Hoxa9+Meis1a Efficiently Transform Hematopoietic Stem Cells but Not Committed Progenitors.

Abstract The homeobox transcription factors Hoxa9 and Meis1a induce the expansion of hematopoietic stem cells (HSC) and cooperate to induce acute myeloid leukemia (AML). We have recently shown that Hoxa9 and Meis1a are part of a self-renewal program found in leukemia stem cells (LSC) isolated from murine leukemias initiated by expression of MLL-AF9 in committed granulocyte macrophage progenitors (GMP). However, it remains unclear whether the Hoxa9-Meis1a complex is sufficient to mimic the leukemogenic effects of MLL-AF9. Therefore we assessed the clonogenic and leukemogenic activity of HSC and GMP transduced with Hoxa9-Meis1a and compared this to MLL-AF9 transduced HSC and GMP. We expressed either Hoxa9-Meis1a or MLL-AF9 in HSC or GMP and sorted single cells into 96 well plates. The data demonstrate that the clonogenic potential of single HSC or GMP expressing Hoxa9-Meis1a or MLL-AF9 are similar and both produce cells that can be replated in vitro for greater than 6 weeks. Remarkably, Hoxa9-Meis1a induces leukemia with high efficiency when expressed in HSC but not committed GMP, while MLL-AF9 can fully transform both HSC and GMP. Next, we identified a population of cells from Hoxa9-Meis1a leukemias that were enriched for LSC. Even though the leukemias were initiated from HSC, the LSC population possessed an immunophenotype and global gene expression program more consistent with differentiated myeloid cells. Further characterization of gene expression in the Hoxa9-Meis1a LSC found that only a subset of the MLL-AF9 self-renewal signature is activated by Hoxa9-Meis1a. These findings show that Hoxa9-Meis1a can efficiently induce LSC from HSC but not GMP. This suggests Hoxa9 and Meis1a induced leukemogenesis is dependent upon cellular context and require programs/pathways active in HSC in order to initiate leukemia. Thus MLL-AF9 must activate pathways critical for LSC development in addition to Hoxa9-Meis1a in order to fully transform committed progenitor cells. Identification of these cooperating pathways should provide insight into MLL-rearranged and other AML.

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In Vitro Self-Renewal Division of Hematopoietic Stem Cells

Journal of Experimental Medicine ◽

10.1084/jem.192.9.1281 ◽

2000 ◽

Vol 192 (9) ◽

pp. 1281-1288 ◽

Cited By ~ 211

Author(s):

Hideo Ema ◽

Hina Takano ◽

Kazuhiro Sudo ◽

Hiromitsu Nakauchi

Keyword(s):

Stem Cells ◽

Cell Division ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Single Cells ◽

Significant Proportion ◽

Self Renewal ◽

Hematopoietic Stem ◽

Culture Effects

Little is known about how hematopoietic stem cells (HSCs) self-renew. We studied the regeneration of HSCs in culture. Effects of various cytokines on cell division of CD34−/low c-Kit+Sca-1+ lineage marker–negative (CD34−KSL) bone marrow cells of the mouse were first evaluated in serum-free single cell culture. We then performed a competitive repopulation assay on divided cells to ask if such cell division involved self-renewal of HSCs. In the presence of stem cell factor (SCF), thrombopoietin (TPO) induced a first cell division of CD34−KSL cells more efficiently than did interleukin (IL)-3 or IL-6. Multilineage repopulating cells were detected in a significant proportion of cells derived from single cells in culture with TPO and SCF, although this culture condition led to a substantial decrease in HSC number. These regenerated repopulating cells could be further transplanted into secondary recipients. When paired daughter cells were separately studied, one of a pair gave rise to repopulating cells with self-renewal potential, suggesting asymmetric self-renewal division. This study provides evidence that one HSC regenerates at least one HSC in culture.

