T(15;17)-PML/RAR-Induced Leukemogenesis: Long-Term Repopulating Hematopoietic Stem Cells as the Initial Target and More Mature Progenitors as the Potential Targets for Final Leukemic Transformation.

Abstract Abstract 3980 Poster Board III-916 Stem cells have been shown to play an important role in the pathogenesis and maintenance of a significant number of malignancies, including leukemias. Similar to normal hematopoiesis the AML cell population is thought to be hierarchically organized. According to this model, only a few stem cells (LSC) are able to initiate and maintain the disease. The inefficient targeting of the leukemic stem cells (LSC) is considered responsible for relapse after the induction of complete hematologic remission (CR) in AML. Acute promyelocytic leukemia (APL) is a subtype of AML characterized by the t(15;17) translocation and expression of the PML/RARα fusion protein. Treatment of APL with all-trans retinoic acid (t-RA) as monotherapy induces CR, but not molecular remission (CMR), followed by relapse within a few months. In contrast arsenic as monotherapy induces high rates of CR and CMR followed by a long relapse-free survival. We recently have shown that in contrast to t-RA, arsenic efficiently targets PML/RAR-positive stem cells, whereas t-RA increases their proliferation. For a better characterization of LSC in APL which has to be targeted for an efficient eradication of the disease we wanted to characterize the leukemia-initiating cell and the cell population able to maintain the disease in vivo. The model was based on a classical transduction/transplantation system of murine Sca1+/lin- HSC combined with a novel approach for the enrichment of transformed cells with long-term stem cell properties. We found that PML/RAR induced leukemia from the Sca1+/lin- HSC with a frequency of 40% and a long latency of 8-12 months independently of its capacity to increase dramatically replating efficiency and CFU-S12 potential as expression of the differentiation block and proliferation potential of derived committed progenitors. Based on the hypothesis that PML/RAR exerts its leukemogenic effects on only a small proportion of the Sca1+1/lin- population, we proceeded to select and to amplify rare PML/RAR-positive cells with the leukemia-initiating potential, by a negative selection of cell populations with proliferation potential without long term stem cell-capacity (LT). Therefore we expressed PML/RAR in Sca1+/lin- cells and enriched this population for LT- (lin-/Sca1+/c-Kit+/Flk2-) and ST-HSC (lin-/Sca1+/c-Kit+/Flk2+). After a passage first in semi-solid medium for 7 days and subsequent transplantation into lethally irradiated mice, cells from the ensuing CFU-S day12 were again transplanted into sublethally recipient mice. After 12 to 36 weeks, 6/6 mice developed acute myeloid leukemia without signs of differentiation in the group transplanted with the lin-/Sca1+/c-Kit+/Flk2- population but not from that transplanted with lin-/Sca1+/c-Kit+/Flk2+ cells. This leukemia was efficiently transplanted into secondary recipients. The primary leukemic cell population gave origin to 6 clearly distinct subpopulations defined by surface marker pattern as an expression of populations with distinct differentiation status, able - after sorting - to give leukemia in sublethally irradiated recipients: Sca1+/c-Kit+/CD34- (LT-HSC), Sca1+/c-Kit+/CD34+ (ST-HSC), Sca1-/c-Kit+, B220lo/GR1+/Mac1+, B220hi/GR1+/Mac1+, B220-/Gr1-/Mac1-. Interestingly, all leukemias from the different population presented an identical phenotype. These findings strongly suggest that there is a difference between a leukemia-initiating (L-IC) and leukemia-maintaining (L-MC) cell population in the murine PML/RAR leukemia model. In contrast to the L-IC, represented by a very rare subpopulation of primitive HSC, recalling a hierarchical stem cell model, the L-MC is represented by a larger cell population with a certain grade of phenotypical heterogeneity, but a high grade of functional homogeneity recalling a stochastic cancer induction model. Disclosures: No relevant conflicts of interest to declare.

