Predictive Progenitor Profiling in Chronic Myelomonocytic Leukemia.

Abstract Chronic myelomonocytic leukemia (CMML) is a unique myeloproliferative disease characterized by marrow dysplasia and an increase in monocytes. The median survival of patients with CMML is short, in part, because CMML is frequently resistant to therapy. In order to provide more effective CMML targeted therapies, a better understanding of the basic biology of CMML progenitors is required. We used FACS analysis and recently identified cell surface markers to identify phenotypic and functional differences between normal and CMML (n=14) bone marrow hematopoietic stem cells and myeloid progenitors. CMML marrow was typified by a reduction in CD34+CD38−CD90+(Thy1)Lin− hematopoietic stem cells and an expansion of CD34+CD38−CD90−Flk2+Lin- cells relative to normal bone marrow. In addition, there was a two-fold expansion in common myeloid progenitors (CMPs) and a corresponding decrease in megakaryocyte-erythroid progenitors (MEPs) suggesting that there was a skew in differentiation toward the myeloid lineage. In contrast to normal bone marrow derived CMPs, CMML CMPs gave rise to myeloid but not erythroid colonies. Moreover, real time quantitative RT-PCR analysis of highly purified FACS-sorted CMML CMPs demonstrated increased expression of two key regulators of myelomonocytic differentiation, PU.1 and c-jun, compared with normal bone marrow. A more detailed understanding of the basic biology of CMML myeloid progenitors and the genes that work in concert to expand them may aid in identifying novel molecular targets for CMML therapy.

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Retention but Significant Reduction of BCR-ABL Transcript in Hematopoietic Stem Cells in Chronic Myelogenous Leukemia after Imatinib Therapy.

Blood ◽

10.1182/blood.v112.11.2116.2116 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2116-2116

Author(s):

Akihiro Abe ◽

Yosuke Minami ◽

Kunio Kitamura ◽

Fumihiko Hayakawa ◽

Yuka Nomura ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cell Sorting ◽

Chronic Phase ◽

Myelogenous Leukemia ◽

Stem Cell Population ◽

Normal Bone Marrow ◽

Hematopoietic Stem ◽

Normal Bone ◽

Myeloid Progenitors

Abstract Chronic myeloid leukemia (CML) is effectively treated with imatinib mesylate (IM), a small molecule inhibitor of the BCR-ABL tyrosine kinase that is expressed in the entire hematopoietic compartment including stem cells (HSC) and progenitors in CML patients. While IM induces disease remission, it does not appear to eradicate BCR-ABL-positive stem cells. We analyzed the HSC/Progenitors profiles using fluorescence-activated cell sorting (FACS) and investigated the minimal residual disease (MRD) in HSC and myeloid progenitors from patients with CML chronic phase (CP) after IM-therapy. HSC identified as CD34+CD38–Lin–, were separated to Thy-1+ (HSC/Thy-1+) and Thy-1– (HSC/Thy-1–) cells. HSC/Thy-1+ (CD34+CD38–Thy-1+Lin–), HSC/Thy-1– (CD34+CD38–Thy-1–Lin–), common myeloid progenitors (CMP: CD34+CD38+IL-3Rα+CD45RA–Lin–), granulocyte–macrophage progenitors (GMP: CD34+CD38+IL-3Rα+CD45RA+Lin–), and megakaryocyte–erythroid progenitors (MEP: CD34+CD38+IL-3Rα–CD45RA– Lin–) were isolated by cell sorting and MRD was quantified with real-time polymerase chain reaction detecting BCR-ABL transcripts. FACS analysis revealed higher levels of the HSC/Thy-1– cells and progenitors (CD34+CD38+Lin– cells) in bone marrow from patients with CML CP than in normal bone marrow although the level of long-term HSC in CML CP was similar with normal bone marrow. After the IM-therapy the proportion of progenitor pools (CD34+Lin– cells) within Lin– were remarkably reduced, especially that of HSC/Thy-1– cells and progenitor cells. The proportion of MEP was increased and that of GMP was decreased in bone marrow from patients with CML CP as compared with their normal counterparts. BCR-ABL positive progenitors in bone marrow were eradicated within 12 month in 5 out of 9 patients. BCR-ABL positive cells, however, remained in the stem cell population. They were positive even after achieving undetectable levels of BCR-ABL transcript in total RNA isolated from the bone marrow. The ratio of BCR-ABL to BCR was significantly decreased with the continuation of imatinib, however the retention of BCR-ABL-positive cells was observed in HSC/Thy-1– or HSC/Thy-1+ populations except one out of 9 cases. These results indicated retention but significant reduction of BCR-ABL positive stem cells in CML during IM-therapy. They also implicate that the sorted and purified stem cells are useful for more sensitive quantification of BCR-ABLpositive MRD.