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Molecular Modulation of Fetal Liver Hematopoietic Stem Cell Mobilization into Fetal Bone Marrow in Mice

Stem Cells International ◽

10.1155/2020/8885154 ◽

2020 ◽

Vol 2020 ◽

pp. 1-12

Author(s):

Huihong Zeng ◽

Jiaoqi Cheng ◽

Ying Fan ◽

Yingying Luan ◽

Juan Yang ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Vascular Endothelial Cells ◽

Fetal Liver ◽

Bone Marrow Niche ◽

Self Renewal ◽

Hematopoietic Stem ◽

Fetal Bone

Development of hematopoietic stem cells is a complex process, which has been extensively investigated. Hematopoietic stem cells (HSCs) in mouse fetal liver are highly expanded to prepare for mobilization of HSCs into the fetal bone marrow. It is not completely known how the fetal liver niche regulates HSC expansion without loss of self-renewal ability. We reviewed current progress about the effects of fetal liver niche, chemokine, cytokine, and signaling pathways on HSC self-renewal, proliferation, and expansion. We discussed the molecular regulations of fetal HSC expansion in mouse and zebrafish. It is also unknown how HSCs from the fetal liver mobilize, circulate, and reside into the fetal bone marrow niche. We reviewed how extrinsic and intrinsic factors regulate mobilization of fetal liver HSCs into the fetal bone marrow, which provides tools to improve HSC engraftment efficiency during HSC transplantation. Understanding the regulation of fetal liver HSC mobilization into the fetal bone marrow will help us to design proper clinical therapeutic protocol for disease treatment like leukemia during pregnancy. We prospect that fetal cells, including hepatocytes and endothelial and hematopoietic cells, might regulate fetal liver HSC expansion. Components from vascular endothelial cells and bones might also modulate the lodging of fetal liver HSCs into the bone marrow. The current review holds great potential to deeply understand the molecular regulations of HSCs in the fetal liver and bone marrow in mammals, which will be helpful to efficiently expand HSCs in vitro.

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Cripto Selectively Expands a Distinct Population of Hematopoietic Stem Cells Expressing the Cell Surface Receptor GRP78 and Strongly Induces An Immature Phenotype In Vivo After Ex Vivo Culture

Blood ◽

10.1182/blood.v116.21.405.405 ◽

2010 ◽

Vol 116 (21) ◽

pp. 405-405

Author(s):

Kenichi Miharada ◽

Göran Karlsson ◽

Jonas Larsson ◽

Emma Larsson ◽

Kavitha Siva ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Distinct Population ◽

Self Renewal ◽

Hematopoietic Stem ◽

Total Bone Marrow

Abstract Abstract 405 Cripto is a member of the EGF-CFC soluble protein family and has been identified as an important factor for the proliferation/self-renewal of ES and several types of tumor cells. The role for Cripto in the regulation of hematopoietic cells has been unknown. Here we show that Cripto is a potential new candidate factor to increase self-renewal and expand hematopoietic stem cells (HSCs) in vitro. The expression level of Cripto was analyzed by qRT-PCR in several purified murine hematopoietic cell populations. The findings demonstrated that purified CD34-KSL cells, known as highly concentrated HSC population, had higher expression levels than other hematopoietic progenitor populations including CD34+KSL cells. We asked how Cripto regulates HSCs by using recombinant mouse Cripto (rmCripto) for in vitro and in vivo experiments. First we tested the effects of rmCripto on purified hematopoietic stem cells (CD34-LSK) in vitro. After two weeks culture in serum free media supplemented with 100ng/ml of SCF, TPO and 500ng/ml of rmCripto, 30 of CD34-KSL cells formed over 1,300 of colonies, including over 60 of GEMM colonies, while control cultures without rmCripto generated few colonies and no GEMM colonies (p<0.001). Next, 20 of CD34-KSL cells were cultured with or without rmCripto for 2 weeks and transplanted to lethally irradiated mice in a competitive setting. Cripto treated donor cells showed a low level of reconstitution (4–12%) in the peripheral blood, while cells cultured without rmCripto failed to reconstitute. To define the target population and the mechanism of Cripto action, we analyzed two cell surface proteins, GRP78 and Glypican-1, as potential receptor candidates for Cripto regulation of HSC. Surprisingly, CD34-KSL cells were divided into two distinct populations where HSC expressing GRP78 exhibited robust expansion of CFU-GEMM progenitor mediated by rmCripto in CFU-assay whereas GRP78- HSC did not respond (1/3 of CD34-KSL cells were GRP78+). Furthermore, a neutralization antibody for GRP78 completely inhibited the effect of Cripto in both CFU-assay and transplantation assay. In contrast, all lineage negative cells were Glypican-1 positive. These results suggest that GRP78 must be the functional receptor for Cripto on HSC. We therefore sorted these two GRP78+CD34-KSL (GRP78+HSC) and GRP78-CD34-KSL (GRP78-HSC) populations and transplanted to lethally irradiated mice using freshly isolated cells and cells cultured with or without rmCripto for 2 weeks. Interestingly, fresh GRP78-HSCs showed higher reconstitution than GRP78+HSCs (58–82% and 8–40%, p=0.0038) and the reconstitution level in peripheral blood increased rapidly. In contrast, GRP78+HSC reconstituted the peripheral blood slowly, still at a lower level than GRP78-HSC 4 months after transplantation. However, rmCripto selectively expanded (or maintained) GRP78+HSCs but not GRP78-HSCs after culture and generated a similar level of reconstitution as freshly transplanted cells (12–35%). Finally, bone marrow cells of engrafted recipient mice were analyzed at 5 months after transplantation. Surprisingly, GRP78+HSC cultured with rmCripto showed higher reconstitution of the CD34-KSL population in the recipients' bone marrow (45–54%, p=0.0026), while the reconstitution in peripheral blood and in total bone marrow was almost the same. Additionally, most reconstituted CD34-KSL population was GRP78+. Interestingly freshly transplanted sorted GRP78+HSC and GRP78-HSC can produce the GRP78− and GRP78+ populations in the bone marrow and the ratio of GRP78+/− cells that were regenerated have the same proportion as the original donor mice. Compared to cultured cells, the level of reconstitution (peripheral blood, total bone marrow, HSC) in the recipient mice was almost similar. These results indicate that the GRP78 expression on HSC is reversible, but it seems to be “fixed” into an immature stage and differentiate with lower efficiency toward mature cells after long/strong exposure to Cripto signaling. Based on these findings, we propose that Cripto is a novel factor that maintains HSC in an immature state and may be a potent candidate for expansion of a distinct population of GRP78 expressing HSC. Disclosures: No relevant conflicts of interest to declare.

