scholarly journals CD66e Enrichment Enhances Repopulation of Human Long-Term Hematopoietic Stem Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2156-2156
Author(s):  
Kuiying Ma ◽  
Riguo Fang ◽  
Lingling Yu ◽  
Yongjian Zhang ◽  
Chao Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Surface Markers ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Differentiation Pattern ◽  
Human Hscs

Abstract Gene-modified hematopoietic stem cells (HSCs) therapy has demonstrated remarkable success for the treatment of inherited blood disorders. As the origin of hematologic hierarchy, HSCs play an essential role in sustaining life-long hematopoiesis. HSCs identification via reliable and robust bio-markers could facilitate the development of HSC gene therapy. Previous studies showed that long-term hematopoietic stem cells (LT-HSCs) were enriched in the Lin -CD34 +CD38 -CD45RA -CD90 +CD49f + population which could support long-term hematopoietic reconstitution. However, several of these surface markers proved to be unreliable when ex vivo culturing, such as CD38 and CD49f. Thus, HSCs characterization is still hindered by lacking bona-fide bio-markers, and consequently identification of long-term HSCs still needs time-consuming in vivo transplantation. To this end, we performed in vitro screening and comprehensive functional evaluation to identify a novel surface marker of human HSCs. During initial screening, a cell surface antigen screen panel (including 242 human cell surface markers) and human CD34 and CD90 antibodies were used to perform flow cytometry analysis on CD34 + HSPCs enriched from umbilical cord blood. Compared with CD34 + cell population, we found that CD66 (a,c,d,e), CD200 and CD48 positive cells were more enriched in CD34 +CD90 + subset. Previous studies indicated that HSCs cannot be maintained during in vitro culturing. By tracking these candidate surface markers based on this principle, CD66e was selected as the potential HSCs bio-marker. Next, we examined the in vivo hematologic repopulating potential of HSCs by limiting dilution assay (LDA) on immune-deficient mouse model. We sorted CD66e + and CD66e - subsets from CD34 +CD90 +CD45RA - subpopulation, and transplanted into irradiated NOD-scid Il2rg −/− (NPG) mice respectively. At week16 post-transplantation, in contrast to the CD66e - group, CD66e + cells exhibited significantly higher reconstitution in peripheral blood (PB), bone marrow (BM) and spleen. Engraftment dynamics revealed that the CD66e - group were only capable of reconstitution 4 weeks post transplantation, even at the highest initial cell dose. Moreover, the CD66e - group displayed impaired multi-lineage differentiation pattern, especially in PB and BM samples, while the CD66e + group presented a robust multi-lineage reconstitution. Notably, LDA results showed that the CD66e + cells within CD34 +CD90 +CD45RA - population contained 1 out of 529 SCID repopulating cells (SRC), almost 60-fold greater than the CD66e - fraction. To further investigate the long-term repopulating potential of the CD66e + cells, we performed the secondary transplantation collected from the BM cells of primary recipients. CD66e + cells presented significant higher repopulating activity than CD66e- subset in the secondary recipients. These findings reveal that the major cells with homing and long-term reconstitution capacity among CD34 +CD90 +CD45RA - cells were CD66e positive. In order to determine the transcriptional profile of CD66e + cells, we performed RNA-sequencing analysis using the population of CD34 + cells, CD34 +CD90 +CD45RA - cells, CD66e + and CD66e - cells within CD34 +CD90 +CD45RA - subset. Remarkably, compared with other groups, the CD66e + cells displayed a bias toward the signature of HSC and early progenitors such as LMPP and CLP. Moreover, gene set enrichment analysis showed that hematopoietic lineage and long-term potentiation-related genes were highly enriched in the CD66e + cells. Further qRT-PCR experiment confirmed that several HSC-related genes were significantly higher expressed in CD34 +CD90 +CD45RA -CD66e + cells, compared to CD66e - population or CD34 + HSPCs, suggesting that the gene expression profile of CD66e + cells is reminiscent of HSC signature. Altogether, we demonstrate that CD66e is a robust functional HSC bio-marker that CD66e-positive population among CD34 +CD90 +CD45RA - cells exhibit typical HSC signature, enhanced in vivo engraftment potential and robust multilineage differentiation pattern, which will provide an invaluable tool to investigate the origin of human HSCs, paving the way for the therapeutic application. Figure 1 Figure 1. Disclosures Fang: EdiGene, Inc.: Current Employment.

Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

2020 ◽  
Vol 15 ◽  
Author(s):  
Fatima Aerts-Kaya
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Stress Conditions ◽  
Stress Factors ◽  
Hematopoietic Stem ◽  
Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

scholarly journals The chemokine GROβ mobilizes early hematopoietic stem cells characterized by enhanced homing and engraftment

Blood ◽  
2007 ◽  
Vol 110 (3) ◽  
pp. 860-869 ◽  
Author(s):  
Seiji Fukuda ◽  
Huimin Bian ◽  
Andrew G. King ◽  
Louis M. Pelus
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem ◽  
Platelet Recovery ◽  
And Function ◽  
Stimulating Factor ◽  
Cxcr4 Axis

Abstract Mobilized peripheral blood hematopoietic stem cells (PBSCs) demonstrate accelerated engraftment compared with bone marrow; however, mechanisms responsible for enhanced engraftment remain unknown. PBSCs mobilized by GROβ (GROβΔ4/CXCL2Δ4) or the combination of GROβΔ4 plus granulocyte colony-stimulating factor (G-CSF) restore neutrophil and platelet recovery faster than G-CSF–mobilized PBSCs. To determine mechanisms responsible for faster hematopoietic recovery, we characterized immunophenotype and function of the GROβ-mobilized grafts. PBSCs mobilized by GROβΔ4 alone or with G-CSF contained significantly more Sca-1+-c-kit+-lineage− (SKL) cells and more primitive CD34−-SKL cells compared with cells mobilized by G-CSF and demonstrated superior competitive long-term repopulation activity, which continued to increase in secondary and tertiary recipients. GROβΔ4-mobilized SKL cells adhered better to VCAM-1+ endothelial cells compared with G-CSF–mobilized cells. GROβΔ4-mobilized PBSCs did not migrate well to the chemokine stromal derived factor (SDF)-1α in vitro that was associated with higher CD26 expression. However, GROβΔ4-mobilized SKL and c-Kit+ lineage− (KL) cells homed more efficiently to marrow in vivo, which was not affected by selective CXCR4 and CD26 antagonists. These data suggest that GROβΔ4-mobilized PBSCs are superior in reconstituting long-term hematopoiesis, which results from differential mobilization of early stem cells with enhanced homing and long-term repopulating capacity. In addition, homing and engraftment of GROβΔ4-mobilized cells is less dependent on the SDF-1α/CXCR4 axis.

Hematopoietic stem cells express Tie-2 receptor in the murine fetal liver

Blood ◽  
2000 ◽  
Vol 96 (12) ◽  
pp. 3757-3762 ◽  
Author(s):  
Hsiang-Chun Hsu ◽  
Hideo Ema ◽  
Mitsujiro Osawa ◽  
Yukio Nakamura ◽  
Toshio Suda ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Receptor Tyrosine Kinase ◽  
Fetal Liver ◽  
Newborn Mice ◽  
Hematopoietic Stem ◽  
Fetal Liver Cells

Tie-2 receptor tyrosine kinase expressed in endothelial and hematopoietic cells is believed to play a role in both angiogenesis and hematopoiesis during development of the mouse embryo. This article addressed whether Tie-2 is expressed on fetal liver hematopoietic stem cells (HSCs) at day 14 of gestation. With the use of anti–Tie-2 monoclonal antibody, its expression was detected in approximately 7% of an HSC population of Kit-positive, Sca-1–positive, lineage-negative or -low, and AA4.1-positive (KSLA) cells. These Tie-2–positive KSLA (T+ KSLA) cells represent 0.01% to 0.02% of fetal liver cells. In vitro colony and in vivo competitive repopulation assays were performed for T+ KSLA cells and Tie-2–negative KSLA (T− KSLA) cells. In the presence of stem cell factor, interleukin-3, and erythropoietin, 80% of T+ KSLA cells formed colonies in vitro, compared with 40% of T− KSLA cells. Long-term multilineage repopulating cells were detected in T+ KSLA cells, but not in T− KSLA cells. An in vivo limiting dilution analysis revealed that at least 1 of 8 T+ KSLA cells were such repopulating cells. The successful secondary transplantation initiated with a limited number of T+ KSLA cells suggests that these cells have self-renewal potential. In addition, engraftment of T+ KSLA cells in conditioned newborn mice indicates that these HSCs can be adapted equally by the adult and newborn hematopoietic environments. The data suggest that T+ KSLA cells represent HSCs in the murine fetal liver.

