CLEC-2 in megakaryocytes is critical for maintenance of hematopoietic stem cells in the bone marrow

Hematopoietic stem cells (HSCs) depend on the bone marrow (BM) niche for their maintenance, proliferation, and differentiation. The BM niche is composed of nonhematopoietic and mature hematopoietic cells, including megakaryocytes (Mks). Thrombopoietin (Thpo) is a crucial cytokine produced by BM niche cells. However, the cellular source of Thpo, upon which HSCs primarily depend, is unclear. Moreover, no specific molecular pathway for the regulation of Thpo production in the BM has been identified. Here, we demonstrate that the membrane protein C-type lectin-like receptor-2 (CLEC-2) mediates the production of Thpo and other factors in Mks. Mice conditionally deleted for CLEC-2 in Mks (Clec2MkΔ/Δ) produced lower levels of Thpo in Mks. CLEC-2–deficient Mks showed down-regulation of CLEC-2–related signaling molecules Syk, Lcp2, and Plcg2. Knockdown of these molecules in cultured Mks decreased expression of Thpo. Clec2MkΔ/Δ mice exhibited reduced BM HSC quiescence and repopulation potential, along with extramedullary hematopoiesis. The low level of Thpo production may account for the decline in HSC potential in Clec2MkΔ/Δ mice, as administration of recombinant Thpo to Clec2MkΔ/Δ mice restored stem cell potential. Our study identifies CLEC-2 signaling as a novel molecular mechanism mediating the production of Thpo and other factors for the maintenance of HSCs.

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TIMP-3 Inhibition of Angiopoietin-1 Signaling Recruits Hematopoietic Stem Cells from the Bone Marrow Niche.

Blood ◽

10.1182/blood.v110.11.1255.1255 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1255-1255

Author(s):

Hideaki Nakajima ◽

Miyuki Ito ◽

David Smookler ◽

Fumi Shibata ◽

Yumi Fukuchi ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Critical Role ◽

Quiescent State ◽

Hematopoietic Stem ◽

Bm Niche ◽

Quiescent Hscs ◽

Angiopoietin 1

Abstract Regulating transition of hematopoietic stem cells (HSCs) between quiescent and cycling states is critical for maintaining homeostasis of blood cell production in the adult bone marrow. Quiescent HSCs are rapidly recruited into the cell cycle when they face hematopoietic demands such as myelosuppression, returning to quiescence once they produce enough progenitors. It was previously shown that quiescent HSCs express Tie2 and that Tie2/angiopoietin-1 (Ang-1) signaling plays a critical role for maintaining HSC quiescence. However, molecular cues for recruiting HSCs from a quiescent state into cycle remain poorly understood. Extracellular signals are often regulated by the extracellular matrix environment, which is modulated by metalloproteinase (MMP) activities. TIMP-3 is an endogenous inhibitor of MMPs, and we have previously proposed that TIMP-3 may play a critical role in HSC physiology. In addition, TIMP-3 has been reported to suppress angiogenesis by inhibiting vascular endothelial growth factor (VEGF) signaling. By analogy with VEGF inhibition, we reasoned that TIMP-3 might suppress Ang-1 signaling in HSC and act as a molecular cue for HSC recruitment. In order to investigate a role of TIMP-3 in the HSC recruitment, we first examined whether TIMP-3 is regulated in the BM upon myelosuppression. Analyses by reverse transcription polymerase chain reaction (RT-PCR) and immunostaining revealed that the injection of 5-fluorouracil (5-FU) or irradiation induced TIMP-3 at the endosteal surface of the BM after 3-days of treatment. We next tested the hypothesis that TIMP-3 might be regulating Ang-1 signals by using cell line models. This revealed that the pre-treatment of cells with TIMP-3 suppressed autophosphorylation of Tie-2 in response to Ang-1. BIAcore and in vitro binding assay revealed that TIMP-3 directly interacted with Ang-1 and Tie-2, indicating that TIMP-3 suppressed Ang-1 signaling through interfering ligand-receptor interaction. Next we examined the effect of TIMP-3 on HSC physiology. TIMP-3 promoted the proliferation of CD34-KSL cells in vitro by approximately 2–3 fold. This was mainly due to the enhanced production of multipotential progenitors from CD34-KSL cells, which was accomplished by an enhanced symmetrical cell division of multipotential progenitors as revealed by paired-daughter cell analysis. Bone marrow transplantation study of TIMP-3-treated CD34-KSL cells showed that they sustained long-term repopulating potential comparable to the control-treated cells. Furthermore, in vivo administration of TIMP-3 into mice accelerated recovery and protected mice from myelosuppression, and in turn, the bone marrow recovery after myelosuppression was delayed in TIMP-3-deficient animals. In summary, TIMP-3 is induced by myelosuppression in the BM niche, stimulates HSC proliferation by inhibiting Ang-1 signaling, and thereby promotes production of multipotential progenitors from HSCs. These results demonstrate that TIMP-3 acts as a molecular cue for recruiting quiescent HSCs from the BM niche.

