Differentiation Profiling of Marrow Stem Cells: A Megakaryopoietic Hotspot and the Continuum Model of Hematopoiesis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4217-4217
Author(s):  
Gerald A. Colvin ◽  
Dooner Gerri ◽  
Delia Demers ◽  
Shiela Pascual ◽  
Samuel Chung ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Stem Cells ◽  
S Phase ◽  
Platelet Glycoprotein ◽  
No Production ◽  
Colony Stimulating Factor ◽  
Hematopoietic Stem ◽  
Steel Factor ◽  
Stimulating Factor

Abstract Hierarchical models of hematopoiesis suppose an ordered system in which stem cells and progenitors with specific fixed differentiation potentials exist. We show here that the potential of marrow stem cells to differentiate changes reversibly with cytokine-induced cell cycle transit. This along with other data strongly suggest that stem cell regulation is not based on the classic hierarchical model, but instead more on a functional continuum We have previously shown that hematopoietic stem cells reversibly shift their engraftment phenotype with cytokine induced cell cycle transit. Further work has shown that adhesion protein, cytokine receptor, gene expression and progenitor phenotypes also shift. Evolving data indicate the phenotype of murine marrow stem cells reversible change with cell cycle transit. Murine experiments have been performed on highly purified, quiescent G0-1 lineagenegativerhodaminelowHoeschtlow (LRH) marrow stem cells. When exposed to thrombopoietin, FLT3-ligand and steel factor, they synchronously pass through cell cycle as measured by propidium iodide, cell doublings and tritiated thymidine. LRH cells enter S-phase in a synchronized fashion by 18 hours, leave S-phase at 40–42 hours and divide between 44–48 hours. The capacity of these cells to respond to a differentiation inductive signal (granulocyte colony-stimulating factor, granulocyte-macrophage colony stimulating factor and steel factor) is altered at different points in cell cycle. Megakaryocyte production is specifically focused at early to mid S-phase, this returned to baseline before the first cell division. Population based cultures after 14-days of differentiation culture produced up to 49% megakaryocytes with stem cells sub-cultured during early-mid S-phase with little to no production with colonies cultured from stem cells in G0-1 or G2 phase at time of differentiation induction signaling. Cell type was confirmed by staining cells with acetylcholinesterase, antibodies to platelet glycoprotein complex IIb/IIIa and von Willebrand’s factor. Evaluation of gene expression at this hotspot showed a marked increase in expression of CD4 with up to 464.2 fold increase above baseline. Sca-1 and transcriptional factor FOG was strikingly amplified at S-phase as well as other relevant markers. While pertinent cytokine receptors were not increased, studies on a clonal level confirm the existence of a reversible megakaryocytic hotspot. Compared with other time-points relating to cell cycle position prior to differentiation sub-culture in one experiment, 33% of clonally derived colonies that grew from early S-phase cells and 10% of colonies that grew from mid S-phase cells had megakaryocytes present two weeks after initiation of culture compared with 0% for G0-1 and G2 cells. Granulocyte differentiation also showed specific differentiation hotspots, but presentation is outside the scope of this abstract. These data indicate that marrow hematopoiesis stem cells exist in a continuum, not in a hierarchy with continuously changing windows of transcriptional opportunity.

Differentiation Profiling of Marrow Stem Cells: A Megakaryocytic Hotspot and the Continuum Model of Hematopoiesis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4776-4776
Author(s):  
Gerald A. Colvin ◽  
Liansheng Liu ◽  
Mark Dooner ◽  
Gerri Dooner ◽  
Kevin Johnson ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Stem Cell ◽  
Stem Cell Differentiation ◽  
S Phase ◽  
Differentiation Potential ◽  
Steel Factor ◽  
A Cell ◽  
Stimulating Factor

