Class I PI3K Is Required for HSC Differentiation

Adult hematopoietic stem cells (HSCs) are a rare and unique population of stem cells that reside in the bone marrow, where they undergo self-renewal and differentiation to maintain the blood system. The maintenance of a proper balance between HSC self-renewal and differentiation requires growth factors, cytokines, and chemokines, most of which activate the phosphoinositide 3-kinase/Protein Kinase B (PI3K/AKT) signaling pathway. Pathologic activation of the AKT pathway is frequently observed in tumors, making it a desirable target for cancer treatment. Since several PI3K inhibitors are now in clinical use, it is critical to determine the roles of PI3K in adult HSCs. However, the specific roles of PI3K in HSC function are poorly understood. Hematopoietic cells express three Class IA catalytic PI3K isoforms (P110α, β, and δ), which can all transduce growth factor and cytokine signals, and can compensate for one another in some cell types. Individual Class 1A PI3K isoforms have unique functions in mature hematopoietic lineages, but are dispensable for HSC function. To uncover the potentially redundant roles of PI3K isoforms in HSCs, we have generated a triple knockout (TKO) mouse model with conditional deletion of p110α and p110β in hematopoietic cells using MX1-Cre, and germline deletion of p110δ. TKO mice develop pancytopenia, which is also observed upon transplantation of TKO bone marrow. Competitive repopulation assays reveal a defect in long-term multi-lineage chimerism. Surprisingly, loss of Class 1A PI3K causes significant expansion of donor-derived long-term (Lin-cKit+Flk2-CD150+CD48-) and short-term (Lin-cKit+Flk2-CD150-CD48-) HSCs in the bone marrow, but not committed progenitors. This phenotype could not be explained by alterations in HSC cell cycling or apoptosis in TKO HSCs. TKO transplant recipients also have dysplastic features in the bone marrow. Methylcellulose plating assays of TKO bone marrow revealed a relative increase in granulocyte erythroid macrophage megakaryocyte (GEMM) colonies and extended serial replating, suggesting increased self-renewal. Thus, our data are consistent with impaired HSC differentiation upon deletion of all Class IA PI3K isoforms, which leads to dysplastic changes. RNA sequencing of sorted long-term HSCs from the bone marrow of TKO transplant recipients revealed the enrichment of human and mouse HSC signatures, and the downregulation of DNA repair gene sets and RNA splicing gene sets in TKO HSCs. Interestingly, we also observed downregulation of autophagy gene sets in TKO HSCs. Macroautophagy has been shown to be essential for the maintenance of HSC metabolism and self-renewal. Analysis of the autophagosomal marker LC3-II in TKO HSCs revealed a decrease in autophagy upon growth factor deprivation. Surprisingly, we observed an increase in MTOR activation in TKO cKit+ bone marrow cells via compensatory signaling through the MAPK pathway. Given that MTOR is a known negative regulator of autophagy, this is consistent with the observed autophagy decrease in TKO HSCs. Additionally, we found that autophagy can still be induced in TKO HSCs with the MTOR inhibitor rapamycin. Furthermore, rapamycin treatment impairs serial replating of TKO bone marrow cells. In conclusion, we found that inactivation of all Class 1A PI3 kinases leads to impaired HSC differentiation, likely due to a defect in autophagy induction in response to growth factor deprivation. Disclosures No relevant conflicts of interest to declare.

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Hepatoma-Derived Growth Factor Is a Novel Factor to Promote the Proliferation of Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v128.22.1471.1471 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1471-1471

Author(s):

Munetada Haruyama ◽

Kozo Yamaichi ◽

Akira Niwa ◽

Megumu K Saito ◽

Tatsutoshi Nakahata

Keyword(s):