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Lin-Sca-1+c-KitlowCD48+CD71+ Cells Are the Engine of Bone Marrow Regeneration

Blood ◽

10.1182/blood.v124.21.5112.5112 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5112-5112

Author(s):

Emanuel Necas ◽

Katarina Szikszai-Forgacova ◽

Katerina Faltusova ◽

Ludek Sefc ◽

Filipp Savvulidi ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Sublethal Dose ◽

Normal Bone Marrow ◽

Early Recovery ◽

Hematopoietic Stem ◽

Normal Bone ◽

Bone Marrow Regeneration

Abstract The hematopoietic tissue is the most suitable tissue for studies into the biology of regeneration. We examined bone marrow regeneration starting from a very low number of repopulating cells. Mice were irradiated by a sublethal dose of 6 Gy and recovery of hematopoiesis was examined in its three basic compartments: stem and progenitor cells, precursors of blood cells, and peripheral blood between days 8 – 90 following irradiation. The irradiation reduced existing pools of progenitors and stem cells to ̴0.1%. We focused on the pools of stem cells and progenitors that were evaluated by several assays. A novel assay based on engraftment of a defined number of transplanted competitors revealed delayed filling of the niches for long-term repopulating cells (LTRCs; stem cells) as compared to filling of the niches for short-term repopulating cells (STRCs; progenitors) by endogenous STRCs and LTRCs generated by cells surviving irradiation. This indicated a very slow recovery of the pool of stem cells, lagging after recovery of the pool of progenitors. This was confirmed by a traditional competitive transplantation assay, as well as by another novel assay that measured the capacity of bone marrow to establish pools of stem cells and progenitors in lethally irradiated mice. Direct assays of progenitors by the spleen colony (CFU-S) and in vitro CFC-GM assays indicated a low frequency of the clonogenic cells in regenerating bone marrow. Productive hematopoiesis was re-established about 10 days after irradiation in presence of very low pools of progenitors and stem cells. Next we examined numbers of immature hematopoietic cells by flow cytometry focused at lineage negative (Lin-), Sca-1 positive (Sca-1+), c-Kit+ cells (LSK cells), characterized further by means of the CD150, CD48 and CD71 antigenic markers. In absolute terms, only the population of LSK CD150+CD48+ recovered and overshot normal values after day 12 following irradiation. In relative terms, the LSK CD48+ cells (CD150+ and CD150-) represented more than 99% of all LSK cells two weeks after irradiation. Also in relative terms, the LSK CD150+CD48- cells, which are highly enriched for hematopoietic stem cells in normal bone marrow, recovered by day 30 and became relatively abundant later on. Another striking change consisted in initial lack and later severe scarcity of cells with the phenotype of LSK CD150-CD48-. These cells, serving as multipotent progenitors in normal bone marrow thus remained severely depleted in regenerating bone marrow during the whole follow-up period. LSK cells were mostly negative for CD71 (transferrin receptor) in normal bone marrow but became highly positive for this marker between days 10 – 20 after irradiation. In conclusion, our semi-quantitative evaluation of regenerating hematopoiesis reveals its highly peculiar features. Those include an early recovery of blood cell production, followed by significant expansion of the Lin-Sca-1+c-KitlowCD48+CD71+ cells in presence of a highly reduced pool of hematopoietic stem cells. Disclosures No relevant conflicts of interest to declare.

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Proliferation and differentiation of highly enriched mouse hematopoietic stem cells and progenitor cells in response to defined growth factors.