Abstract Directed differentiation is defined as the ability to program a stem cell at the most primitive level while it still has its reproductive and full proliferative potential in contrast to ex-vivo expansion where the stem cells are forced into specific lineage commitments, limiting the overall therapeutic utility. Standard hierarchical models of hematopoiesis suppose an ordered system in which stem cells and progenitors with specific fixed differentiation potentials exist. We show here that the potential of marrow stem cells to differentiate changes reversibly with cytokine-induced cell cycle transit. This along with other data strongly suggest that stem cell regulation is not based on the classic hierarchical model, but instead more on a functional continuum and believe that sensitivity to cytokines change as a stem/progenitor cells goes through cell cycle transit. We previously have shown that stem cells reversibly shift their engraftment phenotype with cytokine induced cell cycle transit. Further work has shown that adhesion protein, cytokine receptor, gene expression and progenitor phenotypes also shift. Evolving data indicate the phenotype of murine marrow stem cells reversible change with cell cycle transit. Murine experiments have been performed on highly purified quiescent G0–1 lineagenegativerhodaminelowHoeschtlow (LRH) marrow stem cells. When exposed to thrombopoietin, FLT3-ligand and steel factor, they synchronously pass through cell cycle as measured by propidium iodide, cell doublings and tritiated thymidine. LRH cells enter S-phase in a synchronized fashion by 18 hours, leave S-phase at 40–42 hours and divide between 44–48 hours. The capacity of these cells to respond to a differentiation inductive signal (granulocyte colony-stimulating factor, granulocyte-macrophage colony stimulating factor and steel factor) is altered at different points in cell cycle. We have demonstrated differentiation hotspots on a cell cycle continuum (Exp Heme35:96, 2007). In this work we showed marked but reversible increases in differentiation potential to megakryocyte and granulocytes at different phases of a single cytokine induced cell cycle passage of highly purified quiescent murine LRH marrow stem cells. We have reproducibly induced directed stem cell differentiation by capitalizing on inherent changes in sensitivities to inductive cytokine signals in the context of cell cycle position. We have found that using a differentiation cytokine cocktail of G-CSF at 0.075ng/ml, GM-CSF at 0.0375ng/ml and steel factor at 50ng/ml, we were able to see enhanced megakaryopoiesis occurring 14-days after culture in those LRH stem cells that were in early to mid S-phase at time of inductive signaling. We have now shown that a megakaryocyte hotspot clusters around giving an inductive signal after 32-hours in primary culture; the G1/S interface, and that dramatic reversible changes in differentiation potential occur over half hour time intervals. We have confirmed this data by looking at LRH cells through cell cycle transit after initial cell division showing that a megakaryocyte hotspot occurs in two sequential cell cycles and still tied to S-phase at time of inductive signaling of the daughter cells. This hotspot has been demonstrated on a clonal basis, although the kinetics of the hotspot shifts when clonal as opposed to population studies are carried out. An important issue is whether in vitro cytokine exposure, separate from cell cycle status, determines the existence of the hotspot. To address this, we used Hoechst 33342 dye content to assist in separation of different cell cycle fractions (G0–1, early, mid and late components of S, G2/M) of lineage negative Sca-1+ stem cells, a cycling stem/progenitor cell population in which approximately 20% of the cells are in S-phase at isolation. These cells were only exposed to the differentiation cytokines and showed a megakaryocyte hotspot present in only early S-phase cells after 14-days of culture, showing that in vitro cell cycle phase determined the presence of the hotspot, separate from cytokine exposure. These data indicate that differentiation potential of marrow stem cells exists on a cell cycle related continuum and that this potential can be demonstrated on a single cell basis. Stem cell differentiation hotspots may eventually be utilized to alter repopulation kinetics after bone marrow transplantation improving recovery time of platelets and neutrophils, translating into improved outcomes.

Cyclophosphamide/granulocyte colony-stimulating factor causes selective mobilization of bone marrow hematopoietic stem cells into the blood after M phase of the cell cycle

Blood ◽  
2001 ◽  
Vol 97 (8) ◽  
pp. 2278-2285 ◽  
Author(s):  
Douglas E. Wright ◽  
Samuel H. Cheshier ◽  
Amy J. Wagers ◽  
Troy D. Randall ◽  
Julie L. Christensen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Messenger Rna ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Hematopoietic Stem ◽  
M Phase ◽  
Stimulating Factor