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Differential gene expression profiling of adult murine hematopoietic stem cells

Blood ◽

10.1182/blood.v99.2.488 ◽

2002 ◽

Vol 99 (2) ◽

pp. 488-498 ◽

Cited By ~ 122

Author(s):

In-Kyung Park ◽

Yaqin He ◽

Fangming Lin ◽

Ole D. Laerum ◽

Qiang Tian ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Differential Gene Expression ◽

Molecular Mechanisms ◽

Expression Profiles ◽

Cdna Clones ◽

Self Renewal ◽

Hematopoietic Stem ◽

Differential Gene

Abstract Hematopoietic stem cells (HSCs) have self-renewal capacity and multilineage developmental potentials. The molecular mechanisms that control the self-renewal of HSCs are still largely unknown. Here, a systematic approach using bioinformatics and array hybridization techniques to analyze gene expression profiles in HSCs is described. To enrich mRNAs predominantly expressed in uncommitted cell lineages, 54 000 cDNA clones generated from a highly enriched population of HSCs and a mixed population of stem and early multipotent progenitor (MPP) cells were arrayed on nylon membranes (macroarray or high-density array), and subtracted with cDNA probes derived from mature lineage cells including spleen, thymus, and bone marrow. Five thousand cDNA clones with very low hybridization signals were selected for sequencing and further analysis using microarrays on glass slides. Two populations of cells, HSCs and MPP cells, were compared for differential gene expression using microarray analysis. HSCs have the ability to self-renew, while MPP cells have lost the capacity for self-renewal. A large number of genes that were differentially expressed by enriched populations of HSCs and MPP cells were identified. These included transcription factors, signaling molecules, and previously unknown genes.

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Smad4 is critical for self-renewal of hematopoietic stem cells

Journal of Experimental Medicine ◽

10.1084/jem.20060465 ◽

2007 ◽

Vol 204 (3) ◽

pp. 467-474 ◽

Cited By ~ 88

Author(s):

Göran Karlsson ◽

Ulrika Blank ◽

Jennifer L. Moody ◽

Mats Ehinger ◽

Sofie Singbrant ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

In Vitro Proliferation ◽

Self Renewal ◽

Hematopoietic Stem ◽

Early Embryonic Lethality

Members of the transforming growth factor β (TGF-β) superfamily of growth factors have been shown to regulate the in vitro proliferation and maintenance of hematopoietic stem cells (HSCs). Working at a common level of convergence for all TGF-β superfamily signals, Smad4 is key in orchestrating these effects. The role of Smad4 in HSC function has remained elusive because of the early embryonic lethality of the conventional knockout. We clarify its role by using an inducible model of Smad4 deletion coupled with transplantation experiments. Remarkably, systemic induction of Smad4 deletion through activation of MxCre was incompatible with survival 4 wk after induction because of anemia and histopathological changes in the colonic mucosa. Isolation of Smad4 deletion to the hematopoietic system via several transplantation approaches demonstrated a role for Smad4 in the maintenance of HSC self-renewal and reconstituting capacity, leaving homing potential, viability, and differentiation intact. Furthermore, the observed down-regulation of notch1 and c-myc in Smad4−/− primitive cells places Smad4 within a network of genes involved in the regulation HSC renewal.

