Abnormal Bone Marrow-Derived Mesenchymal Stem Cell: Another Clone in Hematological Malignancy?.

Abstract The growth, differentiation, and survival of normal hematopoietic stem cells as well as bone marrow (BM) cancer cells are regulated closely by the BM microenvironment. In case of lymphoma, recent study has demonstrated that the endothelial cells (not lymphoma cells) in the lymphoma microenvironment have the same chromosomal abnormality to that of lymphoma cells themselves. Besides, in our prior study, the mesenchymal stem cells (MSCs) isolated from BM of patients with various hematological malignancies may have different surface antigens expression as comparing to normal control (Leukemia, 2005, doi:10.1038/sj.leu.2403795, published online 19 May 2005). We therefore hypothesize that the MSCs isolated from BM of patients with hematological malignancies are “different” to those of normal and may probably has the same chromosomal abnormality as to that of the hematological cancer cells. In this study, cytogenetic studies were performed on both BM-blood cells and BM-derived MSCs at the same time by using method of conventional G-banding. At submission of this abstract, we have just examined 4 specimens obtained from patients of various diseases as well as normal adult (see table). Surprisingly, we identified a new chromosomal translocation, t(3;9)(q25;q31), on the BM-derived MSCs of a patient (Case 1 of the table, Figure 1) with acute promyelocytic leukemia harboring t(15;17) on the BM blood cells. Besides, a complex chromosomal abnormalities was found in a male patient of AML (Case 2 of the table, Figure 2). Though he has received a successful allotransplant 2 years ago and the BM blood cells had completely converted to donor type (normal 46 XX), the BM-derived MSCs were still recipient in origin and having a chromosomal abnormality that was different to the BM blood cells (45, -Y) at initial diagnosis of AML. BM-derived MSCs from a patient of CML and normal adult were normal. It is still too early to making a solid conclusion at present and we are now analyzing more specimens to look into the phenomenon we found. However, this is a new field in leukemic research that deserved further investigation and our novel finding indicates that abnormal cells (another clone?) may present in the BM stroma of patients with hematological malignancies. Chromosomes of BM-blood cells and MSCs Case No. Diseases Chromosomes of BM-blood cells Chromosomes of BM-MSCs Case 1 APL, at diagnosis 46 XX, t(15;17)(q21;q11) 46 XX, t(3;9)(q25;q31). (see figure 1) Case 2 AML (45, -Y at diagnosis), after allotransplant Normal 46 XX (male patient, female donor) 46 XY, t(7;22)(p22;q11); del(13)(q12q22); del(15)(q15?). (see figure 2) Case 3 CML, at diagnosis 46 XY, t(9;22)(q34;q11) Normal 46 XY Case 4 Normal adult Normal 46 XY Normal 46 XY Figure Figure

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Identification and Age-dependent Increase of Platelet Biased Human Hematopoietic Stem Cells

10.1101/2022.01.14.475546 ◽

2022 ◽

Author(s):

Merve Aksoz ◽

Grigore-Aristide Gafencu ◽

Bilyana Stoilova Stoilova ◽

Mario Buono ◽

Yiran Meng ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Hematological Malignancies ◽

Hematopoietic Stem ◽

Cell Lineages ◽

Cells Of Origin ◽

Human Individual ◽

Age Dependent ◽

Human Hscs

Hematopoietic stem cells (HSC) reconstitute multi-lineage human hematopoiesis after clinical bone marrow transplantation and are the cells-of-origin of hematological malignancies. Though HSC provide multi-lineage engraftment, individual murine HSCs are lineage-biased and contribute unequally to blood cell lineages. Now, by combining xenografting of molecularly barcoded adult human bone marrow (BM) HSCs and high-throughput single cell RNA sequencing we demonstrate that human individual BM HSCs are also functionally and transcriptionally lineage biased. Specifically, we identify platelet-biased and multi-lineage human HSCs. Quantitative comparison of transcriptomes from single HSCs from young, and aged, BM show that both the proportion of platelet-biased HSCs, and their level of transcriptional platelet priming, increases with age. Therefore, platelet-biased HSCs, as well as their increased prevalence and elevated transcriptional platelet priming during ageing, are conserved between human and murine hematopoiesis.

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Mesenchymal Stem Cells from the Wharton’s Jelly of Umbilical Cord Segments Support the Maintenance of Long Term Culture-Initiating Cells from Cord Blood.

