Life span of multipotential hematopoietic stem cells in vivo.

The findings reported in this study highlight several important features of the development of hematopoietic stem cells after transplantation into irradiated recipients. First, they demonstrate the existence of a class of primitive multipotential stem cells that can function for a significant portion of the lifetime of a mouse (15 mo). In addition, they clearly show that these primitive stem cells can be infected with recombinant retroviruses and thus would be appropriate targets for gene therapy in somatic tissues. Second, our data indicate that the progeny of some, but not all, of the primitive stem cells have fully expanded into the various hematopoietic lineages by 2 mo after reconstitution. Finally, our analysis of the secondary recipients provides strong evidence suggesting that the primitive stem cell population can actually clonally expand. Our current experiments are aimed at determining the extent to which this expansion can occur and whether or not this expansion can be influenced by exogenous factors.

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Novel Lentiviral Vectors Displaying ‘Early-Acting-Cytokines’ Preferentially Promote the Survival and Transduction of NOD/SCID Repopulating Human Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v104.11.2107.2107 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2107-2107

Author(s):

E.L.S. Verhoeyen ◽

Maciej Wiznerowicz ◽

Delphine Olivier ◽

Brigitte Izac ◽

Didier Trono ◽

...

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Stem Cell ◽

Gene Transfer ◽

Hematopoietic Stem Cells ◽

Lentiviral Vectors ◽

Hematopoietic Stem ◽

Efficient Gene ◽

Cd34 Cells

Abstract A major limitation of current generation lentiviral vectors (LVs) is their inability to govern efficient gene transfer into quiescent target cells which hampers their application for hematopoietic stem cell gene therapy. Human CD34+ cells that reside into G0 phase of the cell cycle and thus are quiescent, are indeed higly enriched in hematopoietic stem cells. Here, we designed novel lentiviral vectors that overcome this type of restriction by displaying early-acting-cytokines on their surface. Presentation of a single cytokine, thrombopoietin (TPO), or co-presentation of TPO and stem cell factor (SCF) on the lentiviral vector surface improved gene transfer into quiescent CD34+ cord blood cells by 45-fold and 77-fold, respectively, as compared to conventional lentiviral vectors. Moreover, these new LVs preferentially transduced and promoted the survival of immature resting cells rather than cycling CD34+ cells. Most importantly, the new early-cytokine-displaying lentiviral vectors allowed highly efficient gene transfer in CD34+ immature cells with long-term in vivo NOD/SCID mice repopulating capacity, a hallmark of bona fide HSCs. In conclusion, the novel ‘early-acting cytokines’ displaying LVs described here provide simplified, reproducible gene transfer protocols that ensure efficient gene transfer in hematopoietic stem cells. As such, these novel reagents bring us one step closer to selective in vivo gene therapy.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1801480116 ◽

2019 ◽

Vol 116 (4) ◽

pp. 1447-1456 ◽

Cited By ~ 29

Author(s):

Rong Lu ◽

Agnieszka Czechowicz ◽

Jun Seita ◽

Du Jiang ◽

Irving L. Weissman

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem Cell ◽

Lineage Commitment ◽

Self Renewal ◽

Hematopoietic Stem ◽

Hematopoietic System ◽

Different Levels

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

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Native associations of early hematopoietic stem cells and stromal cells isolated in bone marrow cell aggregates

Blood ◽

10.1182/blood.v83.2.361.bloodjournal832361 ◽

1994 ◽

Vol 83 (2) ◽

pp. 361-369 ◽

Cited By ~ 1

Author(s):

PE Funk ◽

PW Kincade ◽

PL Witte

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Stromal Cells ◽

Stromal Cell ◽

Hematopoietic Cells ◽

Hematopoietic Stem ◽

Factor C

In suspensions of murine bone marrow, many stromal cells are tightly entwined with hematopoietic cells. These cellular aggregations appear to exist normally within the marrow. Previous studies showed that lymphocytes and stem cells adhered to stromal cells via vascular cell adhesion molecule 1 (VCAM1). Injection of anti-VCAM1 antibody into mice disrupts the aggregates, showing the importance of VCAM1 in the adhesion between stromal cells and hematopoietic cells in vivo. Early hematopoietic stem cells were shown to be enriched in aggregates by using a limiting-dilution culture assay. Myeloid progenitors responsive to WEHI-3CM in combination with stem cell factor (c-kit ligand) and B220- B-cell progenitors responsive to insulin-like growth factor-1 in combination with interleukin-7 are not enriched. We propose a scheme of stromal cell-hematopoietic cell interactions based on the cell types selectively retained within the aggregates. The existence of these aggregates as native elements of bone marrow organization presents a novel means to study in vivo stem cell-stromal cell interaction.