Bone Marrow ◽

Growth Factor ◽

Bone Marrow Cells ◽

Ex Vivo ◽

Absolute Number ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

The Mean

Abstract Ex vivo expansion of hematopoietic stem cells (HSCs) is an attractive therapeutic strategy for many hematologic diseases and genetic disorders. Therefore, a variety of ex vivo expansion techniques have been developed, however these systems were not well done to get long term HSCs (LT-HSCs) which have a long term hematopoietic reconstitution ability. As the reasons, it is considered that the factors associating with the proliferation and self-renewal of LT-HSCs have not been clear yet. To obtain the factors to stimulate the proliferation and self-renewal of LT-HSCs, various conditioned media were evaluated. The supernatants of COS-1 cells transfected with cDNA cording for RelA (one of nuclear factor kappa B subunits) stimulated the proliferation of human CD34+ cells derived from umbilical cord blood (UCB) and increased the number of CFU-Mix strongest of all evaluated conditioned media. 60 liters of the supernatants of COS-1 cells transfected RelA genes were separated by column chromatography purifications. LC-MS/MS analysis of the final active fraction provided the information of hepatoma-derived growth factor (HDGF) as a growth factor. HDGF is a 24kD heparin-binding protein and has reported to stimulate the proliferation in various types of cells including fibroblasts, endothelial cells and hepatoma cells, its receptor(s) and signaling remain unclear, moreover, has no known function in hematopoiesis. The recombinant human HDGF indicated the ability to enhance the proliferation of CD34+ cells dose-dependently and increased the number of CFU-Mix in combination with cytokines compared to cytokines alone, especially HDGF showed the strongest synergy effect in a combination with TPO in all combinations of cytokines. Next, uncultured (UC) CD34+ cells, the cells of an equal initial number of CD34+ cells after the serum-free condition cultures in the presence of TPO alone (T), HDGF alone (H) and HDGF+TPO (HT) were transplanted into sublethally irradiated NOG (NOD/Shi-scid,IL-2RγKO) mice. HT increased the number of CD34+CD38- cells compared to UC, T and H. Analysis of CD34+CD38- cells in bone marrow cells of NOG mice 24 weeks after transplantation revealed that the mean of absolute number of CD34+CD38- cells in HT group showed about 4-fold, that in H group showed about 3-fold compared to that in UC group, however, that in T group were not detected.These results indicated that HT increased HSCs including short term and long term HSCs. In order to investigate whether HDGF could increase the number of LT-HSCs, serial transplantation experiment was carried out. Uncultured CD34+ cells and the CD34+ cells cultured with HT were transplanted into sublethally irradiated NOG mice. At 24 weeks after transplantation, the mean of absolute number of CD34+CD38- cells in HT group showed 6-fold compared to that in UC group, a half of total number of bone marrow cells from each mouse in both groups were transplanted into one secondary sublethally irradiated NOG mouse. Analysis of human hematopoietic cells in both group 20 weeks after transplantation revealed that multi-lineage human hematopoietic cells, such as CD3+ cells, CD19+ cells, CD33+ cells, CD235a+ cells, erythrocytes and platelets, were detected in all mice in HT group, but were not detected in all mice in UC group. The mean of absolute number of CD34+CD38- cells in bone marrow of HT group showed 30-fold compared to that of UC group. These results indicated that HDGF could increase the number of LT-HSCs. We showed here that the CD34+ cells cultured with HDGF can be transplanted to secondary hosts to give rise to long-term multilineage repopulation. Thus, HDGF is a novel factor to promote the proliferation of HSCs and plays an important role in hematopoiesis. HDGF will contribute the new HSCs expansion system development by using UCB for hematopoietic stem cell transplantation. Disclosures No relevant conflicts of interest to declare.

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Long-Term Engraftment and Self-Renewal of AML Stem Cells in the Newborn NOD-scid/IL2rgnull Immunodeficient Mouse Model.

Blood ◽

10.1182/blood.v106.11.1261.1261 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1261-1261 ◽

Cited By ~ 2

Author(s):