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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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Establishment of Mouse PMF Model by the Introduction of Constitutive STAT5a Into Purified Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v114.22.3900.3900 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3900-3900

Author(s):

Takafumi Shimizu ◽

Akihiko Ito ◽

Akira Nakagawa ◽

Toshinobu Nishimura ◽

Satoshi Yamazaki ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Active Form ◽

Myeloproliferative Neoplasm ◽

Extramedullary Hematopoiesis ◽

Hematopoietic Stem ◽

Pmf Model ◽

Short Period

Abstract Abstract 3900 Poster Board III-836 Background Polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofiblosis (PMF) are pathologically related and now classified under myeloproliferative neoplasm (MPN). Subsequent studies revealed that MPN is a group of clonal hematopoietic stem cell disorders characterized by proliferation of one or more of the myeloid lineages. The somatic activating mutation in the JAK2 tyrosine kinase, JAK2V617F, is now broadly recognized as a mutation responsible for MPN (Levine R.L. and Gilliland D.G. Blood 2008). Indeed, Most of PV patients, and half of patients with ET or PMF possess this mutation. Recent studies revealed that PV phenotype can be generated in homozygous JAK2V617F transgenic mice, while ET or atypical CML-like marked leukothrombocytosis with mild myelofibrosis can be observed in heterozygous JAK2V617F mice (Tiedt et al, Blood 2008, Shide et al Leukemia 2008). These results indicate that expression levels of JAK2V617F may influence PV and ET phenotypes. On the other hand, typical PMF phenotype has not been generated by the introduction of JAK2V617F. According to the WHO criteria, PMF could be defined as “spent phase of hematopoiesis” with fibrosis formation followed by increased bone marrow cellularity as consequences of granulocytic proliferation and megakaryocyte changes with ineffective hematopoiesis. In this study, we focused on STAT5a, a direct downstream molecule of JAK2, because we previously reported that upon transplantation, purified CD34- lineage- sca-1+ c-Kit+ (CD34-KSL) hematopoietic stem cells (HSCs) transduced with constitutive active form of STAT5A acted as MPN initiating cells causing granulocytosis without erythrocytosis/thrombocytosis (Kato Y. et al, J Exp Med 2005). Based on these observations, we attempted to make PMF model through mimicking typical PMF dynamics; hyper proliferation of HSCs by the introduction of constitutive active STAT5a and following early HSC exhaustion. Materials and Methods CD34-KSL HSCs or CD34+KSL hematopoietic progenitor cells (HPCs) were purified from bone marrow (BM) of C57BL/6 (B6)-Ly5.1 mice. Then, the cells were retrovirally transduced with STAT5a wild-type (wt) or its constitutive active mutant, STAT5a(1*6). The prepared cells were used for methylcellulose assay and were transplanted into lethally irradiated B6-Ly5.2 recipient mice together with 5 × 105 B6-Ly5.1/5.2 competitor BM cells. Peripheral blood (PB) of transplanted mice was monitored biweekly for donor chimerism and lineage deviation using flow cytometry. Subsequently, histrogical analyses of bone marrow and spleen were performed to determine myelofiblosis grade and detecting extramedullar hematopoiesis. Finally, immunohistochemical staining of bone marrow with anti-TGF-b antibody was performed to detect effector cells of myelofibrosis. Results Transplantation of STAT5a (1*6) transduced HSCs resulted in generation of 57 MPN mice (total 83 mice), while no MPN mouse was obtained by STAT5a (1*6) transduced HPCs (total 12 mice). Pathological analysis revealed that majority (70%) of MPN mice had PMF phenotype as defined by leukoerythroblastosis and dacryocytosis without leukothrombocytosis. These mice with PMF phenotype showed marked splenomegaly with extramedullary hematopoiesis, and granulocytic proliferation with megakaryocyte change. In BM, granulocytic proliferation advanced to severe myelofibrosis and osteomyelosclerosis in very short period of time (4 to 8 weeks). Those mice died of hemorrhage induced by pancytopenia within a few months, much faster than the mice with JAK2V617F based PV/ET models. Immunohistological analysis revealed that dominance of Gr-1 / Mac-1 positive granulocytes and CD41 positive small megakaryocytes strongly expressing TGF-beta, a putative inducer of fibroblastosis in BM of PMF mice. Conclusion By transplanting STAT5a(1*6) transduced HSCs, we were able to develop mice with phenotype closely resembling human PMF. Because PMF is rare disease, this animal model should be useful for understanding etiology of PMF, for evaluating existing treatment, and for developing therapeutics targeting STAT5a or its downstream pathway. Disclosures: No relevant conflicts of interest to declare.