Hematopoietic stem cells express Tie-2 receptor in the murine fetal liver

Blood ◽  
2000 ◽  
Vol 96 (12) ◽  
pp. 3757-3762 ◽  
Author(s):  
Hsiang-Chun Hsu ◽  
Hideo Ema ◽  
Mitsujiro Osawa ◽  
Yukio Nakamura ◽  
Toshio Suda ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Receptor Tyrosine Kinase ◽  
Fetal Liver ◽  
Newborn Mice ◽  
Hematopoietic Stem ◽  
Fetal Liver Cells

Abstract Tie-2 receptor tyrosine kinase expressed in endothelial and hematopoietic cells is believed to play a role in both angiogenesis and hematopoiesis during development of the mouse embryo. This article addressed whether Tie-2 is expressed on fetal liver hematopoietic stem cells (HSCs) at day 14 of gestation. With the use of anti–Tie-2 monoclonal antibody, its expression was detected in approximately 7% of an HSC population of Kit-positive, Sca-1–positive, lineage-negative or -low, and AA4.1-positive (KSLA) cells. These Tie-2–positive KSLA (T+ KSLA) cells represent 0.01% to 0.02% of fetal liver cells. In vitro colony and in vivo competitive repopulation assays were performed for T+ KSLA cells and Tie-2–negative KSLA (T− KSLA) cells. In the presence of stem cell factor, interleukin-3, and erythropoietin, 80% of T+ KSLA cells formed colonies in vitro, compared with 40% of T− KSLA cells. Long-term multilineage repopulating cells were detected in T+ KSLA cells, but not in T− KSLA cells. An in vivo limiting dilution analysis revealed that at least 1 of 8 T+ KSLA cells were such repopulating cells. The successful secondary transplantation initiated with a limited number of T+ KSLA cells suggests that these cells have self-renewal potential. In addition, engraftment of T+ KSLA cells in conditioned newborn mice indicates that these HSCs can be adapted equally by the adult and newborn hematopoietic environments. The data suggest that T+ KSLA cells represent HSCs in the murine fetal liver.

scholarly journals Steel factor coordinately regulates the molecular signature and biologic function of hematopoietic stem cells

Blood ◽  
2008 ◽  
Vol 112 (3) ◽  
pp. 560-567 ◽  
Author(s):  
David G. Kent ◽  
Brad J. Dykstra ◽  
Jay Cheyne ◽  
Elaine Ma ◽  
Connie J. Eaves
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Molecular Mechanisms ◽  
Molecular Signature ◽  
Hematopoietic Stem ◽  
Steel Factor ◽  
Biologic Function

Abstract Hematopoietic stem cells (HSCs) regenerated in vivo display sustained differences in their self-renewal and differentiation activities. Variations in Steel factor (SF) signaling are known to affect these functions in vitro, but the cellular and molecular mechanisms involved are not understood. To address these issues, we evaluated highly purified HSCs maintained in single-cell serum-free cultures containing 20 ng/mL IL-11 plus 1, 10, or 300 ng/mL SF. Under all conditions, more than 99% of the cells traversed a first cell cycle with similar kinetics. After 8 hours in the 10 or 300 ng/mL SF conditions, the frequency of HSCs remained unchanged. However, in the next 8 hours (ie, 6 hours before any cell divided), HSC integrity was sustained only in the 300 ng/mL SF cultures. The cells in these cultures also contained significantly higher levels of Bmi1, Lnk, and Ezh2 transcripts but not of several other regulators. Assessment of 21 first division progeny pairs further showed that only those generated in 300 ng/mL SF cultures contained HSCs and pairs of progeny with similar differentiation programs were not observed. Thus, SF signaling intensity can directly and coordinately alter the transcription factor profile and long-term repopulating ability of quiescent HSCs before their first division.