Shuro Yoshida ◽

Fumihiko Ishikawa ◽

Masaki Yasukawa ◽

Toshihiro Miyamoto ◽

Goichi Yoshimoto ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

T Cells ◽

T Cell ◽

Bone Marrow Cells ◽

Scid Mice ◽

Leukemic Cells ◽

Self Renewal ◽

Human T Cells

Abstract Transplantation of human leukemic cells into severe combined immunodeficiency (SCID) mice has been used to analyze developmental mechanisms of human leukemogenesis. Previous models, however, were limited in efficient or long-term engraftment of leukemia initiating cells. Here we report a new SCID model that supports highly efficient long-term engraftment of primary human acute myelogenous leukemia (AML) cells. We have established a novel immune-compromised mouse by backcrossing a complete null mutation of the common cytokine receptor g chain onto NOD-scid mice (NOD/SCID/IL2rgnull mice), and reported that normal human cord blood-derived hematopoietic stem cells efficiently engrafted in newborn NOD/SCID/IL2rgnull mice as compared to NOD/SCID/b2mnull mice (Ishikawa et al, Blood in press). Injection of 5x106 total bone marrow mononuclear cells from primary AML patients (FAB subtypes: M1, M2, M3, M4 and M7) into sublethally-irradiated newborn NOD/SCID/IL2rgnull mice, however, did not result in efficient engraftment of AML cells, while predominant proliferation of human CD4+ and CD8+ T cells was seen. These human T cells expressed CD45RO, and levels of human IFN-g in sera of the recipients significantly elevated, suggesting that human T cells were activated and inhibited the engraftment of human AML cells in the xenogeneic setting. We thus transplanted AML cells after T cell depletion. Strikingly, transplantation of 4x106 T cell-depleted AML bone marrow cells into neonatal NOD/SCID/IL2rgnull mice resulted in the efficient AML engraftment, whose levels were significantly higher than those in transplantation of the same number of T cell-depleted AML cells into NOD/SCID/b2mnull newborns or NOD/SCID/IL2rgnull adults. We also transplanted 103–104 hCD34+hCD38− bone marrow cells purified from AML patients. These low-doses of hCD34+hCD38− cells also successfully engrafted, progressively giving rise to hCD34+hCD38+ and hCD34− leukemic cells over 16 weeks. hCD34+hCD38− cells purified from the bone marrow of primary NOD/SCID/IL2rgnull recipients again reconstituted AML in secondary recipients, indicating that this system supports self-renewal capacity of AML stem cells within the hCD34+hCD38− fraction. Thus, the NOD/SCID/IL2rgnull newborn system provides a powerful model to study human leukemogenesis as well as the interaction between human T cells and AML cells in vivo.

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Myelosuppressive conditioning is required to achieve engraftment of pluripotent stem cells contained in moderate doses of syngeneic bone marrow

Blood ◽

10.1182/blood.v83.4.939.939 ◽

1994 ◽

Vol 83 (4) ◽

pp. 939-948 ◽

Cited By ~ 116

Author(s):

Y Tomita ◽

DH Sachs ◽

M Sykes

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Bone Marrow Cells ◽

Whole Body ◽

Mixed Chimerism ◽

Hematopoietic Stem ◽

Donor Cells ◽

Donor Type ◽

Low Levels

Abstract We have investigated the requirement for whole body irradiation (WBI) to achieve engraftment of syngeneic pluripotent hematopoietic stem cells (HSCs). Recipient B6 (H-2b; Ly-5.2) mice received various doses of WBI (0 to 3.0 Gy) and were reconstituted with 1.5 x 10(7) T-cell-depleted (TCD) bone marrow cells (BMCs) from congenic Ly-5.1 donors. Using anti-Ly-5.1 and anti-Ly-5.2 monoclonal antibodies and flow cytometry, the origins of lymphoid and myeloid cells reconstituting the animals were observed over time. Chimerism was at least initially detectable in all groups. However, between 1.5 and 3 Gy WBI was the minimum irradiation dose required to permit induction of long-term (at least 30 weeks), multilineage mixed chimerism in 100% of recipient mice. In these mice, stable reconstitution with approximately 70% to 90% donor-type lymphocytes, granulocytes, and monocytes was observed, suggesting that pluripotent HSC engraftment was achieved. About 50% of animals conditioned with 1.5 Gy WBI showed evidence for donor pluripotent HSC engraftment. Although low levels of chimerism were detected in untreated and 0.5-Gy-irradiated recipients in the early post-BM transplantation (BMT) period, donor cells disappeared completely by 12 to 20 weeks post-BMT. BM colony assays and adoptive transfers into secondary lethally irradiated recipients confirmed the absence of donor progenitors and HSCs, respectively, in the marrow of animals originally conditioned with only 0.5 Gy WBI. These results suggest that syngeneic pluripotent HSCs cannot readily engraft unless host HSCs sustain a significant level of injury, as is induced by 1.5 to 3.0 Gy WBI. We also attempted to determine the duration of the permissive period for syngeneic marrow engraftment in animals conditioned with 3 Gy WBI. Stable multilineage chimerism was uniformly established in 3-Gy-irradiated Ly-5.2 mice only when Ly-5.1 BMC were injected within 7 days of irradiation, suggesting that repair of damaged host stem cells or loss of factors stimulating engraftment may prevent syngeneic marrow engraftment after day 7.

