Self-Renewal Capacity and Clonal Succession of Haemopoietic Stem Cells In Long-Term Bone Marrow Culture

1985 ◽  
Vol 18 (5) ◽  
pp. 483-491
Author(s):  
J. L. Chertkov ◽  
Nina J. Drize ◽  
Olga A. Gurevitch ◽  
G. A. Udalov
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Culture ◽  
Self Renewal ◽  
Haemopoietic Stem Cells

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

Blood ◽  
2015 ◽  
Vol 125 (17) ◽  
pp. 2678-2688 ◽  
Author(s):  
Marisa Bowers ◽  
Bin Zhang ◽  
Yinwei Ho ◽  
Puneet Agarwal ◽  
Ching-Cheng Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Key Points ◽  
Leukemia Development

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

scholarly journals Development of adult bone marrow stem cells in H-2-compatible and -incompatible mouse fetuses.

1984 ◽  
Vol 159 (3) ◽  
pp. 731-745 ◽  
Author(s):  
R A Fleischman ◽  
B Mintz
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Fetal Liver ◽  
Donor Strain ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Adult Bone Marrow ◽  
Adult Bone

Bone marrow of normal adult mice was found, after transplacental inoculation, to contain cells still able to seed the livers of early fetuses. The recipients' own hematopoietic stem cells, with a W-mutant defect, were at a selective disadvantage. Progression of donor strain cells to the bone marrow, long-term self-renewal, and differentiation into myeloid and lymphoid derivatives was consistent with the engraftment of totipotent hematopoietic stem cells (THSC) comparable to precursors previously identified (4) in normal fetal liver. More limited stem cells, specific for the myeloid or lymphoid cell lineages, were not detected in adult bone marrow. The bone marrow THSC, however, had a generally lower capacity for self-renewal than did fetal liver THSC. They had also embarked upon irreversible changes in gene expression, including partial histocompatibility restriction. While completely allogeneic fetal liver THSC were readily accepted by fetuses, H-2 incompatibility only occasionally resulted in engraftment of adult bone marrow cells and, in these cases, was often associated with sudden death at 3-5 mo. On the other hand, H-2 compatibility, even with histocompatibility differences at other loci, was sufficient to ensure long-term success as often as with fetal liver THSC.

Cellular and molecular basis of haematopoiesis

2020 ◽  
pp. 5172-5181
Author(s):  
Paresh Vyas ◽  
N. Asger Jakobsen
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Cell Number ◽  
Stages Of Development ◽  
Self Renewal ◽  
Haematopoietic System

Haematopoiesis involves a regulated set of developmental stages from haematopoietic stem cells (HSCs) that produce haematopoietic progenitor cells that then differentiate into more mature haematopoietic lineages, which provide all the key functions of the haematopoietic system. Definitive HSCs first develop within the embryo in specialized regions of the dorsal aorta and umbilical arteries and then seed the fetal liver and bone marrow. At the single-cell level, HSCs have the ability to reconstitute and maintain a functional haematopoietic system over extended periods of time in vivo. They (1) have a self-renewing capacity during the life of an organism, or even after transplantation; (2) are multipotent, with the ability to make all types of blood cells; and (3) are relatively quiescent, with the ability to serve as a deep reserve of cells to replenish short-lived, rapidly proliferation progenitors. Haematopoietic progenitor cells are unable to maintain long-term haematopoiesis in vivo due to limited or absent self-renewal. Rapid proliferation and cytokine responsiveness enables increased blood cell production under conditions of stress. Lineage commitment means limited cell type production. The haematopoietic stem cell niche is an anatomically and functionally defined regulatory environment for stem cells modulates self-renewal, differentiation, and proliferative activity of stem cells, thereby regulating stem cell number. Haematopoietic reconstitution during bone marrow transplantation is mediated by a succession of cells at various stages of development. More mature cells contribute to repopulation immediately following transplantation. With time, cells at progressively earlier stages of development are involved, with the final stable repopulation being provided by long-lived, multipotent HSCs. Long-term haematopoiesis is sustained by a relatively small number of HSCs.

scholarly journals Angiogenin Is a Niche-Secreted RNase Which Regulates Hematopoietic Regeneration

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 171-171
Author(s):  
Lev Silberstein ◽  
Kevin Goncalves ◽  
Nicholas Severe ◽  
Guo-fu Hu ◽  
David T. Scadden
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Board Of Directors ◽  
Advisory Committees ◽  
Self Renewal ◽  
Cellular Source ◽  
Equity Ownership ◽  
Ng2 Cells