Blood ◽

10.1182/blood.v108.11.3650.3650 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3650-3650

Author(s):

Kent W. Christopherson ◽

Tiki Bakhshi ◽

Shamanique Bodie ◽

Shannon Kidd ◽

Ryan Zabriskie ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Hematopoietic Stem Cells ◽

Cord Blood ◽

Umbilical Cord ◽

Blood Cells ◽

Hematopoietic Stem ◽

Cord Blood Cells

Abstract Hematopoietic Stem Cells (HSC) are routinely obtained from bone marrow, mobilized peripheral blood, and umbilical Cord Blood. Traditionally, adult bone marrow has been utilized as a source of Mesenchymal Stem Cells (MSC). Bone marrow derived MSC (BM-MSC) have previously been shown to maintain the growth of HSC obtained from cord blood and have been utilized for cord blood expansion purposes. However, the use of a mismatched BM-MSC feeder stromal layer to support the long term culture of cord blood HSC is not ideal for transplant purposes. The isolation of MSC from a novel source, the Wharton’s Jelly of Umbilical Cord segments, was recently reported (Romanov Y, et al. Stem Cells.2003; 21: 105–110) (Lee O, et al. Blood.2004; 103: 1669–1675). We therefore hypothesized that Umbilical Cord derived MSC (UC-MSC) have the ability to support the long term growth of cord blood derived HSC similar to that previously reported for BM-MSC. To test this hypothesis, MSC were isolated from the Wharton’s Jelly of Umbilical Cord segments and defined morphologically and by cell surface markers. UC-MSC were then tested for their ability to support the growth of pooled CD34+ cord blood cells in long term culture - initiating cell (LTC-IC) assays as compared to BM-MSC. We observed that like BM-MSC, CB-MSC express a defined set of cell surface markers. By flow cytometry we determined that that both UC-MSC and BM-MSC are positive for CD29, CD44, CD73, CD90, CD105, CD166, HLA-A and negative for CD45, CD34, CD38, CD117, HLA-DR expression. Utilizing Mitomycin C treated (200 μM, 15 min.) UC-MSC from multiple donors as a feeder layer we observed that UC-MSC have the ability to support the maintenance of long term hematopoiesis during the LTC-IC assay. Specifically, UC-MSC isolated from separate umbilical cord donors support the growth of 69.6±11.9 (1A), 31.7±3.9 (2B), 67.0±13.5 (3A), and 38.5±13.7 (3B) colony forming cells (CFC) per 1×104 CD34+ cord blood cells as compared to 64.0±4.2 CFC per 1×104 CD34+ cord blood cells supported by BM-MSC (Mean±SEM, N=4 separate segments from three different donors). Thus, Umbilical Cord derived Mesenchymal Stem Cells, a recently described novel source of MSC, have the ability to support long term maintenance of Hematopoietic Stem Cells, as defined by the LTC-IC assay. These results may have potential therapeutic application with respect to ex vivo stem cell expansion of Cord Blood Hematopoietic Stem Cells utilizing a Mesenchymal Stem Cell stromal layer. In addition, these data suggest the possibility of co-transplantation of matched Mesenchymal and Hematopoietic Stem Cells from the same umbilical cord and cord blood donor respectively. Lastly, these results describe a novel model system for the future study of the interaction between Cord Blood Hematopoietic Stem Cells and the appropriate supportive microenvironment represented by the Umbilical Cord - Mesenchymal Stem Cells.

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Sparc Is Dispensable for Murine Hematopoiesis, Despite Its Suspected Role in 5q- Myelodysplastic Syndrome

Blood ◽

10.1182/blood.v118.21.4822.4822 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4822-4822

Author(s):

Kavitha Siva ◽

Pekka Jaako ◽

Kenichi Miharada ◽

Emma Rörby ◽

Mats Ehinger ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Knockout Mice ◽

