Bone Marrow Cell Aggregates: A Way of Collecting the Adult Human Hematopoietic Stem Cell Niche

Abstract Hematopoietic stem cell (HSC) function is critical in maintaining hematopoiesis continuously throughout the lifespan of an organism and any change in their ability to self-renew and/or to differentiate into blood cell lineages induces severe diseases. Postnatally, HSC are mainly located in bone marrow where their stem cell fate is regulated through a complex network of local influences, thought to be concentrated in the bone marrow (BM) niche. Despite more than 30 years of research, the precise location of the HSC niche in human BM remains unclear because most observations were obtained from mice models. BM harvesting collects macroscopic coherent tissue aggregates in a cell suspension variably diluted with blood. The qualitative interest of these tissue aggregates, termed hematons, was already reported (first by I. Blaszek's group (Blaszek et al., 1988, 1990) and by our group (Boiret et al., 2003)) yet they remain largely unknown. Should hematons really be seen as elementary BM units, they must accommodate hematopoietic niches and must be a complete ex vivo surrogate of BM tissue. In this study, we analyzed hematons as single tissue structures. Biological samples were collected from i) healthy donor bone marrow (n= 8); ii) either biological samples collected for routine analysis by selecting bone marrow with normal analysis results (n=5); or iii) from spongy bone collected from the femoral head during hip arthroplasty (n=4). After isolation of hematons, we worked at single level, we used immunohistochemistry techniques, scanning electronic microscopy, confocal microscopy, flow cytometry and cell culture. Each hematon constitutes a miniature BM structure organized in lobular form around the vascular tree. Hematons are organized structures, supported by a network of cells with numerous cytoplasmic expansions associated with an amorphous structure corresponding to the extracellular matrix. Most of the adipocytes are located on the periphery, and hematopoietic cells can be observed as retained within the mesenchymal network. Although there is a degree of inter-donor variability in the cellular contents of hematons (on average 73 +/- 10 x103 cells per hematon), we observed precursors of all cell lines in each structure. We detected a higher frequency of CD34+ cells than in filtered bone marrow, representing on average 3% and 1% respectively (p<0.01). Also, each hematon contains CFU-GM, BFU-E, CFU-Mk and CFU-F cells. Mesenchymal cells are located mainly on the periphery and seem to participate in supporting the structure. The majority of mesenchymal cells isolated from hematons (21/24) sustain in vitro hematopoiesis. Interestingly, more than 90% of the hematons studied contained LTC-ICs. Furthermore, when studied using confocal microscopy, a co-localization of CD34+ cells with STRO1+ mesenchymal cells was frequently observed (75% under 10 µm of the nearest STRO-1+ cell, association statistically highly significant; p <1.10-16). These results indicate the presence of one or several stem cell niches housing highly primitive progenitor cells. We are confirming these in vitro data with an in vivo xenotransplantation model. These structures represent the elementary functional units of adult hematopoietic tissue and are a particularly attractive model for studying homeostasis of the BM niche and the pathological changes occurring during disease. Disclosures No relevant conflicts of interest to declare.

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CD34+ Cells Derived from Human Embryonic Stem Cells Demonstrate Hematopoietic Stem Cell Potential In Vitro and in Vivo.

Blood ◽

10.1182/blood.v104.11.564.564 ◽

2004 ◽

Vol 104 (11) ◽

pp. 564-564 ◽

Cited By ~ 2

Author(s):