Abstract Background. Identification of novel niche factors is critical for understanding of regulatory mechanisms which control HSPC cell fate decisions and design of novel pro-regenerative therapies. We have developed a proximity-based differential single cell analysis approach to the study of the bone marrow niche, which showed that individual osteolineage cells located in close proximity to transplanted HSPC are enriched for expression of niche factors, and have previously reported identification of IL18 and Embigin as regulators of HSPC quiescence. Here we describe the results of in vivo validation of Angiogenin (ANG) - the third molecule highlighted using the above strategy - as a potent regulator of HSPC quiescence and regeneration. Results. ANG is a secreted RNase which is known to promote angiogenesis, proliferation of cancer cells and also enhance cell survival in response to stress. Analysis of primitive cells subsets in the ANG knock-out mice (AngKO mice) revealed a 1.4-fold increase in the frequency and absolute number of long-term hematopoietic stem cells (LT-HSCs). Subsequent BrdU incorporation and cell cycle studies demonstrated increased proliferative activity in the primitive HSPC compartment indicating that ANG regulates HSPC quiescence. To confirm these findings functionally and to assess the effect of ANG on self-renewal, we exposed AngKO animals to weekly 5-FU injections and performed serial transplantation experiments of WT LT-HSCs into AngKO hosts. We noted significantly increased mortality of AngKO mice in both experimental settings; in a competitive transplant assay, we observed almost complete absence of engraftment by WT cells in the secondary hosts, in keeping with the exhaustion phenotype. Consistently, exposure of AngKO animals to a different type of hematopoietic stress, such as ageing, resulted in development of peripheral blood cytopenias and marked reduction in the number and frequency of HSPC. ANG is expressed in multiple non-hematopoietic cell types in the bone marrow, including osteoprogenitors, mature osteoblasts and nestin-positive mesenchymal stem cells and NG2-positive arteriolar sheath cells. To establish the predominant cellular source of ANG in the niche, we crossed Ang "floxed" mice with the animals in which tamoxifen-inducible Cre-recombinase was driven by the promoters targeting these cell subsets and examined the effect on hematopoiesis. We found that deletion of ANGfrom Osx+, Nes+ and NG2+ cells resulted in an increase of the number of LT-HSC and more active cycling of LT-HSC, short-term HSC (ST-HSC) and multi-potent progenitors (MPP) while ANGdeletion in mature osteoblasts had no effect on these cell populations, but was associated with an increase in number and more active cycling of common lymphoid progenitors (CLP), as was also seen upon ANGdeletion from Nes+ and NG2+ cells. These results indicate that the target cell population which is regulated by ANG depends on the cellular source. Interestingly, transplantation of WT bone marrow into the animals with Osx-specific ANG deletion resulted in development of macrocytic anemia and neutropenia at 6 months, thus indicating that Angiogenin deficiency in the niche is sufficient for the development of the hematopoietic failure. Impaired long-term reconstitution was also observed when ANG was deleted from Nestin+ mesenchymal stem cells but not col1+ mature osteoblasts in the recipient mice. Our findings that the absence of ANG negatively impacts HSPC self-renewal prompted us to investigate whether exposure of HSPC to recombinant ANG protein will have the opposite effect and enhance hematopoietic regeneration. We therefore treated mouse LT-HSCs with recombinant ANG or vehicle control ex-vivo for 2 hours and competitively transplanted them into lethally irradiated WT recipients. We found that ANG-treated LT-HSCs displayed a significantly higher long-term reconstituting ability, which persisted into the secondary transplants. Similar data were obtained following treatment of CD34+ cord blood cells with human ANG. Conclusion. Our work defines ANG as a previously unrecognized regulator of HSPC quiescence and self-renewal and suggests that it can be explored as a potential therapeutic agent to promote hematopoietic regeneration. Disclosures Scadden: Teva: Consultancy; Apotex: Consultancy; Bone Therapeutics: Consultancy; GlaxoSmithKline: Research Funding; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Fate Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Dr. Reddy's: Consultancy.

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.

Tet2 Uniquely Regulates Self-Renewal and Long Term Repopulating Capacity of Fetal Liver and Bone Marrow Hematopoietic Stem Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4739-4739
Author(s):  
Hiroyoshi Kunimoto ◽  
Yumi Fukuchi ◽  
Masatoshi Sakurai ◽  
Daichi Abe ◽  
Ken Sadahira ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Fetal Liver ◽  
Hematologic Malignancies ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Serial Transplantation

