Cytotoxic lymphocyte maturation factor (interleukin 12) is a synergistic growth factor for hematopoietic stem cells.

The recently cloned cytotoxic lymphocyte maturation factor (interleukin 12 [IL-12]) has been described as a growth factor for mature lymphocytes. The present study investigated whether purified recombinant murine IL-12 (rMuIL-12) also could affect the proliferation of primitive bone marrow progenitor cells. Using a population of Lin-Sca-1+ murine bone marrow stem cells, we now demonstrate that IL-12 is a potent synergistic factor for primitive hematopoietic stem cells. The synergy of IL-12 was observed in single-cell cloning assays, demonstrating that its effects are directly mediated. Specifically, IL-12 enhanced stem cell factor-induced myelopoiesis of Lin-Sca-1+ cells sevenfold, and synergized with colony-stimulating factors (CSFs) to induce proliferation of Lin-Sca-1+ stem cells. IL-12 increased the number of responding progenitor cells as well as the size of the colonies formed. IL-12 also increased colony formation of high proliferative potential colony-forming cells with multiple CSF combinations. The effects of IL-12 were concentration dependent with a 50% effective dose of 2-20 and 20-200 ng/ml, resulting in maximum stimulation. Furthermore, a neutralizing anti-IL-12 antibody blocked the synergistic effects of rMuIL-12. In addition, IL-12 was found to have synergistic effects on more committed bone marrow progenitors as well. Our results therefore suggest that in addition to being a potent lymphopoietic stimulator, IL-12 is a regulator of the growth of hematopoietic stem cells and their myeloid progeny.

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Hemangiogenic Role of ELF4 in Bone Marrow Post Myeloablation.

Blood ◽

10.1182/blood.v108.11.1783.1783 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1783-1783

Author(s):

Mariela Sivina ◽

Takeshi Yamada ◽

Natalie Dang ◽

H. Daniel Lacorazza

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Endothelial Cells ◽

Hematopoietic Stem Cells ◽

Blood Vessels ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Bone Marrow Cells ◽

Hematopoietic Stem ◽

Huvec Cells

Abstract Bone marrow suppression is an important cause of death in patients exposed to radiation or in cancer patients treated with conventional chemotherapeutic agents. Myeloablative treatments (i.e. 5-fluorouracil administration) lead to apoptosis of blood forming cells and to regression of blood vessels in bone marrow. It is well known that hematological recovery post-bone marrow insult depends on the capacity of hematopoietic stem cells to regenerate the entire hematopoietic system, however, the transcriptional machinery involved in the regeneration of sinusoidal blood vessels in bone marrow from endothelial progenitor cells is largely unknown. Endothelial cells express the Tie2 receptor tyrosine kinase (a.k.a. Tek), which is involved in the angiogenic remodeling and vessel stabilization. Gene targeting of Tie2 showed that it is not required for differentiation and proliferation of definitive hematopoietic lineages in the embryo although Tie2 is needed during postnatal bone marrow hematopoiesis. ELF is a subgroup of the ETS family of transcription factors composed by ELF1, ELF2 (a.k.a. NERF), ELF3, ELF4 (a.k.a. MEF) and ELF5. ELF1 and ELF2 have been shown to regulate Tie2 expression in vitro. Recently we showed that ELF4 modulates the exit of hematopoietic stem cells (HSC) from quiescence (Lacorazza et al., Cancer Cell2006, 9:175–187). Given the high homology between ELF1 and ELF4 and the same origin of HSC and endothelial progenitor cells, we hypothesize that ELF4 regulates proliferation and Tie2 expression of endothelial cells. We used a luciferase gene reporter system in COS-7 and HEK cells to examine the capacity of ELF proteins to activate Tie2. ELF4 is the strongest activator of Tie2 expression following the hierarchy ELF4>ELF1>ELF2 variant 1>ELF2 variant 2. Site directed mutagenesis of each of the five ETS-binding sites (EBS) present in the Tie2 promoter shows that ELF4 binds preferentially to EBS 1, 3 and 5. Binding of ELF4 to the Tie2 promoter was confirmed by chromatin immunoprecipitation and EMSA. Although Elf1 gene expression is essentially normal in Elf4−/− bone marrow cells collected after 5-FU treatment, we detected diminished Tie2 expression compared to Elf4+/+ bone marrow cells. The association of this effect to human endothelial cells derived from umbilical cord (HUVEC cells) was investigated. All-trans retinoic acid (ATRA) and vascular-endothelial growth factor (VEGF) induced ELF4 expression in HUVEC cells in a dose and time dependent manner which was followed by increased Tie2 expression, suggesting that expression of ELF4 is modulated by angiogenic signals. Moreover, endothelial cells treated with ATRA showed rapid wound colonization in a wound assay. Expression of the pan-endothelial marker MECA-32 was determined by immunohistochemistry to correlate Tie2 with the regeneration of blood vessels: myeloablated Elf4−/− femurs exhibited a reduction of MECA-32 positive arterioles. Finally, temporal and spatial expression of Tie2 during hematological recovery post ablation was measured in bone marrow using transgenic Tie2-LacZ mice crossed to Elf4−/− mice. Collectively, our data suggests that ELF4 regulates Tie2 expression in endothelial cells but most importantly their proliferative capacity in response to angiogenic signals.