Abstract Cytokine-mobilized peripheral blood hematopoietic stem cells (MPB HSC) are widely used for transplantation in the treatment of malignancies, but the mechanism of HSC mobilization is unclear. Although many HSC in bone marrow (BM) cycle rapidly and expand their numbers in response to cytoreductive agents, such as cyclophosphamide (CY), and cytokines, such as granulocyte colony-stimulating factor (G-CSF), MPB HSC are almost all in the G0 or G1phase of the cell cycle. This has raised the question of whether a subset of noncycling BM HSC is selectively released, or whether cycling BM HSC are mobilized after M phase, but before the next S phase of the cell cycle. To distinguish between these possibilities, mice were treated with one dose of CY followed by daily doses of G-CSF, and dividing cells were marked by administration of bromodeoxyuridine (BrdU) during the interval that BM HSC are expanding. After CY and 4 days of G-CSF, 98.5% of the 2n DNA content long-term repopulating MPB (LT)-HSC stained positively for BrdU, and therefore derived from cells that divided during the treatment interval. Next, LT-HSC from mice previously treated with a single dose of CY, which kills cycling cells, and 3 daily doses of G-CSF, were nearly all killed by a second dose of CY, suggesting that CY/G-CSF causes virtually all LT-HSC to cycle. Analysis of cyclin D2 messenger RNA (mRNA) expression and total RNA content of MPB HSC suggests that these cells are mostly in G1 phase. After CY/G-CSF treatment, virtually all BM LT-HSC enter the cell cycle; some of these HSC then migrate into the blood, specifically after M phase, and are rapidly recruited to particular hematopoietic organs.

scholarly journals Investigating the Possibility of Intervertebral Disc Regeneration Induced by Granulocyte Colony Stimulating Factor-Stimulated Stem Cells in Rats

2011 ◽  
Vol 2011 ◽  
pp. 1-5 ◽  
Author(s):  
Wen-Ching Tzaan ◽  
Hsien-Chih Chen
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Intervertebral Disc ◽  
Bone Marrow Stem Cells ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Hematopoietic Stem ◽  
Disc Regeneration ◽  
Intervertebral Disc Regeneration ◽  
Stimulating Factor

Intervertebral disc (IVD) degeneration is a multifactorial process that is influenced by contributions from genetic predisposition, the aging phenomenon, lifestyle conditions, biomechanical loading and activities, and other health factors (such as diabetes). Attempts to decelerate disc degeneration using various techniques have been reported. However, to date, there has been no proven technique effective for broad clinical application. Granulocyte colony-stimulating factor (GCSF) is a growth factor cytokine that has been shown to enhance the availability of circulating hematopoietic stem cells to the brain and heart as well as their capacity for mobilization of mesenchymal bone marrow stem cells. GCSF also exerts significant increases in circulating neutrophils as well as potent anti-inflammatory effects. In our study, we hypothesize that GCSF can induce bone marrow stem cells differentiation and mobilization to regenerate the degenerated IVD. We found that GCSF had no contribution in disc regeneration or maintenance; however, there were cell proliferation within end plates. The effects of GCSF treatment on end plates might deserve further investigation.

scholarly journals Essential Role for Cyclin D3 in Granulocyte Colony-Stimulating Factor-Driven Expansion of Neutrophil Granulocytes

2006 ◽  
Vol 26 (21) ◽  
pp. 8052-8060 ◽  
Author(s):  
Ewa Sicinska ◽  
Young-Mi Lee ◽  
Judith Gits ◽  
Hirokazu Shigematsu ◽  
Qunyan Yu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Cyclin D3 ◽  
Neutrophil Granulocytes ◽  
Neutrophil Granulocyte ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Hematopoietic Stem ◽  
Stimulating Factor

