Twist-1 Increases Hematopoietic Stem Cell Self-Renewal and Causes Myeloid Skewing

Abstract Twist-1 protein belongs to the large family of basic Helix-Loop-Helix transcription factors with diverse physiological functions in mesoderm-derived tissues. Growing evidences now link Twist-1 to the acquisition of stem-cell-like properties. However, there is little information available regarding its expression pattern and functional role in the hematopoietic system. We analyzed Twist-1 expression patterns in different hematopoietic cell populations from normal mouse bone marrow, and found that Twist-1 was most highly expressed in long-term hematopoietic stem cells (LT-HSCs) but showed a low abundance in more differentiated descendants. To investigate Twist-1 gene function, retroviral-mediated overexpression or removal experiments were performed. Competitive repopulation studies demonstrated that enforced expression of Twist-1 in HSC-enriched Lin−c-Kit+Sca-1+ (LKS) cells resulted in an increase in the size of the G0 population, and in their reconstitution ability after the first and a second transplantation. Conversely, removal of Twist-1 in LKS cells impaired their ability to repopulate. In addition, increased Twist-1 expression caused a shift toward production of myeloid cells. Twist-1 transduction in LKS cells activated myeloid lineage-determining factors PU.1 and GATA-1 and down-regulated lymphoid factor GATA-3 in vitro, suggesting that Twist-1-mediated myeloid skewing occurs in hematopoietic stem and progenitor cells (HSPCs). These findings indicate that Twist-1 is not only involved in the maintenance of HSC dormancy and self-renewal capacity but also implicated in the myeloid lineage fate choice of HSPCs. Exploration of the underlying mechanisms revealed that Twist-1 overexpression lead to altered expression of Runx1/c-Mpl/Tie2 regulatory pathway, and quiescence-associated N-cadherin and Hes1 in LKS cells, which may contribute to the phenotypes observed in Twist-1-overexpressing mice. These studies shed additional light on the mechanisms involved in the maintenance of normal HSC and myeloid lineage differentiation, and may also provide clues to the mechanisms controlling pathogenesis and preservation of leukemic stem cells. Disclosures No relevant conflicts of interest to declare.

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Growth Factor Independence 1b (Gfi1b) Regulates The Commitment, Differentiation and Expansion Of Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v122.21.2433.2433 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2433-2433

Author(s):

Tarik Moroy ◽

Cyrus Khandanpour ◽

Joseph Krongold

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Mouse Bone Marrow ◽

Paracrine Factors ◽

Self Renewal ◽

Hematopoietic Stem

Abstract The efficacy of bone marrow stem cell transplantation is the therapy of choice for many hematopoietic diseases, in particular leukemia and lymphoma. This therapy is critically dependent on the transfer of sufficient numbers of hematopoietic stem cells (HSCs), which possess the capacity for self-renewal and can fully reconstitute the hematopoietic system. As such, the development of techniques for the expansion of fully functional HSCs is of significant clinical interest. By transiently manipulating the factors that govern HSC homeostasis it has been proposed that HSCs can be expanded without the loss of essential stem cell characteristics. Previously we have observed that ablation of the gene encoding the transcription factor Gfi1b in-vivo results in a dramatic expansion and mobilization of hematopoietic stem cells in the bone marrow and periphery. More recent data suggest that the blood mobilization of Gfi1b deficient HSCs is very likely mediated by a deregulation of the integrin expression. These data led us to hypothesize that Gfi1b could be a potential target for ex-vivo treatment and expansion of HSCs. Indeed, when deletion of Gfi1b was induced in whole bone marrow ex-vivo, HSCs showed a significant expansion in both in absolute number and in terms of proportion of bone marrow. We followed HSCs in ex-vivo expansion cultures from mouse bone marrow by tracking expression of the surface marker CD48, which indicates whether an HSC has transitioned to a differentiation committed multi-potent progenitor. We observed that Gfi1b null HSCs expanded without up-regulating CD48 in contrast to wt HSCs. This suggests that Gf11b deficient HSCs underwent symmetric self-renewal type cell divisions at a significantly increased frequency, when compared to wt HSCs. We had previously shown that HSCs lacking Gfi1b cycle at a faster rate than control HSCs. The combination of increased cell division and preferential self-renewal of Gfi1b-/- HSCs indicates that inhibition of Gfi1b may be the ideal strategy for ex-vivo HSC expansion. As well, in accordance with this preference for self-renewal, Gfi1b null HSCs that were cultured under myeloid differentiation conditions remained primarily in an undifferentiated state as defined by a lack of the myeloid surface markers Gr1 and Mac1. These cultures also demonstrated increased long term colony forming capacity versus controls, further supporting an undifferentiated phenotype in Gfi1b-/- cells. Because the stem cell niche is a highly complex and heterogeneous environment we also investigated whether bone marrow in which Gfi1b has been deleted exerts paracrine effects that contributed to HSC expansion. Co-Culture assays demonstrated that Gfi1b-/- bone marrow was able to induce an expansion of progenitors in wild-type bone marrow of more than 10 fold compared to Gfi1b-/+ bone marrow. Interestingly cells co-cultured with Gfi1b null bone marrow also exhibited an overall proliferation advantage after short-term cultures. This suggests that not only does Gfi1b deletion induce HSC expansion via cell intrinsic mechanisms, but also points to the possibility that this occurs through paracrine factors that alter bone marrow homeostasis. Disclosures: No relevant conflicts of interest to declare.