Dan S. Kaufman ◽

Petter S. Woll ◽

Colin H. Martin ◽

Jon L. Linehan ◽

Xinghui Tian

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Es Cells ◽

Es Cell ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Human Es Cells

Abstract We have previously described methods to use either stromal cell co-culture or embryoid-body (EB) formation to support the hematopoietic development of undifferentiated human ES cells (both H1 and H9 cell lines). Either FBS-based media or serum-free media with specific cytokines can be used to derive CD34+ cells, CD45+ cells and hematopoietic progenitors as identified by colony-forming cell (CFC) assays that give rise to mature myeloid, erythroid, and megakaryocytic cells. Genes such as RUNX1, HOXB4, TAL1, and GATA2, all known to be expressed during early hematopoiesis are up-regulated during hematopoietic differentiation of human ES cells. Here, we advance these studies to demonstrate that human ES cell-derived CD34+ cells function as early hematopoietic precursors in surrogate hematopoietic stem cell (HSC) assays. The long-term culture initiating cell (LTC-IC) assay is commonly used to quantify hematopoietic precursors that can be maintained in culture for 5 or more weeks. Human cord blood (CB)-derived CD34+ cells have a LTC-IC frequency of approximately 1:30. We demonstrate LTC-ICs can also be identified from human ES cell-derived CD34+ at a frequency of approximately 1:400. These results suggest CD34+ cells from human ES cells are more heterogeneous than CD34+ cells from CB. Furthermore, we now demonstrate in vitro culture of human ES cell-derived CD34+ cells identify these cells as lymphocyte precursors. Here, we used a natural killer (NK) cell-initiating cell assay (NK-IC) where CD34+ cells are cultured on AFT024 stromal cells in media containing IL15, IL7, and other defined cytokines for 2–4 weeks. Under these conditions, both CB and human ES cell-derived cells give rise to lymphoid cells (NK cells) with over 40% CD45+CD56+ cells. Under alternative culture conditions, CD3+ T cells can also be produced from CD34+ human ES cell-derived cells. Therefore, CD34+ cells derived from human ES cells represent both myeloid and lymphoid precursor cells. Since it is not possible to define a HSC population based solely on in vitro assays, we have examined the potential for human ES cell-derived hematopoietic cells to engraft in sublethally irradiated NOD/SCID mice. Detection of scid-repopulating cells (SRCs) are considered a better surrogate for HSCs. Bone marrow, peripheral blood, and splenocytes were examined for human CD34+ and CD45+ cells 3–6 months after injection of human ES cell-derived blood cells. PCR for human chromosome 17-specific alpha-satellite DNA was also done to confirm the presence of human cells in all mice showing evidence of engraftment. We consistently find stable engraftment with 0.5–3% human CD45+ cells in the bone marrow of these mice. To better define these cells as HSCs, secondary transplants also demonstrate stable engraftment. Importantly, no teratomas are demonstrated in mice injected with differentiated human ES cells. These results demonstrate that HSCs with long-term engraftment and multi-lineage potential can be routinely and efficiently generated from human ES cells.

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Prospective Isolation and Functional Characterization of Human Bone Marrow-Derived Hematopoietic Stem Cell-Supportive Mesenchymal Stromal Cells.

Blood ◽

10.1182/blood.v116.21.3852.3852 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3852-3852

Author(s):

Yoshikazu Matsuoka ◽

Yutaka Sasaki ◽

Masaya Takahashi ◽

Ryusuke Nakatsuka ◽

Yasushi Uemura ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Scid Mice ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cd34 Cells

Abstract Abstract 3852 (Background) The identification of human CD34-negative (CD34−) SCID-repopulating cells (SRCs) provide a new concept for the hierarchy in the human HSC compartment (Blood 101:2924, 2003). Recently, we succeeded to highly purify these CD34-SRCs using 18 lineage specific antibodies (Blood 114:336, 2009). It has been suggested that human hematopoietic stem cell (HSC)-supportive microenvironment exist in the bone marrow (BM), which play a pivotal role in the maintenance of self-renewal capacity and dormancy of primitive HSCs. It was reported that osteoblasts and vascular endothelial cells played an important role to organize HSC niches. However, whether mesenchymal stromal cells (MSCs) contribute to organize HSC niches or not is not clearly understood, because MSCs are heterogeneous population. Therefore, it is important to clarify their origin and functional characteristics. (Objectives) The aim of this study was to prospectively isolate/identify human BM-derived MSCs and investigate their functional characteristics including HSC-supportive abilities. (Results) First, human BM-derived Lin−CD45− cells were subdivided into 4 fractions according to their expression levels of CD271 and SSEA-4 by FACS. We succeeded to isolate 3 MSC lines from these 4 fractions, including CD271+/−SSEA-4+/− cells. Approximately 1 out of 6 CD271+SSEA-4+ (DP) cells could form MSC-derived colony. These DP cells-derived MSCs could differentiate into osteoblasts and chondrocytes, but could not differentiate into adipocytes. In contrast, CD271+SSEA-4− cells and CD271−SSEA-4− cells-derived MSCs could differentiate into three lineages. Then, we assessed CD34− SRC-supportive activity of these 3 MSC lines. First, certain numbers of 18Lin−CD34− cells were cocultured with 3 MSC lines for 1 week, respectively. Next recovered cells were transplanted into NOD/SCID mice by intra-bone marrow injection (IBMI) to investigate SCID-repopulating cell (SRC) activity. After 8 weeks, the highest CD45+ human cell engraftments (0.1 % to 32.4 %, median 8.6 %) were observed in mice received 18Lin−CD34− cells cocultued with DP cells-derived MSCs. As recently reported (Cell Stem Cell 1:635,2007), Lin−CD34+CD38−CD45RA−CD90+ cells contained most primitive human CD34+CD38− SRCs. Very interestingly, these Lin−CD34+CD38−CD45RA−CD90+ cells were generated from the above mentioned cocultures. In order to evaluate SRC activity of these Lin−CD34+CD38−CD45RA−CD90+ cells generated from 18Lin−CD34− cells in vitro, Lin−CD34+CD38−CD45RA−CD90+/− cells were sorted by FACS and then transplanted into NOD/SCID mice by IBMI. Eight weeks after transplantation, 8 out of 16 mice received Lin−CD34+CD38−CD45RA−CD90+ cells (400 to 3000 cells/mouse) were repopulated with human cells. In contrast, only 2 out of 16 mice received Lin−CD34+CD38− CD45RA−CD90− cells (1500 to 7000 cells/mouse) were repopulated. These results demonstrated that human CB-derived 18Lin−CD34− cells could generate very primitive CD34+CD38− SRCs in vitro. (Conclusion) These findings elucidate that human BM-derived DP cell-derived MSCs can support very primitive human CB-derived CD34− SRCs in vitro and suggest that these CD34− SRCs seem to be more immature than CD34+CD38− SRCs. These results provide a new concept of hierarchy in the human primitive HSC compartment. Disclosures: No relevant conflicts of interest to declare.