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Zeb2-Defficiency in the Adult Murine Hematopoietic Precursor Cells Leads to Differentiation Defects in Multiple Hematopoietic Lineages and a Myeloproliferative-Like Phenotype

Blood ◽

10.1182/blood.v120.21.1199.1199 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1199-1199

Author(s):

Tamara Riedt ◽

Steven Goossens ◽

Ines Gütgemann ◽

Carmen Carrillo-Garcia ◽

Hichem D Gallala ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Hematopoietic Cells ◽

Extramedullary Hematopoiesis ◽

Independent Effect ◽

Self Renewal ◽

Hematopoietic Stem ◽

Reticular Fibers

Abstract Abstract 1199 The life long replenishment of highly specialized blood cells by a small number of hematopoietic cells (HSC) requires a strict regulation between self-renewal and differentiation in the immature compartment of the bone marrow. Perturbation of this equilibrium can result in stem cell loss or hematologic malignancies. This balance is at least in part controlled by a network of transcription factors. Zeb2 is a transcriptional repressor and plays an important role during the embryonic development as a modulator of the epithelial to mesenchymal transition (EMT) as well as tumor progression and metastasis. We have previously identified the essential role of Zeb2 in murine embryonic hematopoiesis, where selective Zeb2 deficiency in the hematopoietic stem cells resulted in early lethality around day 12.5. The aim of this study was to analyze whether Zeb2 plays a specific role in the regulation of homeostasis in the adult hematopoietic system. Using the Mx1-Cre based inducible Zeb2 conditional knock out mouse model we analyzed the impact of Zeb2 loss on adult hematopoietic stem cell function. Upon the induction of Zeb2 deletion we found a significant decrease in most cell lineages of the peripheral blood, except the neutrophil granulocytes. However, the reduction of mature cells in the blood was not accompanied by reduced bone marrow cellularity, as the cellularity was similar between Zeb2Δ/Δ Mx1-Cre (Zeb2 conditional KO) mice and the control animals (Zeb2+/+Mx1-Cre). However, in the bone marrow of the Zeb2Δ/Δ Mx1-Cre animals the granulocytic lineage was dominating, whereas other lineages e.g. red blood cell precursors and B-lymphoid precursors were drastically reduced. Histological sections of the bone marrow cavity revealed megacaryocytes with abnormal morphology reflecting maturation defects and an increased production of reticular fibers in the BM of Zeb2Δ/Δ Mx1-Cre mice. In addition Zeb2Δ/Δ Mx1-Cre mice displayed a two to three fold increase in spleen size compared to control animals due to an extramedullary hematopoiesis. Analysis of the primitive hematopoietic compartment in the bone marrow and spleens revealed that Zeb2 deletion resulted in a pronounced increase in the most immature hematopoietic cells, defined as Lin-Sca1+cKit+CD48-CD150+ population, and perturbation in different lineage restricted progenitor subpopulations. No difference in cell cycling or apoptotic rate in the stem cell enriched bone marrow population (Lin-Sca1+cKit+CD48-CD150+) was detectable between the genotypes. Upon transplantation into lethally irradiated wild type recipients, Zeb2 deficient stem cells demonstrated significantly reduced ability to differentiate into multiple hematopoietic lineages indicating a niche independent effect of Zeb2 in promoting differentiation of hematopoietic stem cells. On the molecular level, gene expression analysis of hematopoietic stem and progenitor cells using microarray approach revealed increased transcripts of downstream targets of Wnt/ß-Catenin signaling, suggesting increased Wnt signaling activity in absence of Zeb2 in the hematopoietic compartment, which at least in part might be responsible for the observed phenotype. These data indicate that Zeb2 is involved in the regulation of the balance between self-renewal and differentiation at multiple stages of hematopoietic cell maturation. Furthermore the lack of Zeb2 in the hematopoietic compartment leads to a phenotype that resembles the features of human myeloproliferative disorders, especially the early stages of primary myelofibrosis with dominant granulopoiesis, production of reticular fibers in the bone marrow, and morphological abnormalities in megacaryocytes, accompanied by extramedullary hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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Down-Regulation of Homeobox Gene Ventx Promotes Expansion of Human Bone Marrow Hematopoietic Stem Cells (HSC)