Journal of Experimental Medicine ◽

10.1084/jem.167.6.1825 ◽

1988 ◽

Vol 167 (6) ◽

pp. 1825-1840 ◽

Cited By ~ 38

Author(s):

C E Müller-Sieburg ◽

K Townsend ◽

I L Weissman ◽

D Rennick

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Growth Factors ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

M Cells ◽

Normal Bone Marrow ◽

Hematopoietic Stem ◽

Normal Bone

Three distinct hematopoietic populations derived from normal bone marrow were analyzed for their response to defined growth factors. The Thy-1loT- B- G- M-population, composing 0.2% of bone marrow, is 370-fold enriched for pluripotent hematopoietic stem cells. The two other populations, the Thy-1- T- B- G- M- and the predominantly mature Thy-1+ T+ B+ G+ M+ cells, lack stem cells. Thy-1loT- B- G- M- cells respond with a frequency of one in seven cells to IL-3 in an in vitro CFU-C assay, and give rise to many mixed colonies as expected from an early multipotent or pluripotent progenitor. The Thy-1- T- B- G- M- population also contains progenitor cells which responded to IL-3. However, colonies derived from Thy-1- T- B- G- M- cells are almost exclusively restricted to the macrophage/granulocyte lineages. This indicates that IL-3 can stimulate at least two distinct clonogenic early progenitor cells in normal bone marrow: multipotent Thy-1loT- B- G- M- cells and restricted Thy-1- T- B- G- M- cells. Thy-1loT- B- G- M-cells could not be stimulated by macrophage colony-stimulating factor (M-CSF), granulocyte CSF (G-CSF) or IL-5 (Eosinophil-CSF). The hematopoietic precursors that react to these factors are enriched in the Thy-1- T- G- B- M- population. Thus, multipotent and restricted progenitors can be separated on the basis of the expression of the cell surface antigen Thy-1.

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Poly I:C Stimulates Sca-1 Expression in Murine Bone Marrow and Increases the Number of Phenotypic Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v114.22.2504.2504 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2504-2504

Author(s):

Russell Garrett ◽

Gerd Bungartz ◽

Alevtina Domashenko ◽

Stephen G. Emerson

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Hematopoietic Cells ◽

Poly I:C ◽

Hematopoietic Stem ◽

Marrow Cells ◽

Myeloid Progenitors

Abstract Abstract 2504 Poster Board II-481 Polyinosinic:polycytidlyic acid (poly I:C) is a synthetic double-stranded RNA used to mimic viral infections in order to study immune responses and to activate gene deletion in lox-p systems employing a Cre gene responsive to an Mx-1 promoter. Recent observations made by us and others have suggested hematopoietic stem cells, responding to either poly I:C administration or interferon directly, enter cell cycle. Twenty-two hours following a single 100mg intraperitoneal injection of poly I:C into 10-12 week old male C57Bl/6 mice, the mice were injected with a single pulse of BrdU. Two hours later, bone marrow was harvested from legs and stained for Lineage, Sca-1, ckit, CD48, IL7R, and BrdU. In two independent experiments, each with n = 4, 41 and 33% of Lin- Sca-1+ cKit+ (LSK) IL-7R- CD48- cells from poly I:C-treated mice had incorporated BrdU, compared to 7 and 10% in cells from PBS-treated mice. These data support recently published reports. Total bone marrow cellularity was reduced to 45 and 57% in the two experiments, indicating either a rapid death and/or mobilization of marrow cells. Despite this dramatic loss of hematopoietic cells from the bone marrow of poly I:C treated mice, the number of IL-7R- CD48- LSK cells increased 145 and 308% in the two independent experiments. Importantly, the level of Sca-1 expression increased dramatically in the bone marrow of poly I:C-treated mice. Both the percent of Sca-1+ cells and the expression level of Sca-1 on a per cell basis increased after twenty-four hours of poly I:C, with some cells acquiring levels of Sca-1 that are missing from control bone marrow. These data were duplicated in vitro. When total marrow cells were cultured overnight in media containing either PBS or 25mg/mL poly I:C, percent of Sca-1+ cells increased from 23.6 to 43.7%. Within the Sca-1+ fraction of poly I:C-treated cultures, 16.7% had acquired very high levels of Sca-1, compared to only 1.75% in control cultures. Quantitative RT-PCR was employed to measure a greater than 2-fold increase in the amount of Sca-1 mRNA in poly I:C-treated cultures. Whereas the numbers of LSK cells increased in vivo, CD150+/− CD48- IL-7R- Lin- Sca-1- cKit+ myeloid progenitors almost completely disappeared following poly I:C treatment, dropping to 18.59% of control marrow, a reduction that is disproportionately large compared to the overall loss of hematopoietic cells in the marrow. These cells are normally proliferative, with 77.1 and 70.53% accumulating BrdU during the 2-hour pulse in PBS and poly I:C-treated mice, respectively. Interestingly, when Sca-1 is excluded from the analysis, the percent of Lin- IL7R- CD48- cKit+ cells incorporating BrdU decreases following poly I:C treatment, in keeping with interferon's published role as a cell cycle repressor. One possible interpretation of these data is that the increased proliferation of LSK cells noted by us and others is actually the result of Sca-1 acquisition by normally proliferating Sca-1- myeloid progenitors. This new hypothesis is currently being investigated. Disclosures: No relevant conflicts of interest to declare.