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Inducible SBDS Deficiency Impairs Bone Marrow Niche Function to Engraft Donor Hematopoietic Stem Cell after Transplantation

Blood ◽

10.1182/blood-2019-121763 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3199-3199

Author(s):

Ji Zha ◽

Lori Kunselman ◽

Hongbo Michael Xie ◽

Brian Ennis ◽

Jian-Meng Fan ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Mouse Model ◽

Board Of Directors ◽

Hematopoietic Stem Cell ◽

Graft Failure ◽

Advisory Committees ◽

Hematopoietic Stem ◽

Bm Niche ◽

Deficient Mice

Hematopoietic stem cell (HSC) transplantation (HSCT) is required for curative therapy for patients with high-risk hematologic malignancies, and a number of non-malignant disorders including inherited bone marrow failure syndromes (iBMFS). Strategies to enhance bone marrow (BM) niche capacity to engraft donor HSC have the potential to improve HSCT outcome by decreasing graft failure rates and enabling reduction in conditioning intensity and regimen-associated complications. Several studies in animal models of iBMFS have demonstrated that BM niche dysfunction contributes to both the pathogenesis of iBMFS, as well as impaired graft function after HSCT. We hypothesize that such iBMFS mouse models are useful tools for discovering targetable niche elements critical for donor engraftment after HSCT. Here, we report the development of a novel mouse model of Shwachman-Diamond Syndrome (SDS) driven by conditional Sbds deletion, which demonstrates profound impairment of healthy donor hematopoietic engraftment after HSCT due to pathway-specific dysfunctional signaling within SBDS-deficient recipient niches. We first attempted to delete Sbds specifically in mature osteoblasts by crossing Sbdsfl/flmice with Col1a1Cre+mice. However, the Col1a1CreSbdsExc progenies are embryonic lethal at E12-E15 stage due to developmental musculoskeletal abnormalities. Alternatively, we generated an inducible SDS mouse model by crossing Sbdsfl/flmice with Mx1Cre+ mice, and inducing Sbds deletion in Mx1-inducible BM hematopoietic and osteolineage niche cells by polyinosinic-polycytidilic acid (pIpC) administration. Compared with Sbdsfl/flcontrols, Mx1CreSbdsExc mice develop significantly decreased platelet counts, an inverted peripheral blood myeloid/lymphoid cell ratio, and reduced long-term HSC within BM, consistent with stress hematopoiesis seen in BMF and myelodysplastic syndromes. To assess whether inducible SBDS deficiency impacts niche function to engraft donor HSC, we transplanted GFP+ wildtype donor BM into pIpC-treated Mx1CreSbdsExc mice and Sbdsfl/flcontrols after 1100 cGy of total body irradiation (TBI). Following transplantation, Mx1CreSbdsExc recipient mice exhibit significantly higher mortality than controls (Figure 1). The decreased survival was related to primary graft failure, as Mx1CreSbdsExc mice exhibit persistent BM aplasia after HSCT and decreased GFP+ reconstitution in competitive secondary transplantation assays. We next sought to identify the molecular and cellular defects within BM niche cells that contribute to the engraftment deficits in SBDS-deficient mice. We performed RNA-seq analysis on the BM stromal cells from irradiated Mx1CreSbdsExc mice versus controls, and the results revealed that SBDS deficiency in BM niche cells caused disrupted gene expression within osteoclast differentiation, FcγR-mediated phagocytosis, and VEGF signaling pathways. Multiplex ELISA assays showed that the BM niche of irradiated Mx1CreSbdsExc mice expresses lower levels of CXCL12, P-selectin and IGF-1, along with higher levels of G-CSF, CCL3, osteopontin and CCL9 than controls. Together, these results suggest that poor donor HSC engraftment in SBDS-deficient mice is likely caused by alterations in niche-mediated donor HSC homing/retention, bone metabolism, host monocyte survival, signaling within IGF-1 and VEGF pathways, and an increased inflammatory state within BM niches. Moreover, flow cytometry analysis showed that compared to controls, the BM niche of irradiated Mx1CreSbdsExc mice contained far fewer megakaryocytes, a hematopoietic cell component of BM niches that we previously demonstrated to be critical in promoting osteoblastic niche expansion and donor HSC engraftment. Taken together, our data demonstrated that SBDS deficiency in BM niches results in reduced capacity to engraft donor HSC. We have identified multiple molecular and cellular defects in the SBDS-deficient niche contributing to this phenotype. Such niche signaling pathway-specific deficits implicate these pathways as critical for donor engraftment during HSCT, and suggest their potential role as targets of therapeutic approaches to enhance donor engraftment and improve HSCT outcome in any condition for which HSCT is required for cure. Disclosures Olson: Merck: Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria.