Peripheral Blood ◽

Blood Cells ◽

Lethal Dose ◽

Wild Type ◽

Hematopoietic Stem ◽

Normal Peripheral Blood ◽

Expression Levels

Abstract Abstract 4822 Hematopoiesis is a complex process where a limited number of stem cells give rise to all mature blood cells. It involves interplay of several factors, many of which are yet to be identified. In a search for novel regulators of hematopoiesis, we chose to study SPARC (Secreted Protein Acidic and Rich in Cysteine, also known as Osteonection and BM40) because it is downregulated upon hematopoietic differentiation (Bruno et al., Mol Cell Biol, 2004) and might therefore play a role in the regulation of hematopoietic stem cells (HSC). SPARC is a matricellular protein that forms a major component of bone and is ubiquitously expressed in a variety of tissues. It is the founding member of a family of SPARC-like proteins. Several publications have indicated an important role for SPARC in hematopoiesis. In particular – knockdown of SPARC in zebrafish embryos resulted in an altered number of circulating blood cells, and a knockout mouse model showed thrombocytopenia and reduced erythroid colony formation. We carried out an in depth phenotypic and functional analysis of the hematopoietic system of SPARC knockout mice; using it as a model to gain insight into the role of SPARC in hematopoiesis. These mice are viable and fertile but show severe osteopenia and age-onset cataract at about six months of age. They also show an altered response to tumour growth and wound healing. We used mice (129SVJ background) (Gilmour et al. EMBO, 1998) that were less than six months old. These mice had normal peripheral blood counts and the bone marrow and spleen showed no alterations in morphology or cellularity. A detailed phenotypic analysis of precursors within the bone marrow showed no significant differences in myelo-erythroid precursors as compared to wild types (n=6). Though in vitro, the precursors showed lower ability to form BFU-E (n=5, p=0.048). In transplantations of lethally irradiated recipient mice, SPARC knockout cells gave rise to multi-lineage long-term reconstitution. Also, when competed with wild type cells, they provided reconstitution as well as their wild type counterparts. When SPARC knockout mice (n=8) were transplanted with wild type cells, there was normal reconstitution, indicating that a SPARC deficient niche can fully support normal hematopoiesis. We also tested if SPARC deficient mice respond differently to hematopoietic stress. We subjected mice (n=7) to sub lethal dose of irradiation and to experimentally induced anemia (n=7) and followed recovery by analyzing peripheral blood counts. In both SPARC knockouts and wild type mice, the blood counts recovered in a similar fashion. In conclusion, we find that SPARC is dispensable for murine hematopoiesis. It is possible that there are compensatory mechanisms involving other members of the SPARC family that ultimately lead to normal hematopoiesis in the murine model. In humans, SPARC maps to the deleted region in 5q MDS and has been reported to be 71 % down regulated in patient samples (Lehmann et al. Leukemia, 2007). It is the most prominent gene that is up regulated in response to lenalidomide, a drug that inhibits the malignant clone (Pellagatti et al. PNAS, 2007). SPARC is thus increasingly speculated to be involved in the pathophysiology of this hematopoetic disease. We analysed the expression levels of SPARC mRNA in the hematopoietic stem/progenitor cell compartment and found high expression levels in the CD34+ fraction of human cord blood cells. In contrast, there is very low level of SPARC expression in all compartments of murine HSCs. Therefore SPARC function may play a more important role in human hematopoiesis than in murine blood cell regulation. Disclosures: No relevant conflicts of interest to declare.

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Long-Term Follow-Up of Serially Transplanted Mice Receiving Extended Reduced Intensity Selection of MGMT-P140K Expressing Murine Repopulating Stem Cells Demonstrates Stable Oligo- to Polyclonal Hematopoiesis without Clonal Exhaustion.

Blood ◽

10.1182/blood.v106.11.1286.1286 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1286-1286

Author(s):

Claudia Ball ◽

Manfred Schmidt ◽

Ingo Pilz ◽

Monika Schrempp ◽

Christof von Kalle ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Gene Transfer ◽