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Generation and Analysis of a Novel Mouse Model for Chronic Myelomonocytic Leukemia (CMML) with Acquired Expression of c-CBLQ367P

Blood ◽

10.1182/blood.v124.21.4602.4602 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4602-4602

Author(s):

Yuichiro Nakata ◽

Takeshi Ueda ◽

Akiko Nagamachi ◽

Linda Wolff ◽

Ogawa Seishi ◽

...

Keyword(s):

Stem Cells ◽

Mouse Model ◽

Hematopoietic Stem Cells ◽

Myeloid Cells ◽

Ring Finger ◽

B Cell Lymphoma ◽

Chronic Myelomonocytic Leukemia ◽

Hematopoietic Stem ◽

Myelomonocytic Cells ◽

Myelomonocytic Leukemia

Abstract Myelodysplastic syndromes (MDS) are disorders originated from hematopoietic stem cells (HSCs), which are characterized by ineffective hematopoiesis, dysplasia mainly in the myeloid lineage, and high progression ratio to acute myeloid leukemia (AML). Recently, we identified mutations of the c-CBL (Casitas B cell lymphoma, a cellular homologue of v-CBL) gene in patients with MDS and myeloproliferative neoplasms (MPN). The mutations are detected in about 8% of the patients with the highest frequency in chronic myelomonocytic leukemia (CMML) cases with acquired uniparental disomy (aUPD) at 11q. c-CBL encodes a RING finger-based E3 ubiquitin ligase that negatively regulates receptor-mediated intracellular signaling. c-CBL is highly expressed in HSCs, strongly suggesting that it functions as a fine regulator of hematopoietic homeostasis. In fact, c-CBL knockout (KO) mice showed a myeloproliferative phenotype, owing to the hyper-responsiveness of HSCs to cytokine stimulation and subsequent augmented hematopoietic progenitor pool. In addition, c-CBL knockin (KI) mice harboring a mutation in the RING finger domain in one allele and a null mutation in the other allele exhibit an MPD-like disease and eventually progress to AML. These findings indicate that dysfunction of c-CBL perturbs normal hematopoietic development and contributes to hematopoietic abnormalities, but the precise leukemogenic mechanism(s) remains elusive. To gain insights into this issue and to create a novel animal model for mutated c-CBL-harboring leukemia, we generated conditional knock-in (cKI) mice that express wild-type c-CBL at steady state and inducibly express c-CBLQ367P, which was identified in patients with chronic myelomonocytic leukemia (CMML). After induced expression of c-CBLQ367P, the cKI mice exhibited a rapid and sustained increase in myelomonocytic cells with dysplasia in the peripheral blood and splenic enlargement with proliferation of myeloid cells, which closely resemble to the phenotype of human CMML. The bone marrow (BM) was hypercellular with predominance of myeloid cells, and increased number of HSC subpopulations and early myeloid progenitors were observed. In addition, phosphorylation of AKT, STAT3 and STAT5 was detected in long-term hematopoietic stem cells (LT-HSCs) of c-CBLQ367P cKI mice, indicating that PI3K/AKT and JAK/STAT signaling pathways are activated in c-CBLQ367P LT-HSCs. Moreover, competitive repopulation assays revealed that mice transplanted with c-CBLQ367P LT-HSCs showed significantly higher donor-derived chimerism than those transplanted with control LT-HSCs and displayed expansion of myelomonocytic cells as observed in c-CBLQ367P cKI mice, indicating that c-CBLQ367P conferred a proliferative advantage to LT-HSCs and that the phenotypes observed in c-CBLQ367P cKI mice were hematopoietic cell-intrinsic. CMML is known to progress to AML, possibly with additional genetic aberrations. To investigate the mechanism(s) underlying the disease evolution, we performed retroviral insertional mutagenesis using MOL4070A, a derivative of Moloney murine leukemia virus capable of inducing myeloid diseases. Almost all MOL4070A-infected c-CBLQ367P cKI mice developed AML, while no disease was observed in virus-injected control mice. Inverse PCR method identified Evi1 gene as a common integration site in the diseased mice and high Evi1 expression was detected in Evi1-integrated tumors. Mice transplanted with Evi1-transduced c-CBLQ367P cKI c-kit-positive BM cells developed AML at a high frequency and in a shortened period as compared to those transplanted with Evi1-transduced control cells. Taken together, we demonstrated that acquired expression of c-CBLQ367P plays a causative role in the development of CMML by activating PI3K/AKT and JAK/STAT pathways in HSCs and found that Evi1 overexpression cooperates with c-CBLQ367P to develop AML. Our mouse model provides a powerful tool for understanding of the pathogenesis of CMML and for developing novel therapeutic strategies. Disclosures No relevant conflicts of interest to declare.

