells was not dependent on age of the NIPA ko mice, suggesting a role for NIPA in stem cell activation or regeneration. This statement was studied in vitro by methylcellulose assays with 10 000 BM cells seeded in methylcellulose with cytokines and replated for three times after 10 days. Nipa deficient hematopoietic progenitors showed a reduced ability to proliferate and differentiate into colonies compared to their controls with an increasing difference after each replating (p(third replating) < 0.0001). Dynamic cell cycle analysis of seeded BM cells with BRDU and PI uncovered delayed cell cycle progress and mitotic entry in NIPA-/- BM cells in contrast to wt BM cells. Using competitive BM transplantation assay we investigated the role of NIPA for hematopoietic reconstitution in vivo. These experiments showed that NIPA-/- BM cells were severely deficient in hematopoietic recovery as recipient mice of NIPA-/- BM cells showed only half the amount of donor-derived peripheral blood cells in contrast to recipient mice of wt BM cells after 4, 11, 17 and over 23 weeks after transplantation. Furthermore NIPA-/- cells contributed only 7% in BM of transplanted mice 6 month after transplantation compared to 33% in recipients transplanted with wt BM cells (p<0.005). To further explore this defect in hematopoietic repopulation capacity and apply to more primitive progenitors serial transplantation assays were conducted with LSK cells transplanted together with support BM cells. Taken together our results demonstrate a critical role of NIPA in regulating the primitive hematopoietic compartment as a regulator of self-renewal, cycle capacity and HSC expansion. Disclosures: No relevant conflicts of interest to declare.

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ETO2 Controls Hematopoietic Stem Cell Expansion.

Blood ◽

10.1182/blood.v114.22.396.396 ◽

2009 ◽

Vol 114 (22) ◽

pp. 396-396

Author(s):

Stephane Barakat ◽

Julie Lambert ◽

Guy Sauvageau ◽

Trang Hoang

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Short Term ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Abstract 396 Hematopoietic stem cells that provide short term reconstitution (ST-HSCs) as well as hematopoietic progenitors expand from a small population of long term hematopoietic stem cells (LT-HSCs) that are mostly dormant cells. The mechanisms underlying this expansion remain to be clarified. SCL (stem cell leukemia), is a bHLH transcription factor that controls HSC quiescence and long term competence. Using a proteomics approach to identify components of the SCL complex in erythroid cells, we and others recently showed that the ETO2 co-repressor limits the activity of the SCL complex via direct interaction with the E2A transcription factor. ETO2/CBF2T3 is highly homologous to ETO/CBFA2T1 and both are translocation partners for AML1. We took several approaches to identify ETO2 function in HSCs. We initially found by Q-PCR that ETO2 is highly expressed in populations of cells enriched in short-term HSC (CD34+Flt3-Kit+Sca+Lin-) and lympho-myeloid progenitors (CD34+Flt3+Kit+Sca+Lin-) and at lower levels in LT-HSCs (CD34-Kit+Sca+Lin- or CD150+CD48-Kit+Sca+Lin-). Next, the role of ETO2 was studied by overexpression or downregulation combined with transplantation in mice. Ectopic ETO2 expression induces a 100 fold expansion of LT-HSCs in vivo in transplanted mice associated with differentiation blockade in all lineages, suggesting that ETO2 overexpression overcomes the mechanisms that limit HSC expansion in vivo. We are currently testing the role of the NHR1 domain of ETO2 in this expansion. Conversely, shRNAs directed against ETO2 knock down ET02 levels in Kit+Sca+Lin- cells, causing a ten-fold decrease in this population after transplantation, associated with reduced short-term reconstitution in mice. Finally, proliferation assays using Hoechst and CFSE indicate that ETO2 downregulation affects cell division (CFSE) and leads to an accumulation of Kit+Sca+Lin-cells in G0/G1 state (Hoescht). In conclusion, we show that ETO2 is highly expressed in ST-HSCs and lymphoid progenitors, and controls their expansion by regulating cell cycle entry at the G1-S checkpoint. In addition, ETO2 overexpression converts the self-renewal of maintenance into self-renewal of expansion in LT-HSCs. Disclosures: No relevant conflicts of interest to declare.