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Reversibility of CD34 expression on human hematopoietic stem cells that retain the capacity for secondary reconstitution

Blood ◽

10.1182/blood-2002-01-0025 ◽

2003 ◽

Vol 101 (1) ◽

pp. 112-118 ◽

Cited By ~ 71

Author(s):

Mo A. Dao ◽

Jesusa Arevalo ◽

Jan A. Nolta

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cell Surface ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Cd34 Cells ◽

Cd34 Expression

Abstract The cell surface protein CD34 is frequently used as a marker for positive selection of human hematopoietic stem/progenitor cells in research and in transplantation. However, populations of reconstituting human and murine stem cells that lack cell surface CD34 protein have been identified. In the current studies, we demonstrate that CD34 expression is reversible on human hematopoietic stem/progenitor cells. We identified and functionally characterized a population of human CD45+/CD34− cells that was recovered from the bone marrow of immunodeficient beige/nude/xid (bnx) mice 8 to 12 months after transplantation of highly purified human bone marrow–derived CD34+/CD38− stem/progenitor cells. The human CD45+ cells were devoid of CD34 protein and mRNA when isolated from the mice. However, significantly higher numbers of human colony-forming units and long-term culture-initiating cells per engrafted human CD45+ cell were recovered from the marrow of bnx mice than from the marrow of human stem cell–engrafted nonobese diabetic/severe combined immunodeficient mice, where 24% of the human graft maintained CD34 expression. In addition to their capacity for extensive in vitro generative capacity, the human CD45+/CD34− cells recovered from thebnx bone marrow were determined to have secondary reconstitution capacity and to produce CD34+ progeny following retransplantation. These studies demonstrate that the human CD34+ population can act as a reservoir for generation of CD34− cells. In the current studies we demonstrate that human CD34+/CD38− cells can generate CD45+/CD34− progeny in a long-term xenograft model and that those CD45+/CD34− cells can regenerate CD34+ progeny following secondary transplantation. Therefore, expression of CD34 can be reversible on reconstituting human hematopoietic stem cells.

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In vivo and in vitro stem cell function of c-kit- and Sca-1-positive murine hematopoietic cells

Blood ◽

10.1182/blood.v80.12.3044.bloodjournal80123044 ◽

1992 ◽

Vol 80 (12) ◽

pp. 3044-3050 ◽

Cited By ~ 2

Author(s):