scholarly journals Anti-CD117 antibody depletes normal and myelodysplastic syndrome human hematopoietic stem cells in xenografted mice

Blood ◽  
2019 ◽  
Vol 133 (19) ◽  
pp. 2069-2078 ◽  
Author(s):  
Wendy W. Pang ◽  
Agnieszka Czechowicz ◽  
Aaron C. Logan ◽  
Rashmi Bhardwaj ◽  
Jessica Poyser ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Mouse Models ◽  
Hematopoietic Cell Transplantation ◽  
Normal Donor ◽  
Hematopoietic Stem ◽  
Increased Risk ◽  
Human Hscs

Abstract The myelodysplastic syndromes (MDS) represent a group of clonal disorders that result in ineffective hematopoiesis and are associated with an increased risk of transformation into acute leukemia. MDS arises from hematopoietic stem cells (HSCs); therefore, successful elimination of MDS HSCs is an important part of any curative therapy. However, current treatment options, including allogeneic hematopoietic cell transplantation (HCT), often fail to ablate disease-initiating MDS HSCs, and thus have low curative potential and high relapse rates. Here, we demonstrate that human HSCs can be targeted and eliminated by monoclonal antibodies (mAbs) that bind cell-surface CD117 (c-Kit). We show that an anti-human CD117 mAb, SR-1, inhibits normal cord blood and bone marrow HSCs in vitro. Furthermore, SR-1 and clinical-grade humanized anti-human CD117 mAb, AMG 191, deplete normal and MDS HSCs in vivo in xenograft mouse models. Anti-CD117 mAbs also facilitate the engraftment of normal donor human HSCs in MDS xenograft mouse models, restoring normal human hematopoiesis and eradicating aggressive pathologic MDS cells. This study is the first to demonstrate that anti-human CD117 mAbs have potential as novel therapeutics to eradicate MDS HSCs and augment the curative effect of allogeneic HCT for this disease. Moreover, we establish the foundation for use of these antibody agents not only in the treatment of MDS but also for the multitude of other HSC-driven blood and immune disorders for which transplant can be disease-altering.

Human reconstituting hematopoietic stem cells up-regulate Fas expression upon active cell cycling but remain resistant to Fas-induced suppression

Blood ◽  
2003 ◽  
Vol 102 (1) ◽  
pp. 118-126 ◽  
Author(s):  
Ingunn Dybedal ◽  
Liping Yang ◽  
David Bryder ◽  
Ingbritt Aastrand-Grundstrom ◽  
Karin Leandersson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Severe Combined Immunodeficiency ◽  
Constitutive Expression ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Nonobese Diabetic

Abstract The Fas receptor and its ligand have been implicated in mediating the bone marrow (BM) suppression observed in graft-versus-host disease and a number of other BM-failure syndromes. However, previous studies have suggested that Fas is probably not expressed on human hematopoietic stem cells (HSCs), but up-regulated as a consequence of their commitment and differentiation, suggesting that progenitors or differentiated blood cells, rather than HSCs, are the targets of Fas-mediated suppression. The present studies confirm that candidate HSCs in human cord blood and BM lack constitutive expression of Fas, but demonstrate that Fas expression on CD34+ progenitor and stem cells is correlated to their cell cycle and activation status. With the use of recently developed in vitro conditions promoting HSC self-renewing divisions, Fas was up-regulated on virtually all HSCs capable of multilineage reconstituting nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice in vivo, as well as on long-term culture-initiating cells (LTC-ICs). Similarly, in vivo cycling of NOD-SCID repopulating cells upon transplantation, resulted in up-regulation of Fas expression. However, repopulating HSCs expressing high levels of Fas remained highly resistant to Fas-mediated suppression, and HSC function was compromised only upon coactivation with tumor necrosis factor. Thus, reconstituting human HSCs up-regulate Fas expression upon active cycling, demonstrating that HSCs could be targets for Fas-mediated BM suppression. (Blood. 2003;102: 118-126)

scholarly journals Phenotypic and functional characterization of competitive long-term repopulating hematopoietic stem cells enriched from 5-fluorouracil- treated murine marrow