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Clinical stem-cell sources contain CD8+CD3+ T-cell receptor–negative cells that facilitate bone marrow repopulation with hematopoietic stem cells

Blood ◽

10.1182/blood-2007-02-076000 ◽

2008 ◽

Vol 111 (3) ◽

pp. 1735-1738 ◽

Cited By ~ 10

Author(s):

Stephanie Bridenbaugh ◽

Linda Kenins ◽

Emilie Bouliong-Pillai ◽

Christian P. Kalberer ◽

Elena Shklovskaya ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

T Cell ◽

T Cell Receptor ◽

Cell Receptor ◽

Hematopoietic Stem ◽

Cd8 Cells ◽

Human Cd34 ◽

Cd34 Cells

Abstract Clinical observations in patients undergoing bone marrow transplantation implicate the involvement of CD8+ cells in promoting the stem-cell engraftment process. These findings are supported by mouse transplant studies, which attributed the engraftment-facilitating function to subpopulations of murine CD8+ cells, but the analogous cells in humans have not been identified. Here, we report that clinical stem-cell grafts contain a population of CD8α+CD3ϵ+ T-cell receptor– negative cells with an engraftment facilitating function, named candidate facilitating cells (cFCs). Purified cFC augmented human hematopoiesis in NOD/SCID mice receiving suboptimal doses of human CD34+ cells. In vitro, cFCs cocultured with CD34+ cells increased hematopoietic colony formation, suggesting a direct effect on clonogenic precursors. These results provide evidence for the existence of rare human CD8+CD3+TCR− cells with engraftment facilitating properties, the adoptive transfer of which could improve the therapeutic outcome of stem-cell transplantation.

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UM171 Regulates the Hematopoietic Differentiation of Human Acquired Aplastic Anemia-Derived Induced Pluripotent Stem Cells

Blood ◽

10.1182/blood-2019-127466 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2500-2500

Author(s):

Tellechea Maria Florencia ◽

Flavia S. Donaires ◽

Tiago C. Silva ◽

Lilian F. Moreira ◽

Yordanka Armenteros ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Pluripotent Stem Cells ◽

Patient Specific ◽

Hematopoietic Differentiation ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Induced Pluripotent