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A Functional In Vivo RNAi screen Involving Jumonji C Domain Containing Candidates Unravels Kdm5b As a Negative Modulator of Hematopoietic Stem Cell Self-Renewal

Blood ◽

10.1182/blood.v118.21.385.385 ◽

2011 ◽

Vol 118 (21) ◽

pp. 385-385

Author(s):

Sonia Cellot ◽

Kristin J Hope ◽

Martin Sauvageau ◽

Jalila Chagraoui ◽

Eric Deneault ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Cell Expansion ◽

Rt Pcr ◽

Self Renewal ◽

Hematopoietic Stem ◽

Stem Cell Expansion ◽

Time Point

Abstract Abstract 385 Epigenetic modifications influence chromatin accessibility, impacting on cell fate decisions, such as stem cell self-renewal and differentiation, in both normal and leukemic stem cells (LSC). To investigate the putative role of histone demethylases (HDM) in modulating primary hematopoietic stem cell (HSC) fate, an in vivo functional screen was performed, using an RNAi based strategy, involving 25 members of the Jumonji (JmjC) domain protein family. As a first step, expression profile studies of these gene candidates were undertaken. Transcripts of all these enzymes were detected in isolated HSC populations (frequency 1:2) from fetal liver (n=1) and bone marrow (n=2), except for Hairless. As compared to unsorted bone marrow (BM), stem cells harboured higher expression of Jarid1b (relative-fold enrichment (RQ) of 3.9±1.7), Jmjd2d (RQ3.8±1.9), and Jhdm1b (3.1±1.7). Next, 5shRNA were designed against each of the 25 JmjC containing proteins, and cloned into a retroviral LMP vector encoding GFP to permit tracking of transduced cells in vivo. HSC-enriched CD150+CD48−Lin−cells (∼60 LT-HSC) were infected over 5 days by co-culture with retroviral producer cells in an arrayed 96-well format, with one shRNA per well. Directly after infection, the in vivo reconstituting potential of ¼ of each well was evaluated through duplicate competitive repopulation assays involving the co-transplantation of 1.5 × 105 congenic BM competitor cells into irradiated recipients. The remaining cell fraction served to asses gene transfer by GFP epifluorescence measurements, and RNA isolated from sorted GFP+ cells was used to evaluate gene knockdown levels by Q-RT-PCR analysis. Blood reconstitution was evaluated at an early (4wks) and late time point (16–20wks), tracking the contribution of the donor CD45.1+ transduced (GFP+) cells to recipient hematopoiesis over time. As baseline references, sh-RNA to Luciferase (no effect) and the histone acetyl transferase Myst3 (stem cell loss) were used, as well as Hoxb4 over-expression (stem cell expansion). The primary screen, followed by validation experiments, unravelled one positive (Jhdm1f/Phf8) and two negative (Jarid1b, Hif1an) regulators of HSC activity. The strongest impact was seen with Jarid1b knockdown, and the resulting gain in HSC activity. As a confirmation step, cells were kept in culture for one week, to better contrast an increase in HSC activity, compared to control HSC. After 7 days in vitro, 1/8 equivalents of single well cultures were transplanted into 3 mice, and blood reconstitution levels serially assessed. Cells transduced with sh-RNA against Jarid1b contributed more significantly to host hematopoiesis than sh-RNA Luciferase transduced cells (58±16% vs 26±3% GFP), or Hoxb4 over-expressing cells (37±2% GFP), at comparable gene transfer rates, at the 16 week time point and beyond (3 independent experiments). Long-term HSC frequencies were evaluated from these cultures, and found to be 6–10 fold increased in shJari1d1b-cell cultures. In long-term recipients, differentiation potential of these cells was preserved, as evidenced by CD4+CD8+ thymic cells, B220+ splenic cells and CD11b+ bone marrow cells in the GFP positive contingent. Clonality studies on DNA isolated from these sorted populations confirmed oligoclonality of the stem cell expansion, and HSC pluripotency. There were no cases of leukemic transformation in all of the transplant recipients (n>30). As assessed by Q-RT-PCR, levels of HoxA5, HoxA9, HoxA10 and CxCl5 were increased in day7 sh3Jarib1b-cells (vs ctl), while the levels of the tumor suppressors Cav1, Sash1 and Egr1 were decreased. A more detailed assessment of the HoxA cluster revealed predominant expression of 5' cluster genes in expanding shJarib1b-cells, from HoxA5 to HoxA11, with a concomitant increase in the level of H3K4 tri-methylation, as assessed by ChIP-CHIP. In conclusion, HDM of the JmjC family can modulate HSC activity, both positively and negatively. These data suggest that the H3K4 demethylase Jarid1b (KDM5b) restrains stem cell self-renewal, acting as a co-repressor, possibly via epigenetic regulation of the HoxA gene cluster, among other target genes. This observation could be further exploited as an HSC expansion strategy. Disclosures: No relevant conflicts of interest to declare.