Abstract Abstract 4739 Ten-eleven-translocation 2 (TET2) gene is one of the frequent targets of mutation in various hematologic malignancies. These observations suggest critical roles of TET2 dysfunction in their molecular pathogenesis. To investigate physiological roles of TET2 in hematopoiesis, we previously analyzed fetal liver (FL) hematopoiesis of Tet2 gene-trap (Tet2gt) mice and showed that Tet2gt/gt FL cells displayed enhanced self-renewal and long term repopulating (LTR) capacity with expansion of Lineage(−)Sca-1(+)c-Kit(+) (LSK) and common myeloid progenitor (CMP) fractions. However, there remain several questions unanswered. First, self-renewal capacity was examined only by using bulk FL cells and therefore effects of Tet2 loss on purified cell populations such as hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs) remain elusive. Second, because other groups have reported myeloid transformation in Tet2 conditional knockout mice, it is possible that Tet2 loss confers self-renewal capacity to non-self-renewing myeloid progenitors such as CMPs. Third, effects of Tet2 haploinsufficiency on adult hematopoiesis was not examined using purified HSCs or HPCs. To address these issues, we analyzed E14.5 FL and adult bone marrow (BM) cells from Tet2gt mice. We first performed serial replating assay of FL-LSK cells in methylcellulose containing interleukin (IL)-3, IL-6, stem cell factor (SCF) and erythropoietin (Epo). In this assay, Tet2gt/gt FL-LSK cells showed significantly higher replating capacity as compared to that of WT cells. Interestingly, Tet2gt/gt FL-LSK cells formed various types of colonies including granulocyte-macrophage (GM) and erythrocyte-megakaryocyte (EM) colonies, whereas WT FL-LSK cells generated only GM colonies at the second time of replating, showing that multipotent differentiation capacity was maintained in Tet2gt/gt cells even in the presence of lineage-acting cytokines. Next we examined the self-renewal capacity of highly purified FL-HSCs (CD34+LSK or CD150+LSK cells) by competitive repopulation assay. As expected, the recipients of Tet2gt/gt CD34+LSK cells showed significantly higher donor chimerism in peripheral blood as compared to those receiving WT cells. Furthermore, CD150+LSK cells from Tet2+/gt and Tet2gt/gt FLs demonstrated higher peripheral blood repopulation in the secondary and tertiary recipient mice as compared to that of WT recipients in serial transplantation assay. These results indicate that the enhanced self-renewal and LTR capacity of Tet2-mutant FL cells was uniquely associated with highly purified HSCs. This conclusion also applied to the BM LSK cells from adult mice, since Tet2+/gt BM LSK cells also showed significantly higher peripheral blood contribution compared to the WT cells in serial transplantation assays. This result demonstrates that Tet2 haploinsufficiency is sufficient to confer the enhanced self-renewal and LTR capacity to HSCs in adult hematopoiesis. Lastly, we examined self-renewal capacity of FL CMPs by serial replating assay. Interestingly, Tet2gt/gt FL CMP cells displayed increased replating capacity as compared to WT cells. However, in vivo repopulation assay using Tet2+/+, Tet2+/gt, and Tet2gt/gt FL CMP cells showed no significant difference in peripheral blood chimerism among these recipients. Taken together, enhanced self-renewal and LTR capacity by Tet2 ablation is uniquely associated with HSCs in FL and adult BM, but not with myeloid progenitors, indicating that Tet2 regulates self-renewal program intrinsic to HSCs. In addition, Tet2 haploinsufficiency is sufficient to enhance self-renewal and LTR capacity of HSCs, which explains pathological relation between high incidence of heterozygous TET2 mutations and hematologic malignancies. Disclosures: No relevant conflicts of interest to declare.

Vascular Niche-Derived Angiocrine Factors Specify and Maintain Hematopoietic Stem Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. SCI-25-SCI-25
Author(s):  
Shahin Rafii ◽  
Jason M. Butler ◽  
Ginsberg Michael ◽  
Jennifer L Gori ◽  
Hans-Peter Kiem ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Pluripotent Stem Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Hematopoietic Recovery ◽  
Vascular Niche ◽  
Angiocrine Factors