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Canonical Notch Signaling Is Dispensable for the Maintenance of Adult Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v106.11.267.267 ◽

2005 ◽

Vol 106 (11) ◽

pp. 267-267 ◽

Cited By ~ 37

Author(s):

Ivan Maillard ◽

Seth E. Pross ◽

Olga Shestova ◽

Hong Sai ◽

Jon C. Aster ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Notch Signaling ◽

Transcriptional Activation ◽

Cell Function ◽

Fetal Life ◽

Hematopoietic Stem

Abstract Canonical Notch signaling operates through a highly conserved pathway that regulates the differentiation and homeostasis of hematopoietic cells. Ligand-receptor binding initiates proteolytic release of the Notch intracellular domain (ICN) which migrates to the nucleus, binds the transcription factor CSL/RBPJk and activates target genes through the recruitment of transcriptional coactivators of the Mastermind-like family (MAML). Notch signaling is essential for the emergence of hematopoietic stem cells (HSCs) during fetal life, but its effects on adult HSCs are controversial. In gain-of-function experiments, activation of Notch signaling in adult HSCs increased their self-renewal potential in vitro and in vivo. However, loss-of-function studies have provided conflicting results as to the role of physiological Notch signaling in HSC maintenance and homeostasis. To address this question, we expressed DNMAML1, a GFP-tagged pan-inhibitor of Notch signaling, in mouse HSCs. We have shown previously that DNMAML1 interferes with the formation of the ICN/CSL/MAML transcriptional activation complex and blocks signaling from all four Notch receptors (Notch1-4) (Maillard, Blood 2004). Transfer of DNMAML1-transduced bone marrow (BM) as compared to control GFP-transduced BM into lethally irradiated recipients gave rise to similar long-term stable expression of GFP for at least 6 months after transplant. DNMAML1 and GFP-transduced cells contributed equally to all hematopoietic lineages, except to the T cell and marginal zone B cell lineages, which are Notch-dependent. Expression of DNMAML1 did not affect the size of the BM progenitor compartment (Lin negative, Sca-1 positive, c-Kit high, or LSK cells), or the proportion of LSK cells that were negative for Flt3 and L-Selectin expression (containing long-term HSCs). The stem cell function of DNMAML1-transduced LSK cells was further assessed with in vivo competitive repopulation assays in lethally irradiated recipients. DNMAML1 and GFP-transduced LSK cells competed equally well with wild-type BM, as judged by their contribution to the myeloid lineage up to 4 months post-transplant, through two successive rounds of transplantation. Our data indicate that canonical Notch signaling is dispensable for the maintenance of stem cell function in adult HSCs.

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In Vivo Fate Tracing Studies Using the SCL Stem Cell Enhancer: Embryonic Hematopoietic Stem Cells Significantly Contribute to Adult Hematopoiesis.

Blood ◽

10.1182/blood.v104.11.559.559 ◽

2004 ◽

Vol 104 (11) ◽

pp. 559-559

Author(s):