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MiR-150 Suppresses MLL-AF9 Associated Leukemia Through Simultaneously Targeting Multiple Oncogenes,

Blood ◽

10.1182/blood.v118.21.3461.3461 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3461-3461

Author(s):

Beiyan Zhou

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Target Genes ◽

Conflicts Of Interest ◽

Expression Patterns ◽

Gene Profiling ◽

Fusion Genes ◽

Leukemic Stem Cells ◽

Hematopoietic Stem ◽

Mll Gene

Abstract Abstract 3461 The mixed lineage leukemia (MLL) gene codes for an evolutionarily conserved histone methyltransferase that is crucial for early hematopoiesis. As a result of a chromosomal translocation involving locus 11q23 results in formation of chimeras composed of the 5' part of the MLL gene fused with more than 60 partner genes lead to disruption of normal function of MLL as a histone methytransferase and acquisition of transcriptional properties conferred by the partner genes. MLL fusion genes (MLL-FG) are often the causal mutations for aggressive acute myeloid and lymphoid leukemias (AML and ALL) that correlated with poor prognosis. In order to treat or even eliminate MLL-associated leukemias, extensive studies on the regulatory mechanism underlying MLL associated transformation and progression have been carried out. Leukemic stem cells (LSC) can derive from either hematopoietic stem or progenitor cells with the recruitment of MLL-fusion genes (MLL-FG) and wild type MLL protein. We report that miR-150, a key hematopoietic regulatory microRNA (miRNA) and one of the most downregulated miRNAs in MLL-associated leukemias, acts as a tumor suppressor to block the leukemogenic potency of leukemic stem cells. When expression of miR-150 was restored, a significantly suppressed leukemic stem cell potency of MLL-AF9 cells was observed both in vivo and in vitro. Gene profiling analysis demonstrated that elevated miR-150 altered various aspects of gene expression patterns in MLL-AF9 cells, including stem cell signatures, cancer pathways, and cell survival. By screening more than 30 predicted target genes, we identified multiple leukemia-associated oncogenes as bona fide miR-150 targets, and knockdown of these genes by shRNAs recapitulated the tumor suppressive effects observed after ectopically expression of miR-150 in MLL-AF9 cells. Disclosures: No relevant conflicts of interest to declare.

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MiR-150 Inhibits MLL-AF9 Associated Leukemia By Suppressing Leukemic Stem Cells

Blood ◽

10.1182/blood.v122.21.3764.3764 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3764-3764

Author(s):