S Okada ◽

H Nakauchi ◽

K Nagayoshi ◽

S Nishikawa ◽

Y Miura ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Cell Function ◽

Bone Marrow Cells ◽

Synergistic Effects ◽

Single Cells ◽

Hematopoietic Stem

c-kit is expressed on hematopoietic stem cells and progenitor cells, but not on lymphohematopoietic differentiated cells. Lineage marker- negative, c-kit-positive (Lin-c-kit+) bone marrow cells were fractionated by means of Ly6A/E or Sca-1 expression. Lin-c-kit+Sca-1+ cells, which consisted of 0.08% of bone marrow nucleated cells, did not contain day-8 colony-forming units-spleen (CFU-S), but 80% were day-12 CFU-S. One hundred cells rescued the lethally irradiated mice and reconstituted hematopoiesis. On the other hand, 2 x 10(3) of Lin-c- kit+Sca-1- cells formed 20 day-8 and 11 day-12 spleen colonies, but they could not rescue the lethally irradiated mice. These data indicate that Lin-c-kit+Sca-1+ cells are primitive hematopoietic stem cells and that Sca-1-cells do not contain stem cells that reconstitute hematopoiesis. Lin-c-kit+Sca-1+ cells formed no colonies in the presence of stem cell factor (SCF) or interleukin-6 (IL-6), and only 10% of them formed colonies in the presence of IL-3. However, approximately 50% of them formed large colonies in the presence of IL-3, IL-6, and SCF. Moreover, when single cells were deposited into culture medium by fluorescence-activated cell sorter clone sorting system, 40% of them proliferated on a stromal cell line (PA-6) and proliferated for more than 2 weeks. In contrast, 15% of the Lin-c- kit+Sca-1-cells formed colonies in the presence of IL-3, but no synergistic effects were observed in combination with SCF plus IL-6 and/or IL-3. Approximately 10% proliferated on PA-6, but most of them degenerated within 2 weeks. The population ratio of c-kit+Sca-1+ to c-kit+Sca-1- increased 2 and 4 days after exposure to 5-fluorouracil (5-FU). These results are consistent with the relative enrichment of highly proliferative colony-forming cells by 5-FU. These data show that, although c-kit is found both on the primitive hematopoietic stem cells and progenitors, Sca-1+ cells are more primitive and respond better than Sca-1- cells to a combination of hematopoietic factors, including SCF and stromal cells.

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Characterization of Hematopoietic and Non-Hematopoietic Stem/Progenitor Cells in Freshly Isolated Adult Human Bone Marrow Using an 8-Color Flow Cytometric Assay.

Blood ◽

10.1182/blood.v110.11.4045.4045 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4045-4045

Author(s):

Ferda Tekinturhan ◽

Ludovic Zimmerlin ◽

Vera S. Donnenberg ◽

Melanie E. Pfeifer ◽

Darlene A. Monlish ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Endothelial Progenitor Cells ◽

Progenitor Cell ◽

P Value ◽

Hematopoietic Stem ◽

Adult Human

Abstract Bone marrow (BM) contains hematopoietic stem cells (HSCs), which can give rise to all mature blood cells and marrow stromal cells as well. Recently, it has been shown that non-hematopoietic stem/progenitor cells which can differentiate into non-hematopoietic tissues also reside in the BM. Although culture expanded cells have been studied in great detail, little is known about the phenotype and quantity of these cells in freshly harvested adult human BM. The aim of this study is to isolate and characterize hematopoietic and non-hematopoietic stem/progenitor cells in adult human BM by comparing two different isolation techniques and their effects on the yield of hematopoietic, mesenchymal and endothelial stem/progenitor cell populations. BM samples were collected mechanically from isolated rib specimens obtained during lung resection (n=10), or from BM aspirates harvested from the humerus of orthopedic patients (n=17). BM mononuclear cells were purified on a Ficoll/Hypaque density gradient and stained simultaneously using CD105 FITC, CD73 PE, CD34 ECD, CD90 PE.Cy5, CD117 PE.Cy7, CD133 APC, CD45 APC.Cy7 and DAPI as a marker of nucleated cells. 2–15 million cells per sample were acquired on a Dako CyAn cytometer and the data were analyzed offline using prototype analytical software (Venturi, Applied Cytometry Systems). The significant difference in the percentage of the CD45 − singlets (non-hematopoietic cells) between BM aspirates and rib-derived samples indicates hemodilution in the bone marrow aspirates. Although we have observed a slight difference in the mean of hematopoietic stem cell content between samples, it was not statistically significant. According to our results, the quantity of mesenchymal stem cells was higher in rib-derived BM than BM aspirates (p value=0.028). The expression of some stem/progenitor cell markers, such as CD90 (Thy-1), CD117 (c-Kit) and CD133 remained similar for all cell types. Our results are shown in the table below. Surface Antigens RibBM (n=10)¥ BMA (n=17)¥ p Value % % Total Cells CD45- of nucleated cells 15.3 ± 7.9 5.7 ± 5.2 0.004 CD34+ Hematopoietic Stem Cells (HSCs)* CD34 of CD45+ 1.7 ± 1.48 2.6 ± 2.0 0.883 CD117 74.6 ± 31.3 53.3 ± 18.8 0.073 CD90 60.3 ± 44.5 35.9 ± 36.5 0.134 CD133 70.3 ± 31.8 62.3 ± 21.4 0.443 Endothelial Progenitor Cells (EPCs)* EPCs of nucleated cells 0.05 ± 0.03 0.12 ± 0.2 0.323 CD117 81.3 ± 29.8 78.1 ± 20.2 0.746 CD90 66.7 ± 39.7 53.7 ± 31.4 0.356 CD133 45.9 ± 32.7 33.9 ± 22.0 0.265 Mesenchymal Stem Cells (MSCs)* MSCs of nucleated cells 0.086 ± 0.14 0.008 ± 0.01 0.028 CD117 60.2 ± 36.8 49.8 ± 34.3 0.471 CD90 66.0 ± 27.7 65.7 ± 29.1 0.981 CD133 37.8 ± 27.4 39.9 ± 28.9 0.857 RibBM: Rib-derived BM, BMA: Bone Marrow Aspirate ¥Data are given as mean ± SD. *CD90, CD117 and CD133 expressions are shown for each stem/progenitor fraction: Hematopoietic stem cells (CD34 + CD45 + and light scatter properties according to the ISHAGE protocol), endothelial progenitor cells (CD34bright CD45 − CD105 +) and mesenchymal stem cells (CD34 − CD45 − CD73 + CD105 +).