Blood Cells ◽

Bone Marrow Cells ◽

Hematopoietic Stem ◽

Reduced Intensity ◽

Selection Of

Abstract In vivo selection of gene modified hematopoietic stem cells permanently increases the relative proportion of blood cells that carry a therapeutic transgene despite initially low gene transfer efficiency, thereby decreasing the likelihood of insertional mutagenesis and avoiding the need of myeloablative conditioning regimens. P140K Mutant O6-methylguanine-DNA methyltransferase (MGMT) enzyme confers resistance to the combination of the MGMT inhibitor O(6)-benzylguanine (O(6)BG) and nitrosourea drugs such as 1,3-bis-(2 chloroethyl)-1-nitrosourea (BCNU). We have previously shown that reduced intensity and toxicity BCNU/O6-BG selection allows efficient selection of MGMT-P140K expressing oligoclonal murine hematopoiesis. Nevertheless, whether long-term selection and the associated proliferative stress impairs long-term differentiation and proliferation of MGMT-P140K expressing stem cell clones is currently unknown and remains a major concern in the clinical application of MGMT selection. To address this question, serial transplantations of murine MGMT-P140K expressing hematopoiesis combined with repeated administrations of O6-BG and BCNU were done. After ex vivo gene transfer of an MGMT/IRES/eGFP encoding retroviral vector, bone marrow cells were transplanted into syngeneic C57 BL/6J mice and primary, secondary and tertiary recipient mice were subsequently treated every four weeks in order to exaggerate potential effects on long-term clonal behaviour. Lineage contribution of the transduced hematopoiesis was monitored by FACS over a total of 14 rounds of selection and clonality by LAM-PCR over a total of 12 rounds of selection. In primary mice the percentage of transduced blood cells increased from 4.7 ± 0.8 % to 36.4 ± 9.8 % (n=12) and in secondary mice from 29.9 ± 7.2 % to 65.1 ± 8.7 % (n=18) after selection without persisting peripheral blood cytopenia. Lineage analysis showed an unchanged multilineage differentiation potential of transduced cells in 1st, 2nd and 3rd generation animals. LAM PCR analysis of peripheral blood samples revealed stable oligo- to polyclonal hematopoiesis in primary and secondary mice. Evidence for predominant clones or clonal exhaustion was not observed despite up to 12 rounds of BCNU/O6-BG treatment. Interestingly, pairs of secondary transplanted mice that received bone marrow cells from identical donors showed very similar clonal composition, engraftment kinetics under selection and lineage contribution of the transduced hematopoiesis, indicating extensive self-renewal of transplantable stem cells in the primary mice resulting in a net symmetric refilling of the stem cell compartment. In summary, we demonstrate that even extended selection of MGMT-P140K expressing hematopoietic stem cells by repetitive chemotherapy does not affect their differentiation or proliferation potential and does not result in clonal exhaustion. Our results have important implications for the clinical use of MGMT selection strategies for the amplification of a limited number of gene corrected clones in clinical gene therapy.

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Homeostatic Role of the Krüppel-Like Factor 4 (KLF4) in Proliferation and Differentiation of Primitive Hematopoietic Progenitor Cells.

Blood ◽

10.1182/blood.v114.22.1497.1497 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1497-1497 ◽

Cited By ~ 2

Author(s):

Chun Shik Park ◽

Takeshi Yamada ◽

H. Daniel Lacorazza

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Progenitor Cells ◽

Peripheral Blood ◽

Blood Cells ◽

Proliferative Potential ◽

Dependent Manner ◽

Hematopoietic Stem ◽

Deficient Mice

Abstract Abstract 1497 Poster Board I-520 KLF4 is a tumor suppressor in the gastrointestinal tract known to induce cell cycle arrest in a cell context dependent manner. We recently reported that KLF4 maintains quiescence of T lymphocytes downstream of T-cell receptor signaling (Yamada et al., Nature Immunology, 2009). The role of KLF4 in reprogramming adult somatic cells into pluripotent stem cells along with Oct3/4, c-Myc and Sox2 suggests that KLF4 restricts proliferation of undifferentiated cells. In spite of a redundant role of KLF4 in fetal liver hematopoietic stem cells (HSC), its role in the maintenance of adult bone marrow HSCs has not been studied yet. To study the role of KLF4 in the hematopoietic system we used gain- and loss-of-function mouse models. Retroviral transfer of KLF4 into wild type bone marrow (BM) cells led to significant reduction of colony forming units (CFU) in methylcellulose cultures due to increased apoptosis and lower proliferation. Then, Mx1-Cre was used to induce deletion of Klf4-floxed mice by polyI:C administration. Analysis of peripheral blood cells up to 6-9 months post polyI:C administration showed significant reduction of monocytes, as previously reported, and expansion of CD8+CD44+ T cells due to their increased proliferative potential. BM cells from Klf4-deficient mice exhibited increased number of myeloid progenitor cells measured by flow cytometry (Lin-Sca-1-c-kit+FcRII/III+CD34+ cells), CFU and CFU-S8. Cytoablation with 5-fluorouracil (5-FU) showed lower nadir of peripheral white blood cells in Klf4-deficient mice compared to control mice. In spite of normal multilineage reconstitution in BM transplants experiments, competitive reconstitution with Klf4-deficient and normal BM cells resulted in reduced contribution of Klf4-deficient cells to peripheral blood, likely due to homing and proliferative differences. Collectively, our data shows that KLF4 has an important role in function of hematopoietic stem and progenitor cells. Disclosures: No relevant conflicts of interest to declare.