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Age-Associated Characteristics of Murine Hematopoietic Stem Cells

Journal of Experimental Medicine ◽

10.1084/jem.192.9.1273 ◽

2000 ◽

Vol 192 (9) ◽

pp. 1273-1280 ◽

Cited By ~ 477

Author(s):

Kazuhiro Sudo ◽

Hideo Ema ◽

Yohei Morita ◽

Hiromitsu Nakauchi

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Lymphoid Cells ◽

Differentiation Potential ◽

Self Renewal ◽

Hematopoietic Stem ◽

Functional Changes

Little is known of age-associated functional changes in hematopoietic stem cells (HSCs). We studied aging HSCs at the clonal level by isolating CD34−/lowc-Kit+Sca-1+ lineage marker–negative (CD34−KSL) cells from the bone marrow of C57BL/6 mice. A population of CD34−KSL cells gradually expanded as age increased. Regardless of age, these cells formed in vitro colonies with stem cell factor and interleukin (IL)-3 but not with IL-3 alone. They did not form day 12 colony-forming unit (CFU)-S, indicating that they are primitive cells with myeloid differentiation potential. An in vivo limiting dilution assay revealed that numbers of multilineage repopulating cells increased twofold from 2 to 18 mo of age within a population of CD34−KSL cells as well as among unseparated bone marrow cells. In addition, we detected another compartment of repopulating cells, which differed from HSCs, among CD34−KSL cells of 18-mo-old mice. These repopulating cells showed less differentiation potential toward lymphoid cells but retained self-renewal potential, as suggested by secondary transplantation. We propose that HSCs gradually accumulate with age, accompanied by cells with less lymphoid differentiation potential, as a result of repeated self-renewal of HSCs.

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Microrna 199b Regulates Mouse Hematopoietic Stem Cells Maintenance

Blood ◽

10.1182/blood-2019-126283 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3704-3704

Author(s):

Aldona A Karaczyn ◽

Edward Jachimowicz ◽

Jaspreet S Kohli ◽

Pradeep Sathyanarayana

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Quiescent State ◽

Differentiation Potential ◽

Self Renewal ◽

Hematopoietic Stem

The preservation of hematopoietic stem cell pool in bone marrow (BM) is crucial for sustained hematopoiesis in adults. Studies assessing adult hematopoietic stem cells functionality had been shown that for example loss of quiescence impairs hematopoietic stem cells maintenance. Although, miR-199b is frequently down-regulated in acute myeloid leukemia, its role in hematopoietic stem cells quiescence, self-renewal and differentiation is poorly understood. Our laboratory investigated the role of miR-199b in hematopoietic stem and progenitor cells (HSPCs) fate using miR-199b-5p global deletion mouse model. Characterization of miR-199b expression pattern among normal HSPC populations revealed that miR-199b is enriched in LT-HSCs and reduced upon myeloablative stress, suggesting its role in HSCs maintenance. Indeed, our results reveal that loss of miR-199b-5p results in imbalance between long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells (ST-HSCs) and multipotent progenitors (MMPs) pool. We found that during homeostasis, miR-199b-null HSCs have reduced capacity to maintain quiescent state and exhibit cell-cycle deregulation. Cell cycle analyses showed that attenuation of miR-199b controls HSCs pool, causing defects in G1-S transition of cell cycle, without significant changes in apoptosis. This might be due to increased differentiation of LT-HSCs into MPPs. Indeed, cell differentiation assay in vitro showed that FACS-sorted LT-HSCs (LineagenegSca1posc-Kitpos CD48neg CD150pos) lacking miR-199b have increased differentiation potential into MPP in the presence of early cytokines. In addition, differentiation assays in vitro in FACS-sorted LSK population of 52 weeks old miR-199b KO mice revealed that loss of miR-199b promotes accumulation of GMP-like progenitors but decreases lymphoid differentiation, suggesting that miR199b may regulate age-related pathway. We used non-competitive repopulation studies to show that overall BM donor cellularity was markedly elevated in the absence of miR-199b among HSPCs, committed progenitors and mature myeloid but not lymphoid cell compartments. This may suggest that miR-199b-null LT-HSC render enhanced self-renewal capacity upon regeneration demand yet promoting myeloid reconstitution. Moreover, when we challenged the self-renewal potential of miR-199b-null LT-HSC by a secondary BM transplantation of unfractionated BM cells from primary recipients into secondary hosts, changes in PB reconstitution were dramatic. Gating for HSPCs populations in the BM of secondary recipients in 24 weeks after BMT revealed that levels of LT-HSC were similar between recipients reconstituted with wild-type and miR-199b-KO chimeras, whereas miR-199b-null HSCs contributed relatively more into MPPs. Our data identify that attenuation of miR-199b leads to loss of quiescence and premature differentiation of HSCs. These findings indicate that loss of miR-199b promotes signals that govern differentiation of LT-HSC to MPP leading to accumulation of highly proliferative progenitors during long-term reconstitution. Hematopoietic regeneration via repopulation studies also revealed that miR-199b-deficient HSPCs have a lineage skewing potential toward myeloid lineage or clonal myeloid bias, a hallmark of aging HSCs, implicating a regulatory role for miR-199b in hematopoietic aging. Disclosures No relevant conflicts of interest to declare.