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Acute exercise mobilizes hematopoietic stem and progenitor cells and alters the mesenchymal stromal cell secretome

Journal of Applied Physiology ◽

10.1152/japplphysiol.00925.2015 ◽

2016 ◽

Vol 120 (6) ◽

pp. 624-632 ◽

Cited By ~ 27

Author(s):

Russell Emmons ◽

Grace M. Niemiro ◽

Olatomide Owolabi ◽

Michael De Lisio

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Stromal Cells ◽

Acute Exercise ◽

Marrow Cell ◽

Exercise Bout ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

Transplantation of hematopoietic stem and progenitor cells (HSPC), collected from peripheral blood, is the primary treatment for many hematological malignancies; however, variable collection efficacy with current protocols merits further examination into factors responsible for HSPC mobilization. HSPCs primarily reside within the bone marrow and are regulated by mesenchymal stromal cells (MSC). Exercise potently and transiently mobilizes HSPCs from the bone marrow into peripheral circulation. Thus the purpose of the present study was to evaluate potential factors in the bone marrow responsible for HSPC mobilization, investigate potential sites of HSPC homing, and assess changes in bone marrow cell populations following exercise. An acute exercise bout increased circulating HSPCs at 15 min (88%, P < 0.001) that returned to baseline at 60 min. Gene expression for HSPC homing factors (CXCL12, vascular endothelial growth factor-a, and angiopoietin-1) were increased at 15 min in skeletal muscle and HSPC content was increased in the spleen 48 h postexercise (45%, P < 0.01). Acute exercise did not alter HSPCs or MSCs quantity in the bone marrow; however, proliferation of HSPCs (40%, P < 0.001), multipotent progenitors (40%, P < 0.001), short-term hematopoietic stem cells (61%, P < 0.001), long-term hematopoietic stem cells (55%, P = 0.002), and MSCs (20%, P = 0.01) increased postexercise. Acute exercise increased the content of the mobilization agent granulocyte-colony stimulating factor, as well as stem cell factor, interleukin-3, and thrombopoeitin in conditioned media collected from bone marrow stromal cells 15 min postexercise. These findings suggest that the MSC secretome is responsible for HSPC mobilization and proliferation; concurrently, HSPCs are homing to extramedullary sites following exercise.

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BCR-ABL enhances differentiation of long-term repopulating hematopoietic stem cells

Blood ◽

10.1182/blood-2009-04-215376 ◽

2010 ◽

Vol 115 (16) ◽

pp. 3185-3195 ◽

Cited By ~ 64

Author(s):

Mirle Schemionek ◽

Christian Elling ◽

Ulrich Steidl ◽

Nicole Bäumer ◽

Ashley Hamilton ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Chronic Myeloid Leukemia ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Hematopoietic Stem ◽