Aplastic anemia (AA) is characterized by a hypoplastic bone marrow associated with low peripheral blood counts. In acquired cases, the immune system promotes hematopoietic stem and progenitor cell (HSPC) depletion by the action of several pro-inflammatory Th1 cytokines. The current treatment options for severe cases consist of sibling-matched allogeneic hematopoietic stem cell transplantation (HSCT) and immunosuppressive therapy (IST) with anti-thymocyte globulin, cyclosporine, and eltrombopag. However, most patients are not eligible for HSCT and, although about 85% of patients respond to IST with eltrombopag, a proportion of patients eventually relapse, requiring further therapies. Failure to respond adequately to immunosuppression may be attributed to the scarcity of HSPCs at the time of diagnosis. Induced pluripotent stem cells (iPSCs) are potentially an alternative source of patient-specific hematopoietic cells. Patient-specific HSPCs derived from in vitro iPSC differentiation may serve as a tool to study the disease as well as a source of hematopoietic tissue for cell therapies. The pyrimidoindole molecule UM171 induces ex vivo expansion of HSCs of human cord and peripheral blood and bone marrow, but the pathways modulated by this molecule are not well understood. Here we evaluated the hematopoietic differentiation potential of iPSCs obtained from patients with acquired AA. We further determined the effects of UM171 on this differentiation process. First, we derived iPSCs from 3 patients with acquired AA after treatment (1 female; average age, 31 years; 2 partial responders, 1 complete responder) and 3 healthy subjects (3 females; average age, 61 years) and induced differentiation in vitro through the embryoid body system in cell feeder and serum-free medium supplemented with cytokines. The hematopoietic differentiation of healthy-iPSCs yielded 19% ± 8.1% (mean ± SEM) of CD34+cells after 16 days in culture, in contrast with 11% ± 4.9% of CD34+cells obtained from the differentiation of AA-iPSCs, which corresponds to a 1.7-fold reduction in CD34+cell yield. The total number of erythroid and myeloid CFUs was lower in the AA-iPSC group as compared to healthy-iPSCs (12±4.2 vs.24±7.2; respectively; p<0.03). These findings suggest that erythroid-derived AA-iPSC have an intrinsic defect in hematopoietic differentiation. Next, we tested whether UM171 modulated hematopoietic differentiation of AA-iPSCs. We found that UM171 significantly stimulated the differentiation of both healthy and AA-iPSCs. In the healthy-iPSC group, the percentage of CD34+cells was 1.9-fold higher when treated with UM171 compared to controls treated with DMSO (37% ± 7.8% vs.19% ± 8.1%; respectively; p<0.03) and in AA-iPSCs the increase was 3.9-fold (45% ± 11% vs. 11% ± 4.9%; p<0.07). The clonogenic capacity of progenitors to produce erythroid and myeloid colonies also was augmented in both groups in comparison to DMSO (28±11 vs. 23±7.2) for healthy-iPSCs and for AA-iPSCs (23±8.5 vs. 12±4.2, p<0.06). We then investigated the molecular pathways influenced by UM171. The transcriptional profile of differentiated CD34+cells showed that UM171 up-regulated genes involved in early hematopoiesis from mesoderm (BRACHYURY and MIXL1) and primitive streak specification (APELA and APLNR), to hemangioblasts and primitive hematopoietic progenitor commitment (TDGF1, SOX17, and KLF5). We also observed the up-regulation of pro-inflammatory NF-kB activators (MAP4K1, ZAP70, and CARD11) and the anti-inflammatory gene PROCR, a marker of cultured HSCs and an NF-kB inhibitor. This balanced network has been previously suggested to be modulated by UM171 (Chagraoui et. al. Cell Stem Cell 2019). Taken together, our results showed that acquired AA-iPSCs may have intrinsic defects that impair hematopoietic differentiation in vitro. This defect may be atavic to the cell or, alternatively, the consequence of epigenetic changes in erythroid precursors provoked by the immune attack. In addition, our findings demonstrate that UM171 significantly stimulate the hematopoietic differentiation of AA-iPSCs and identified a novel molecular mechanism for UM171 as an enhancer of early hematopoietic development programs. These observations may be valuable for improving the achievement of de novo hematopoietic cells. Disclosures No relevant conflicts of interest to declare.

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Abstract 156: Angiotensin Ii Regulates Hematopoietic Stem Cell Proliferation, Differentiation, and Engraftment Efficacy

Hypertension ◽

10.1161/hyp.62.suppl_1.a156 ◽

2013 ◽

Vol 62 (suppl_1) ◽

Author(s):