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“Hierarchical” Induction and “Stochastic” Maintenance of Leukemia in An In Vivo Model of t(6;9) Positive Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v114.22.2953.2953 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2953-2953

Author(s):

Claudia Oancea ◽

Brigitte Rüster ◽

Jessica Roos ◽

Anjali Dubey ◽

Hannelore Held ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Population ◽

Conflicts Of Interest ◽

Age Of Onset ◽

Cell Model ◽

In Vivo Model ◽

Hematopoietic Stem

Abstract Abstract 2953 Poster Board II-929 Stem cells have been shown to play an important role in the pathogenesis and maintenance of a significant number of malignancies, including leukemias. Similar to normal hematopoiesis the AML cell population is thought to be hierarchically organized. According to this model, only a few stem cells (LSC) are able to initiate and maintain the disease. The inefficient targeting of the leukemic stem cells (LSC) is considered responsible for relapse after the induction of complete hematologic remission (CR) in AML. t(6;9)-positive AML is classified as a separate entity, because of its young age of onset and poor prognosis. The t(6;9) associated fusion protein is DEK/CAN. Assuming that in AML the genetic aberration, here the t(6;9) and the expression of DEK/CAN, represents the initiation event of the leukemogenic process we wanted i.) to disclose its effects on the biology of primitive hematopoietic stem cells (HSC) and its leukemogenic potential and ii.) to characterize the leukemia-initiating cell and the cell population able to maintain the disease in vivo. The model was based on a classical transduction/transplantation system of murine Sca1+/lin- HSC combined with a novel approach for the enrichment of transformed cells with long-term stem cell properties. We found that i.) DEK/CAN induced leukemia from the Sca1+/lin− HSC with a frequency of 20% and a long latency of 8-12 months. ii.) DEK/CAN did not efficiently block the differentiation of committed progenitors; iii.) DEK/CAN increased number of colony forming cells in Sca1+/lin− HSC which did not exhibit increased replating efficiency as compared to controls; iv.) DEK/CAN augmented ST-HSC potential but not LT-HSC of murine Sca1+/lin− HSCs, most likely due to its incapacity to up-regulate p21Cip1/Waf1 expression. Based on the hypothesis that DEK/CAN exerts its leukemogenic effects on only a small proportion of the Sca1+1/lin- population, we proceeded to select and to amplify rare DEK/CAN-positive cells with the leukemia-initiating potential, by a negative selection of cell populations with proliferation potential without long term stem cell-capacity (LT). Therefore we expressed DEK/CAN in Sca1+/lin− cells and enriched this population for LT- (lin−/Sca1+/c-Kit+/Flk2−) and ST-HSC (lin−/Sca1+/c-Kit+/Flk2+). After a passage first in semi-solid medium for 7 days and subsequent transplantation into lethally irradiated mice, cells from the ensuing CFU-S day12 were again transplanted into sublethally recipient mice. We here report that i.) after 4 to 42 weeks, 6/6 mice developed AML without signs of differentiation in the group transplanted with the lin−/Sca1+/c-Kit+/Flk2− population but not from that transplanted with lin−/Sca1+/c-Kit+/Flk2+ cells; ii.) the DEK/CAN-induced AML was efficiently transplanted into secondary recipients exhibiting a very aggressive clinical picture; iii.) the leukemic cell population gave origin to four different clearly distinct subpopulations defined by surface marker pattern as an expression of populations with distinct differentiation status, all able - after sorting - to give leukemia in sublethally irradiated recipients: lin−/Sca1+/c-Kit+/CD34− (LT) lin−/Sca1+/c-Kit+/CD34+ (ST), Sca1−/c-Kit+/Mac1+/Gr1+, Sca1−/c-Kit+/Mac1−/Gr1−. These findings strongly suggest that there is a difference between a leukemia-initiating (L-IC) and leukemia-maintaining (L-MC) cell population in the murine DEK/CAN leukemia model. In contrast to the L-IC, represented by a very rare subpopulation of primitive HSC, recalling a hierarchical stem cell model, the L-MC is represented by a larger cell population with a certain grade of phenotypical heterogeneity, but a high grade of functional homogeneity recalling a stochastic cancer induction model. Disclosures: No relevant conflicts of interest to declare.

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A Novel Small Molecule CXCR4 Antagonist Potently Mobilizes Hematopoietic Stem Cells in Mice and Monkeys

10.21203/rs.3.rs-49226/v1 ◽

2020 ◽

Author(s):

Xiao Fang ◽

Xiong Fang ◽

Yujia Mao ◽

Aaron Ciechanover ◽

Yan Xu ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Small Molecule ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem ◽