Multipotent Progenitor Cells

Abstract In a previously developed inducible transgenic mouse model of chronic myeloid leukemia, we now demonstrate that the disease is transplantable using BCR-ABL+ Lin−Sca-1+c-kit+ (LSK) cells. Interestingly, the phenotype is more severe when unfractionated bone marrow cells are transplanted, yet neither progenitor cells (Lin−Sca-1−c-kit+), nor mature granulocytes (CD11b+Gr-1+), nor potential stem cell niche cells (CD45−Ter119−) are able to transmit the disease or alter the phenotype. The phenotype is largely independent of BCR-ABL priming before transplantation. However, prolonged BCR-ABL expression abrogates the potential of LSK cells to induce full-blown disease in secondary recipients and increases the fraction of multipotent progenitor cells at the expense of long-term hematopoietic stem cells (LT-HSCs) in the bone marrow. BCR-ABL alters the expression of genes involved in proliferation, survival, and hematopoietic development, probably contributing to the reduced LT-HSC frequency within BCR-ABL+ LSK cells. Reversion of BCR-ABL, or treatment with imatinib, eradicates mature cells, whereas leukemic stem cells persist, giving rise to relapsed chronic myeloid leukemia on reinduction of BCR-ABL, or imatinib withdrawal. Our results suggest that BCR-ABL induces differentiation of LT-HSCs and decreases their self-renewal capacity.

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Reversible Expansion of Hematopoietic Stem Cells (HSC) and CML-Like Disease in Transgenic Mice Expressing BCR-ABL under the Control of the SCL 3′ Enhancer.

Blood ◽

10.1182/blood.v104.11.715.715 ◽

2004 ◽

Vol 104 (11) ◽

pp. 715-715

Author(s):

Steffen Koschmieder ◽

Berthold Goettgens ◽

Pu Zhang ◽

Tajhal Dayaram ◽

Kristin Geary ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Transgenic Mice ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Peripheral Blood ◽

Molecular Mechanisms ◽

Leukemic Cells ◽

Hematopoietic Stem ◽

Double Transgenic Mice

Abstract Chronic myeloid leukemia (CML) is a malignant disorder originating from the transformation of hematopoietic stem cells (HSC) by the BCR-ABL oncogene. Using the tet-off system, we have generated double-transgenic mice in which BCR-ABL is expressed under the control of the murine SCL 3′ enhancer, which targets expression to the vast majority of HSC and progenitors. After induction of BCR-ABL, all mice developed progressive chronic neutrophilia and leukocytosis (20–40 K/ul), and the animals died or were sacrificed in moribund condition within 58+/−28 days. Upon necropsy, bone marrow granulocytic hyperplasia, splenomegaly as well as organ infiltration by leukemic cells (liver, kidney, lung, small intestine, skin) were found. In addition, 31% of the mice subsequently developed ALL or lymphomas. BCR-ABL mRNA and protein expression were demonstrated in the affected organs. Expression of the transactivating transgene tTA was high in HSC, CMP, and CLP, but low in GMP and MEP, as assessed by real-time PCR, suggesting that the SCL 3′ enhancer indeed directed BCR-ABL expression to the most primitive hematopoietic cells within the bone marrow. The percentage of HSC in the bone marrow was expanded 7- and 26-fold in double-transgenic as compared to single-transgenic or wild-type control mice within 12 and 21 days, respectively, after BCR-ABL induction. GMP were increased 2- and 3-fold while the number of CMP was decreased 2-fold after 12 days but was increased 1.5-fold after 21 days. MEP were decreased 3-fold at both time points. In keeping with these results, the percentage of Ter-119 positive erythroid cells was decreased while the percentage of Gr-1 positive granulocytic cells was increased in the bone marrow. To assess reversibility of the phenotype, we readministered tetracycline to abrogate BCR-ABL expression. Double-transgenic mice showed rapid clinical improvement, reversion of neutrophilia and leukocytosis, normalization of Gr-1/Mac-1 positive cells in the peripheral blood and spleen, and reversion of splenomegaly. In addition, in mice that had developed lymphoblastic disease, readministration of tetracycline led to disappearance of lymphomas and of B220/BP-1 positive lymphoblastic cells in the peripheral blood. Furthermore, expansion of the HSC compartment in the bone marrow was also reversible, and the percentage of HSC decreased to levels observed in control mice. Repeated induction of BCR-ABL expression by removal of tetracycline led to reappearance of the myeloid and lymphoid phenotype. Again, the disease was reversible, and none of the animals relapsed while on tetracycline, suggesting that the phenotype remained completely dependent on the expression of the oncogene. In conclusion, we present a model of BCR-ABL mediated CML-like disease with expansion of phenotypic hematopoietic stem cells and myeloid progenitor cells in the bone marrow. The target cell population in this model closely resembles the origin of transformation in patients with CML, allowing for in vivo monitoring of early molecular mechanisms of BCR-ABL transformation. We are currently studying the function of the expanded HSC and progenitor cells in transplantation experiments.

