scholarly journals The replication rate of human hematopoietic stem cells in vivo

Blood ◽  
2011 ◽  
Vol 117 (17) ◽  
pp. 4460-4466 ◽  
Author(s):  
Sandra N. Catlin ◽  
Lambert Busque ◽  
Rosemary E. Gale ◽  
Peter Guttorp ