In Vivo Pre-Instructed HSCs Robustly Execute Asymmetric Cell Divisions In Vitro

Hematopoietic stem cells (HSCs) are responsible for life-long production of all mature blood cells. Under homeostasis, HSCs in their native bone marrow niches are believed to undergo asymmetric cell divisions (ACDs), with one daughter cell maintaining HSC identity and the other committing to differentiate into various mature blood cell types. Due to the lack of key niche signals, in vitro HSCs differentiate rapidly, making it challenging to capture and study ACD. To overcome this bottleneck, in this study, we used interferon alpha (IFNα) treatment to ”pre-instruct” HSC fate directly in their native niche, and then systematically studied the fate of dividing HSCs in vitro at the single cell level via time-lapse analysis, as well as multigene and protein expression analysis. Triggering HSCs’ exit from dormancy via IFNα was found to significantly increase the frequency of asynchronous divisions in paired daughter cells (PDCs). Using single-cell gene expression analyses, we identified 12 asymmetrically expressed genes in PDCs. Subsequent immunocytochemistry analysis showed that at least three of the candidates, i.e., Glut1, JAM3 and HK2, were asymmetrically distributed in PDCs. Functional validation of these observations by colony formation assays highlighted the implication of asymmetric distribution of these markers as hallmarks of HSCs, for example, to reliably discriminate committed and self-renewing daughter cells in dividing HSCs. Our data provided evidence for the importance of in vivo instructions in guiding HSC fate, especially ACD, and shed light on putative molecular players involved in this process. Understanding the mechanisms of cell fate decision making should enable the development of improved HSC expansion protocols for therapeutic applications.

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Early megakaryocyte lineage-committed progenitors in adult mouse bone marrow

10.1101/2021.08.06.455158 ◽

2021 ◽

Author(s):

Zixian Liu ◽

Jinhong Wang ◽

Miner Xie ◽

Peng Wu ◽

Yao Ma ◽

...

Keyword(s):

Bone Marrow ◽

Single Cell ◽

Pcr Analysis ◽

Mouse Bone Marrow ◽

Hematopoietic Stem ◽

Colony Assay ◽

Megakaryocyte Progenitors ◽

Cell Colony

Hematopoietic stem cells (HSCs) have been considered to progressively lose their self-renewal and differentiation potentials prior to the commitment to each blood lineage. However, recent studies have suggested that megakaryocyte progenitors are generated at the level of HSCs. In this study, we newly identified early megakaryocyte lineage-committed progenitors (MgPs) in CD201-CD48- cells and CD48+ cells separated from the CD150+CD34-Kit+Sca-1+Lin- HSC population of the bone marrow in C57BL/6 mice. Single-cell transplantation and single-cell colony assay showed that MgPs, unlike platelet-biased HSCs, had little repopulating potential in vivo, but formed larger megakaryocyte colonies in vitro (on average eight megakaryocytes per colony) than did previously reported megakaryocyte progenitors (MkPs). Single-cell RNA-sequencing supported that these MgPs lie between HSCs and MkPs along the megakaryocyte differentiation pathway. Single-cell colony assay and single-cell RT-PCR analysis suggested the coexpression of CD41 and Pf4 is associated with megakaryocyte colony-forming activity. Single-cell colony assay of a small number of cells generated from single HSCs in culture suggested that MgPs are not direct progeny of HSCs. In this study, we propose a differentiation model in which HSCs give rise to MkPs through MgPs.