Joachim R. Gothert ◽

Sonja E. Gustin ◽

Mark A. Hall ◽

Anthony R. Green ◽

Berthold Gottgens ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

De Novo ◽

Genetic Manipulation ◽

Fetal Liver ◽

Hematopoietic Stem ◽

Reporter Mice ◽

First Time

Abstract Evidence for the direct lineage relationship between embryonic and adult hematopoietic stem cells (HSCs) in the mouse is primarily indirect. In order to study this relationship in a direct manner we expressed the tamoxifen-inducible Cre-ERT-recombinase under the control of the SCL-stem-cell-enhancer in transgenic mice (HSC-SCL-Cre-ERT). To determine functionality, HSC-SCL-Cre-ERT transgenics were bred with the Cre-reporter-mice ROSA26R and R26R-EYFP. Flow-cytometric and transplantation studies revealed tamoxifen-dependent recombination occurring in more than 90% of adult long-term HSCs, whereas the targeted proportion within mature progenitor populations was significantly lower. Moreover, the transgene was able to irreversibly tag embryonic HSCs on days 10 and 11 of gestation. These cells contributed to bone marrow hematopoiesis five months later. In order to investigate whether the de novo HSC-generation is completed during embryogenesis, HSC-SCL-Cre-ERT marked fetal liver cells were transplanted into adult recipients. Strikingly, the proportion of marked cells within the transplanted and the in vivo-remaining HSC-compartment was not different, implying that no further HSC-generation occurred during late fetal and neonatal stages of development. These data demonstrate for the first time the direct lineage relationship between mid-gestation embryonic and adult HSCs in the mouse. Additionally, the HSC-SCL-Cre-ERT mice will provide a valuable tool to achieve temporally controlled genetic manipulation of HSCs.

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Differential Expression of SLAM Family Receptor Markers in Normal Human Hematopoietic Stem Cells and Their Malignant Counterpart in MDS and AML.

Blood ◽

10.1182/blood.v108.11.1897.1897 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1897-1897

Author(s):

Ramon V. Tiu ◽

Jennifer J. Powers ◽

Abdo Haddad ◽

Ying Jiang ◽

Jaroslaw P. Maciejewski

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Cell Population ◽

Stem Cell Population ◽

Leukemic Stem Cell ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Slam Family

Abstract Members of the signaling lymphocytic activation molecule (SLAM) family, including CD150, CD48 and CD244, were shown to precisely distinguish more committed lineage restricted progenitor cells from pluripotent and multipotent murine hematopoietic stem cells (HSC; Kiel et al; 2005 Cell). Similar SLAM profiles may also be present on HSC subsets in humans. We hypothesized that these SLAM markers may be indicators not only of stem cell potential in normal hematopoiesis but also distinguish a subset of the most immature malignant precursors of leukemia. In agreement with the concept of a “cancer stem cell,” the presence of leukemic stem cell population may be an indicator of important clinical and biological properties. We first tested the distribution of CD150, CD48 and CD244 antigens on human CD34+ cells derived from 7 control individuals using 4-color flow cytometry. CD34+ cells were measured in the blast gate based on side scatter and CD45 expression. Within CD34+ blasts, expression of CD48, CD150, and CD244 was detected on 16.71%±9.69, 6.53%±2.93, and 26.92%±6.95 of cells respectively. Subsequently, we investigated SLAM expression in 9 immature leukemic cell lines, including KG-1, K562, U937, HEL, HL60, MKN-95, NB-4, Kasumi and UT7, and found increased expression of SLAM markers in KG-1 (CD48+, CD150+, CD244+) and Kasumi (CD48−, CD150−, CD244+). Consequently, none of the leukemic cells showed pluripotent/multipotent SLAM profiles. We then compared the SLAM marker expression on blasts from patients with AML and MDS with that of CD34+ cells from normal controls. We studied a total of 28 patients: 11 MDS (2 low grade, 5 advanced MDS, 3 MDS/MPD overlap) and 10 AML (FAB: 3 M1, 2 M2, 1 M3, 2 M4/M4E0 and 2 M6). In our cohort, 8/10 AML patients expressed one of the three SLAM markers; 6/10 were CD150−CD48−CD244+ (63.57%±6.96) and 2/10 were CD150+CD48−CD244−(46%±10.96) suggestive of the presence of either pluripotent or multipotent leukemic stem cell phenotype. In the MDS cohort, 8/11 patients expressed one of three SLAM markers, 7/11 were CD150−CD48−CD244+ (41.21% ± 8.9) and 1/11 were CD150+CD48−CD244− (1.26%±0.59) again consistent with a profile derived from either pluripotent or multipotent stem cells. None of the MDS and AML patients had either co-expression of CD244 and CD48 or increased expression of CD48 alone. Two of the M1 type AML patients with CD150−CD48−CD244+ phenotype received prior chemotherapy and achieved complete remission on bone marrow biopsy and flow cytometry using traditional blast markers. In some, we conclude that the SLAM receptor markers may be associated with certain types of leukemic blasts and may be useful in the identification of leukemic stem cell population in both MDS and AML.

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Cell-Intrinsic Heterogeneity Plays An Important Role in the Development of Clonal Dominance after Retroviral Gene Transfer into Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v112.11.819.819 ◽

2008 ◽

Vol 112 (11) ◽

pp. 819-819

Author(s):

Olga S. Kustikova ◽

Bernhard Schiedlmeier ◽

Martijn H. Brugman ◽

Maike Stahlhut ◽

Zhixiong Li ◽

...