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Phenotypic and Functional Changes Induced at the Clonal Level in Hematopoietic Stem Cells After 5-Fluorouracil Treatment

Blood ◽

10.1182/blood.v89.10.3596 ◽

1997 ◽

Vol 89 (10) ◽

pp. 3596-3606 ◽

Cited By ~ 195

Author(s):

Troy D. Randall ◽

Irving L. Weissman

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Cell Population ◽

Stem Cell Population ◽

Normal Bone Marrow ◽

Hematopoietic Stem ◽

Normal Bone

Abstract A significant fraction of hematopoietic stem cells (HSCs) have been shown to be resistant to the effects of cytotoxic agents such as 5-fluorouracil (5-FU), which is thought to eliminate many of the rapidly dividing, more committed progenitors in the bone marrow and to provide a relatively enriched population of the most primitive hematopoietic progenitor cells. Although differences between 5-FU–enriched progenitor populations and those from normal bone marrow have been described, it remained unclear if these differences reflected characteristics of the most primitive stem cells that were revealed by 5-FU, or if there were changes in the stem-cell population itself. Here, we have examined some of the properties of the stem cells in the bone marrow before and after 5-FU treatment and have defined several activation-related changes in the stem-cell population. We found that long-term reconstituting stem cells decrease their expression of the growth factor receptor c-kit by 10-fold and increase their expression of the integrin Mac-1 (CD11b). These changes begin as early as 24 hours after 5-FU treatment and are most pronounced within 2 to 3 days. This activated phenotype of HSCs isolated from 5-FU–treated mice is similar to the phenotype of stem cells found in the fetal liver and to the phenotype of transiently repopulating progenitors in normal bone marrow. We found that cell cycle is induced concomitantly with these physical changes, and within 2 days as many as 29% of the stem-cell population is in the S/G2/M phases of the cell cycle. Furthermore, when examined at a clonal level, we found that 5-FU did not appear to eliminate many of the transient, multipotent progenitors from the bone marrow that were found to be copurified with long-term repopulating, activated stem cells. These results demonstrate the sensitivity of the hematopoietic system to changes in its homeostasis and correlate the expression of several important surface molecules with the activation state of HSCs.