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Interleukin-3 supports expansion of long-term multilineage repopulating activity after multiple stem cell divisions in vitro

Blood ◽

10.1182/blood.v96.5.1748 ◽

2000 ◽

Vol 96 (5) ◽

pp. 1748-1755 ◽

Cited By ~ 90

Author(s):

David Bryder ◽

Sten E. W. Jacobsen

Keyword(s):

Bone Marrow Cells ◽

Ex Vivo ◽

Calf Serum ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cell Divisions ◽

Stem Cell Divisions

Abstract Although long-term repopulating hematopoietic stem cells (HSC) can self-renew and expand extensively in vivo, most efforts at expanding HSC in vitro have proved unsuccessful and have frequently resulted in compromised rather than improved HSC grafts. This has triggered the search for the optimal combination of cytokines for HSC expansion. Through such studies, c-kit ligand (KL), flt3 ligand (FL), thrombopoietin, and IL-11 have emerged as likely positive regulators of HSC self-renewal. In contrast, numerous studies have implicated a unique and potent negative regulatory role of IL-3, suggesting perhaps distinct regulation of HSC fate by different cytokines. However, the interpretations of these findings are complicated by the fact that different cytokines might target distinct subpopulations within the HSC compartment and by the lack of evidence for HSC undergoing self-renewal. Here, in the presence of KL+FL+megakaryocyte growth and development factor (MGDF), which recruits virtually all Lin−Sca-1+kit+ bone marrow cells into proliferation and promotes their self-renewal under serum-free conditions, IL-3 and IL-11 revealed an indistinguishable ability to further enhance proliferation. Surprisingly, and similar to IL-11, IL-3 supported KL+FL+MGDF-induced expansion of multilineage, long-term reconstituting activity in primary and secondary recipients. Furthermore, high-resolution cell division tracking demonstrated that all HSC underwent a minimum of 5 cell divisions, suggesting that long-term repopulating HSC are not compromised by IL-3 stimulation after multiple cell divisions. In striking contrast, the ex vivo expansion of murine HSC in fetal calf serum-containing medium resulted in extensive loss of reconstituting activity, an effect further facilitated by the presence of IL-3.

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Combined Single Cell Lineage and Transcriptome Sequencing Unveils Cell-Autonomous Regulators of Hematopoietic Stem Cell Fate

Blood ◽

10.1182/blood-2019-123447 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 446-446

Author(s):

Alejo E Rodriguez-Fraticelli ◽

Caleb S Weinreb ◽

Allon Moshe Klein ◽

Shou-Wen Wang ◽

Fernando D Camargo

Keyword(s):

Single Cell ◽

Cell Fate ◽

Conflicts Of Interest ◽

Large Fraction ◽

Cell Lineage ◽

Gene Signature ◽

Hematopoietic Stem ◽

Cell State

Blood regeneration upon transplantation relies on the activity of long-term repopulating hematopoietic stem cells (LT-HSCs). One of the major controversies in hematopoiesis relates to the apparently different properties that HSCs have in transplantation versus unperturbed settings. In unperturbed steady state hematopoiesis, the most potent HSCs appear to be mostly dormant, and only producing platelet-lineage cells. In turn, upon transplant, even a single transplanted HSC can actively divide and regenerate hundreds of millions of blood progenitors of all lineages. It would thus appear that HSCs have different fundamental properties in each study system. However, most transplantation studies have only tracked the lineage output of the transplanted HSC clones, and rarely the regeneration of the HSC compartment itself. In addition, clonal assays have not been performed at sufficient resolution to fully capture the diversity and clonal complexity of the regenerated HSC compartment. Here, we have used expressible barcodes, which can be sequenced in conventional single cell RNAseq assays, to simultaneously record the functional outcomes and transcriptional states of thousands of HSCs. Our analysis revealed multiple clonal HSC behaviors following transplantation that drastically differ in their differentiation activity, lineage-bias and self-renewal. Surprisingly, we witnessed a large fraction of clones that efficiently repopulate the HSC compartment but show limited contribution to differentiated progeny. Furthermore, these inactive clones have increased competitive multilineage serial repopulating capacity, implying that shortly after transplant a subset of clones reestablishes the native-like LT-HSC behaviors. Our results also argue that this clonal distribution of labor is controlled by cell autonomous, heritable properties (i.e. the epigenetic cell state). Then, using only our clonal readouts to segregate single HSC transcriptomes, we unveiled the transcriptional signatures that associated with unique HSC outcomes (platelet bias, clonal expansion, dormancy, etc.) and unraveled, for the first time, a gene signature for functional long-term serially repopulating clones. We interrogated the drivers of this cell state using an in vivo inducible CRISPR screening and identified 5 novel regulators that are required to regenerate the HSC compartment in a cell autonomous fashion. In conclusion, we demonstrate that functional LT-HSCs share more similar properties in native and transplantation hematopoiesis than previously expected. Consequently, we unveil a definition of the essential, common functional properties of HSCs and the molecular programs that control them. Figure 1 Disclosures No relevant conflicts of interest to declare.