ABSTRACT The proliferation of neutrophil granulocyte lineage is driven largely by granulocyte colony-stimulating factor (G-CSF) acting via the G-CSF receptors. In this study, we show that mice lacking cyclin D3, a component of the core cell cycle machinery, are refractory to stimulation by the G-CSF. Consequently, cyclin D3-null mice display deficient maturation of granulocytes in the bone marrow and have reduced levels of neutrophil granulocytes in their peripheral blood. The mutant mice are unable to mount a normal response to bacterial challenge and succumb to microbial infections. In contrast, the expansion of hematopoietic stem cells and lineage-committed myeloid progenitors proceeds relatively normally in mice lacking cyclin D3, revealing that the requirement for cyclin D3 function operates at later stages of neutrophil development. Importantly, we verified that this requirement is specific to cyclin D3, as mice lacking other G1 cyclins (D1, D2, E1, or E2) display normal granulocyte counts. Our analyses revealed that in the bone marrow cells of wild-type mice, activation of the G-CSF receptor leads to upregulation of cyclin D3. Collectively, these results demonstrate that cyclin D3 is an essential cell cycle recipient of G-CSF signaling, and they provide a molecular link of how G-CSF-dependent signaling triggers cell proliferation.

scholarly journals Basic fibroblast growth factor mediates its effects on committed myeloid progenitors by direct action and has no effect on hematopoietic stem cells

Blood ◽  
1995 ◽  
Vol 86 (6) ◽  
pp. 2123-2129 ◽  
Author(s):  
AC Berardi ◽  
A Wang ◽  
J Abraham ◽  
DT Scadden
Keyword(s):  
Stem Cells ◽  
Growth Factor ◽  
Hematopoietic Stem Cells ◽  
Fibroblast Growth Factor ◽  
Colony Formation ◽  
Fibroblast Growth ◽  
Colony Stimulating Factor ◽  
Hematopoietic Stem ◽  
Myeloid Progenitor ◽  
Stimulating Factor

Basic fibroblast growth factor or fibroblast growth factor-2 (FGF) has been shown to affect myeloid cell proliferation and hypothesized to stimulate primitive hematopoietic cells. We sought to evaluate the effect of FGF on hematopoietic stem cells and to determine if FGF mediated its effects on progenitor cells directly or through the induction of other cytokines. To address the direct effects of FGF, we investigated whether FGF induced production of interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha, IL-6, granulocyte colony- stimulating factor, or granulocyte-macrophage colony-stimulating factor by two types of accessory cells, bone marrow (BM) fibroblasts and macrophages. We further evaluated whether antibodies to FGF-induced cytokines affected colony formation. To determine if FGF was capable of stimulating multipotent progenitors, we assessed the output of different colony types after stimulation of BM mononuclear cells (BMMC) or CD34+ BMMC and compared the effects of FGF with the stem cell active cytokine, kit ligand (KL). In addition, a subset of CD34+ BMMC with characteristics of hematopoietic stem cells was isolated by functional selection and their response to FGF was evaluated using proliferation, colony-forming, and single-cell polymerase chain reaction (PCR) assays. We determined that FGF had a stimulatory effect on the production of a single cytokine, IL-6, but that the effects of FGF on colony formation were not attributable to that induction. FGF was more restricted in its in vitro effects on BM progenitors than KL was, having no effect on erythroid colony formation. FGF did not stimulate stem cells and FGF receptors were not detected on stem cells as evaluated by single-cell reverse transcription PCR. In contrast, FGF receptor gene expression was detected in myeloid progenitor populations. These data support a directly mediated effect for FGF that appears to be restricted to lineage-committed myeloid progenitor cells. FGF does not appear to modulate the human hematopoietic stem cell.

scholarly journals Inhibition of day-12 spleen colony-forming units by a monoclonal antibody to the murine macrophage/monocyte colony-stimulating factor receptor

Blood ◽  
1995 ◽  
Vol 85 (10) ◽  
pp. 2731-2734 ◽  
Author(s):  
GL Gilmore ◽  
RK Shadduck
Keyword(s):  
Stem Cells ◽  
Growth Factor ◽  
Hematopoietic Stem Cells ◽  
Growth Factor Receptor ◽  
Hematopoietic Growth Factor ◽  
Colony Stimulating Factor ◽  
Hematopoietic Stem ◽  
Colony Forming Units ◽  
Stimulating Factor ◽  
Specific Control