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Potential Involvement of BIRC5 in Maintaining Pluripotency and Cell Differentiation of Human Stem Cells

Oxidative Medicine and Cellular Longevity ◽

10.1155/2019/8727925 ◽

2019 ◽

Vol 2019 ◽

pp. 1-8 ◽

Cited By ~ 7

Author(s):

Paulina Gil-Kulik ◽

Arkadiusz Krzyżanowski ◽

Ewa Dudzińska ◽

Jolanta Karwat ◽

Piotr Chomik ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cell Differentiation ◽

Peripheral Blood ◽

Survivin Expression ◽

Regulatory Function ◽

Normal Bone Marrow ◽

Human Stem Cells ◽

Hematopoietic Stem ◽

Normal Bone

The BIRC5 gene encodes a survivin protein belonging to class III of inhibitors of apoptosis, IAP. This protein serves a dual role. First, it regulates cell death, and second, it is an important regulator of mitosis progression, although its physiological regulatory function has not been fully understood. Many studies have shown and confirmed that survivin is practically absent in mature tissues in nature, while its overexpression has been reported in many cancerous tissues. There is little information about the significance of BIRC5 expression in normal adult human stem cells. This paper presents the study and analysis of survivin expression at the transcription level using qPCR method, in hematopoietic stem cells from peripheral blood mobilized with a granulocyte growth factor, adherent cells derived from the umbilical cord, and normal bone marrow stem cells. The expression of this gene was also examined in the blood of normal healthy individuals. The results of the analysis have shown that the more mature the cells are, the lower the expression of the BIRC5 gene is. The lowest expression has been found in peripheral blood cells, while the highest in normal bone marrow cells. The more the CD34+ and CD105 cells in the tested material are, the higher the BIRC5 expression is. Stem cells from cell culture show higher BIRC5 expression. The study confirms the involvement of BIRC5 from the IAP family in many physiological processes apart from apoptosis inhibition. The possible effect of BIRC5 on cell proliferation; involvement in cell cycle, cell differentiation, survival, and maintenance of stem cells; and the possible effect of IAP on the antineoplastic properties of mesenchymal stem cells have been demonstrated. Our research suggests that BIRC5 may be responsible for the condition of stem cell pluripotency and its high expression may also be responsible for the dedifferentiation of tumor cells.

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Mesenchymal Stem Cells from Patients 1 Year Following Autologous Stem Cell Transplantation Have a Pro-Inflammatory and Senescent Phenotype Compromising the Support of Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v124.21.4368.4368 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4368-4368

Author(s):