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Runx1 Is Required for Notch-Induced Specification of Megakaryocyte Development from Early Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood.v112.11.1370.1370 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1370-1370

Author(s):

Melanie G Cornejo ◽

Thomas Mercher ◽

Joseph D. Growney ◽

Jonathan Jesneck ◽

Ivan Maillard ◽

...

Keyword(s):

Notch Signaling ◽

Cell Fate ◽

Stromal Cells ◽

Gfp Expression ◽

Hematopoietic Stem ◽

Cell Fate Decisions ◽

Insight Into ◽

Megakaryocyte Differentiation

Abstract The Notch signaling pathway is involved in a broad spectrum of cell fate decisions during development, and in the hematopoietic system, it is known to favor T cell- vs B cell lineage commitment. However, its role in myeloid lineage development is less well understood. We have shown, using heterotypic co-cultures of murine primary hematopoietic stem cells (Lin-Sca-1+ckit+ HSCs) and OP9 stromal cells expressing the Notch ligand Delta1 (OP9-DL1), that Notch signaling derived from cell non-autonomous cues acts as a positive regulator of megakaryocyte fate from LSK cells. Bone marrow transplantation experiments with a constitutively active Notch mutant resulted in enhanced megakaryopoiesis in vivo, with increased MEP numbers and megakaryocyte colony formation. In contrast, expression of dnMAML using a conditional ROSA26 knock-in mouse model significantly impaired megakaryopoiesis in vivo, with a marked decrease in megakaryocyte progenitors. In order to understand the cellular differentiation pathways controlled by Notch, we first examined the ability of various purified progenitor populations to differentiate toward megakaryocytes upon Notch stimulation in vitro. We observed that CMP and MEP, but not GMP, can engage megakaryopoiesis upon Notch stimulation. Our results were consistent with expression analysis of Notch signaling genes in these purified progenitors and were supported by the observation that transgenic Notch reporter mice display higher levels of reporter (i.e. GFP) expression in HSC and MEP, vs. CMP and GMP in vivo. Furthermore, purified progenitors with high GFP expression gave rise to increased numbers of megakarocyte-containing colonies when plated in vitro compared to GFP-negative progenitors. In addition, further purification of the HSC population into long-term (LT), short-term (ST), and lymphoid-primed myeloid progenitors (LMPP) before plating on OP9-DL1 stroma showed that LMPP have a reduced ability to give rise to megakaryocytes compared to the other two populations. These data support the hypothesis that there is an early commitment to erythro/megakaryocytic fate from HSC prior to lymphoid commitment. To gain insight into the molecular mechanism underlying Notch-induced megakaryopoiesis, we performed global gene expression analysis that demonstrated the engagement of a megakaryopoietic transcriptional program when HSC were co-cultured with OP9-DL1 vs. OP9 stroma or OP9-DL1 treated with gamma-secretase inhibitor. Of interest, Runx1 was among the most upregulated genes in HSC co-cultured on OP9-DL1 stroma. To assess whether Notch signaling engages megakaryocytic fate through induction of Runx1, we plated HSC from Runx1 −/− mice on OP9-DL1 stroma. Compared to WT cells, Runx1 −/− HSC had a severely reduced ability to develop into CD41+ cells. In contrast, overexpression of Runx1 in WT HSC was sufficient to induce megakaryocyte fate on OP9 stroma without Notch stimulation. Together, our results indicate that Notch pathway activation induced by stromal cells is an important regulator of cell fate decisions in early progenitors. We show that Notch signaling is upstream of Runx1 during Notch-induced megakaryocyte differentiation and that Runx1 is an essential target of Notch signaling. We believe that these results provide important insight into the pathways controlling megakaryocyte differentiation, and may have important therapeutic potential for megakaryocyte lineage-related disorders.