Abstract Organ-specific endothelial cells (ECs) are both conduits for delivery of nutrients and also establish an instructive vascular niche. The vascular niche produces paracrine factors, (i.e., angiocrine factors), that balance self-renewal and differentiation of hematopoietic stem/progenitor cells (HSPCs) (1,2). Activation of Akt-mTOR pathway in sinusoidal ECs (SECs) stimulates physiological expression of angiocrine factors, including Kit-ligand, Notch-ligands, Wnts, FGFs, BMPs and TGFb, that expand long-term repopulating HSPCs. Activation of MAPkinase in ECs upregulates expression of GM-CSF, M-CSF, IL6, IL7, SDF-1 and G-CSF (..others) to accelerate HSPC multi-lineage differentiation. We developed an ex vivo vascular niche in which HSPC/EC co-cultures are maintained and expanded in serum-free conditions. This vascular niche platform produces physiologic levels of angiocrine factors that balance expansion/differentiation of human cord blood, mobilized peripheral blood, and steady state bone marrow HSPCs that maintain their ability to reconstitute hematopoiesis in vivo. In contrast to our vascular platform, co-culture with bone marrow-derived mesenchymal does not support long-term expansion of HSPCs. In collaboration with Drs. Kiem and Gori at Hutchinson Cancer Center, we have shown that ECs expand repopulating nonhuman primate marrow-derived HSPCs. Transplantation of the vascular-niche expanded gene-modified HSPCs reconstituted long-term multi-lineage hematopoiesis in autologous transplantation setting in nonhuman primates. Importantly, intravenous co-infusion of the vascular niche with HSPCs did not cause infusional toxicity. Vascular niche-expanded HSPCs supported robust hematopoietic recovery underscoring the essential function of vascular niche-signals in hematopoietic reconstitution without provoking fibrosis (3). The ECs also supplies key signals that induce emergence of HSPCs from hemogenic ECs. To prove this point, we transduced adult human or mouse ECs with Runx1/Spi1/Gfi1/FosB transcription factors along with vascular niche-induction allowing for conversion of these ECs into stable and long-term engraftable HSPCs, including functional immune cells (4). Importantly, transition through a pluripotent state results in poorly engraftable hematopoietic cells that are unstable and upon exposure to pathophysiological stressors differentiate aberrantly into other cell-types. Remarkably, signals from vascular niche support specification of repopulating multipotent-HSPCs from both human and nonhuman primate pluripotent stem cells (5). In summary, we developed and characterized a vascular niche platform that provides physiologically relevant levels of key angiocrine factors that stimulate safe clinical-scale expansion of authentic adult, cord blood, and primitive HSPCs under GMP-grade culture conditions. We are currently translating the vascular niche platform to the clinical setting, to evaluate the potential of co-transplantation of HSPCs with vascular niche cells to reconstruct injured EC niches thereby accelerating short- and long-term hematopoietic recovery. This first-in-man clinical application will set the stage for repopulation with true hematopoietic stem cells, thereby enabling use of a vascular niche for treatment of a wide range of acquired, inherited, and malignant hematopoietic diseases. 1. Butler JM …… Rafii S. Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells. Cell Stem Cell, 3:251-64, 2010. 2. Nolan D........Rafii S. Molecular and cellular signatures of tissue-specific vascular heterogeneity in organ maintenance and regeneration. Developmental Cell, 26(2):204-19, 2013. 3. Ding BS …..Rafii S. Divergent angiocrine signals from vascular niche balance liver regeneration and fibrosis.Nature 505(7481):97-102, 2014. 4. Sandler VM, Lis R ...... Butler JM, Scandura JM, Rafii S. Reprogramming of Human Endothelium Into Engraftable Hematopoietic Progenitors by Vascular Niche Induction.Nature, 511(7509):312-8, 2014. 5. Gori J., Butler JM, .....Rafii S, Kiem HP. Vascular niche promotes hematopoietic multipotent progenitor formation from pluripotent stem cells. Journal of Clinical Investigation, 125(3): 1243-54, 2015. Disclosures Rafii: Angiocrine Bioscience: Consultancy, Equity Ownership.

scholarly journals Deterministic regulation of hematopoietic stem cell self-renewal and differentiation

Blood ◽  
2002 ◽  
Vol 100 (4) ◽  
pp. 1302-1309 ◽  
Author(s):  
Christa E. Müller-Sieburg ◽  
Rebecca H. Cho ◽  
Marilyn Thoman ◽  
Becky Adkins ◽  
Hans B. Sieburg
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Lymphoid Cells ◽  
The Self ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Direct Corollary ◽  
Kinetics Of ◽  
Adult Bone

Most current theories assume that self-renewal and differentiation of hematolymphoid stem cells (HSCs) is randomly regulated by intrinsic and environmental influences. A direct corollary of these tenets is that self-renewal will continuously generate functionally heterogeneous daughter HSCs. Decisions about self-renewal versus commitment are made by individual, single HSCs and, thus, require examination on the clonal level. We followed the behavior of individual, clonally derived HSCs through long-term, serial repopulation experiments. These studies showed that daughter HSCs derived from individual clones were remarkably similar to each other in the extent and kinetics of repopulation. Moreover, daughter HSCs within a clone showed equivalent contributions to the myeloid or lymphoid lineages. Lineage contribution could be followed because of the discovery of a new subset of HSCs that gave rise stably to skewed ratios of myeloid and lymphoid cells. Overall, the data argue that self-renewal does not contribute to the heterogeneity of the adult HSC compartment. Rather, all HSCs in a clone follow a predetermined fate, consistent with the generation-age hypothesis. By extension, this suggests that the self-renewal and differentiation behavior of HSCs in adult bone marrow is more predetermined than previously thought.

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