Stromal Microenvironment ◽

Stromal Cell Derived Factor ◽

Hsc Transplantation

Abstract Background Hematopoietic stem cell (HSC) transplantation is an effective treatment strategy for many types of diseases. Peripheral blood (PB) is the most commonly used source of bone marrow (BM)-derived stem cells for current HSC transplantation. However, PB usually contains very few HSCs under normal conditions, as these cells are normally retained within the BM. This retention depends on the interaction between the CXC chemokine receptor 4 (CXCR4) expressed on the HSCs and its natural chemokine ligand, stromal cell-derived factor (SDF)-1α (also named CXCL12) present in the BM stromal microenvironment. In clinical practice, blocking this interaction with a CXCR4 antagonist can induce the rapid mobilization of HSCs from the BM into the PB.Methods C3H/HEJ, DBA/2, CD45.1+, CD45.2+ mice and monkeys were employed in colony-forming unit (CFU) assays, flow cytometry assays, and competitive/non-competitive transplantation assays, to assess the short-term mobilization efficacy of HF51116 and the long-term repopulating (LTR) ability of HSCs. Kinetics of different blood cells and the concentration of HF51116 in PB were also explored by blood routine examinations and pharmacokinetic assays. Results In this paper, we report that a novel small molecule CXCR4 antagonist, HF51116, which was designed and synthesized by our laboratory, can rapidly and potently mobilize HSCs from BM to PB in mice and monkeys. HF51116 not only mobilized HSCs when used alone but also synergized with the mobilizing effects of granulocyte-colony stimulating factor (G-CSF) after co-administration. Following mobilization by HF51116 and G-CSF, the long-term repopulating (LTR) and self-renewing HSCs were sufficiently engrafted in primary and secondary lethally irradiated mice and were able to rescue and support long-term mouse survival. In monkeys, HF51116 exhibited strong HSC mobilization activity and quickly reached the highest in vivo blood drug concentration. Conclusions These results demonstrate that HF51116 is a new promising stem cell mobilizer which specifically targets CXCR4 and merits further preclinical and clinical studies.

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Characterization of the Putative Stem Cells From Umbilical Cord Blood.

Blood ◽

10.1182/blood.v114.22.4231.4231 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4231-4231

Author(s):

Amos S. Gaikwad ◽

Michael Cubbage ◽

Tatiana Goltsova ◽

Christopher Threeton ◽

Maria Ty ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cord Blood ◽

Cell Population ◽

Cell Biology ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Side Population ◽

Hematopoietic Stem ◽

Cell Fractions

Abstract Abstract 4231 Cord blood (CB) is a rich source of hematopoietic stem cells (HSC) with long-term repopulating activity necessary for allogeneic stem cell transplantation. CD34+ stem cells are considered sufficient for transplantation, however recent progress in stem cell biology indicates that cells with other surface markers such as CD133 or cells expressing high aldehyde dehydrogenase activity with low side scatter (ALDHhigh/SSClow) or a rare side population (SP) of cells that exclude the Hoechst 33342 vital dye via multi drug transporters have been shown to possess stem cell properties. We characterized CD34+, CD133+, ALDH+ and SP in mononuclear cells (MNC) isolated from human CB. While the SP cell population is rare and detectable in few CB-MNC examined, we found abundant CD34+ and CD133+ cells (1.0+/-0.5 and 0.8+/-0.4 per 100 CD45+ MNC cells, respectively) following the ISHAGE protocol. A distinct ALDH+ cell population (median of 0.26%; range of 0.1 to 0.5%) was also present in all of the CB-MNC analyzed. Over 90% of the ALDH+ cells were also CD34+ and CD133+. The ability of CB-MNC to form colonies in methocult semi-solid media supplemented with cytokines yielded myeloid, lymphoid and erythroid colonies. The clonogenic potential of CB-MNC ranged from 16-48%. We are assessing the colony forming ability of purified stem cell fractions using flow cytometry. The clonogenic efficiency of these individual putative stem cells will be discussed. Disclosures: No relevant conflicts of interest to declare.

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A novel small molecule CXCR4 antagonist potently mobilizes hematopoietic stem cells in mice and monkeys

10.21203/rs.3.rs-49226/v2 ◽

2020 ◽

Author(s):

Xiao Fang ◽

Xiong Fang ◽

Yujia Mao ◽

Aaron Ciechanover ◽

Yan Xu ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Small Molecule ◽

Cxcr4 Antagonist ◽

Hematopoietic Stem ◽

Stromal Microenvironment ◽

Stromal Cell Derived Factor ◽

Hsc Transplantation

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An Ex Vivo Bioengineered Human Liver Microenvironment to Support Hematopoietic Stem Cell Maintenance and Expansion

Blood ◽

10.1182/blood-2020-141437 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 9-10

Author(s):