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Efficient retroviral gene transfer to purified long-term repopulating hematopoietic stem cells

Blood ◽

10.1182/blood.v84.1.74.74 ◽

1994 ◽

Vol 84 (1) ◽

pp. 74-83 ◽

Cited By ~ 36

Author(s):

SJ Szilvassy ◽

S Cory

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Cell Biology ◽

Bone Marrow Cells ◽

High Titer ◽

Marker Gene ◽

Mouse Bone Marrow ◽

Hematopoietic Stem

Abstract Efficient gene delivery to multipotential hematopoietic stem cells would greatly facilitate the development of effective gene therapy for certain hematopoietic disorders. We have recently described a rapid multiparameter sorting procedure for significantly enriching stem cells with competitive long-term lymphomyeloid repopulating ability (CRU) from 5-fluorouracil (5-FU)-treated mouse bone marrow. The sorted cells have now been tested as targets for retrovirus-mediated delivery of a marker gene, NeoR. They were cocultured for 4 days with fibroblasts producing a high titer of retrovirus in medium containing combinations of the hematopoietic growth factors interleukin-3 (IL-3), IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF) and then injected into lethally irradiated recipients, together with sufficient “compromised” bone marrow cells to provide short-term support. Over 80% of the transplanted mice displayed high levels (> or = 20%) of donor- derived leukocytes when analyzed 4 to 6 months later. Proviral DNA was detected in 87% of these animals and, in half of them, the majority of the hematopoietic cells were marked. Thus, infection of the stem cells was most effective. The tissue and cellular distribution of greater than 100 unique clones in 55 mice showed that most sorted stem cells had lymphoid as well as myeloid repopulating potential. Secondary transplantation provided strong evidence for infection of very primitive stem cells because, in several instances, different secondary recipients displayed in their marrow, spleen, thymus and day 14 spleen colony-forming cells the same proviral integration pattern as the primary recipient. Neither primary engraftment nor marking efficiency varied for stem cells cultured in IL-3 + IL-6, IL-3 + IL-6 + KL, IL-3 + IL-6 + LIF, or all four factors, but those cultured in IL-3 + IL-6 + LIF appeared to have lower secondary engraftment potential. Provirus expression was detected in 72% of the strongly marked mice, albeit often at low levels. Highly efficient retroviral marking of purified lymphomyeloid repopulating stem cells should enhance studies of stem cell biology and facilitate analysis of genes controlling hematopoietic differentiation and transformation.

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Host Conditioning With 5-Fluorouracil and kit-Ligand to Provide for Long-Term Bone Marrow Engraftment

Blood ◽

10.1182/blood.v89.7.2376 ◽

1997 ◽

Vol 89 (7) ◽

pp. 2376-2383 ◽

Cited By ~ 25

Author(s):