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Interleukin-3 supports expansion of long-term multilineage repopulating activity after multiple stem cell divisions in vitro

Blood ◽

10.1182/blood.v96.5.1748.h8001748_1748_1755 ◽

2000 ◽

Vol 96 (5) ◽

pp. 1748-1755 ◽

Cited By ~ 5

Author(s):

David Bryder ◽

Sten E. W. Jacobsen

Keyword(s):

Bone Marrow Cells ◽

Ex Vivo ◽

Calf Serum ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cell Divisions ◽

Stem Cell Divisions

Although long-term repopulating hematopoietic stem cells (HSC) can self-renew and expand extensively in vivo, most efforts at expanding HSC in vitro have proved unsuccessful and have frequently resulted in compromised rather than improved HSC grafts. This has triggered the search for the optimal combination of cytokines for HSC expansion. Through such studies, c-kit ligand (KL), flt3 ligand (FL), thrombopoietin, and IL-11 have emerged as likely positive regulators of HSC self-renewal. In contrast, numerous studies have implicated a unique and potent negative regulatory role of IL-3, suggesting perhaps distinct regulation of HSC fate by different cytokines. However, the interpretations of these findings are complicated by the fact that different cytokines might target distinct subpopulations within the HSC compartment and by the lack of evidence for HSC undergoing self-renewal. Here, in the presence of KL+FL+megakaryocyte growth and development factor (MGDF), which recruits virtually all Lin−Sca-1+kit+ bone marrow cells into proliferation and promotes their self-renewal under serum-free conditions, IL-3 and IL-11 revealed an indistinguishable ability to further enhance proliferation. Surprisingly, and similar to IL-11, IL-3 supported KL+FL+MGDF-induced expansion of multilineage, long-term reconstituting activity in primary and secondary recipients. Furthermore, high-resolution cell division tracking demonstrated that all HSC underwent a minimum of 5 cell divisions, suggesting that long-term repopulating HSC are not compromised by IL-3 stimulation after multiple cell divisions. In striking contrast, the ex vivo expansion of murine HSC in fetal calf serum-containing medium resulted in extensive loss of reconstituting activity, an effect further facilitated by the presence of IL-3.

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Stromal Expression of Jagged 1 Promotes Colony Formation by Fetal Hematopoietic Progenitor Cells

Blood ◽

10.1182/blood.v92.5.1505 ◽

1998 ◽

Vol 92 (5) ◽

pp. 1505-1511 ◽

Cited By ~ 121

Author(s):

Philip Jones ◽

Gill May ◽

Lyn Healy ◽

John Brown ◽

Gerald Hoyne ◽

...

Keyword(s):

Progenitor Cells ◽

Cell Fate ◽

Stromal Cell ◽

Fetal Liver ◽

Culture Conditions ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem

Abstract The Notch signaling system regulates proliferation and differentiation in many tissues. Notch is a transmembrane receptor activated by ligands expressed on adjacent cells. Hematopoietic stem cells and early progenitors express Notch, making the stromal cells which form cell-cell contacts with progenitor cells candidate ligand-presenting cells in the hematopoietic microenvironment. Therefore, we examined primary stromal cell cultures for expression of Notch ligands. Using reverse transcription-polymerase chain reaction, in situ hybridization, immunohistochemistry, and Western blotting, we demonstrate expression of Jagged 1 in primary stromal cultures. To investigate if the stromal expression of Jagged 1 has functional effects on hematopoietic progenitors, we cultured CD34+, c-kit+ hematopoietic progenitor cells derived from the aorto gonadal mesonephros region of day 11 mouse embryos on the Jagged 1− stromal cell line S17 and on S17 cells engineered to express Jagged 1. The presence of Jagged 1 increased the number of colonies formed in subsequent methylcellulose culture fourfold. Larger increases in colony numbers were observed under the same culture conditions with CD34+, c-kit+ hematopoietic progenitor cells derived from d11 fetal liver. These results obtained in vitro table Jagged 1 as a candidate regulator of stem cell fate in the context of stromal microenvironments in vivo. © 1998 by The American Society of Hematology.