Na Yoon Paik ◽

Grace E. Brown ◽

Lijian Shao ◽

Kilian Sottoriva ◽

James Hyun ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Fetal Liver ◽

Human Umbilical Cord ◽

Hematopoietic Stem ◽

Main Site

Over 17,000 people require bone marrow transplants annually, based on the US department of Health and Human Services (https://bloodcell.transplant.hrsa.gov). Despite its high therapeutic value in treatment of cancer and autoimmune disorders, transplant of hematopoietic stem cells (HSC) is limited by the lack of sufficient source material due primarily inadequate expansion of functional HSCs ex vivo. Hence, establishing a system to readily expand human umbilical cord blood or bone marrow HSCs in vitro would greatly support clinical efforts, and provide a readily available source of functional stem cells for transplantation. While the bone marrow is the main site of adult hematopoiesis, the fetal liver is the primary organ of hematopoiesis during embryonic development. The fetal liver is the main site of HSC expansion during hematopoietic development, furthermore the adult liver can also become a temporary extra-medullary site of hematopoiesis when the bone marrow is damaged. We have created a bioengineered micropatterned coculture (MPCC) system that consists of primary human hepatocytes (PHHs) islands surrounded and supported by 3T3-J2 mouse embryonic fibroblasts. Long-term establishment of stable PHH-MPCC allows us to culture and expand HSC in serum-free medium supplemented with pro-hematopoietic cytokines such as stem cell factor (SCF) and thrombopoietin (TPO). HSCs cultured on this PHH-MPCC microenvironment for two weeks expanded over 200-fold and formed tight clusters around the periphery of the PHH islands. These expanded cells also retained the expression of progenitor markers of Lin-, Sca1+, cKit+, as well as the long-term HSC phenotypic markers of CD48- and CD150+. In addition to the phenotypic analysis, the expanded cells were transplanted into lethally irradiated recipient mice to determine HSC functionality. The expanded cells from the PHH-MPCC microenvironment were able to provide multi-lineage reconstitution potential in primary and secondary transplants. With our bioengineered MPCC system, we further plan to scale up functional expansion of human HSC ex vivo and to better understand the mechanistic, cell-based niche factors that lead to maintenance and expansion HSC. Disclosures No relevant conflicts of interest to declare.

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Canonical Notch Signaling Is Dispensable for the Maintenance of Adult Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v106.11.267.267 ◽

2005 ◽

Vol 106 (11) ◽

pp. 267-267 ◽

Cited By ~ 37

Author(s):

Ivan Maillard ◽

Seth E. Pross ◽

Olga Shestova ◽

Hong Sai ◽

Jon C. Aster ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Notch Signaling ◽

Transcriptional Activation ◽

Cell Function ◽

Fetal Life ◽

Hematopoietic Stem

Abstract Canonical Notch signaling operates through a highly conserved pathway that regulates the differentiation and homeostasis of hematopoietic cells. Ligand-receptor binding initiates proteolytic release of the Notch intracellular domain (ICN) which migrates to the nucleus, binds the transcription factor CSL/RBPJk and activates target genes through the recruitment of transcriptional coactivators of the Mastermind-like family (MAML). Notch signaling is essential for the emergence of hematopoietic stem cells (HSCs) during fetal life, but its effects on adult HSCs are controversial. In gain-of-function experiments, activation of Notch signaling in adult HSCs increased their self-renewal potential in vitro and in vivo. However, loss-of-function studies have provided conflicting results as to the role of physiological Notch signaling in HSC maintenance and homeostasis. To address this question, we expressed DNMAML1, a GFP-tagged pan-inhibitor of Notch signaling, in mouse HSCs. We have shown previously that DNMAML1 interferes with the formation of the ICN/CSL/MAML transcriptional activation complex and blocks signaling from all four Notch receptors (Notch1-4) (Maillard, Blood 2004). Transfer of DNMAML1-transduced bone marrow (BM) as compared to control GFP-transduced BM into lethally irradiated recipients gave rise to similar long-term stable expression of GFP for at least 6 months after transplant. DNMAML1 and GFP-transduced cells contributed equally to all hematopoietic lineages, except to the T cell and marginal zone B cell lineages, which are Notch-dependent. Expression of DNMAML1 did not affect the size of the BM progenitor compartment (Lin negative, Sca-1 positive, c-Kit high, or LSK cells), or the proportion of LSK cells that were negative for Flt3 and L-Selectin expression (containing long-term HSCs). The stem cell function of DNMAML1-transduced LSK cells was further assessed with in vivo competitive repopulation assays in lethally irradiated recipients. DNMAML1 and GFP-transduced LSK cells competed equally well with wild-type BM, as judged by their contribution to the myeloid lineage up to 4 months post-transplant, through two successive rounds of transplantation. Our data indicate that canonical Notch signaling is dispensable for the maintenance of stem cell function in adult HSCs.