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The Endothelial Antigen ESAM Marks Hematopoietic Stem Cells throughout Life

Blood ◽

10.1182/blood.v112.11.727.727 ◽

2008 ◽

Vol 112 (11) ◽

pp. 727-727 ◽

Cited By ~ 1

Author(s):

Takafumi Yokota ◽

Kenji Oritani ◽

Stefan Butz ◽

Koichi Kokame ◽

Paul W Kincade ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Endothelial Cell ◽

Hematopoietic Stem Cells ◽

Blood Cells ◽

Fetal Liver ◽

Side Population ◽

Expression Profiles ◽

Hematopoietic Stem

Abstract Hematopoietic stem cells (HSC) are an important cell type with the capacity for self-renewal as well as differentiation into multi-lineage blood cells, maintaining the immune system throughout life. Many studies have attempted to identify unique markers associated with these extremely rare cells. In bone marrow of adult mice, the Lin-c-kitHi Sca1+ CD34−/Lo Thy1.1Lo subset is known to include HSC with long-term repopulating capacity. However, several of these parameters differ between strains of mice, change dramatically during developmental age and/or are expressed on many non-HSC during inflammation. Efficient HSC-based therapies and the emerging field of regenerative medicine will benefit from learning more about what defines stem cells. We previously determined that the most primitive cells with lymphopoietic potential first develop in the paraaortic splanchnopleura/aorta-gonad-mesonephros (AGM) region of embryos using Rag1/GFP knock-in mice. We also reported that Rag1/GFP-c-kitHi Sca1+ cells derived from E14.5 fetal liver (FL) reconstituted lympho-hematopoiesis in lethally irradiated adults, while Rag1/GFPLo c-kitHi Sca1+ cells transiently contributed to T and B lymphopoiesis. To extend those findings, microarray analyses were conducted to search for genes that characterize the initial transition of fetal HSC to primitive lymphopoietic cells. The comparisons involved mRNA from Rag1Lo ckitHi Sca1+, early lymphoid progenitors (ELP) and the HSC-enriched Rag1-ckitHi Sca1+ fraction isolated from E14.5 FL. While genes potentially related to early lymphopoiesis were discovered, our screen also identified genes whose expression seemed to correlate with HSC. Among those, endothelial cell-selective adhesion molecule (ESAM) attracted attention because of its conspicuous expression in the HSC fraction and sharp down-regulation on differentiation to ELP. ESAM was originally identified as an endothelial cell-specific protein, but expression on megakaryocytes and platelets was also reported (J. Biol. Chem., 2001, 2002). Flow cytometry analyses with anti-ESAM antibodies showed that the HSC-enriched Rag1-c-kitHi Sca1+ fraction could be subdivided into two on the basis of ESAM levels. The subpopulation with the high density of ESAM was enriched for c-kitHi Sca1Hi cells, while ones with negative or low levels of ESAM were found in the c-kitHi Sca1Lo subset. Among endothelial-related antigens on HSC, CD34 and CD31/PECAM1 were uniformly present on Rag1-c-kitHi Sca1+ cells in E14.5 FL and neither resolved into ESAMHi and ESAM−/Lo fractions. Expression profiles of Endoglin and Tie2 partially correlate with ESAM. The primitive ESAMHi fraction uniformly expressed high levels of Endoglin and Tie2, but many of the more differentiated ESAM−/Lo cells still retained the two markers. ESAM expression correlated well with HSC activity. Cells in the ESAMHi Rag1-ckitHi Sca1+ fraction formed more and larger colonies than those in the ESAM-/Lo Rag1-ckitHi Sca1+ fraction. Particularly, most CFU-Mix, primitive progenitors with both myeloid and erythroid potential, were found in the ESAMHi fraction. In limiting dilution stromal cell co-cultures, we found that 1 in 2.1 ESAMHi Rag1-ckitHi Sca1+ cells and 1 in 3.5 ESAM−/Lo Rag1-ckitHi Sca1+ cells gave rise to blood cells. However, while only 1 in 125 ESAM−/Lo Rag1-ckitHi Sca1+ cells were lymphopoietic under these conditions, 1 in 8 ESAMHi Rag1-ckitHi Sca1+ cells produced CD19+ B lineage cells. In long-term reconstituting assays, ESAMHi Rag1-ckitHi Sca1+ cells contributed highly to the multi-lineage recovery of lympho-hematopoiesis in recipients, but no chimerism was detected in mice transplanted with ESAM−/Lo Rag1-ckitHi Sca1+ cells. These results suggested that HSC in E14.5 FL are exclusively present in the ESAMHi fraction. Tie2+ c-kit+ lympho-hematopoietic cells of E10.5 AGM also expressed high levels of ESAM. Furthermore, ESAM expression in adult bone marrow was detected on primitive progenitors and cells in the side population within the Lin-ckitHi Sca1+ fraction. Interestingly, the expression was up-regulated in aged mice. Based on these observations, we conclude that ESAM marks HSC throughout life in mice. We also observed that many of human cord blood CD34+ CD38− cells express ESAM, suggesting potential application for the purification of human HSC.