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Insertional Mutagenesis ◽

Cell Potential ◽

Hematopoietic Stem ◽

Long Term Follow Up ◽

Stem Cell Potential

Abstract The development of clonal imbalance after transplantation of genetically modified hematopoietic cells is a cause of concern in the long-term follow-up of patients undergoing gene therapy for the treatment of severe or acquired hematopoietic disorders. We and others have previously described how insertional proto-oncogene dysregulation by transgene integration may provoke clonal restriction and leukemia, thus becoming a dose-limiting toxicity of gene therapy. When targeting populations enriched for or depleted from hematopoietic stem cells (HSC) in the C57Bl6 CD45 chimerism model, we found that intrinsic stem cell potential is a conditio sine qua non for the establishment of expanding insertional mutants. Mice observed for 6–7 months after co-transplantation of gene-modified cells and non-transduced fresh competitor cells were monitored in regular intervals of 6 weeks and the emergence of dominant clones was assessed by flow cytometry in combination with an LM-PCR procedure validated on mixtures of polyclonal and oligoclonal DNA. Dominant clones originating after gammaretroviral insertion in the Evi1 locus reproducibly occurred with a frequency of 1:10,000 when targeting multipotent LSK cells or short-term repopulating HSC (LSK CD34+ CD135−), but no such events were detected in the progeny of >1 million Sca1- Lin- c-Kit+ (LK) cells or ~75,000 multipotent progenitor cells (MPP, LSK CD34+ CD135+). Dominant clones originating from multipotent cells and displaying insertional upregulation of Evi1 showed greatly diminished T lymphopoiesis in vivo, formally demonstrating transforming events. Residual progeny of MPP or LK cells was detected in transplanted animals with insertional events in proto-oncogenes, but these clones were unable to expand to significant levels of hematopoiesis (>1%). Targeting HSC-enriched cell populations (LSK CD34+ CD135− or LSK CD34− CD135−), a comparison of gamma-retroviral transduction conditions in a 5 days serum-free culture period and lentiviral transduction in a 20h protocol revealed that the latter conditions significantly improved chimerism with a greatly increased clonal diversity in the first 8 weeks of repopulation. However, after lentiviral transduction clonal dominance progressively developed over an observation time of 6 months, although there was no evidence for insertional proto-oncogene upregulation as the underlying cause even when using a lentiviral vector with a strong internal enhancer-promoter capable of insertional long-distance effects. Our study suggests two important conclusions: (1) Insertional mutagenesis in gene therapy is unlikely to endow differentiating progenitor cells with (leukemogenic) stem cell potential and (2) clonal restriction developing in the long-term follow-up after transplantation of gene-modified hematopoietic stem cells is not necessarily a side effect of insertional mutagenesis, but may also reflect classical “gene marking” of a stem cell clone with a strong intrinsic potential for competitive dominance.

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In Vitro and In Vivo Evidence That Umbilical Cord Blood (UCB)-Derived CD45-/SSEA-4+/OCT-4+/CD133+/CXCR4+/Lin- Very Small Embryonic/Epiblast Like Stem Cells (VSELs) Do Not Contain Clonogenic Hematopoietic Progenitors but Are Highly Enriched in More Primitive Stem Cells - Novel View On Hierarchy of UCB Stem Cell Compartment.

Blood ◽

10.1182/blood.v114.22.35.35 ◽

2009 ◽

Vol 114 (22) ◽

pp. 35-35 ◽

Cited By ~ 2

Author(s):

Ewa K. Zuba-Surma ◽

Izabela Klich ◽

Marcin Wysoczynski ◽

Nicholas J Greco ◽

Mary J. Laughlin ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Cord Blood ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Hematopoietic Stem ◽