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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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Enrichment of human hematopoietic stem cell activity in the CD34+Thy- 1+Lin- subpopulation from mobilized peripheral blood

Blood ◽

10.1182/blood.v85.2.368.368 ◽

1995 ◽

Vol 85 (2) ◽

pp. 368-378 ◽

Cited By ~ 136

Author(s):

L Murray ◽

B Chen ◽

A Galy ◽

S Chen ◽

R Tushinski ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Population ◽

Peripheral Blood ◽

Colony Stimulating Factor ◽

In Vivo Models ◽

Hematopoietic Stem ◽

Stimulating Factor ◽

Mobilized Peripheral Blood

Abstract The number of CD34+ cells in the peripheral blood of cancer patients is known to be increased following the administration of high dose chemotherapy and hematopoietic growth factors. These so-called peripheral blood stem cell grafts are now frequently used for autologous transplantation of patients with malignancies. In this report, we address the question of whether true long-term repopulating pluripotent hematopoietic stem cells (PHSC) are mobilized into peripheral blood following chemotherapy plus granulocyte/macrophage colony-stimulating factor (GM-CSF) or granulocyte colony-stimulating factor (G-CSF) mobilization. We have examined the presence of stem cells in mobilized peripheral blood (MPB) by using an antibody to the human Thy-1 molecule to stain the CD34+Lineage- (Lin-) population. The kinetics of mobilization of CD34+Thy-1+ Lin- cells into peripheral blood were studied, and the percentage of cells with this phenotype was found to vary widely depending on the day of leukapheresis. A CD34+Thy- 1+Lin- cell population, potentially containing PHSCs, was isolated by fluorescence activated cell sorting (FACS) and analyzed for activity. The multilineage differentiative capacity of this candidate stem cell- containing population in MPB was determined using an in vitro long-term culture system, in which cobblestone area formation was used as a means of detecting PHSCs. We also measured repopulating capacity by using two in vivo models in which severe combined immunodeficiency (SCID)-hu mice were implanted with human fetal bone or thymus grafts. Using these assays, we show that the highest frequency of cobblestone area-forming cells (CAFC) after 7 weeks of culture was observed in a subpopulation of CD34+Lin- cells, which expressed low levels of Thy-1. This cell population was capable of producing both B and myeloid cells, and maintaining CD34+Lin- cells in these long term cultures. Moreover, the CD34+Thy-1+Lin- cell subset possessed a higher ability to engraft and to demonstrate multilineage differentiative potential at 8 weeks in the SCID-hu bone assay. However, in the SCID-hu thymus model, both Thy-1+ and Thy-1- subpopulations were capable of donor T-cell engraftment at 6 weeks, suggesting the presence of cells capable of initiating T lymphopoiesis in both populations.(ABSTRACT TRUNCATED AT 400 WORDS)

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Identification of hematopoietic stem cell subsets on the basis of their primitiveness using antibody ER-MP12

Blood ◽

10.1182/blood.v85.4.952.bloodjournal854952 ◽

1995 ◽

Vol 85 (4) ◽

pp. 952-962 ◽

Cited By ~ 37

Author(s):