Ronald van Os ◽

Donald Dawes ◽

John M.K. Mislow ◽

Alice Witsell ◽

Peter M. Mauch

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Bone Marrow Cells ◽

Mixed Chimerism ◽

Kit Ligand ◽

Donor Bone Marrow ◽

Before And After ◽

Total Body ◽

Marrow Cells

Abstract Administration of kit-ligand (KL) before and after doses of 5-fluorouracil (5-FU) results in marrow failure in mice, presumably because of enhanced KL-induced cycling of stem cells, which makes them more susceptible to the effects of 5-FU. In attempt to capitalize on this effect on stem cells, we studied the ability of KL and 5-FU to allow stable donor engraftment of congenically marked marrow in a C57BL/6 (B6) mouse model. KL was administered subcutaneously at 50 μg/kg, 21 hours and 9 hours before and 3 hours after each of two doses of 5-FU (125 mg/kg) given 7 days apart to B6-recipients. Animals then received three injections of 107 congenic B6-Gpi-1a-donor bone marrow cells at 24, 48, and 72 hours after the second 5-FU dose. A separate group of animals received a single dose of either 1 × 107 or 3 × 107 donor marrow cells 24 hours after the last 5-FU dose. The level of engraftment was measured from Gpi-phenotyping at 1, 3, 6, and 8 months in red blood cells (RBCs) and at 8 months by phenotyping cells from the thymus, spleen, and marrow. Percent donor engraftment in RBCs appeared stable after 6 months. The percent donor engraftment in RBCs at 8 months was significantly higher in KL + 5-FU prepared recipients (33.0 ± 2.7), compared with 5-FU alone (18.5 ± 2.6, P < .0005), or saline controls (17.8 ± 1.7, P < .0001). In an additional experiment, granulocyte colony-stimulating factor (100 μg/dose) was added to a reduced dose of KL (12.5 μg/dose); engraftment was similar to KL alone. At 8 months after transplantation the levels of engraftment in other tissues such as bone marrow, spleen, and thymus correlated well with erythroid engraftment to suggest that multipotent long-term repopulating stem cells had engrafted in these animals. There are concerns for the toxicity of total body irradiation (TBI)- or busulfan-based regimens in young recipients of syngeneic or transduced autologous marrow who are transplanted for correction of genetic disease. In these recipients complete donor engraftment may not be needed. The results with KL and 5-FU are encouraging for the further refinement of non-TBI, nonbusulfan techniques to achieve stable mixed chimerism.

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Platelet Factor 4 and Other CXC Chemokines Support the Survival of Normal Hematopoietic Cells and Reduce the Chemosensitivity of Cells to Cytotoxic Agents

Blood ◽

10.1182/blood.v89.7.2328 ◽

1997 ◽

Vol 89 (7) ◽

pp. 2328-2335 ◽

Cited By ~ 58

Author(s):

Zhong Chao Han ◽

Min Lu ◽

Junmin Li ◽

Mai Defard ◽

Bernadette Boval ◽

...

Keyword(s):

Bone Marrow ◽

Growth Factor ◽

Cell Lines ◽

Cancer Cell ◽

Bone Marrow Cells ◽

Hematopoietic Cells ◽

Platelet Factor 4 ◽

Cytotoxic Agents ◽

Platelet Factor ◽

Cd34 Cells

Abstract The effects of platelet factor 4 (PF4) on the viability and chemosensitivity of normal hematopoietic cells and cancer cell lines were studied to determine the mechanisms whereby PF4 functions as either an inhibitor or a protector and to evaluate its clinical significance. Two other chemokines, interleukin-8 (IL-8) and neutrophil-activating peptide-2 (NAP-2), were also studied in comparison to PF4. Using a tetrazolium salt assay for cell viability, we observed that PF4 at 1 to 50 μg/mL supported the viability of normal human bone marrow cells. Approximately 45% of cells cultured for 48 hours survived, whereas 80% or more survived in the presence of PF4 5 μg/mL. PF4 also supported the viability of CD34+ cord blood (CB) cells and protected them from apoptosis induced by transforming growth factor β1 (TGFβ1) and cytotoxic drugs. Pretreatment of CD34+ cells by PF4, but not by TGFβ1, caused an increase in the number of megakaryocyte colonies after these cells were replated in secondary cultures. Flow cytometry analysis showed that when CD34+ cells were preincubated with PF4 or TGFβ1 for 12 days in hematopoietic growth factor–rich medium, an increased number of remaining CD34+ cells was observed only for PF4-treated cells. Furthermore, PF4 significantly reduced the chemosensitivity of bone marrow cells, as shown by its ability to increase the 50% inhibition concentration (IC50) of several cytotoxic agents. Like PF4, IL-8 and NAP-2 at 0.1, 0.6, and 1 μg/mL supported the survival of myeloid progenitors, including colony-forming units granulocyte, erythroblast, monocyte, megakaryocyte (CFU-GEMM), CFU-megakaryocyte (CFU-MK), CFU–granulocyte/macrophage (CFU-GM), and burst-forming units–erythroblast (BFU-E), and reduced their sensitivity to the toxicity of etoposide (ETP). Protamine sulfate at 1 to 100 μg/mL showed no such activity of PF4. Interestingly, the three chemokines failed to affect significantly the viability and chemosensitivity of three leukemic and two other tumor cell lines. Based on these results, we conclude for the first time that PF4 and IL-8 and NAP-2 support the survival of normal hematopoietic precursors and protect them from the toxicity of chemotherapeutic agents. Because such activities are unique to normal hematopoietic cells but not to the cancer cell lines evaluated, a potential clinical application of these molecules in the treatment of cancer is suggested.