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Analysis of cardiac differentiation at single cell resolution reveals a requirement of hypertrophic signaling for HOPX transcription

10.1101/229294 ◽

2017 ◽

Author(s):

Clayton E Friedman ◽

Quan Nguyen ◽

Samuel W Lukowski ◽

Han Sheng Chiu ◽

Abbigail Helfer ◽

...

Keyword(s):

Stem Cells ◽

Single Cell ◽

Cell Fate ◽

Heart Development ◽

Time Course ◽

Cardiac Differentiation ◽

Transcriptional Networks ◽

Hypertrophic Signaling

AbstractDifferentiation into diverse cell lineages requires the orchestration of gene regulatory networks guiding diverse cell fate choices. Utilizing human pluripotent stem cells, we measured expression dynamics of 17,718 genes from 43,168 cells across five time points over a thirty day time-course of in vitro cardiac-directed differentiation. Unsupervised clustering and lineage prediction algorithms were used to map fate choices and transcriptional networks underlying cardiac differentiation. We leveraged this resource to identify strategies for controlling in vitro differentiation as it occurs in vivo. HOPX, a non-DNA binding homeodomain protein essential for heart development in vivo was identified as dys-regulated in in vitro derived cardiomyocytes. Utilizing genetic gain and loss of function approaches, we dissect the transcriptional complexity of the HOPX locus and identify the requirement of hypertrophic signaling for HOPX transcription in hPSC-derived cardiomyocytes. This work provides a single cell dissection of the transcriptional landscape of cardiac differentiation for broad applications of stem cells in cardiovascular biology.

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Ex-Vivo Expansion of Marrow Repopulating Cells by Chromatin Modifying Agents Is Associated with Modulation of Expression of Genes Implicated in HSC Self-Renewal.

Blood ◽

10.1182/blood.v108.11.1670.1670 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1670-1670

Author(s):

Hiroto Araki ◽

Kazumi Yoshinaga ◽

Ronald Hoffman ◽

Piernicola Boccuni ◽

Nadim Mahmud

Keyword(s):