Hematopoietic Activity

Abstract Abstract 35 Recently, we identified in umbilical cord blood (UCB) a population of very small embryonic/epiblast-like (VSEL) stem cells (Leukemia 2007;21:297–303) that are i) smaller than erythrocytes, ii) SSEA-4+/Oct-4+/CD133+/CXCR4+/Lin−/CD45−, iii) respond to SDF-1 gradient and iv) possess large nuclei containing primitive euchromatin. We have demonstrated in vitro that UCB-derived VSELs did not reveal hematopoietic activity freshly after isolation, but grow hematopoietic colonies following co-culture/activation over OP-9 cells. To investigate the hierarchy of UCB-derived, CD45 negative VSELs, we employed staining with Aldefluor - detecting aldehyde dehydrogenase (ALDH), the enzyme expressed in primitive hematopoietic cells. Subsequently, we sorted CD45−/CD133+/ALDHhigh and CD45−/CD133+/ALDHlow sub-fractions of VSELs from UCB samples and established that freshly sorted from UCB VSELs in contrast to sorted CD45+/ CD133+/ALDHhigh and CD45+/CD133+/ALDHlow hematopoietic stem cells (HSC) did not grow colonies in vitro. However, when CD45− VSELs were activated/expanded over OP-9 stroma cells, they exhibit hematopoietic potential and grew in routine methylcellulose cultures hematopoietic colonies composed of CD45+ cells. Interestingly, while CD45−/CD133+/ALDHhigh VSELs gave raise to hematopoietic colonies after the first replating, the formation of colonies by CD45−/CD133+/ALDHlow VSELs was somehow delayed, what suggest that they needed more time to acquire hematopoietic commitment. Thus our in vitro data indicate that both populations of CD45− cells may acquire hematopoietic potential; however hematopoietic specification is delayed for CD45−/CD133+/ALDHlow cells, suggesting their more primitive nature. In parallel, real time PCR analysis confirmed that while freshly isolated CD45−/CD133+/ALDHhigh VSELs express more hematopoietic transcripts (e.g., c-myb, 80.2±27.4 fold difference), CD45−/CD133+/ALDHlow exhibit higher levels of pluripotent stem cell markers (e.g., Oct-4, 119.5±15.5 fold difference as compared to total UCB mononuclear cells) (Figure 1 panel A). Next hematopoietic potential of UCB-derived VSELs was tested in vivo after transplantation into NOD/SCID mice (Figure 1 panel B and C). We noticed that both CD45−/CD133+/ALDHhigh and CD45−/CD133+/ALDHlow VSELs, give rise to human lympho-hematopoietic chimerism in lethally irradiated NOD/SCID mice as assayed 4–6 weeks after transplantation. The level of human hematopoietic CD45+ cells in murine peripheral blood (PB), bone marrow (BM) and spleen (SP) were comparable for both transplanted UCB-VSELs fractions - 7.1±2.9% (PB), 23.2±0.2% (SP) and 25.2±1.0% (BM). In conclusion, our data suggest that freshly isolated very small CD45 negative UCB-VSELs are depleted from clonogeneic progenitors, however they are highly enriched for primitive HSC. Based on our in vitro and in vivo data we postulate following hierarchy of hematopoietic stem cells in UCB (from most primitive to more differentiated) i) CD45−/CD133+/ALDHlow, ii) CD45−/CD133+/ALDHhigh , iii) CD45+/CD133+/ALDHlow and iv) CD45−/CD133+/ALDHhigh. We also postulate that as we have already shown for murine BM-derived VSELs, human UCB-derived CD45 negative VSELs correspond to a population of most primitive long term repopulating HSC (LT-HSC). Of note, we also found that currently employed, routine UCB processing strategies may lead up to ∼50% unwanted loss of these small cells that are endowed with such remarkable hematopoietic activity! Disclosures: No relevant conflicts of interest to declare.

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Heterogeneity Of Leukemic Stem Cell Capacity Of BCR-ABL+ Long-Term Hematopoietic Stem cells In CML Is Associated With Variability In MPL Expression

Blood ◽

10.1182/blood.v122.21.516.516 ◽

2013 ◽

Vol 122 (21) ◽

pp. 516-516

Author(s):