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Implication of the Distinct Differentiation Pathway of Megakaryocytes From Hematopoietic Stem Cells in the Mouse Bone Marrow

Blood ◽

10.1182/blood.v120.21.1204.1204 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1204-1204

Author(s):

Hidekazu Nishikii ◽

Kenji Matsushita ◽

Yosuke Kanazawa ◽

Yasuhisa Yokoyama ◽

Takayasu Kato ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Dominant Negative Mutant ◽

Mouse Bone Marrow ◽

Alpha Cells ◽

Hematopoietic Stem ◽

Deficient Mice

Abstract Abstract 1204 Background. Hematopoietic progenitor cells are the progeny of hematopoietic stem cells (HSC) that coordinate the production of precise number of mature blood cells of diverse functional lineages. Megakaryocytes (Meg) are mapped at the downstream of bilineage progenitors for erythroid and megakaryocyte (MEP) in the most widely accepted scenarios, although different notions have also been suggested. Thrombopoietin (TPO) is thought to be the master cytokine for megakaryopoiesis. In mice lacking cMpl, the receptor for TPO, production of platelets and Meg is severely impaired. However, Meg are known to be still present in the bone marrow of these mice. These findings suggested that TPO independent signaling for Meg differentiation would exist. Purpose. To clarify the differentiation pathway of the Meg lineage, we focused on GPIb (CD42)-V-IX complex, expression of which has not been characterized in any progenitor cells whereas it is well known to be expressed on mature Meg and platelets. We also investigated how TPO-cMpl signaling would affect at MEP or pure megakaryocyte progenitor (MKP) stage using the cMpl deficient mice. Results and Discussion. GPIb alpha (CD42b) was expressed on 3–6 % of a mouse bone marrow population characterized as common myeloid progenitors (CMP), i.e., Lin-c-Kit+Sca1-CD34+CD16/32low cells. The GPIb alpha+ CMP (thereafter designated 34-alpha) population also expresses CD9, SLAM1, and CD41. These 34-alpha cells showed a restricted differentiation capacity to the mature Meg in in vitro culture. By intravenously infusing 34-alpha cells derived from CAG promoter-driven GFP-expressing mice into sublethally irradiated syngenic mice, GFP-expressing platelets were generated in vivo. Thus, we designate the 34-alpha cells as 34-alpha MKP. Gene expression analysis also supported that 34-alpha MKP has a restricted capacity of megakaryopoiesis. In vitro colony-forming assay and short-term liquid culture assay suggested that they are not derived from MEP but from the SLAM1+Flt3-c-Kit+Sca1+Lin- population, which highly contain HSC. When experimental thrombocytopenia was induced by injecting 5-fluorouracil into mice, the frequency of 34-alpha MKP was rapidly increased compared to that of MEP. These data imply a distinct pathway of Meg differentiation, which originates at the proximity of HSC. We next investigated whether generation of 34-alpha MKP and MEP is differently impaired in cMpl-deficient mice. The frequency of MEP was only mildly reduced. In contrast, 34-alpha MKP were much severely reduced. Notably, in vitro Meg differentiation was markedly impaired from both MEP and 34-alpha MKP derived from cMpl-deficient mice. These data suggested that discordance between Meg and platelet production is caused by the different dependence on TPO-cMpl signaling between the pathways generating MEP and 34-alpha MKP from HSC. We also found that Hes1, a transcription factor that is the best characterized effector functioning downstream of the Notch signaling pathway, is highly expressed in 34-alpha MKP. Conversely, Meg differentiation was abrogated by retroviral transduction of a dominant-negative mutant of Hes1. Taken together, our data imply the presence of two distinct Meg differentiation pathways from HSC and further suggest that the dependency of TPO-cMpl signaling is different in these pathways and Notch-Hes signaling plays an additional role in them. Disclosures: No relevant conflicts of interest to declare.