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A Novel Perspective on Stem Cells, Tissue Senescence and Aging: Pluripotent Very Small Embryonic-Like Stem Cells (VSELs) and Hematopoietic Stem Cells (HSCs) Are Prematurely Depleted in Adult Bone Marrow (BM) in Response to Prolonged Growth Hormone/Insulin-Like Growth Factor-1 Signaling (IIS) and Chronic Increase in Caloric Uptake

Blood ◽

10.1182/blood.v118.21.4818.4818 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4818-4818

Author(s):

Magda Kucia ◽

Janina Ratajczak ◽

Dong-Myung Shin ◽

Rui Liu ◽

Michal Masternak ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Growth Hormone ◽

Growth Factor ◽

Transgenic Mice ◽

Hematological Malignancies ◽

Caloric Intake ◽

Hematopoietic Stem ◽

Igf I ◽

Dwarf Mice

Abstract Abstract 4818 Background. Caloric uptake elevates the plasma level of growth hormone (GH), which subsequently stimulates release of insulin-like growth factor-1 (IGF-1) from the liver. Evidence is accumulating that an increase in caloric intake, which leads to prolonged growth hormone (GH) and insulin/insulin-like growth factors signaling (IIS), accelerates aging. On the other hand, caloric restriction and a resulting decrease in IIS has the opposite effect and extends lifespan (Nature 2010;464:504). In support of this finding, mice with low circulating IGF-1 levels (Laron, Ames, and Snell dwarfs) live much longer than their normal littermates and, conversely, mice with high levels of circulating IGF-1 (e.g., transgenic mice that overexpress bovine growth hormone [bGH]) have significantly reduced life span. Aim of Hypothesis. To explain these phenomena, we hypothesized that prolonged IIS prematurely depletes adult tissues of very small embryonic-like stem cells (VSELs), which are the most developmentally primitive adult tissue-residing pluripotent stem cells (Leukemia 2006;20:857). We envision that VSELs play an important role in rejuvenation of the pool of tissue-committed stem cells and, as we observed previously, the number of these cells in murine BM decreases with age. We demonstrated that in bone marrow (BM), VSELs give rise to long-term repopulating hematopoietic stem cells (LT-HSCs) (Leukemia 2011; doi:10.1038/leu.2011.73, Exp. Hematology 2011;39:225–237). As previously reported, VSELs are kept quiescent in BM and protected from premature depletion by erasure of the somatic imprint in differentially methylated regions (DMRs) of some paternally imprinted genes involved in IIS (e.g., Igf2-H19 and RasGRF1). Results. In the current study, we provide direct evidence that the number of VSELs deposited in BM during ontogenesis is related to plasma IIS signaling, which is affected by the GH/IGF-1 level. In particular, mice with elevated IGF-I level in plasma due to expression of the bovine GH transgene and wild type mice injected for a sustained period with porcine GH both exhibit significant decreases in the number of VSELs and HSCs in BM compared to control animals. These decreases were paralleled by epigenetic changes in Igf2-H19 and RasGRF1 loci in which DMRs became hypermethylated over time. These changes in methylation lead to increases in IGF-2 and RasGRF1 expression and may explain why bovine GH transgenic mice have an increase in IIS and a significantly reduced life span. Conversely, mice with low circulating plasma IGF-1 levels (Laron and Ames dwarf mice ) have higher numbers of VSELs and HSCs in BM that, in contrast to aged-matched normal littermates, are maintained at high levels even into advanced age. The molecular signature of VSELs in these animals revealed prolonged retention of hypomethylation in the DMRs within the Igf2-H19 and RasGRF1 loci, which attenuated IIS signaling in these cells. The number of VSELs, however, decreased in these animals after prolonged treatment with porcine GH or human recombinant IGF-I. Conclusions. Our data shed new light on the relationships between senescence, high GH level, prolonged IIS, and depletion of VSELs and LT-HSCs. Accordingly, we propose a new paradigm in which chronic IIS (e.g., due to chronic high caloric intake and the resulting elevated GH and IGF-1 levels) prematurely depletes VSELs in BM, which leads to a decrease in the number of LT-HSCs. By contrast, caloric restriction and a decrease in IIS may delay the age-dependent elimination of VSELs from BM. This study also indicates that GH-based anti-aging therapies need careful re-evaluation of their potentially uncontrolled stimulation of VSELs in BM and downstream effects on hematopoiesis and the development of hematological malignancies. In support of this concern, elevated IIS may lead to hematological malignancies (uncontrolled proliferation of VSELs), while, by contrast, it is known that Laron dwarf mice and Laron dwarf patients, which have a GH-receptor deficiency and low plasma IGF-1 levels, do not develop leukemias. Disclosures: Ratajczak: Neostem Inc: Consultancy, Research Funding.