Patali S Cheruku ◽

Marina Bousquet ◽

Guoqing Zhang ◽

Guangtao Ge ◽

Wei Ying ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Progenitor Cells ◽

Colony Formation ◽

Conflicts Of Interest ◽

Expression Patterns ◽

Gene Profiling ◽

Leukemic Stem Cells ◽

Hematopoietic Stem ◽

Signature Genes

Abstract Leukemic stem cells (LSCs) are derived from hematopoietic stem or progenitor cells and often share gene expression patterns and specific pathways. Characterization and mechanistic studies of LSCs are critical as they are responsible for the initiation and potential relapse of leukemias, however the overall framework, including epigenetic regulation, is not yet clear. We previously identified microRNA-150 (miR-150) as a critical regulator of mixed lineage leukemia (MLL) -associated leukemias by targeting oncogenes. Our additional results suggest that miR-150 can inhibit LSC survival and disease initiating capacity by suppressing more than 30% of “stem cell signature genes,” hence altering multiple cancer pathways and/or stem cell identities. MLL-AF9 cells derived from miR-150 deficient hematopoietic stem/progenitor cells displayed significant proliferating advantage and enhanced leukemic colony formation. Whereas, with ectopic miR-150 expression, the MLL-AF9 associated LSC population (defined as Lin-ckit+sca1- cells) was significantly decreased in culture. This is further confirmed by decreased blast leukemic colony formation in vitro. Furthermore, restoration of miR-150 levels in transformed MLL-AF9 cells, which often display loss of miR-150 expression in AML patients with MLL-fusion protein expressing, completely blocked the myeloid leukemia development in a transplantation mouse model. Gene profiling analysis demonstrated that an increased level of miR-150 expression down regulates 30 of 114 stem cell signature genes by more than 1.5 fold, partially mediated by the suppressive effects of miR-150 on CBL, c-Myb and Egr2 oncogenes. In conclusion, our results suggest that miR-150 is a potent MLL-AF9 leukemic inhibitor that may act by suppressing the survival and leukemic initiating potency of MLL-AF9 LSCs. Disclosures: No relevant conflicts of interest to declare.

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Roles of Ring1A/B on Stem Cell Potential of MOZ and Other Acute Myeloid Leukemias,

Blood ◽

10.1182/blood.v118.21.3998.3998 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3998-3998

Author(s):

Haruko Shima ◽

Mika Shino ◽

Kazutsune Yamagata ◽

Yukiko Aikawa ◽

Haruhiko Koseki ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Differentiation ◽

Conflicts Of Interest ◽

Expression Profiles ◽

Leukemia Stem Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cell Functions ◽

Acute Myeloid

Abstract Abstract 3998 Leukemia and other cancers possess self-renewing stem cells that help maintain cancer. Chromosomal translocations are often involved in the development of human acute myeloid leukemia (AML). The monocytic leukemia zinc finger (MOZ) gene is one of the targets of such translocations. While MOZ is essential for the self-renewal of hematopoietic stem cells, the leukemia associated MOZ-fusion proteins enable the transformation of non–self-renewing myeloid progenitors into leukemia stem cells. Ring1A and Ring1B are catalytic subunits of the polycomb-group repressive complex 1 (PRC1) complex containing Bmi1, and PRC1 complex plays an important role in the regulation of stem cell self-renewal. Using Ring1A-null and Ring1B-conditional deficient mice, we showed that Ring1A/B are required for continuous colony forming ability that is enabled by MOZ-TIF2 and other AML-associated fusions such as MLL-AF10, AML1-ETO, and PML-RARα. Furthermore, MOZ-TIF2- and MLL-AF10-induced AML development in mice were prevented by Ring 1A/B deficiency. To clarify the mechanism of stemness regulation in AML stem cells by PRC1 complex, we compared gene expression profiles of Ring1A/B deleted and non-deleted MOZ-TIF2-induced AML cells. As expected, Ink4a/Arf, a known major target of PRC1 complex involved in stem cell functions, was derepressed by deletion of Ring1A/B. Although deletion of Ink4a/Arf in Ring1A/B deficient AML cells partially restored colony formation ability, it was not substantial to initiate leukemia in recipient mice. Among several target genes which were derepressed by deletion of Ring1A/B, we focused on “Stemness inhibitory factor (SIF)”, known to be required for cell differentiation and morphogenesis in some specific organs. Enforced expression of SIF in MOZ-TIF2-induced AML cells stimulated differentiation of AML progenitors into macrophages. On the other hand, knock-down of SIF blocked cell differentiation block and restored the immortalizing ability of MOZ-TIF2-induced AML progenitors, despite of the absence of Ring1A/B. Collectively, our data demonstrate that Ring 1A/B play crucial roles in the maintenance of AML stem cells through repression of SIF, which strongly promote differentiation of leukemia stem cells. Disclosures: No relevant conflicts of interest to declare.