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STS1 and STS2 Are Important Regulators Of Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v122.21.1186.1186 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1186-1186

Author(s):

Jing Zhang ◽

Hubert Serve ◽

Christian H. Brandts

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cell Surface ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Wild Type ◽

Short Term ◽

Double Knockout ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

Abstract The receptor tyrosine kinases FLT3 and KIT are highly expressed on the surface of leukemic blasts in most patients with acute myeloid leukemia. Although about one third of patients display activating mutations in FLT3 (and more rarely in KIT), the majority of patients have no mutations in FLT3 or KIT. Previously, we demonstrated that Cbl functions as the E3 ligase for both FLT3 and KIT, and that ligase-inactivating mutations of Cbl stabilize FLT3 and KIT on the cell surface by preventing endocytosis and degradation (Sargin et al, Blood 2007). Furthermore, we demonstrated that expression of E3-ligase deficient Cbl mutants led to the development of a myeloproliferative disease in a murine bone marrow transplantation model (Bandi et al, Blood 2009). However, Cbl mutations are rarely found in AML. Here, we investigated the role of the Cbl regulators suppressors of T-cell signaling 1 and 2(STS1 and STS2) in stabilizing wild-type FLT3 and KIT on the cell surface of hematopoietic stem and progenitor cells (HSPCs). STS1 is ubiquitously expressed, while STS2 expression is restricted to the hematopoietic tissue. STS1 and STS2 constitutively bind to Cbl, while their binding to FLT3 and KIT is dependent on ligand-activation by FL and SCF, respectively. Interestingly, STS1 (but not STS2) functions as a tyrosine phosphatase for both ligand-activated FLT3 and KIT. This required the PGM domain of STS1, as PGM point mutant of STS1 did not dephosphorylate FLT3 or KIT. In line with this, knockdown of STS1 using stably expressing shRNA constructs showed a significant hyperphosphorylation of FLT3 and KIT. By using STS1/STS2 single and double knockout mice, we analyzed the effects of STS1 and STS2 on hematopoietic stem and progenitor cells in vivo. We found that deficiency of STS1 causes an increase of both absolute number and frequency of LSK (lineage marker-, KIT+, Sca1+) cells, which contain HSPCs. This phenotype was even more pronounced in STS1 and STS2 double knockout (dKO) mice, and is mainly attributable to the short term hematopoietic stem cell (ST-HSC) and multipotent progenitor (MPP) cell population, as defined by both standard and SLAM markers. Colony assays using primary bone marrow cells revealed a significantly higher colony forming ability in STS1-KO and dKO cells compared to wild type (wt) cells, particularly after serial replating. A careful analysis of the cells derived from methylcellulose culture revealed an increased proportion of immature (Mac1- CD48+ CD16/32-) cells in STS1-KO and dKO cells. Competitive repopulation assays showed an advantage for dKO cells when compared to wt, suggesting that the LT-HSC compartment is also affected. Even more pronounced were the differences in CFU-S assays (colony forming units spleen), displaying significantly more colonies of dKO compared to wt donor cells, functionally demonstrating a significantly increased ST-HSC / MPP population in dKO donors. A detailed analysis of the downstream signaling events demonstrated that loss of STS1 specifically causes an activated PI3-Kinase / AKT pathway. In summary, our data demonstrates that STS1 functions as a phosphatase of FLT3 and KIT and, using genetic mouse models, indicates a critical role in the maintenance and proliferation of long-term and short-term hematopoietic stem cells. This may also affect sensitivity to kinase inhibitors. Disclosures: No relevant conflicts of interest to declare.

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Critical Role Of Casein Kinase (Ck)1α Heterozygote Gene Inactivation In The Clonal Advantage Of Hematopoietic Stem Cells In Del(5q) MDS

Blood ◽

10.1182/blood.v122.21.98.98 ◽

2013 ◽

Vol 122 (21) ◽

pp. 98-98

Author(s):

Rebekka K. Schneider ◽

Dirk Heckl ◽

Marcus Järås ◽

Lisa Chu ◽

McConkey Marie ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Hematopoietic Stem Cell ◽