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In vivo and in vitro stem cell function of c-kit- and Sca-1-positive murine hematopoietic cells

Blood ◽

10.1182/blood.v80.12.3044.bloodjournal80123044 ◽

1992 ◽

Vol 80 (12) ◽

pp. 3044-3050 ◽

Cited By ~ 2

Author(s):

S Okada ◽

H Nakauchi ◽

K Nagayoshi ◽

S Nishikawa ◽

Y Miura ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Cell Function ◽

Bone Marrow Cells ◽

Synergistic Effects ◽

Single Cells ◽

Hematopoietic Stem

c-kit is expressed on hematopoietic stem cells and progenitor cells, but not on lymphohematopoietic differentiated cells. Lineage marker- negative, c-kit-positive (Lin-c-kit+) bone marrow cells were fractionated by means of Ly6A/E or Sca-1 expression. Lin-c-kit+Sca-1+ cells, which consisted of 0.08% of bone marrow nucleated cells, did not contain day-8 colony-forming units-spleen (CFU-S), but 80% were day-12 CFU-S. One hundred cells rescued the lethally irradiated mice and reconstituted hematopoiesis. On the other hand, 2 x 10(3) of Lin-c- kit+Sca-1- cells formed 20 day-8 and 11 day-12 spleen colonies, but they could not rescue the lethally irradiated mice. These data indicate that Lin-c-kit+Sca-1+ cells are primitive hematopoietic stem cells and that Sca-1-cells do not contain stem cells that reconstitute hematopoiesis. Lin-c-kit+Sca-1+ cells formed no colonies in the presence of stem cell factor (SCF) or interleukin-6 (IL-6), and only 10% of them formed colonies in the presence of IL-3. However, approximately 50% of them formed large colonies in the presence of IL-3, IL-6, and SCF. Moreover, when single cells were deposited into culture medium by fluorescence-activated cell sorter clone sorting system, 40% of them proliferated on a stromal cell line (PA-6) and proliferated for more than 2 weeks. In contrast, 15% of the Lin-c- kit+Sca-1-cells formed colonies in the presence of IL-3, but no synergistic effects were observed in combination with SCF plus IL-6 and/or IL-3. Approximately 10% proliferated on PA-6, but most of them degenerated within 2 weeks. The population ratio of c-kit+Sca-1+ to c-kit+Sca-1- increased 2 and 4 days after exposure to 5-fluorouracil (5-FU). These results are consistent with the relative enrichment of highly proliferative colony-forming cells by 5-FU. These data show that, although c-kit is found both on the primitive hematopoietic stem cells and progenitors, Sca-1+ cells are more primitive and respond better than Sca-1- cells to a combination of hematopoietic factors, including SCF and stromal cells.

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Enhanced Self-Renewal of Hematopoietic Stem Cells Mediated by the Polycomb Gene Product, Bmi-1.