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The Mechanism By Which Mutant Nucleophosmin (NPM1) Creates Leukemic Self-Renewal Is Readily Reversed

Blood ◽

10.1182/blood.v128.22.444.444 ◽

2016 ◽

Vol 128 (22) ◽

pp. 444-444 ◽

Cited By ~ 3

Author(s):

Xiaorong Gu ◽

Reda Z. Mahfouz ◽

Quteba Ebrahem ◽

Francis Enane ◽

Tomas Radivoyevitch ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hox Genes ◽

Cell Model ◽

Expression Patterns ◽

Monocytic Differentiation ◽

Self Renewal ◽

Produce Cell ◽

Hematopoietic Stem ◽

Renewal Program

Abstract Acute myeloid leukemia (AML) is self-renewal by immature myeloid precursors that fail to differentiate. An influential 'leukemia stem cell' model thus proposes that leukemogenic proteins augment or introduce a stem cell-like self-renewal program into cells, e.g., by upregulating signaling or transcription factors (TF) emblematic of stem cells (e.g., HOX). We investigated how the most recurrently mutated protein in AML, mutant nucleophosmin (mNPM1), causes leukemic cell expansion. The results challenge this model, but most importantly, open the door to rational targeted therapy for mNPM1 AML. One way of examining for stem cell programs in AML cells is to look at expression patterns of master TF that regulate expression of hundreds of genes and dictate cell fates. Of these select TF, the master TF that create hematopoietic stem cells (HLF etc.) are minimally or not expressed. Instead, there are very high levels of the master TF that drive monocyte and granulocyte lineage fates, PU.1 (SPI1) and CEBPA. Clearly, however, the lineage-programs intended by PU.1/CEBPA are inefficiently executed if at all - mNPM1 AML patient bone marrows had 85-97% cells with a granulocyte-monocyte progenitor phenotype, accumulated at the expense of downstream mature cells (Quek et al, JEM 2016). This aggregation at a lineage-committed, intermediate, naturally proliferative level of the hematopoietic hierarchy suggests an alternative model - instead of introducing a poorly-defined stem cell self-renewal program, mutant proteins disable differentiation programs which usually quench MYC-driven proliferation intrinsic to lineage-progenitors. To better understand how mNPM1 interacts with cellular machinery, we used mass-spectrometry to comprehensively document the protein interactions of endogenous NPM1 in AML cell nuclear and cytoplasmic fractions, the first analysis of this kind. Notably, the NPM1 protein interactome was enriched for PU.1. Critically, wild-type (wt) NPM1/PU.1 was in the nucleus of wtNPM1 AML cells, but mNPM1/PU.1 was in the cytoplasm of mNPM1 AML cells. This was evident clearly also by Western blot of cell fractions and by IF microscopy of primary AML cells and cell lines. Is cytoplasmic dis-location of PU.1 sufficient to explain persistent hematopoietic precursor proliferation? We used murine Pu.1 knock-out hematopoietic precursors transduced to express Pu.1 fused with the estrogen receptor (Pu.1-ER) to answer this question - Pu.1 relocation from the cytoplasm to the nucleus by tamoxifen triggered monocytic differentiation that terminated proliferation. Moreover, Pu.1-ER cells, like mNPM1 AML cells, highly express Hox genes, rapidly suppressed upon Pu.1 relocation to the nucleus. Thus, Pu.1 dominantly controls Hox and proliferation, as befitting of a master TF, and persistent HOX expression, like persistent progenitor proliferation, can be caused by Pu.1 loss-of-function. Protein macromolecules like NPM1 require transport factors to exit (exportins) the nucleus. A specific exportin, XPO1, was the major exportin found in the NPM1 interactome. XPO1 interactions with transported cargo can be inhibited by the small molecule drug KPT330. KPT330 10-20 nM rapidly re-located mNPM1 and PU.1 to the nucleus, downregulated MYC, upregulated p27/CDKN1B, upregulated monocyte but not granulocyte differentiation markers, induced morphologic changes of monocyte differentiation, and terminated proliferation of mNPM1 AML cells. The same low nanomolar treatment did not induce differentiation of wtNPM1 AML cells (THP1). Moreover, these KPT330 levels are not toxic to normal hematopoiesis (also shown by others). Thus, rather than gain-of-function of elusive stem cell-like self-renewal, the most frequently mutated protein in AML creates self-renewal by disabling a differentiation program that quenches intrinsic MYC-driven proliferation of lineage-progenitors. These observations are a mechanistic rationale to select refractory/relapsed mNPM1 AML patients for treatment with low well-tolerated doses of KPT330, with a defined molecular pharmacodynamic objective of returning PU.1 to the nucleus, to produce cell cycle exits by differentiation rather than p53-mediated apoptosis (to address chemotherapy resistance), to spare precious normal HSC (good therapeutic index), and directly reverse the basis for leukemic self-renewal (proliferation without differentiation). Figure. Figure. Disclosures Landesman: Karyopharm Therapeutics Inc: Employment, Other: stockholder. Saunthararajah:EpiDestiny: Consultancy, Other: patents around decitabine and tetrahydrouridine.