Seungbum Kim ◽

Edward W Scott ◽

Mohan K Raizada

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Angiotensin Ii ◽

Hematopoietic Stem Cell ◽

Peripheral Blood ◽

Ang Ii ◽

Hematopoietic Stem ◽

Proliferation And Differentiation ◽

Important Regulator

INTRODUCTION: Emerging evidence indicates that differentiation and mobilization of hematopoietic stem cell (HSC) are critical in the development and establishment of hypertension-linked vascular pathophysiology. This, coupled with the intimate involvement of a hyperactive renin-angiontensin system in hypertension, led us to propose the hypothesis that chronic angiotensin II (Ang II) infusion would regulate HSC proliferation and differentiation at the bone marrow level. METHODS: 1) Ang II was chronically infused into C57BL6 mice using mini-osmotic pumps (1500ng/kg/min) for 3 weeks. This resulted in an increase in MAP of 45mmHg. Bone marrow, peripheral blood and splenocytes from control and Ang II-treated mice were analyzed using FACS. 2) 0.5-3 X10 4 GFP + Sca-1 + , c-Kit + , Lin - (SKL) HSC were pre-incubated with Ang II for 24h in vitro (100μg/ml), rinsed and injected into lethally irradiated C57BL6 mice. Donor derived GFP + cells were analyzed by FACS and histology to evaluate engraftment efficiency. RESULTS: We observed a 32% decrease of HSCs in the bone marrow of Ang II treated mice. In addition, there was an 29-52% increase in the number of CX3CR1+/Gr-1- monocyte in the peripheral blood and spleen. These changes in HSC and myeloid cells were blocked by co-treatment of Losartan (60mg/kg/day, ip injection). Next, we investigated if Ang II affects HSC homing and engraftment efficacy, which are critical steps in successful bone marrow transplantation. We observed a significant delay of the homing GFP+ SKL cells that were pre-treated with Ang II in lethally irradiated recipient mice. In addition, the SKL cells treated with Ang II failed to efficiently engraft to the innate osteoblastic niche. Consistent with this observation, colony formation unit-Spleen (CFU-S) in the Ang II infused recipients was reduced to 65% compared to control mice. CONCLUSION: These observations demonstrate that hypertension induced by chronic Ang II infusion significantly impairs the engraftment ability of HSC in the bone marrow, which appears to be mediated by the AT1R on HSC and that Ang II accelerates HSC differentiation into myeloid lineage. These multifaceted roles of Ang II indicate that Ang II acts as an important regulator of HSC in the bone marrow.

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The Roles of Pten in Hematopoietic Stem Cell Regulation and Leukemogenesis.

Blood ◽

10.1182/blood.v106.11.468.468 ◽

2005 ◽

Vol 106 (11) ◽

pp. 468-468

Author(s):

Jiwang Zhang ◽

Xi He ◽

Sach Jayasinghe ◽

Jason Ross ◽

Jeff Haug ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

B Lymphocyte ◽

Human Tumor ◽

Leukemic Cells ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Cell Counts

Abstract Pten was the first phosphatase identified as a tumor suppressor and one of the most frequently mutated genes involved in human tumor/cancer. Pten, involved in regulation of both PI3K/Akt and MEK/Erk activity, is downstream of growth factor, cytokine, integrin and cadherin signaling pathways and therefore plays important roles in cell growth, survival, differentiation, metabolism and migration. Although Pten mutation is not common in leukemic cells, phosphorylated Pten (p-Pten), which represents the inactive form of Pten, has been observed in a majority of acute myeloid leukemias that are associated with poor clinical outcomes. To explore the role of Pten in hematopoietic stem cell (HSC) regulation and leukemogenesis, we generated an interferon-inducible Pten knockout mouse by crossing Mx1Cre mice with Ptenloxp mice. All of the mutant mice developed myeloproliferative disorder characterized by increased peripheral white blood cell counts, hyperproliferative macrophages and granulocytes in bone marrow and spleen, and multiple tissue infiltration by myeloid cells. The HSC number was decreased in the bone marrow but mobilized and expanded in the spleen. Extra-medullar hematopoiesis was shown by dramatically increased myeloid and erythroid progenitors in the spleen. B lymphocyte differentiation was blocked at the common lymphoid progenitor stage, while the T cell number was increased in all hematopoietic tissues. Compared to wild type, Pten mutant HSCs and progenitor cells were highly proliferative, forming larger colonies in an in vitro culture study. However, Pten mutant HSCs showed reduced competency in repopulation assay after in vivo bone marrow transplantation. Our study demonstrates that Pten plays important roles in restricting HSC activation, proliferation and mobilization. Pten also plays a role in HSC lineage decision by favoring myeloid differentiation at the expense of B lymphocyte lineage.