Blood ◽

10.1182/blood.v104.11.1686.1686 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1686-1686

Author(s):

Hideyuki Oguro ◽

Atsushi Iwama ◽

Hiromitsu Nakauchi

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Target Genes ◽

Ex Vivo ◽

Self Renewal ◽

Hematopoietic Stem ◽

Ex Vivo Culture ◽

Deficient Mice

Abstract The Polycomb group (PcG) proteins form multiprotein complexes that play an important role in the maintenance of transcriptional repression of target genes. Loss-of-function analyses show abnormal hematopoiesis in mice deficient for PcG genes including Bmi-1, Mph-1/Rae28, M33, Mel-18, and Eed, suggesting involvement of PcG complexes in the regulation of hematopoiesis. Among them, Bmi-1 has been implicated in the maintenance of hematopoietic and leukemic stem cells. In this study, detailed RT-PCR analysis of mouse hematopoietic cells revealed that all PcG genes encoding components of the Bmi-1-containing complex, such as Bmi-1, Mph1/Rae28, M33, and Mel-18 were highly expressed in CD34−c-Kit+Sca-1+Lin− (CD34−KSL) hematopoietic stem cells (HSCs) and down-regulated during differentiation in the bone marrow. These expression profiles support the idea of positive regulation of HSC self-renewal by the Bmi-1-containing complex. To better understand the role of each component of the PcG complex in HSC and the impact of forced expression of PcG genes on HSC self-renewal, we performed retroviral transduction of Bmi1, Mph1/Rae28, or M33 in HSCs followed by ex vivo culture. After 14-day culture, Bmi-1-transduced but not Mph1/Rae28-transduced cells contained numerous high proliferative potential-colony forming cells (HPP-CFCs), and presented an 80-fold expansion of colony-forming unit-neutrophil/macrophage/Erythroblast/Megakaryocyte (CFU-nmEM) compared to freshly isolated CD34−KSL cells. This effect of Bmi-1 was comparable to that of HoxB4, a well-known HSC activator. In contrast, forced expression of M33 reduced proliferative activity and caused accelerated differentiation into macrophages, leaving no HPP-CFCs after 14 days of ex vivo culture. To determine the mechanism that leads to the drastic expansion of CFU-nmEM, we employed a paired daughter cell assay to see if overexpression of Bmi-1 promotes symmetric HSC division in vitro. Forced expression of Bmi-1 significantly promoted symmetrical cell division of daughter cells, suggesting that Bmi-1 contributes to CFU-nmEM expansion by promoting self-renewal of HSCs. Furthermore, we performed competitive repopulation assays using transduced HSCs cultured ex vivo for 10 days. After 3 months, Bmi-1-transduced HSCs manifested a 35-fold higher repopulation unit (RU) compared with GFP controls and retained full differentiation capacity along myeloid and lymphoid lineages. As expected from in vitro data, HSCs transduced with M33 did not contribute to repopulation at all. In ex vivo culture, expression of both p16INK4a and p19ARF were up-regulated. p16INK4aand p19ARF are known target genes negatively regulated by Bmi-1, and were completely repressed by transducing HSCs with Bmi-1. Therefore, we next examined the involvement of p19ARF in HSC regulation by Bmi-1 using p19ARF-deficient and Bmi-1 and p19ARF-doubly deficient mice. Although bone marrow repopulating activity of p19ARF-deficient HSCs was comparable to that of wild type HSCs, loss of p19ARF expression partially rescued the defective hematopoietic phenotypes of Bmi-1-deficient mice. In addition, transduction of Bmi-1 into p19ARF-deficient HSCs again enhanced repopulating capacity compared with p19ARF-deficient GFP control cells, indicating the existence of additional targets for Bmi-1 in HSCs. Our findings suggest that the level of Bmi-1 is a critical determinant for self-renewal of HSC and demonstrate that Bmi-1 is a novel target for therapeutic manipulation of HSCs.