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Gpx3 Determines Competitiveness of Normal and Leukemic Stem Cells.

Blood ◽

10.1182/blood.v116.21.1587.1587 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1587-1587 ◽

Cited By ~ 1

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.

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The Role of Mel18 Overlaps with BMI1 In the Self-Renewal of Human Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v116.21.2619.2619 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2619-2619

Author(s):

Yasmin Reyal ◽

Dominique Bonnet

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cord Blood ◽

Conflicts Of Interest ◽

Expression Patterns ◽

Polycomb Group ◽

Self Renewal ◽

Hematopoietic Stem ◽

Starting Point

Abstract Abstract 2619 Complex mechanisms regulate the ability of hematopoietic stem cells (HSCs) to self-renew, some of which may be exploited by leukemic stem cells. BMI1, a member of the polycomb group (PcG) proteins is known to be a positive regulator of this process, largely by repressing the p16/INK4a locus. However the role of other PcG proteins is unclear. We initially screened HSCs and progenitor populations from umbilical cord blood (CB), for the expression patterns of a number of PcG genes. Levels of expression were heterogeneous, indicating that there may be different roles for different PcG in HSCs versus progenitors. As a starting point we have focused on Mel18 (PCGF2) as it has been suggested in murine hematopoiesis that it acts to counteract BMI1. Lineage negative CB cells were transduced with lentiviral vectors expressing shRNA against Mel18, BMI1 and a control sequence. Specific knockdown by these constructs was confirmed at the RNA level to be at least 80% for both genes and was verified at the protein level by Western blot. Our data indicates that knockdown of Mel18 impairs the proliferation of primitive cord blood cells in both stromal-dependent and -independent culture, in a similar manner to BMI1. Furthermore Mel18 deficiency impedes both primary and secondary colony formation of all myeloid lineages in methylcellulose. These findings have been confirmed in vivo with significant reduction in engraftment of CB lineage negative cells in NOD/SCID mice at twelve weeks. We are investigating whether over expression of Mel18 can rescue BMI1 deficient cells to establish if these homologous genes have redundant functions. The data so far suggests that BMI1 is not unique in its role in HSC self - renewal, and Mel18 may share overlapping functions. This highlights a possible difference between human and murine hematopoiesis. Moreover it is likely that other members of the PcG family are also important in human HSCs. It will be of interest to investigate whether like BMI1, they are also implicated in the maintenance of the leukemic stem cell. Disclosures: No relevant conflicts of interest to declare.

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Vascular Pericytes Sustain Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v118.21.2394.2394 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2394-2394 ◽

Cited By ~ 2

Author(s):

Mirko Corselli ◽

Chintan Parekh ◽

Elisa Giovanna Angela Montelatici ◽

Arineh Sahghian ◽

Wenyuan Wang ◽

...

Keyword(s):