Bin Zhang ◽

Yin Wei Ho ◽

Wei Tong ◽

Ling Li ◽

Ravi Bhatia

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Growth ◽

Hematopoietic Stem Cells ◽

Myelogenous Leukemia ◽

Hematopoietic Stem ◽

Nsg Mice ◽

Donor Cells

Abstract In chronic myelogenous leukemia (CML), in vivo long-term repopulating and leukemia stem cell (LSC) capacity is restricted to a small population of BCR-ABL+ long-term hematopoietic stem cells (LTHSC). Using an inducible transgenic SCL-tTA/BCR-ABL mouse model of CML, we have shown that leukemic cells with long-term repopulating and leukemia-initiating capacity have the Lin-Sca-1+Kit+Flt3-CD150+CD48- phenotype, also characteristic of normal LTHSC. Limiting dilution transplantation studies show that frequency of cells with LTHSC phenotype with long-term engraftment capacity (1:6) is considerably higher than those with leukemia-initiating capacity (1:80) suggesting that only some LTHSC may have LSC capacity (Cancer Cell 21:577, 2012). To further evaluate the basis for heterogeneity in LSC potential of BCR-ABL+ LTHSC, SCL-tTA/BCR-ABL mice were crossed with GFP expressing mice to allow tracking of donor cells, and a cohort of mice were transplanted with limiting numbers of GFP+LTHSC (200 per mouse) and followed for engraftment of GFP+ cells and development of CML (WBC>10,000/ul). Only 11 of 20 mice developed CML, whereas 9 mice showed long term engraftment without development of CML. GFP+ LTHSC selected from primary recipients were transplanted into secondary recipients (200 per mouse). Seven of 17 mice receiving cells from mice with CML also developed CML after the second transplant, whereas none of the mice receiving cells from non-CML mice developed CML, suggesting the distinction between leukemogenic versus non-leukemogenic LTHSC was maintained after transplantation. LTHSC isolated from primary recipients were also analyzed for expression of several HSC-regulatory genes by multiplex Q-PCR using the Fluidigm system. On hierarchical clustering, LTHSC from mice developing CML clustered separately from LTHSC from mice without CML. Amongst cell surface expressed genes, expression of the thrombopoietin (TPO) receptor MPL (p=0.006) and CD47 (p=0.006) was significantly increased in LTHSC from mice developing CML. We did not see significant differences in BCR-ABL expression in LTHSC from mice with or without CML. We further analyzed the relationship of MPL expression with CML LTHSC function. CML LTHSC (n=6) expressing high levels of MPL (MPLhi, top 10% based on MPL expression) showed significantly increased cell growth (p<0.0001) and CFC potential (p=0.0007) when cultured with TPO (10ng/ml) compared to LTHSC expressing low levels of MPL (MPLlo, lowest 10% based on MPL expression), as well as significantly increased cell growth (p=0.005) and CFC (p=0.03) compared to normal MPLhi LTHSC. Following transplantation, MPLhi LTHSC (200 per mouse) generated significantly higher short-term (4 wks, p=0.008) and long-term (16 wks, p=0.003) engraftment of donor cells compared to MPLlo LTHSC. Seven of 16 mice receiving MPLhi LTHSC developed CML compared to only 1 out of 17 mice receiving MPLlo LTHSC. We next evaluated heterogeneity of MPL expression in LTHSC (CD34+CD38-CD90+ cells) from CML patients and normal subjects. As was seen in murine studies, human CML MPLhi LTHSC cultured with TPO (10ng/ml) showed increased cell growth (p<0.0001) and CFC frequency (p=0.02) compared to CML MPLlo LTHSC, and significantly increased cell growth (p<0.0001) and CFC generation (p=0.02) compared to normal MPLhi LTHSC. Both baseline and TPO stimulated p-Stat3/5 levels were significantly higher in human CML MPLhi LTHSC compared with MPLlo LTHSC (p<0.0001), and in CML compared to normal MPLhi LTHSC. Interestingly p-Stat5 response peaked at 1 hour in CML LTHSC compared to 20 minutes in normal LTHSC, further indicating alterations in MPL signaling in CML LTHSC. Transplantation of CML MPLhi LTHSC (3x104 cells/mouse) into NSG mice resulted in higher engraftment of human myeloid cells in BM at both 4 and 16 weeks (p<0.05) compared with MPLlo LTHSC. Normal MPLhi LTHSC also showed higher engraftment in NSG mice at 4 and 16 weeks compared with MPLlo cells. Our studies indicate that CML LTHSC represent a heterogeneous population with varying LSC capacity. Heterogeneity in LSC capacity is associated with variability in expression of MPL. Higher levels of MPL expression in CML LTHSC are associated with significantly increased Stat3/5 signaling, in vitro and in vivo growth, and LSC capacity. These results identify MPL as a key regulator of LSC potential of BCR-ABL+ LTHSC and a potential target for LSC-directed therapeutics. Disclosures: No relevant conflicts of interest to declare.

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