Ex Vivo ◽

Trichostatin A ◽

Donor Cell ◽

Characteristic Functional ◽

Self Renewal ◽

Hematopoietic Stem ◽

Immunodeficient Mice ◽

Cell Divisions

Abstract Human hematopoietic stem cells (HSCs) exposed to cytokine combinations in vitro rapidly divide and lose their characteristic functional properties presumably due to the alteration of a genetic program which determines the properties of HSC. In order to expand the number of HSC present in a single unit of cord blood (CB) ex vivo, self-renewal type of HSC division must occur. We hypothesize that in vitro culture conditions result in the silencing of genes crucial for HSC maintenance and that silencing of these genes can be circumvented by addition of chromatin modifying agents. We have attempted to reverse the silencing of the genes crucial for HSC self-renewal which apparently occurs during the ex vivo culture by treatment of CD34+ cells with the chromatin modifying agents, 5-aza-2-deoxycytidine (5azaD) and trichostatin A (TSA). In our current studies, we have investigated the mechanism of expansion of SRC following treatment with chromatin modifying agents in the culture. We demonstrate that all CD34+CD90+ cells treated with 5azaD/TSA and cytokines after 9 days of incubation divide, but to a lesser degree than cells exposed to cytokines alone. CD34+CD90+ cells exposed to the chromatin modifying agents are capable of producing greater numbers of primitive multipotential progenitors and also form cobblestone areas. When CD34+CD90+ cells that had undergone extensive number of cell divisions (5–10) in vitro in the presence of cytokines alone were re-isolated by FACS and transplanted into immunodeficient mice, donor cell chimerism was not detectable (0 of 5 mice). By contrast, 5azaD/TSA treated cells that had undergone similar numbers of cell divisions retain their marrow repopulating potential (3 of 6 mice). To test whether chromatin modifying agents treated cells following culture possess long-term in vivo repopulation potential, we have performed secondary NOD/SCID assay. Five of six secondary NOD/SCID mice receiving bone marrow from primary mice engrafted with cells treated with 5azaD/TSA resulted in human cell engraftment, indicating that these cells are capable of secondary reconstitution. To understand the molecular mechanism responsible for the expansion of HSC observed following 5azaD/TSA treatment, we examined transcription levels of several genes and their products (i.e., HOXB4, Bmi-1 and P21) implicated in self-renewal of HSC using real-time quantitative PCR and Western blot. The expression of these genes and their products were up-regulated in CB cells treated with 5azaD/TSA. We have also compared the efficacy of an additional HDAC inhibitor valproic acid (VPA) in order to determine its ability to expand HSC ex vivo. VPA was capable of dramatic expansion of CD34+CD90+ cells as well as progenitor cells but was unable to expand SRC. However, unlike the culture exposed to cytokines alone VPA treatment resulted in maintenance of SRC numbers. Currently, we are investigating key candidate genes accountable for the expansion of SRC using a global microarray approach analyzing cells exposed to various chromatin modifying agents in conjunction with their in vivo functional potential. In summary, our data suggest that the loss of SRC can be circumvented by the use of chromatin modifying agents in the culture which results in a slower rate of cell division and is associated with higher expression of a group of HSC regulatory genes.

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Functional analysis of the Human Pbx Interacting Protein (HPIP) in Human Hematopoiesis

Blood ◽

10.1182/blood.v112.11.2430.2430 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2430-2430

Author(s):

Pawandeep Kaur ◽

Christiane Stadler ◽

Farid Ahmed ◽

Monica Cusan ◽

R. Keith Humphries ◽

...

Keyword(s):

Cord Blood ◽

Cell Fate ◽

Constitutive Expression ◽

Homeodomain Protein ◽

Acute Lymphocytic Leukaemia ◽

Human Cord Blood ◽

Interacting Protein ◽

Hematopoietic Stem

Abstract The recognition of novel proteins that regulate human hematopoietic stem cell and early progenitor cell fate is a prime objective in experimental and clinical hematology. Human hematopoietic PBX interacting protein (HPIP), with no significant homology to known proteins, is a 731 amino acid protein, discovered as a novel interacting partner of the PBX homeodomain protein. HPIP has been implicated as a nuclear-cytoplasmic shuttle molecule and shown to have the capacity to bind to the cytoskeleton. It also inhibits the ability of PBX-HOX heterodimers to bind to target sequences and strongly inhibits the transactivation activity of E2A-PBX1 [t(1;19) translocation, which occurs in 25% of pediatric pre-B cell acute lymphocytic leukaemia] (Abramovich C. et al JBC, 2000; Oncogene, 2002). It is highly expressed in human CD34+ progenitor cells, but is silenced in differentiated cells. To gain further insights into the possible functional role of HPIP and its domains and its possible role in a common pathway with HOX transcription co-factor PBX1, HPIP cDNA was cloned in pMSCV-IRES-YFP cassette. Umbilical cord blood enriched with CD34+ population of stem cells was obtained to perform in vitro and in vivo experiments. Mutants, with deletions of the microtubule binding region (ΔMBR-HPIP), and nuclear receptor and PBX1 interacting motif (ΔNRPID-HPIP) were generated and tested in vitro and in vivo. The constitutive expression of HPIP wt and ΔMBR-HPIP in human cord blood cells (CD34+) enhanced erythroid colony formation in CFC assay (p=0.008, n=6) while the ΔNRPID-HPIP mutant nullified the effect. Both mutants of HPIP augmented significantly, the formation of primitive colonies (GEMM and GM) in methylcellulose assay (p≤0.01, n=6) as compared to YFP control and HPIP wt. In replating CFC assays ΔNRPID-HPIP showed an increased number of myeloid colonies (p≤0.01, n=6) and GM (p=ns) colonies but a decrease in granulocytic colonies (p≤0.05, n=6) compared to YFP control and HPIP wt