Stem Cells ◽

Adipose Tissue ◽

Stem Cell ◽

Stromal Cells ◽

Conflicts Of Interest ◽

Cell Interactions ◽

Lymphoid Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Abstract 2394 Mesenchymal stromal (or stem-) cells (MSC) are culture-selected, heterogeneous supporting cells that can differentially regulate hematopoietic stem cell (HSC) behavior in vitro. The elusive identity of native MSC has obscured the contribution, if any, of these cells to HSC support in vivo. Having previously demonstrated that vascular pericytes (ubiquitous cells encircling endothelial cells in capillaries and microvessels) are ancestors of human MSC, we now hypothesize that pericytes are a critical component of the HSC “niche”. Consequently, pericyte isolation from total stroma would allow to develop co-culture systems for human HSC maintenance. In the present study, human cord blood CD34+ cells were cultured onto confluent human pericytes isolated from adipose tissue as CD146+CD34-CD45-CD56- cells. Co-culture of CD34+ cells on pericytes, for up to 6 weeks in the absence of any added growth factor, produced significantly i) higher numbers of CD45+ and CD34+ cells (p<0.05), ii) higher percentages of primitive CD34+CD33-CD10-CD19- progenitors (p<0.05), iii) higher percentages of single- and multi-lineage CFU (p<0.05) and iv) lower percentages of mature myeloid and lymphoid cells (p<0.05), compared to control co-cultures on unfractionated adipose stromal cells (ASC) (n=10 individual experiments, n=4 biological replicates). Most importantly, only pericytes could maintain HSC with self-renewal and long-term repopulating potential, as demonstrated by transplantation into primary and secondary NOD/SCID/IL2Rg−/− mouse recipients (n=3 individual experiments). In the latter setting, none of the mice receiving CD34+ cells co-cultured with ASC engrafted (n=10), whereas all recipients of CD34+ cells cultured in the presence of pericytes developed lympho-myeloid hematopoietic human cells (n=10). Altogether, these results support the hypothesis that pericytes maintain hematopoietic cell stemness. Conversely, unfractionated stromal cell cultures may promote HSC differentiation at the expense of self-renewal. Both tentative scenarios were explored. Co-cultures with pericytes in a transwell system revealed that cell-to-cell contact is required for HSC survival. Since Notch signaling regulates stem cell maintenance by inhibiting cell differentiation through cell-cell interactions, we hypothesized that pericytes purified from total stroma express specific Notch ligands. As shown by qPCR, the expression of Jagged-1 is 2 fold higher in pericytes compared to unfractionated ASC. Addition of a Notch inhibitor (DAPT) to pericyte/HSC co-cultures resulted in the significant reduction of CFU numbers (p<0.05) and increase in B-cell development. Furthermore, increased myeloid differentiation was observed when ASC conditioned medium was added to pericytes/HSC co-cultures. In conclusion, we demonstrate that vascular pericytes sustain HSC by promoting survival and preventing differentiation via cell-to-cell interactions involving Notch activation, whereas unfractionated stroma promotes HSC differentiation through a paracrine mechanism. We thus infer that HSC-supporting stromal cells are not confined within blood-forming organs (similar observations, not reported here, have been made on skeletal muscle pericytes). This novel concept is not easy to reconcile with normal hematopoiesis, but may be highly relevant in the context of the dissemination of malignant hematopoietic cells. Of important note, adipose tissue used in this study represents a convenient, safe and often abundant source of autologous therapeutic cells. Therefore, human fat-derived pericytes emerge as a candidate cell product for HSC ex vivo manipulation in the clinic. Disclosures: No relevant conflicts of interest to declare.

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Stem Cell Programs Are Retained In Human Leukemic Lymphoblasts With Distinct Immunophenotypes

Blood ◽

10.1182/blood.v122.21.4995.4995 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4995-4995

Author(s):

Dengli Hong

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Conflicts Of Interest ◽

Molecular Model ◽

Normal Population ◽

Lymphoblastic Leukemia ◽

Bioinformatic Analysis ◽

Self Renewal ◽

Hematopoietic Stem ◽

Renewal Program

Abstract Leukemic lymphoblasts within different immunophenotypic populations possess stem cell Property. It remains largely unknown however whether self-renewal Program is retained from stem cells or endowed to Progenitors by leukemogenic molecules. We have addressed this issue in the context of TEL-AML1-associated acute lymphoblastic leukemia (ALL) by profiling a refined Program edited from genes essential for self-renewal of hematopoietic stem cells and B-cell development. Bioinformatic analysis shows that ALL populations are loosely clustered and all most close to the normal population that contains early lymphoid Progenitors, indicating that immunophenotypes do not reflect maturation stages in ALL and that self-renewal Program might be retained from stem cells. Results of assessing “first hit” function of TEL-AML1 in different populations of normal cells demonstrate the molecular model. Therefore, our studies reveal a leukemogenic scenario of human ALL that programs of stem cells are sustained in distinct fractions by leukemogenic mutations. Disclosures: No relevant conflicts of interest to declare.

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