Carin L.E. Hazenberg ◽

Fiona A.J. van den Heuvel ◽

Ben N.G. Giepmans ◽

Jan Jacob Schuringa ◽

Edo Vellenga

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Growth Factor ◽

Stem Cell Transplantation ◽

Autologous Stem Cell Transplantation ◽

Normal Bone Marrow ◽

Hematopoietic Stem ◽

Bone Marrow Biopsies ◽

Normal Bone

Abstract Autologous stem cell transplantation (ASCT) is a frequently applied treatment modality for patients with multiple myeloma (MM) and (relapsing) malignant lymphomas. Normal peripheral blood cell counts are usually observed 1 year post ASCT although the hematopoietic stem cell (HSC) compartment is severely impaired reflected by reduced HSC frequency and quiescence (Haematologica 2013;98:1264). Since HSCs interact intensively with the surrounding microenvironment in the bone marrow and strongly depend on these cells for a proper function, we studied the mesenchymal stem cell (MSC) compartment in patients 1 year post ASCT. We generated a biobank with patient material acquired 1 year after ASCT. Immunohistological studies of bone marrow biopsies post ASCT showed increased expression of CD271 (Nerve Growth Factor Receptor, NGFR) compared to normal bone marrow (NBM, 11.26%±1.2 of bone marrow area versus 1.87%±0.9, p<0.0001) while no difference was observed for the percentage of nestin+ or perivascular CD146+ (Melanoma Cell Adhesion Molecule, MCAM) cells. In addition an increase in CD271+-multilocular adipocytes was noted, reflecting a difference in preferential MSC differentiation. Subsequently MSCs were cultured from the CD34- fraction of the bone marrow mononuclear cells, obtained from post ASCT patients (n=11) and compared to healthy subjects (n=17). MSCs were selected by their plastic-adherency and subsequently replated to generate MSCs. Cultured MSCs from post ASCT and NBM had similar population doubling times (1.92±0.22 and 3.52±1.02 in P4 (passage 4) respectively). In addition no difference in cell surface expression of CD146 and CD271 was demonstrated on MSCs post ASCT as compared to NBM. However, the post ASCT MSCs showed a change in morphology at early passages (P3-4) and premature exhaustion of growth in 45% of the studied patients (n=11) at P5, in contrast to 18% from NBM (n=11). B-galactosidase staining of post ASCT MSCs was increased in P5 and P6 compared to NBM MSCs (20.08%±3.0 vs 9.9%±1.1, p=0.04). To study the functionality of these MSCs, post ASCT MSCs from a low passage (P3 or P4) were used for co-culture experiments with CD34+ cord blood cells in the presence of cytokines SCF, FLT3 and TPO. Co-cultures with MSCs from different post ASCT patients showed a large variation in number of cobblestone-area forming cells (CAFCs, range: 11-163, mean: 81.3±16.0) as well as the size of cobblestone area. This reflects the diversity in HSC support by post ACST MSCs and concurs with the diversity found between patients in the clinical setting. Finally gene profiling performed on cultured post ASCT (n=10) and NBM (n=9) MSCs in early passages (P2 and P3) showed upregulation of proinflammatory genes such as interleukin-6 (IL6) and genes involved in Notch and Transforming Growth Factor-ß (TGF-B) signaling such as Hairy and Enhancer of Split-1 (HES1), and Bone Morphogenetic Protein (BMP)1 and BMP4. These findings were confirmed by quantitative PCR. Foxc1 expression, recently linked to maintenance of hematopoietic stem and progenitor cells, was significantly increased in post ASCT MSCs. Collectively, these data indicate changes in the bone marrow niche, especially in the mesenchymal (CD271+) compartment, inducing premature exhaustion and affecting their supportive role for the HSCs. This damage to the niche may account for the reduced bone marrow reserve observed in patients and generate insight into putative therapeutic targets for improving transplantation strategies. Figure 1a,b,c. Figure 1a,b,c. CD271+ expression is significantly increased in post ASCT bone marrow biopsies (b) compared to normal bone marrow (NBM, a). Quantification of CD271 expression in percentage of total bone marrow area by ImageJ software. * p<0.0001 Figure 2a,b,c,d. Figure 2a,b,c,d. Similar expression of CD146 and CD271 on NBM and post ASCT MSCs. Ns: not significant Premature exhaustion of growth in post ASCT (45%) vs NBM (18%) MSCs before P6 Significant increase in B-galactosidase staining in post ASCT MSCs in P5-P6. * p<0.05 Changed morphology of post ASCT MSCs in vitro, representative example in P4 Disclosures No relevant conflicts of interest to declare.

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Phenotypic and Functional Changes Induced at the Clonal Level in Hematopoietic Stem Cells After 5-Fluorouracil Treatment

Blood ◽

10.1182/blood.v89.10.3596.3596_3596_3606 ◽

1997 ◽

Vol 89 (10) ◽

pp. 3596-3606 ◽

Cited By ~ 9

Author(s):