Blood ◽

10.1182/blood.v118.21.1272.1272 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1272-1272

Author(s):

Hong (Jenny) Gao ◽

Xiaoming Wu ◽

Yan Sun ◽

Jiayun Lu ◽

Leslie E Silberstein ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Cell Fate ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Great Promise ◽

Down Regulation ◽

Hematopoietic Stem ◽

Proliferation And Differentiation

Abstract Abstract 1272 Hematopoietic stem cells (HSC) give rise to mature cells of all lineages of blood and immune systems. HSC transplantation has shown great promise in the treatment of malignancies, reconstitution of hematopoietic systems and HSC-based gene therapy. Cell intrinsic factors/pathways have been the targets of intensive investigation for its potential application in HSC expansion. Over the past decades, several critical cell fate determination pathways, such as the Wnt signaling pathways and senescence pathways have been implicated in the proliferation and differentiation of HSC. Moreover, overexpression of HoxB4 and BMI1 was found to be able to expand human HSC 2∼3 folds. Nevertheless, the regulatory mechanisms of HSC proliferation and differentiation remain incompletely understood and safe and efficacious expansion of human HSC remains as a fundamental challenge that limits the clinical application of HSC-based therapy. VentX is a human homologue of the Xenopus homeobox protein Xom of the BMP4 signaling pathway. Using Xenopus model and methods of reverse genetics, our recent work showed that VentX is a LEF/TCF associated Wnt repressor and an activator of senescence pathways. VentX expression is highly regulated and restricted in hematopoietic cells and serves a major regulator of hematopoietic cell differentiation. To explore the potential role of VentX in proliferation and differentiation of HSC during hematopoiesis, we quantified VentX expression during hematopoiesis, using qRT-PCR methods and examined the effects of altered VentX expression on HSC properties in vitro and in vivo. Our data showed that VentX expression is significantly up-regulated during oncogenesis of hematopioetic cells. We demonstrated that lentiviral knockdown of VentX allowed for more than 5 fold ex vivo expansion of human HSC with balanced lineage development. Importantly, transient knockdown of VentX by siRNA also led to expansion of HSC. The effect of VentX down-regulation on the expansion of human HSC was also demonstrated by enhanced engraftment in the SCID/NODγ2null mouse model. Consistent with its role as a novel regulator of HSC, overexpression of VentX significantly inhibited clonal genesis of HSC. Mechanistically, we demonstrated that VentX controls the expression of cell cycle regulators downstream of the Wnt and senescence pathways, such as the C-myc, CyclinD1 and p21. In summary, using methods of reverse genetic and developmental modeling, we identified VentX as a novel regulator for expansion of human BM HSC. The results of our investigations provide novel insight in regulating HSC proliferation and differentiation. In addition, the findings that transient down-regulation of VentX by SiRNA lead to efficient expansion of bone marrow HSC suggests that VentX may serve as a novel target for safe expansion of HSC for its potential clinical applications. Disclosures: No relevant conflicts of interest to declare.

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Transplanted adult hematopoietic stems cells differentiate into functional endothelial cells

Blood ◽

10.1182/blood-2003-05-1684 ◽

2004 ◽

Vol 103 (1) ◽

pp. 13-19 ◽

Cited By ~ 188

Author(s):

Alexis S. Bailey ◽

Shuguang Jiang ◽

Michael Afentoulis ◽

Christina I. Baumann ◽

David A. Schroeder ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Endothelial Cells ◽