JC van der Loo ◽

WA Slieker ◽

D Kieboom ◽

RE Ploemacher

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cell ◽

Antigen Expression ◽

Chimeric Mouse ◽

Hematopoietic Stem ◽

Colony Forming Units

Monoclonal antibody ER-MP12 defines a novel antigen on murine hematopoietic stem cells. The antigen is differentially expressed by different subsets in the hematopoietic stem cell compartment and enables a physical separation of primitive long-term repopulating stem cells from more mature multilineage progenitors. When used in two-color immunofluorescence with ER-MP20 (anti-Ly-6C), six subpopulations of bone marrow (BM) cells could be identified. These subsets were isolated using magnetic and fluorescence-activated cell sorting, phenotypically analyzed, and tested in vitro for cobblestone area-forming cells (CAFC) and colony-forming units in culture (CFU-C; M/G/E/Meg/Mast). In addition, they were tested in vivo for day-12 spleen colony-forming units (CFU-S-12), and for cells with long-term repopulating ability using a recently developed alpha-thalassemic chimeric mouse model. Cells with long-term repopulation ability (LTRA) and day-12 spleen colony-forming ability appeared to be exclusively present in the two subpopulations that expressed the ER-MP12 cell surface antigen at either an intermediate or high level, but lacked the expression of Ly- 6C. The ER-MP12med20- subpopulation (comprising 30% of the BM cells, including all lymphocytes) contained 90% to 95% of the LTRA cells and immature day-28 CAFC (CAFC-28), 75% of the CFU-S-12, and very low numbers of CFU-C. In contrast, the ER-MP12hi20- population (comprising 1% to 2% of the BM cells, containing no mature cells) included 80% of the early and less primitive CAFC (CAFC-5), 25% of the CFU-S-12, and only 10% of the LTRA cells and immature CAFC-28. The ER-MP12hi cells, irrespective of the ER-MP20 antigen expression, included 80% to 90% of the CFU-C (day 4 through day 14), of which 70% were ER-MP20- and 10% to 20% ER-MP20med/hi. In addition, erythroblasts, granulocytes, lymphocytes, and monocytes could almost be fully separated on the basis of ER-MP12 and ER-MP20 antigen expression. Functionally, the presence of ER-MP12 in a long-term BM culture did not affect hematopoiesis, as was measured in the CAFC assay. Our data demonstrate that the ER-MP12 antigen is intermediately expressed on the long-term repopulating hematopoietic stem cell. Its level of expression increases on maturation towards CFU-C, to disappear from mature hematopoietic cells, except from B and T lymphocytes.

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Short-Term Hematopoietic Stem Cells (ST-HSC) Have Full Long-Term Capacity with Sustained but Reduced Potential Compared with LT-HSC.

Blood ◽

10.1182/blood.v114.22.2550.2550 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2550-2550

Author(s):

Gerald A. Colvin ◽

David Berz ◽

Peter J. Quesenberry ◽

Elaine Papa ◽

Liansheng Liu

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Cell Number ◽

Large Animal ◽

Short Term ◽

Hematopoietic Stem ◽

One Year

Abstract Abstract 2550 Poster Board II-527 We evaluated the hypothesis that there was a homing defect between long-term (LT) hematopoietic stem cell (HSC) (KLS-Flk2-) and ST-HSC (KLS-Flk2+) that explained differences in engraftment potential and duration. Short-term HSC by definition have limited self-renewal capacity, generally described as giving rise to lymphohematopoiesis for 4–12 weeks before senescence. We performed three large animal engraftment studies into lethally ablated mice (950cGy split dose) looking at engraftment of both ST and LT-HSC cells delivered via intravenous, intraperitoneal and intra-femoral route. Two-hundred or 500 cells derived from B6/SJL mice were administered to each animal along with 300,000 recipient (C57/BLK) whole bone marrow cells for radioprotection following prior published studied [PNAS:98;14541, Stem Cells 24:1087] with optimization of flourochromes for better discrimination with our Cytopeia sorter. The animals were serially transplanted after eight months or one year to secondary recipients. In our hands, the ST-HSC engrafted animals did not lose chimerism over time. Review of the literature revealed that there were not confirmatory studies from those published from the initial one publication describing the ST-HSC. We found the ST-HSC were not short-term and persisted for one year in primary recipients and at least 3 months in secondary recipients. Engraftment kinetics favored LT-HSC over ST-HSC with engraftment examples at one year of 62% compared with 30% respectively when administered intravenously, 10% verses 4% given intra-femoral and 0.5% verse 0.3% given intraperitoneal. Chimerism was on average 50% better for the LT-HSC when compared with the ST-HSC and was irrespective of route proving that the differences seen are not due to homing deficiency but rather intrinsic differences in the two stem cell pools. Prior studies gave a maximum of 100 cells. Cell number was purposely increased for better differentiate of subtle differences in engraftment kinetics for statistical reasons. To avoid contamination of Flk2+ cells in the Flk2- cohort and vise-versa, discrimination of the gates were enhanced from that which was published prior. Double sorting of the cells confirmed that there was no appreciable cross contamination but obviously we cannot totally rule that out as a potentially confounding factor. In conclusion we found that ST-HSC as described have long-term capacity with intrinsic differences in engraftment potential that is not driven by a homing defect. Disclosures: No relevant conflicts of interest to declare.

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