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Evidence of both ontogeny and transplant dose-regulated expansion of hematopoietic stem cells in vivo

Blood ◽

10.1182/blood.v88.8.2852.bloodjournal8882852 ◽

1996 ◽

Vol 88 (8) ◽

pp. 2852-2858 ◽

Cited By ~ 121

Author(s):

R Pawliuk ◽

C Eaves ◽

RK Humphries

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Fetal Liver ◽

Complete Recovery ◽

Self Renewal ◽

Hematopoietic Stem ◽

Adult Mice

Recent assessment of the long-term repopulating activity of defined subsets of hematopoietic cells has offered new insights into the characteristics of the transplantable stem cells of this system; however, as yet, there is very little known about mechanisms that regulate their self-renewal in vivo. We have now exploited the ability to quantitate these cells using the competitive repopulating unit (CRU) assay to identify the role of both intrinsic (ontological) and extrinsic (transplanted dose-related) variables that may contribute to the regulation of CRU recovery in vivo. Ly5.1 donor cells derived from day-14.5 fetal liver (FL) or the bone marrow (BM) of adult mice injected 4 days previously with 5-fluorouracil were transplanted at doses estimated to contain 10, 100, or 1,000 long-term CRU into irradiated congenic Ly5.2 adult recipient mice. Eight to 12 months after transplantation, there was a complete recovery of BM cellularity and in vitro clonogenic progenitor numbers and a nearly full recovery of day-12 colony-forming unit-spleen numbers irrespective of the number or origin of cells initially transplanted. In contrast, regeneration of Ly5.1+ donor-derived CRU was incomplete in all cases and was dependent on both the origin and dose of the transplant, with FL being markedly superior to that of adult BM. As a result, the final recovery of the adult marrow CRU compartment ranged from 15% to 62% and from 1% to 18% of the normal value in recipients of FL and adult BM transplantation, respectively, with an accompanying maximum CRU amplification of 150-fold for recipients of FL cells and 15-fold for recipients of adult BM cells. Interestingly, the extent of CRU expansion from either source was inversely related to the number of CRU transplanted. These data suggest that recovery of mature blood cell production in vivo may activate negative feedback regulatory mechanisms to prematurely limit stem cell self-renewal ability. Proviral integration analysis of mice receiving retrovirally transduced BM cells confirmed regeneration of totipotent lymphomyeloid repopulating cells and provided evidence for a greater than 300-fold clonal amplification of a single transduced stem cell. These results highlight the differential regenerative capacities of CRU from fetal and adult sources that likely reflect intrinsic, genetically defined determinants of CRU expansion but whose contribution to the magnitude of stem cell amplification ultimately obtained in vivo is also strongly influenced by the initial number of CRU transplanted. Such findings set the stage for attempts to enhance CRU regeneration by administration of agents that may enable full expression of regenerative potential or through the expression of intracellular gene products that may alter intrinsic regenerative capacity.