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p53 Co-Activator Aspp1 Induces Apoptosis in Damaged Hematopoietic Stem Cells and Prevents Malignant Transformation

Blood ◽

10.1182/blood.v124.21.603.603 ◽

2014 ◽

Vol 124 (21) ◽

pp. 603-603 ◽

Cited By ~ 1

Author(s):

Masayuki Yamashita ◽

Eriko Nitta ◽

Toshio Suda

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Bone Marrow ◽

Dna Damage ◽

Hematopoietic Stem Cells ◽

Hematological Malignancies ◽

Aberrant Methylation ◽

Self Renewal ◽

Hematopoietic Stem ◽

Apoptotic Genes

Abstract Accumulation of DNA damage in hematopoietic stem cells (HSCs) is associated with aging, bone marrow failure and development of hematological malignancies. Mutation accumulation in HSCs precedes the development of leukemia and lymphoma, and these “pre-leukemic HSCs” can survive after chemotherapy, contributing to the relapse of the disease. Thus, understanding for the DNA damage response at a HSC level is a matter of critical importance for lifelong hematopoiesis, yet the protection mechanism for HSCs from DNA damage accumulation remains to be elucidated. During our study on the response of HSCs to ionizing radiation (IR), we have detected higher responsiveness of HSCs to DNA damage compared with committed progenitor cells: higher p53 activation was observed in HSC-enriched LSK (Lin-Sca1+cKit+) cells and LT-HSCs (CD150+CD41-CD48-LSK) than in myeloid progenitor-enriched LKS- cells. Of note, when treated with 4 Gy IR, LSK cells exhibited stronger upregulation of pro-apoptotic genes Bax, Noxa and Puma compared with LKS- cells, whereas upregulation of survival-contributing p21 and Mdm2 genes was comparable between the two populations. Corresponding to such characteristic behavior, we have identified apoptosis-stimulating protein of p53 1 (Aspp1) as a novel specific regulator of HSCs that provides HSCs with high sensitivity to apoptosis. We found that mRNA and protein of Aspp1 were specifically detected in LSK cells and LT-HSCs. To uncover the roles of Aspp1 in the regulation of HSCs, we evaluated HSCs of adult Aspp1 knockout (KO) mice. These mutant mice exhibited a major increase in the absolute number of LSK cells (1.5-fold; P<0.05) and LT-HSCs (2-fold; P<0.0005). Furthermore, self-renewal capacity of Aspp1-null HSCs was significantly enhanced as measured by serial competitive bone marrow (BM) transplantation assays (P<0.01). To assess the cause of enhanced self-renewal of Aspp1-null HSCs, we examined gene expression profile of Aspp1-null LSK cells before and after BM transplantation using multiplex quantitative RT-PCR array. Aspp1-null LSK cells showed higher expression of multiple quiescence-related genes including Tek, Mpl and Ndn. In line with this, Ki67 staining revealed that Aspp1-null LSK cells showed resistance to the loss of quiescence after serial BM transplantation (P<0.01), and Aspp1 KO mice showed accelerated recovery of peripheral blood and BM when treated with a single dose of 5-FU (P<0.05). Moreover, when serially transplanted or subjected to 4 Gy IR in vivo, Aspp1-null LSK cells exhibited higher resistance to apoptosis which was detected as decreased proportion of Annexin V-positive cells (P<0.05). Gene expression analysis consistently revealed that the induction of pro-apoptotic genes Bax, Noxa and Puma was impaired in irradiated Aspp1-null LSK cells. As a result of the reduced apoptosis, Aspp1-null LSK cells exhibited the tendency to retain persistent DNA damage after genotoxic stress as assessed by γH2AX and 53BP1 foci (chi-square test, P<0.05). Importantly, by breeding Aspp1 KO mice with Mx1-Cre mice and p53flox/flox mice, we verified that Aspp1 synergized with p53 to regulate self-renewal and genomic integrity of HSCs beyond its canonical p53-dependent function. Aspp1 loss further enhanced self-renewal capacity of HSCs in a p53-null background when assayed by serial BM transplantation (P<0.05). Likewise, Aspp1 deficiency further accentuated the accumulation of DNA damage after IR exposure in the absence of p53 (P<0.05). Consequently, whereas approximately half of the recipients receiving p53-null LSK cells died of thymic lymphoma, the recipient mice transplanted with LSK cells deficient for both Aspp1 and p53 were 100% lethal within 6 months after BM transplantation (log-rank test, P<0.01). These mice succumbed to hematological malignancies, mostly T-cell acute lymphoblastic lymphoma and leukemia (ALL) (88%) but also B-cell (6%) and myeloid (6%) malignancies. Taken together, our study demonstrates that Aspp1 attenuates HSC quiescence and induces apoptosis in damaged HSCs, in both p53-dependent and -independent manners, thereby inhibiting the development of leukemia and lymphoma in conjunction with p53 in HSCs. As loss of Aspp1 expression due to aberrant methylation of its promoter has already been proven to be an independent poor prognosis factor in ALL patients, Aspp1 may be a potential target for stem cell-directed therapy of leukemia and lymphoma. Disclosures No relevant conflicts of interest to declare.