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Disruption of Tet2 Leads to Enhanced Self-Renewal and Competitive Repopulating Capacity of Fetal Liver Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v118.21.2331.2331 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2331-2331

Author(s):

Hiroyoshi Kunimoto ◽

Yumi Fukuchi ◽

Masatoshi Sakurai ◽

Ken Sadahira ◽

Yasuo Ikeda ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Gene Trap ◽

Wild Type ◽

Self Renewal ◽

Hematopoietic Stem ◽

Myeloid Malignancies ◽

Serial Transplantation

Abstract Abstract 2331 TET2 (ten-eleven-translocation 2) gene has been reported to be frequently mutated in various human myeloid malignancies, including myeloproliferative neoplasms, myelodysplastic syndromes, acute myeloid leukemia, and chronic myelomonocytic leukemia. These observations suggest critical roles of TET2 dysfunction in molecular pathogenesis of myeloid malignancies. Recent studies using conditional knockout mouse model indicated that mouse Tet2 loss leads to clonal dominance of adult hematopoietic stem cells (HSCs) in competitive repopulation assay. However, self-renewal capacity of adult HSCs has never been addressed precisely by serial transplantation assay. In addition, the effect of Tet2 loss on hematopoietic stem/ progenitor cells was examined only in the BM, but not in the fetal livers (FLs). Since FL HSCs and adult HSCs differ in several aspects of their phenotypes and functions, we speculated that Tet2 might be involved differently in the regulation of FL and adult hematopoiesis. To address this issue, we analyzed E14.5 FL cells from Tet2 gene-trap (Tet2gt) mice. In these mice, gene trap-cassette was inserted into the second intron, just before the first coding exon. RT-PCR analysis showed that over 99% of Tet2 mRNAs from endogenous promoter were trapped by the gene-trap cassette in Tet2gt/gt mice, showing that Tet2gt allele can be considered as a null allele. Initial analysis showed that Tet2gt/gt embryos developed normally, but most Tet2gt/gt mice were perinatally lethal. Total numbers of FL cells and the numbers of committed progenitors in FLs as revealed by colony assays were not significantly different between each genotype. Interestingly, Tet2gt/gt embryos displayed significant increase in lineage (Lin)(-)Sca-1(+)c-Kit(+)(LSK) fraction compared to wild type (WT) (Tet2+/+) littermate (2.42±0.66% vs. 1.17±0.18%, p=0.02). In addition, common myeloid progenitor (CMP) fraction (IL7Rα(-), Lin(-), Sca-1(-), C-Kit(+), CD34(+), FcgRII/ III(low)) was significantly increased in Tet2gt/gt FLs compared to WT (9.04±1.09% vs. 6.26±0.53%, p=0.008). In serial transplantation assays, donor cells derived from Tet2+/gt and Tet2gt/gt FLs showed significantly higher peripheral blood chimerism in secondary and tertiary recipient mice as compared to that of WT cells, showing that disruption of Tet2 leads to the enhanced self-renewal capacity of FL HSCs. Moreover, donor-derived HSC fraction (CD34−LSK cells) was significantly expanded in the recipients of Tet2gt/gt FL cells, suggesting that increased self-renewal capacity is cell intrinsic to Tet2gt/gt HSCs. We have also examined differentiation of Tet2-mutant FL cells in the recipients' peripheral blood, and found that Tet2gt/gt cells displayed impaired differentiation to Gr-1(+)CD11b(+) mature granulocytes (WT vs. Tet2gt/gt = 5.02±1.35% vs. 11.5±3.09% in the primary recipients) and slight, but significant increase of B cells. Liquid culture of FL cells with cocktails of cytokines in vitro demonstrated that Tet2gt/gt FL cells retained higher percentage and number of LSK, Lin- and c-Kit+ cells after the culture for 7-days compared to WT cells, showing enhanced resistance of Tet2gt/gt cells to differentiative stimuli in in vitro culture. It is of note that Tet2+/gt mice showed a significant increase in hematopoietic stem/progenitor fraction (LSK) in the BM compared to wild type littermate (0.48±0.11% vs. 0.32±0.04%, p=0.04). However, they presented no signs of extramedullary hematopoiesis such as splenomegaly and expansion of LSK cells in spleens during an observation up to 35-weeks. Taken together, we demonstrate that Tet2 critically regulates self-renewal and long-term repopulating capacity of FL HSCs and has pleiotropic functions in myeloid and lymphoid differentiation. These data strongly indicate that Tet2 is an essential regulator of BM and FL hematopoiesis. In addition, enhanced HSC self-renewal, expansion of HPC and myeloid progenitors and perturbed myeloid differentiation induced by TET2 ablation likely to set molecular basis for myeloid transformation, which explains high incidence of loss-of-function mutations of TET2 in myeloid malignancies. Disclosures: No relevant conflicts of interest to declare.

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