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Screen for Small Molecules Capable of Expanding Human Hematopoietic Stem Cell Ex Vivo

Blood ◽

10.1182/blood.v118.21.1919.1919 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1919-1919

Author(s):

Iman Hatem Fares ◽

Jalila Chagraoui ◽

Jana Krosl ◽

Denis-Claude Roy ◽

Sandra Cohen ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Fold Increase ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Abstract 1919 Hematopoietic stem cell (HSC) transplantation is a life saving procedure whose applicability is restricted by the lack of suitable donors, by poor responsiveness to mobilization regimens in preparation of autologous transplantations, by insufficient HSC numbers in individual cord blood units, and by the inability to sufficiently amplify HSCs ex vivo. Characterization of Stemregenin (SR1), an aryl hydrocarbon receptor (AHR) antagonist that promotes HSC expansion, provided a proof of principle that low molecular weight (LMW) compounds have the ability to promote HSC expansion. To identify novel putative agonists of HSC self-renewal, we initiated a high throughput screen (HTS) of a library comprising more than 5,000 LMW molecules using the in vitro maintenance of the CD34+CD45RA- phenotype as a model system. Our study was based on the fact that mobilized peripheral blood-derived CD34+CD45RA- cells cultured in media supplemented with: stem cell factor, thrombopoietin, FLT3 ligand and interleukin 6, would promote the expansion of mononuclear cells (MNC) concomitant with a decrease in CD34+CD45RA- population and HSC depletion. LMW compounds preventing this loss could therefore act as agonists of HSC expansion. In a 384-well plate, 2000 CD34+cells were initially cultured/well in 50μl medium comprising 1μM test compounds or 0.1% DMSO (vehicle). The proportions of CD34+CD45RA− cells were determined at the initiation of experiment and after a 7-day incubation. Six of 5,280 LMW compounds (0.11%) promoted CD34+CD45RA− cell expansion, and seventeen (0.32%) enhanced differentiation as determined by the increase in proportions of CD34−CD45RA+ cells compared to control (DMSO). The 6 LMW compounds promoting expansion of the CD34+CD45RA− cell population were re-analyzed in a secondary screen. Four out of these 6 molecules suppressed the transcriptional activity of AHR, suggesting that these compounds share the same molecular pathway as SR1 in stimulating HSC expansion, thus they were not further characterized. The remaining 2 compounds promoted, similar to SR1 or better, a 10-fold and 35-fold expansion of MNC during 7 and 12-day incubations, respectively. The expanded cell populations comprised 65–75% of CD34+ cells compared to 12–30% determined for DMSO controls. During 12-day incubation with these compounds, the numbers of CD34+ cells increased ∼25-fold over their input values, or ∼ 6-fold above the values determined for controls. This expansion of CD34+ cells was associated with a ∼5-fold increase in the numbers of multilineage CFC (granulocyte, erythroid, monocyte, and megakaryocyte, or CFU-GEMM) compared to that found in DMSO control cultures. The ability of the 2 newly identified compounds to expand functional HSCs is currently being evaluated in vivo usingimmunocompromised mice. In conclusion, results of our initial screen suggest that other mechanism, besides inhibition of AhR, are at play for expansion of human HSC. Disclosures: No relevant conflicts of interest to declare.

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Functional Significance of MPL Expression in the Human Primitive Hematopoietic Stem Cell Compartment

Blood ◽

10.1182/blood.v120.21.1195.1195 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1195-1195 ◽

Cited By ~ 2

Author(s):

Masaya Takahashi ◽

Yoshikazu Matsuoka ◽

Ryuji Iwaki ◽

Ryusuke Nakatsuka ◽

Tatsuya Fujioka ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Human Cell ◽