Primitive hematopoietic stem cells differentiate into committed progenitors that are thought to selectively express hematopoietic growth factor receptor(s), thereby acquiring hematopoietic growth factor responsiveness. To assess whether hematopoietic stem cells express hematopoietic growth factor receptors, the progenitor activity of bone marrow (BM) fractions, isolated by expression of receptors for macrophage/monocyte colony-stimulating factor (M-CSF), were examined. Recovery of day-12 spleen colony-forming units (CFU-S) is diminished in both M-CSF receptor-positive (M-CSFR+) and M-CSFR-fractions, indicating antibody inhibition of day-12 CFU-S. Incubation of BM cells with antibody without fractionation inhibits 50% to 60% of day-12 CFU-S. This inhibition is specific (control antibodies have no effect) and reversible by removal of bound antibody at low pH. Incubating BM cells with control or antireceptor antibody does not affect day-8 CFU-S, which are predominantly erythroid. Treating sublethally irradiated mice with antibody inhibits endogenous day-12 CFU-S. These results indicate that some early progenitors express M-CSFRs, and blocking M-CSFRs inhibits the ability of these progenitors to form colonies, possibly because of inactivation caused by prolonged receptor blockade.

scholarly journals Efficient retrovirus transduction of mouse pluripotent hematopoietic stem cells mobilized into the peripheral blood by treatment with granulocyte colony-stimulating factor and stem cell factor

Blood ◽  
1994 ◽  
Vol 84 (5) ◽  
pp. 1482-1491 ◽  
Author(s):  
DM Bodine ◽  
NE Seidel ◽  
MS Gale ◽  
AW Nienhuis ◽  
D Orlic
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Stem Cell Factor ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Hematopoietic Stem ◽  
Stimulating Factor

Abstract Cytokine-mobilized peripheral blood cells have been shown to participate in hematopoietic recovery after bone marrow (BM) transplantation, and are proposed to be useful targets for retrovirus- mediated gene transfer protocols. We treated mice with granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) to mobilize hematopoietic progenitor cells into the peripheral blood. These cells were analyzed for the number and frequency of pluripotent hematopoietic stem cells (PHSC). We found that splenectomized animals treated for 5 days with G-CSF and SCF showed a threefold increase in the absolute number of PHSC over normal mice. The number of peripheral- blood PHSC increased 250-fold from 29 per untreated mouse to 7,200 in peripheral-blood PHSC in splenectomized animals treated for 5 days with G-CSF and SCF. Peripheral blood PHSC mobilized by treatment with G-CSF and SCF were analyzed for their ability to be transduced by retroviral vectors. Peripheral-blood PHSC from splenectomized animals G-CSF and SCF were transduced with a recombinant retrovirus containing the human MDR-1 gene. The frequency of gene transfer into peripheral blood PHSC from animals treated for 5 and 7 days was two-fold and threefold higher than gene transfer into PHSC from the BM of 5-fluorouracil-treated mice (P < .01). We conclude that peripheral blood stem cells mobilized by treatment with G-CSF and SCF are excellent targets for retrovirus- mediated gene transfer.

scholarly journals Sequencing of RNA in single cells reveals a distinct transcriptome signature of hematopoiesis in GATA2 deficiency

2020 ◽  
Vol 4 (12) ◽  
pp. 2702-2716
Author(s):  
Zhijie Wu ◽  
Shouguo Gao ◽  
Carrie Diamond ◽  
Sachiko Kajigaya ◽  
Jinguo Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Stem Cells ◽  
Chromosomal Abnormalities ◽  
Single Cells ◽  
Trisomy 8 ◽  
Broad Perspective ◽  
Hematopoietic Stem ◽  
Gata2 Deficiency ◽  
Additional Chromosomal Abnormalities