Hematopoietic Stem ◽

Myeloid Progenitor Cells ◽

Stem And Progenitor Cells ◽

A Cell

Abstract Casein kinase 1α (Ck1α) is a serine/threonine kinase located in the common deleted region (5q32) in del(5q) myelodysplastic syndrome (MDS). Ck1α is a regulator of the canonical WNT signaling pathway and may play a role in the clonal advantage of del(5q) cells. In addition, we identified CK1α as a therapeutic target in myeloid malignancies in an in vivo RNA interference screen, and haploinsufficiency for CK1α could further sensitize del(5q) cells to CK1α inhibition. To explore the biology and therapeutic potential of CK1α in MDS, we generated a conditional Ck1α knockout mouse model. Conditional homozygous inactivation of Ck1α resulted in bone marrow failure, ablation of hematopoietic stem and progenitor cells, a severe anemia and rapid lethality within 7-12 days, confirming that Ck1α is essential for hematopoietic stem and progenitor cell survival. In contrast, mice with haploinsufficiency of Ck1α developed a hypercellular bone marrow, as is typical in MDS, a significantly elevated white blood cell count (p=0.002) and normal hemoglobin levels. The hematopoietic stem cells (LSK, LT-HSC, ST-HSC) as well as progenitor cells (LK, pre-GMP, GMP, pre-CFU-e, CFU-e, pre-megakaryocytes-erythrocytes) were not affected by Ck1α haploinsufficiency 14 days after induction. Only the megakaryocytic progenitor cells (p=0.04) were significantly reduced. This finding was in line with severe dysplasia and hypolobulated micromegakaryocytes observed in the bone marrow, another typical histomorphological feature of del(5q) MDS. In long-term experiments up to 8 months, the survival of mice with Ck1α haploinsufficiency was not impaired, although we observed an exhaustion of the stem cell pool with significant reduction of ST-HSC (p<0.001), LT-HSC (p=0.003), and MPP (p=0.007). We were able to demonstrate that this significant reduction is a cell-extrinsic effect. In transplantation and HSC repopulation assays, an intact HSC function and even a significant expansion of hematopoietic stem cells and progenitor cells with Ck1α haploinsufficiency was confirmed in comparison to MxCre controls (LSK p=0.019; LK p=0.035; CMP p=0.036; GMP p=0.027; MEP p=0.005), suggesting a repopulation advantage of HSC with Ck1α haploinsufficiency. In contrast, Ck1α homozygous deletion leads to a cell-autonomous, p53-mediated HSC failure in transplantation assays. To dissect the mechanism of hematopoietic stem cell expansion in Ck1α haploinsufficiency on the one hand and the hematopoietic stem cell ablation after Ck1α ablation on the other hand, we analyzed regulatory mechanisms including proliferation and apoptosis in LK cells (myeloid progenitor cells) and LSK cells (enriched for hematopoietic stem cells). Ablation of Ck1α led to a significant increase (p=0.001) in the number of LSK and LK in the S/M/G2 phase, accompanied by a significant reduction in the G0/G1 fraction, suggesting their exit from quiescence. Ck1α haploinsufficiency led to a significant increase in the fraction of cycling cells in myeloid progenitor cells (LK, p=0.052), the quiescent hematopoietic stem cells were not significantly affected. In Western Blots of ckit+ hematopoietic stem and progenitor cells, a significant increase of intracellular ß-catenin levels was detected in both Ck1α haploinsufficient and even stronger in Ck1α ablated cells, accompanied by an exit from stem cell quiescence shown by loss of p21-mediated growth arrest and up-regulation of phosphorylated retinoblastoma protein indicating cell cycle progression from G0 to G1 in comparison to the MxCre+ control cells. Ck1α ablation led to p53-mediated apoptosis in stem and progenitor cells (Annexin V/7-AAD). In Ck1α haploinsufficient cells, apoptosis was not significantly induced in neither LK cells or in LSK cells although p53 induction was observed in the bone marrow. Taken together, our results indicate that Ck1α is essential for hematopoietic stem and progenitor cell survival, but that Ck1α haploinsufficiency does not decrease, and may increase, hematopoietic stem cell function. This finding highlights the potential of preferential elimination of the del(5q) hematopoietic stem cells through Ck1α inhibtion and thus provides a potential therapeutic window. Consistent with this hypothesis, targeting the haploinsufficient kinase activity in vitro with the Ck1α small molecule inhibitor D4476, selectively targets CK1α haploinsufficient cells relative to wild-type cells. Disclosures: Järås: Cantargia: Equity Ownership.

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