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Neogenin-1 distinguishes between myeloid-biased and balancedHoxb5+mouse long-term hematopoietic stem cells

10.1101/608398 ◽

2019 ◽

Author(s):

Gunsagar S. Gulati ◽

Monika Zukowska ◽

Joseph Noh ◽

Allison Zhang ◽

Rahul Sinha ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Blood Cells ◽

Surface Marker ◽

Cycle Phase ◽

Hematopoietic Stem ◽

Prospective Isolation

ABSTRACTHematopoietic stem cells (HSCs) self-renew and generate all blood cells. Recent studies with single-cell transplants (1–3) and lineage tracing (4, 5) suggest that adult HSCs are diverse in their reconstitution and lineage potentials. However, prospective isolation of these subpopulations has remained challenging. Here, we identify Neogenin-1 (NEO1) as a unique surface marker on a fraction of mouse HSCs labeled withHoxb5, a specific reporter of long-term HSCs (LT-HSCs) (6). We show that NEO1+Hoxb5+LT-HSCs expand with age and respond to myeloablative stress, while NEO1−Hoxb5+LT-HSCs exhibit no significant change in number. NEO1+Hoxb5+LT-HSCs are more often in the G2/S cell cycle phase compared to NEO1−Hoxb5+LT-HSCs in both young and old bone marrow. Upon serial transplantation, NEO1+Hoxb5+LT-HSCs exhibit myeloid-biased differentiation and reduced reconstitution, while NEO1−Hoxb5+LT-HSCs are lineage-balanced and stably reconstitute recipients. Gene expression comparison reveals increased expression of cell cycle genes and evidence of lineage-priming in the NEO1+fraction. Finally, transplanted NEO1+Hoxb5+LT-HSCs rarely generate NEO1−Hoxb5+LT-HSCs, while NEO1−Hoxb5+LT-HSCs repopulate both LT-HSC fractions. This supports a model in which dormant, balanced, NEO1−Hoxb5+LT-HSCs can hierarchically precede active, myeloid-biased NEO1+Hoxb5+LT-HSCs.SIGNIFICANCE STATEMENTHematopoietic stem cells (HSCs) are rare cells that have the unique ability to regenerate themselves and produce all blood cells throughout life. However, HSCs are functionally heterogeneous and several studies have shown that HSCs can differ in their contribution to major blood lineages. In this study, we discovered that the surface marker, Neogenin-1, can divide mouse HSCs into two subpopulations—one that is more active but biased towards producing myeloid cells and another that is more dormant and capable of equally producing all blood lineages. Neogenin-1 reveals the diversity and hierarchical relationship of HSCs in the mouse bone marrow, enables the prospective isolation of myeloid-biased and balanced HSCs, and opens opportunities to do the same in humans.

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