Abstract Constitutional GATA2 deficiency caused by heterozygous germline GATA2 mutations has a broad spectrum of clinical phenotypes, including systemic infections, lymphedema, cytopenias, and myeloid neoplasms. Genotype–phenotype correlation is not well understood mechanistically in GATA2 deficiency. We performed whole transcriptome sequencing of single hematopoietic stem and progenitor cells from 8 patients, who had pathogenic GATA2 mutations and myelodysplasia. Mapping patients’ cells onto normal hematopoiesis, we observed deficiency in lymphoid/myeloid progenitors, also evident from highly constrained gene correlations. HSPCs of patients exhibited distinct patterns of gene expression and coexpression compared with counterparts from healthy donors. Distinct lineages showed differently altered transcriptional profiles. Stem cells in patients had dysregulated gene expression related to apoptosis, cell cycle, and quiescence; increased expression of erythroid/megakaryocytic priming genes; and decreased lymphoid priming genes. The prominent deficiency in lympho-myeloid lineages in GATA2 deficiency appeared at least partly due to the expression of aberrant gene programs in stem cells prior to lineage commitment. We computationally imputed cells with chromosomal abnormalities and determined their gene expression; DNA repair genes were downregulated in trisomy 8 cells, potentially rendering these cells vulnerable to second-hit somatic mutations and additional chromosomal abnormalities. Cells with complex cytogenetic abnormalities showed defects in genes related to multilineage differentiation and cell cycle. Single-cell RNA sequencing is powerful in resolving transcriptomes of cell subpopulations despite a paucity of cells in marrow failure. Our study discloses previously uncharacterized transcriptome signatures of stem cells and progenitors in GATA2 deficiency, providing a broad perspective of potential mechanisms by which germline mutations modulate early hematopoiesis in a human disease. This trial was registered at www.clinicaltrials.gov as NCT01905826, NCT01861106, and NCT00001620.

MicroRNA-29a Is An Oncogene in Acute Myeloid Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 683-683
Author(s):  
Christopher Y. Park ◽  
Yoon-Chi Han ◽  
Govind Bhagat ◽  
Jian-Bing Fan ◽  
Irving L Weissman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Hematopoietic Stem Cells ◽  
Mirna Expression ◽  
Myeloid Leukemia ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Abstract microRNAs (miRNAs) are short, non-protein encoding RNAs that bind to the 3′UTR’s of target mRNAs and negatively regulate gene expression by facilitating mRNA degradation or translational inhibition. Aberrant miRNA expression is well-documented in both solid and hematopoietic malignancies, and a number of recent miRNA profiling studies have identified miRNAs associated with specific human acute myeloid leukemia (AML) cytogenetic groups as well as miRNAs that may prognosticate clinical outcomes in AML patients. Unfortunately, these studies do not directly address the functional role of miRNAs in AML. In fact, there is no direct functional evidence that miRNAs are required for AML development or maintenance. Herein, we report on our recent efforts to elucidate the role of miRNAs in AML stem cells. miRNA expression profiling of AML stem cells and their normal counterparts, hematopoietic stem cells (HSC) and committed progenitors, reveals that miR-29a is highly expressed in human hematopoietic stem cells (HSC) and human AML relative to normal committed progenitors. Ectopic expression of miR-29a in mouse HSC/progenitors is sufficient to induce a myeloproliferative disorder (MPD) that progresses to AML. During the MPD phase of the disease, miR-29a alters the composition of committed myeloid progenitors, significantly expedites cell cycle progression, and promotes proliferation of hematopoietic progenitors at the level of the multipotent progenitor (MPP). These changes are manifested pathologically by marked granulocytic and megakaryocytic hyperplasia with hepatosplenomegaly. Mice with miR-29a-induced MPD uniformly progress to an AML that contains a leukemia stem cell (LSC) population that can serially transplant disease with as few as 20 purified LSC. Gene expression analysis reveals multiple tumor suppressors and cell cycle regulators downregulated in miR-29a expressing cells compared to wild type. We have demonstrated that one of these genes, Hbp1, is a bona fide miR-29a target, but knockdown of Hbp1 in vivo does not recapitulate the miR-29a phenotype. These data indicate that additional genes are required for miR-29a’s leukemogenic activity. In summary, our data demonstrate that miR-29a regulates early events in normal hematopoiesis and promotes myeloid differentiation and expansion. Moreover, they establish that misexpression of a single miRNA is sufficient to drive leukemogenesis, suggesting that therapeutic targeting of miRNAs may be an effective means of treating myeloid leukemias.

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