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Enforced Expression of the GATA-2 Transcription Factor Blocks Normal Hematopoiesis

Blood ◽

10.1182/blood.v93.2.488 ◽

1999 ◽

Vol 93 (2) ◽

pp. 488-499 ◽

Cited By ~ 157

Author(s):

Derek A. Persons ◽

James A. Allay ◽

Esther R. Allay ◽

Richard A. Ashmun ◽

Donald Orlic ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Transcription Factor ◽

Blood Cell ◽

Fluorescent Protein ◽

Bone Marrow Cells ◽

Hematopoietic Cells ◽

Immunoblot Analysis ◽

Marrow Cells

Abstract The zinc finger transcription factor GATA-2 is highly expressed in immature hematopoietic cells and declines with blood cell maturation. To investigate its role in normal adult hematopoiesis, a bicistronic retroviral vector encoding GATA-2 and the green fluorescent protein (GFP) was used to maintain the high levels of GATA-2 that are normally present in primitive hematopoietic cells. Coexpression of the GFP marker facilitated identification and quantitation of vector-expressing cells. Bone marrow cells transduced with the GATA-2 vector expressed GFP as judged by flow cytometry and GATA-2 as assessed by immunoblot analysis. A 50% to 80% reduction in hematopoietic progenitor-derived colony formation was observed with GATA-2/GFP-transduced marrow, compared with marrow transduced with a GFP-containing vector lacking the GATA-2 cDNA. Culture of purified populations of GATA-2/GFP-expressing and nonexpressing cells confirmed a specific ablation of the colony-forming ability of GATA-2/GFP-expressing progenitor cells. Similarly, loss of spleen colony-forming ability was observed for GATA-2/GFP-expressing bone marrow cells. Despite enforced GATA-2 expression, marrow cells remained viable and were negative in assays to evaluate apoptosis. Although efficient transduction of primitive Sca-1+Lin- cells was observed with the GATA-2/GFP vector, GATA-2/GFP-expressing stem cells failed to substantially contribute to the multilineage hematopoietic reconstitution of transplanted mice. Additionally, mice transplanted with purified, GATA-2/GFP-expressing cells showed post-transplant cytopenias and decreased numbers of total and gene-modified bone marrow Sca-1+ Lin−cells. Although Sca-1+ Lin− bone marrow cells expressing the GATA-2/GFP vector were detected after transplantation, no appreciable expansion in their numbers occurred. In contrast, control GFP-expressing Sca-1+Lin− cells expanded at least 40-fold after transplantation. Thus, enforced expression of GATA-2 in pluripotent hematopoietic cells blocked both their amplification and differentiation. There appears to be a critical dose-dependent effect of GATA-2 on blood cell differentiation in that downregulation of GATA-2 expression is necessary for stem cells to contribute to hematopoiesis in vivo.

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In vitro proliferation of primitive hemopoietic stem cells supported by stromal cells: evidence for the presence of a mechanism(s) other than that involving c-kit receptor and its ligand.