Blood ◽  
1993 ◽  
Vol 81 (9) ◽  
pp. 2310-2320 ◽  
Author(s):  
SJ Szilvassy ◽  
S Cory
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Functional Characterization ◽  
Significant Proportion ◽  
Cell Types ◽  
Hematopoietic Stem ◽  
Clonogenic Cell

Lymphomyeloid stem cells from the bone marrow of C57BL/6 mice treated with 5-fluorouracil (5-FU) were characterized with respect to 12 parameters using fluorescence-activated cell sorting and a competitive long-term repopulation assay. Stem cells were larger than lymphocytes and exhibited side light-scatter characteristic of blast cells. Most expressed low levels of Thy-1.2, high levels of Sca-1 (Ly6-A/E), H-2Kb, and AA4.1 antigens and stained brightly with rhodamine-123. Significantly, most long-term repopulating cells also expressed CD4, some at high density. In addition, a significant proportion displayed low to medium levels of the “lineage-specific” markers CD45R (B220), Gr- 1, and TER-119. A simple and rapid multiparameter sorting procedure enriched the stem cells 100-fold and substantially removed most other clonogenic cell types, including day 12 spleen colony-forming cells. Cells able to generate cobblestone colonies on stromal cells in vitro were co-enriched. Lethally irradiated mice transplanted with limiting numbers of the sorted stem cells did not survive unless cotransplanted with “compromised” marrow cells prepared by prior serial transplantation and shown to be depleted of long-term repopulating activity. A significant number of recipients transplanted with 25 to 100 sorted cells contained donor-derived B and T lymphocytes and granulocytes in their peripheral blood for at least 6 months. Limiting dilution analysis in vivo indicated that the frequency of competitive long-term repopulating units (CRU) in the sorted population was at least 1 in 60 cells. The calculated frequency of CRU was largely independent of the time of recipient analysis between 10 and 52 weeks, indicating that highly enriched stem cells can be recruited relatively early in certain transplant settings. This simple enrichment and assay strategy for repopulating hematopoietic stem cells should facilitate further analysis of their regulation in vivo.