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An RNA Interference Screen Identifies the Cell Fate Determinants Msi2 and Prox1 as Novel Regulators of Hematopoietic Stem Cell Self-Renewal.

Blood ◽

10.1182/blood.v114.22.394.394 ◽

2009 ◽

Vol 114 (22) ◽

pp. 394-394

Author(s):

Kristin J Hope ◽

Sonia Cellot ◽

Stephen Ting ◽

Guy Sauvageau

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Fate ◽

Self Renewal ◽

Hematopoietic Stem ◽

Control Shrna ◽

Fate Determinants ◽

Cell Fate Determinants

Abstract Abstract 394 Hematopoietic stem cells (HSC) can not yet be unambiguously prospectively identified, a fact which has made it difficult to determine whether a segregation of cell fate determinants underlies the asymmetric/symmetric self-renewal of these cells or whether deregulation of such determinants could contribute to the pathogenesis of hematopoietic malignancies by inducing constitutive symmetric self-renewal divisions. We have addressed these questions through a functional genetics approach taking advantage of systematic RNAi to evaluate the function of conserved polarity factors and cell fate determinants in HSCs. From a list of 72 of such factors identified in the literature, 30 murine homologues were chosen based on their differentially higher level of expression in HSC-enriched populations as measured by qRT-PCR. For each candidate we designed 3 unique short hairpin RNA (shRNA) encoding retroviral constructs also carrying EGFP for the purposes of following transduced cells. Primitive hematopoietic cells enriched for HSC were infected at high efficiency with the library in an arrayed 96-well format and their in vivo reconstituting potential was then evaluated through competitive repopulating unit assays. Genes for which shRNA vectors altered late transplant EGFP levels below or above thresholds as defined by a control shRNA to luciferase were considered as hits. Using this approach, we identified and comprehensively validated 4 genes, including the RNA binding protein Msi2, for which shRNA-mediated depletion dramatically impairs repopulation but does not induce cell death or a cell cycle block. Importantly, we show that the loss in the repopulating ability of these shRNA transduced cells is mediated at the stem cell level and is not due to progenitor or downstream cell toxicity or to any defect in the process of bone marrow homing. Subsequent expression profiling indicated that Msi2 is also upregulated in HOXB4-overexpressing symmetrically expanding HSC in line with our findings that it functions as a positive HSC regulator and further suggesting that it represents a potential novel HSC marker. As well as finding HSC agonists, the RNAi screen identified the homeodomain containing transcription factor Prox1 as a negative HSC regulator since its shRNA-mediated transcript loss consistently led to the dramatic in vivo accumulation of EGFP+ transduced cells. Grafts comprised of Prox1 shRNA-transduced cells did not exhibit any lineage skewing however, repeatedly contained an average of 10-fold more primitive Lin-Sca+CD150+48- cells as compared to non-transduced donor cells within the same recipient or to control shRNA-luciferase grafts indicating Prox1 knockdown leads to a significant in vivo expansion of phenotypic HSCs. Moreover, following a 7 day in vitro culture, cells infected with shRNAs to Prox1 were both morphologically and immunophenotypically more primitive than control cells and when transplanted at this time yielded a significantly enhanced engraftment level relative to control shRNAs (51+/-6% GFP vs 8+/-3% GFP). These results further suggest that Prox1 reduction by RNAi expands functional HSCs in vitro. Together these findings have identified conserved cell fate determinants as important and novel regulators of murine hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

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