Endothelial Cell ◽

Hematopoietic Stem Cells ◽

Blood Vessels ◽

Cell Potential ◽

Hematopoietic Stem ◽

Adult Bone Marrow ◽

Adult Bone

Abstract During early embryogenesis, blood vessels and hematopoietic cells arise from a common precursor cell, the hemangioblast. Recent studies have identified endothelial progenitor cells in the peripheral blood, and there is accumulating evidence that a subset of these cells is derived from precursors in the bone marrow. Here we show that adult bone marrow–derived, phenotypically defined hematopoietic stem cells (c-kit+, Sca-1+, lineage–) give rise to functional endothelial cells. With the exception of the brain, donor-derived cells are rapidly integrated into blood vessels. Durably engrafted endothelial cells express CD31, produce von Willebrand factor, and take up low-density lipoprotein. Analysis of DNA content indicates that donor-derived endothelial cells are not the products of cell fusion. Self-renewal of stem cells with hematopoietic and endothelial cell potential was revealed by serial transplantation studies. The clonal origin of both hematopoietic and endothelial cell outcomes was established by the transfer of a single cell. These results suggest that adult bone marrow–derived hematopoietic stem cells may serve as a reservoir for endothelial cell progenitors.

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Hematopoietic Stem Cell Purification Leads to Loss of a Stem Cell Population within the Lineage Positive Cellular Fraction

Blood ◽

10.1182/blood.v126.23.4756.4756 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4756-4756

Author(s):

Laura R. Goldberg ◽

Mark Dooner ◽

Elaine Papa ◽

Mandy Pereira ◽

Del Tatto Michael ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Peripheral Blood ◽

Stem Cell Marker ◽

Stem Cell Population ◽

Cell Potential ◽

Hematopoietic Stem ◽

Stem Cell Potential

Abstract Background: Hematopoietic stem cells (HSCs) have tremendous self-renewal and differentiation capacity. The majority of murine hematopoietic stem cell studies have focused on rare purified populations of HSCs, conventionally described as negative for lineage-specific markers and positive for particular cell surface epitope profiles, including c-Kit, Sca-1, and CD150. However, our data indicate that such purifications lead to the loss of a significant population of actively cycling marrow cells with long-term multi-lineage stem cell potential. In the studies presented here, we tested the hypothesis that this discarded stem cell population lies, in part, within the lineage positive (Lin+) fraction of marrow. Methods: We flushed whole bone marrow (WBM) from B6.SJL mice and incubated it with allophycocyanin-tagged antibodies against erythroid (TER119), myeloid (CD11b, GR1), B-lymphoid (B220), or T-lymphoid (CD3, CD4, CD8) markers. Different doses of each specific Lin+ subset isolated by fluorescence activated cell sorting were competitively engrafted into lethally irradiated C57BL/6 host mice. At 1,3, and 6 months post-transplant, peripheral blood was analyzed for donor contribution to chimerism and lineage specificity. Results: Although typically considered to be without stem cell activity, we found that all Lin+ sub-fractions upon single sorting were able to contribute to marrow repopulation in competitive bone marrow transplants. For example, when lethally irradiated recipient mice received 3x105 C57BL/6J competitive whole bone marrow cells in combination with single-sorted GR1+ ± CD11b+ cells (2x106 cells/mouse), peripheral blood showed 15% donor chimerism at 6 months. Similarly, if single sorted CD3+ ±CD4+ ±CD8+ cells (70,000 cells/mouse), B220+ cells (1x106 cells/mouse), or Ter119+ cells (1x106 cells/mouse) were competitively engrafted with 3x105 C57BL/6 WBM cells, the donor Lin+ sub-fractions contributed to 2%, 15%, and 35% peripheral blood chimerism at 6 months post-transplant, respectively. This contribution was multi-lineage in all cases. When we performed double sorting of the Lin+ subsets, there was a dramatically reduced engraftment capacity between 1-6% donor chimerism for all subgroups. However, we do not think the loss of stem cell capacity with double sorting seen in these studies is due merely to the loss of classical hematopoietic stem cells (Lineage-/stem cell marker+). In our earlier studies, we showed that the total Lin+ population contains long-term multi-lineage engraftment capacity due almost entirely to actively cycling cells. Therefore, if the engraftment capacity within the single sorted Lin+ sub-fractions was due solely to the presence of classical HSCs lost with double sorting, the engraftment capacity found within the Lin+ compartment should be due only to quiescent cells in keeping with the cell cycle status of engrafting highly purified stem cells. Conclusions: Based on these data, we predict that a cycling population of stem cells exists within this single sorted, Lin+ enriched fraction discarded with conventional HSC purification. Future studies are ongoing to further characterize the subsets of Lin+ cells that both remain Lin+ and are found to be Lin- upon double sorting. We will analyze these populations for engraftment capacity, concomitant stem cell marker expression and cell cycle status, in order to fully characterize the total stem cell potential within whole bone marrow that is not included in the purified HSC populations. Disclosures No relevant conflicts of interest to declare.

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