Journal of Experimental Medicine ◽

10.1084/jem.176.2.351 ◽

1992 ◽

Vol 176 (2) ◽

pp. 351-361 ◽

Cited By ~ 44

Author(s):

H Kodama ◽

M Nose ◽

Y Yamaguchi ◽

J Tsunoda ◽

T Suda ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Bone Marrow Cells ◽

Hemopoietic Stem Cells ◽

In Vitro Proliferation ◽

Kit Receptor ◽

Colony Forming Units ◽

Marrow Cells

The preadipose cell line, PA6, can support long-term hemopoiesis. Frequency of the hemopoietic stem cells capable of sustaining hemopoiesis in cocultures of bone marrow cells and PA6 cells for 6 wk was 1/5.3 x 10(4) bone marrow cells. In the group of dishes into which bone marrow cells had been inoculated at 2.5 x 10(4) cells/dish, 3 of 19 dishes (16%) contained stem cells capable of reconstituting erythropoiesis of WBB6F1-W/Wv mice, indicating that PA6 cells can support the proliferation of primitive hemopoietic stem cells. When the cocultures were treated with an antagonistic anti-c-kit monoclonal antibody, ACK2, only a small number of day 12 spleen colony-forming units survived; and hemopoiesis was severely reduced. However, when the cocultures were continued with antibody-free medium, hemopoiesis dramatically recovered. To examine the proliferative properties of the ACK2-resistant stem cells, we developed a colony assay system by modifying our coculture system. Sequential observations of the development of individual colonies and their disappearance demonstrated that the stem cells having higher proliferative capacity preferentially survive the ACK2 treatment. Furthermore, cells of subclones of the PA6 clone that were incapable of supporting long-term hemopoiesis expressed mRNA for the c-kit ligand. These results suggest that a mechanism(s) other than that involving c-kit receptor and its ligand plays an important role in the survival and proliferation of primitive hemopoietic stem cells.

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Osteoblast ablation reduces normal long-term hematopoietic stem cell self-renewal but accelerates leukemia development

Blood ◽

10.1182/blood-2014-06-582924 ◽

2015 ◽

Vol 125 (17) ◽

pp. 2678-2688 ◽

Cited By ~ 64

Author(s):

Marisa Bowers ◽

Bin Zhang ◽

Yinwei Ho ◽

Puneet Agarwal ◽

Ching-Cheng Chen ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Hematopoietic Stem Cell ◽

Self Renewal ◽

Hematopoietic Stem ◽

Key Points ◽

Leukemia Development

Key Points Bone marrow OB ablation leads to reduced quiescence, long-term engraftment, and self-renewal capacity of hematopoietic stem cells. Significantly accelerated leukemia development and reduced survival are seen in transgenic BCR-ABL mice following OB ablation.

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Cord blood transplantation provides better reconstitution of hematopoietic reservoir compared with bone marrow transplantation

Blood ◽

10.1182/blood-2003-03-0720 ◽

2003 ◽

Vol 102 (3) ◽

pp. 1138-1141 ◽

Cited By ~ 53

Author(s):

Francesco Frassoni ◽

Marina Podestà ◽

Rita Maccario ◽

Giovanna Giorgiani ◽

Gabriele Rossi ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cord Blood ◽

Mononuclear Cells ◽

Cord Blood Transplantation ◽

Hematopoietic Progenitor Cell ◽

Transplant Recipients ◽

Platelet Recovery ◽

Blood Transplantation

Abstract Delayed hematopoietic recovery is the main factor precluding a wider use of cord blood (CB) transplants. We hypothesized that this delayed engraftment might not be related to an insufficient number of stem cells in the graft, but to an intrinsic difficulty of these cells to undergo differentiation. To test our hypothesis, 2 groups of children were compared; 12 received a CB transplant and 12 an adult bone marrow (BM) transplant. We studied neutrophil and platelet recovery and, at a median time of approximately 1 year after transplantation, the frequency of colony-forming cells (CFCs) and long-term culture initiating cells (LTC-ICs) in the BM of the 2 groups. Recipients of BM transplants received 1-log more cells and had significantly faster neutrophil and platelet recovery. Conversely, the frequency of committed and early progenitors was significantly higher in the BM of children given CB cells compared with BM transplant recipients (median count of CFC/2 × 104 BM mononuclear cells, 20 versus 11, P = .007; median count of LTC-IC/106 BM mononuclear cells, 8.2 versus 0.2 P = .001). CB, but not adult BM stem cells, can better restore the host hematopoietic progenitor cell reservoir; the delayed engraftment after CB transplantation may reflect the difficulty of CB progenitors to reprogram themselves toward differentiation.

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