Functional Significance ◽

The Other ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Nog Mice ◽

Cell Repopulation

Abstract Abstract 1195 Background: In the murine primitive hematopoietic stem cell (HSC) compartment, MPL/thrombopoietin (THPO) signaling plays an important role for the maintenance of adult quiescent HSCs. However, the role of MPL/THPO signaling in the human primitive HSC compartment has not yet been elucidated. We have previously identified very primitive human cord blood (CB)-derived CD34-negative (CD34−) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) using the intra-bone marrow injection (IBMI) method (Blood 2003:101;2924). Recently, we developed a high-resolution purification method for the primitive CD34− SRCs using 18 lineage (Lin)-specific antibodies (Exp Hematol 2011:39:203). In this study, we investigated the functional significance of the MPL expression in the human primitive CD34− SRCs (HSCs). Materials and Methods: First, we sorted 18Lin−CD34+/−MPL+/−cells by FACS. Thereafter, these four fractions of cells were analyzed for their hematopoietic colony-forming capacities (CFCs), the maintenance/production of CD34+ cells in cocultures with human bone marrow-derived mesenchymal stromal cells (DP MSCs) (Blood 2010:116:1575). Finally, these four fractions of cells were transplanted by IBMI into NOD/Shi-scid/IL-2R ƒÁcnull (NOG) mice to investigate their long-term (LT) repopulating capacities. We performed primary, secondary, and tertiary transplantations for up to 18 months. Results: The CFCs of highly purified 18Lin−CD34−cells were quite unique, since they yielded mainly erythroid-bursts (BFU-E) and erythro/megakaryocytes-containing mixed colonies (CFU-EM). Interestingly, they showed very weak myeloid CFCs. Moreover, 200 18Lin−CD34−MPL+cells yielded about 150 colonies, consisting of 20% BFU-E, 20% colony-forming unit-megakaryocytes, and 60% CFU-EM in the presence of 10% platelet-poor plasma, THPO, IL-3, and erythropoietin. The phenotypic and functional characteristics of the 18Lin−CD34+/−MPL+/−cells were further investigated by cocultures with DP MSCs. After 7 days of coculturing the 18Lin−CD34+MPL+/−cells, the total number of cells was observed to expand by about 500 times, 40 % of which consisted of CD34+ cells. On the other hand, the 18Lin−CD34−MPL+/−cells expanded only by about 100 times, 10 to 20% of which consisted of CD34+ cells. Interestingly, the lineage differentiation potentials of the observed 18Lin−CD34+/−MPL+/−cells were significantly different. The percentages of myeloid cells generated from the 18Lin−CD34+MPL+/−cells were significantly greater than those of the 18Lin−CD34−MPL+/−cells. On the contrary, 18Lin−CD34−MPL+cells generated significantly higher percentages of CD41+ cells compared to the other fractions. These observations suggested that the differentiation potentials of 18Lin−CD34+/−MPL+/− cells were different and that these four cell populations contained distinct classes of hematopoietic progenitors as well as HSCs. We next investigated the SRC activities of the 18Lin−CD34+/−MPL+/−cells using NOG mice. In the primary recipient mice, all 18 mice (9 for each cell type) received CD34+MPL+/−SRCs were highly repopulated with human CD45+ cells. On the other hand, 11/13 mice receiving CD34−MPL+SRCs and 12/12 mice receiving CD34−MPL−SRCs showed human cell repopulation. Interestingly, these CD34+/−MPL+/−SRCs showed different repopulation kinetics. The CD34+MPL+ SRCs showed a rapid and sustained repopulating pattern (Fig.1-A). In contrast, the repopulation rates in the mice receiving CD34+MPL− and CD34−MPL+/− SRCs gradually increased, and thereafter reached a high level of repopulation at 18–24 weeks after the transplantation (Fig.1-A, B). All of the primary recipient mice that received CD34+MPL+/− SRCs showed a secondary repopulating capacity. However, only the mice that received CD34+MPL− SRCs showed a tertiary repopulating capacity. Very interestingly, the primary recipient mice that received CD34−MPL− SRCs showed a distinct secondary repopulating capacity. Tertiary transplantation experiments in these mice are now underway in our laboratory. Conclusion: These results clearly indicated that both the CD34+/− SRCs not expressing MPL sustained a LT (> 1 year) human cell repopulation in NOG mice. Therefore, these findings suggest that the functional significance of the MPL expression in the human primitive HSCs is different from that in murine primitive HSCs. Disclosures: No relevant conflicts of interest to declare.

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Culture from human bone marrow of blast progenitor cells with an extensive proliferative capacity

Blood ◽

10.1182/blood.v69.3.804.804 ◽

1987 ◽

Vol 69 (3) ◽

pp. 804-808 ◽

Cited By ~ 39

Author(s):

SD Rowley ◽

SJ Sharkis ◽

C Hattenburg ◽

LL Sensenbrenner

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Human Bone ◽

Human Bone Marrow ◽

Vitro Assay ◽

Hematopoietic Stem ◽

Blast Cells ◽

Normal Human

Abstract The investigation of human hematopoiesis is limited by the lack of an in vitro assay for the most primitive hematopoietic stem cell. In this report, we describe the culture from normal human bone marrow of unique colonies of morphologically immature cells with scanty, agranular, cytoplasm and a primitive nucleus with nucleoli. These “blast” cells demonstrate a significant ability for the generation of secondary colonies of multiple lineages, including additional blast cell colonies. These colonies are detected at various times during the culture period of up to 28 days. Neither the time of appearance in primary culture nor any feature of the morphological appearance of the blast cells is correlated with replating ability or the differentiation pathway followed. The progenitor cell giving rise to these colonies may represent the earliest pluripotent hematopoietic stem cell yet grown in culture.

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