Troy D. Randall ◽

Irving L. Weissman

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Cell Population ◽

Stem Cell Population ◽

Normal Bone Marrow ◽

Hematopoietic Stem ◽

Normal Bone

A significant fraction of hematopoietic stem cells (HSCs) have been shown to be resistant to the effects of cytotoxic agents such as 5-fluorouracil (5-FU), which is thought to eliminate many of the rapidly dividing, more committed progenitors in the bone marrow and to provide a relatively enriched population of the most primitive hematopoietic progenitor cells. Although differences between 5-FU–enriched progenitor populations and those from normal bone marrow have been described, it remained unclear if these differences reflected characteristics of the most primitive stem cells that were revealed by 5-FU, or if there were changes in the stem-cell population itself. Here, we have examined some of the properties of the stem cells in the bone marrow before and after 5-FU treatment and have defined several activation-related changes in the stem-cell population. We found that long-term reconstituting stem cells decrease their expression of the growth factor receptor c-kit by 10-fold and increase their expression of the integrin Mac-1 (CD11b). These changes begin as early as 24 hours after 5-FU treatment and are most pronounced within 2 to 3 days. This activated phenotype of HSCs isolated from 5-FU–treated mice is similar to the phenotype of stem cells found in the fetal liver and to the phenotype of transiently repopulating progenitors in normal bone marrow. We found that cell cycle is induced concomitantly with these physical changes, and within 2 days as many as 29% of the stem-cell population is in the S/G2/M phases of the cell cycle. Furthermore, when examined at a clonal level, we found that 5-FU did not appear to eliminate many of the transient, multipotent progenitors from the bone marrow that were found to be copurified with long-term repopulating, activated stem cells. These results demonstrate the sensitivity of the hematopoietic system to changes in its homeostasis and correlate the expression of several important surface molecules with the activation state of HSCs.

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Loss Of p53 Promotes Transformation Of Oncogenic Nras-Induced Chronic Myelomonocytic Leukemia To An Acute Phase

Blood ◽

10.1182/blood.v122.21.3790.3790 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3790-3790

Author(s):

Jingfang Zhang ◽

Yangang Liu ◽

Guangyao Kong ◽

Yuan-I Chang ◽

Erik A. Ranheim ◽

...

Keyword(s):

Bone Marrow ◽

Median Survival ◽

Bone Marrow Cells ◽

Chronic Myelomonocytic Leukemia ◽

Self Renewal ◽

Hematopoietic Stem ◽

Myelomonocytic Leukemia ◽

Acute Myeloid ◽

Marrow Cells ◽

Myeloid Progenitors

Abstract Chronic myelomonocytic leukemia (CMML) primarily occurs in the elderly with the median age ranging from 65 to 75 years. As defined by WHO, CMML is characterized by persistent monocytosis in peripheral blood, hepatosplenomegaly, and the absence of BCR-ABL fusion gene. CMML is a devastating cancer for multiple reasons, one of which is that approximately 20% of CMML cases evolve into acute myeloid leukemia (AML) soon after their first diagnosis. However, little is known about the cellular and molecular mechanisms underlying this malignant transformation. Recently, our lab developed a CMML mouse model induced by oncogenic NrasG12D/+ expressed from its endogenous locus. Above 90% of recipient mice with NrasG12D/+ bone marrow cells developed CMML-like phenotypes with a median survival of ∼56 weeks. Interestingly, none of these mice spontaneously transform to AML. To identify the pathogenetic origins underlying CMML transformation to AML, we further deleted p53 expression in NrasG12D/+ bone marrow cells using p53fl/fl allele and Mx1-Cre because deletion of p53 is a common genetic event observed in oncogenic Ras-driven cancers. We found that ERK1/2 is significantly hyperactivated in NrasG12D/+; p53-/- hematopoietic stem/progenitor cells (enriched for myeloid progenitors) in the absence of cytokines or in the presence of low concentration of GM-CSF. Concomitantly, the mutant myeloid progenitors show significantly increased self-renewal in a serial replating assay in vitro. We transplanted NrasG12D/+, p53-/-, or NrasG12D/+; p53-/- bone marrow cells into lethally irradiated mice. Unlike recipients with p53-/- cells that died of a T-cell disease with 100% penetrance and a median survival of 24 weeks, ∼70% of recipients with NrasG12D/+; p53-/- cells died of AML or acute myeloid sarcoma with a median survival of 16 weeks. These malignant myeloid diseases are transplantable in secondary recipients. Interestingly, only mutant hematopoietic stem cells (HSCs) could initiate and maintain leukemia phenotypes in the NrasG12D/+ induced CMML model, whereas both NrasG12D/+; p53-/- HSCs and myeloid progenitors could initiate AML or acute myeloid sarcoma. Our results indicate that deletion of p53 cooperates with NrasG12D/+ mutation to transform CMML into an acute phase. This malignant transformation is initiated by mutant myeloid progenitors, which show increased self-renewal and potentially serve as leukemia initiating cells. Disclosures: No relevant conflicts of interest to declare.

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