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Nucleotide Receptor P2Y14 Modulates Hematopoietic Stem Cell Response to Tissue Injury Altering Stem Cell Preservation and Tissue Recovery.

Blood ◽

10.1182/blood.v108.11.679.679 ◽

2006 ◽

Vol 108 (11) ◽

pp. 679-679 ◽

Cited By ~ 1

Author(s):

Yoriko Saito ◽

Eyal Attar ◽

Samyukta Jana ◽

David Dombkowski ◽

Viktor Janzen ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cell ◽

Self Renewal ◽

Hematopoietic Stem ◽

Chemical Injury ◽

Cell Cycle Entry ◽

Induced Apoptosis ◽

Lethal Irradiation

Abstract P2 receptors are functionally diverse cell surface receptors that bind nucleotides adenine (ADP, ATP) and uridine (UDP, UTP). P2Y receptors are metabotropic G protein-coupled receptors that mediate vascular and immune responses to injury. We previously reported the differential expression cloning of the UTP-glycoconjugate receptor, P2Y14 from quiescent primary human bone marrow (BM) hematopoietic stem cells (HSCs). Using P2Y14−/− mice, we now report that the presence of P2Y14 protects HSCs from apoptosis in the face of cytotoxic chemical injury. P2Y14 null mice develop normally and showed no significant differences in peripheral blood cell counts, BM cellularity or the absolute number/proportion of lin−cKit+Sca1+ (LKS+) and CD34−/lowLKS+ (34-LKS+) cells compared to their wildtype littermates. Similarly, cell cycle status, in vitro colony-forming cell (CFC) capacity, in vivo homing and in vivo colony-forming unit-spleen (CFU-S) function were unaffected. Since the role of nucleotide receptors in injury response have been reported, we examined BM HSC content following IP injection of 200mg/kg cyclophosphamide (CTX) and found that the relative protection of LKS+ and 34-LKS+ cells from CTX-induced apoptosis was lost in P2Y14 null animals (WT LKS+: 12.7% AnnexinV+7AAD-, KO LKS+ 36.8% AnnexinV+7AAD−, n=5 each, p=0.004; WT 34-LKS+: 13.2% AnnexinV+7AAD−, KO LKS+ 38.7% AnnexinV+7AAD−, n=5 each, p=0.007). In addition, the kinetics of long-term myeloid recovery after a single injection of 5-Fluorouracil (5FU) IP 150mg/kg was significantly more accentuated in P2Y14 null animals, with significantly greater peripheral blood Gr-1+ cell count at days 21–56 post-injection (n=10 each, p=0.009). When sorted BM LKS+ cells were exposed in vitro to UDP-glucose, a putative P2Y14 ligand known to be released from cytoplasm during cellular injury, BrDU incorporation was significantly reduced (n=3 each, p<0.05), suggesting that P2Y14 activation with UDP-glucose reduces HSC cell cycle entry in response to injury. While these in vivo models examine HSC response to injury to both BM microenvironment and the HSCs themselves, when uninjured HSCs were reintroduced into injured microenvironment in the setting of hematopoietic reconstitution following lethal irradiation, P2Y14 null BM HSCs performed better in serial transplantation (n=10 each, p<0.01 for primary, secondary and tertiary transplantation), showing greater reconstitution and self-renewal capacity compared with WT littermates. From these findings, we propose that P2Y14 protects HSCs from chemical injury by acting as a sensor for metabolic “danger signal” in the form of released intracellular UDP-glucose during acute chemical injury in the BM and maintaining relative resistance of HSCs to toxin-induced apoptosis by restricting cell cycle entry. In the setting of injury exclusive to BM microenvironment (HSC transplantation), P2Y14 null HSCs, unable to detect UDP-glucose, respond to highly proliferative environment following lethal irradiation, resulting in greater reconstitution and self-renewal.

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