Differential Expression of c-Kit Identifies Hematopoietic Stem Cells with Variable Self-Renewal Potential.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2325-2325
Author(s):  
Joseph Yusup Shin ◽  
Wenhuo Hu ◽  
Christopher Y. Park
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Differential Expression ◽  
Peripheral Blood ◽  
Post Transplantation ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Abstract 2325 Hematopoietic stem cells (HSC) can be identified on the basis of differential cell surface protein expression, such that 10 out of 13 purified HSC (Lin−c-Kit+Sca-1+CD150+CD34−FLK2−) exhibit long-term reconstitution potential in single-cell transplants. HSCs express c-Kit, and interactions between c-Kit and its ligand, stem cell factor, have been shown to be critical for HSC self-renewal; however, HSCs express a log-fold variation in c-Kit levels. We hypothesized that differing levels of c-Kit expression on HSCs may identify functionally distinct classes of HSCs. Thus, we measured the function and cellular characteristics of c-Kithi HSCs and c-Kitlo HSCs (defined as the top 30% and bottom 30% of c-Kit expressors, respectively), including colony formation, cell cycle status, lineage fates, and serial engraftment potential. In methylcellulose colony assays, c-Kithi HSCs formed 5-fold more colonies than c-Kitlo HSCs (P=0.01), as well as 4-fold more megakaryocyte colonies in vitro. c-Kithi HSC were 2.4-fold enriched for cycling cells (G2-S-M) in comparison to c-Kitlo HSC as assessed by flow cytometry in vivo (15.4% versus 6.4%, P=0.001). Lethally irradiated mice competitively transplanted with 400 c-Kitlo HSCs and 300,000 competitor bone marrow cells exhibited increasing levels of donor chimerism, peaking at a mean of 80% peripheral blood CD45 chimerism by 16 weeks post-transplantation, whereas mice transplanted with c-Kithi HSCs reached a mean of 20% chimerism (p<0.00015). Evaluation of the bone marrow revealed an increase in HSC chimerism from 23% to 44% in mice injected with c-Kitlo HSCs from weeks 7 to 18, while HSC chimerism decreased from 18% to 3.0% in c-Kithi HSC-transplanted mice (P<0.00021). Levels of myeloid chimerism in the bone marrow and peripheral blood were not significantly different during the first 4 weeks following transplantation between mice transplanted with c-Kithi or c-Kitlo HSCs, and evaluation of HSC bone marrow lodging at 24 hours post-transplantation demonstrated no difference in the number of c-Kithi and c-Kitlo HSCs, indicating that differential homing is not the reason for the observed differences in long-term engraftment. Donor HSCs purified from mice transplanted with c-Kithi HSC maintained higher levels of c-Kit expression compared to those from mice injected with c-Kitlo HSC by week 18 post-transplantation (P=0.01). Secondary recipients serially transplanted with c-Kithi HSC exhibited a chimerism level of 40% to 3% from week 4 to 8 post-secondary transplant, whereas chimerism levels remained at 6% in mice injected with c-Kitlo HSC. These results indicate that c-Kithi HSCs exhibit reduced self-renewal capacity compared with c-Kitlo HSCs, and that the differences in c-Kithi and c-Kitlo HSC function are cell-intrinsic. Analysis of transplanted HSC fates revealed that c-Kithi HSCs produced two-fold more pre-megakaryocyte-erythroid progenitors and pluriploid megakaryocytes compared to their c-Kitlo counterparts in vivo, suggesting a megakaryocytic lineage bias in c-Kithi HSC. Consistent with this finding, the transplanted c-Kithi HSC gave rise to 10-fold more platelets and reached a maximum platelet output two days earlier than c-Kitlo HSC. To determine the potential mechanisms underlying the transition from c-Kitlo to c-Kithi HSCs, we assessed the activity of c-Cbl, an E3 ubiquitin ligase known to negatively regulate surface c-Kit expression in a Src-dependent manner. Flow cytometric analysis revealed 6-fold more activated c-Cbl in freshly purified c-Kitlo HSC compared to c-Kithi HSC (P=0.02), suggesting that functional loss of c-Cbl increases c-Kit expression on c-Kitlo HSCs. Mice treated for nine days with Src inhibitors, which inhibit c-Cbl activity, experienced a 1.5-fold and 2-fold increase in the absolute number of c-Kithi HSCs (P=0.067) and megakaryocyte progenitors (P=0.002), respectively. Thus, c-Cbl loss likely promotes the generation of c-Kithi HSCs. In summary, differential expression of c-Kit identifies HSC with distinct functional attributes with c-Kithi HSC exhibiting increased cell cycling, megakaryocyte lineage bias, decreased self-renewal capacity, and decreased c-Cbl activity. Since c-Kitlo HSC represent a population of cells enriched for long-term self-renewal capacity, characterization of this cell population provides an opportunity to better understand the mechanisms that regulate HSC function. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Role of Apoptosis in the Regulation of Hematopoietic Stem Cells

2000 ◽  
Vol 191 (2) ◽  
pp. 253-264 ◽  
Author(s):  
Jos Domen ◽  
Samuel H. Cheshier ◽  
Irving L. Weissman
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Cells ◽  
Wild Type ◽  
Direct Role ◽  
Hematopoietic Stem

Hematopoietic stem cells (HSC) give rise to cells of all hematopoietic lineages, many of which are short lived. HSC face developmental choices: self-renewal (remain an HSC with long-term multilineage repopulating potential) or differentiation (become an HSC with short-term multilineage repopulating potential and, eventually, a mature cell). There is a large overcapacity of differentiating hematopoietic cells and apoptosis plays a role in regulating their numbers. It is not clear whether apoptosis plays a direct role in regulating HSC numbers. To address this, we have employed a transgenic mouse model that overexpresses BCL-2 in all hematopoietic cells, including HSC: H2K-BCL-2. Cells from H2K-BCL-2 mice have been shown to be protected against a wide variety of apoptosis-inducing challenges. This block in apoptosis affects their HSC compartment. H2K-BCL-2–transgenic mice have increased numbers of HSC in bone marrow (2.4× wild type), but fewer of these cells are in the S/G2/M phases of the cell cycle (0.6× wild type). Their HSC have an increased plating efficiency in vitro, engraft at least as well as wild-type HSC in vivo, and have an advantage following competitive reconstitution with wild-type HSC.

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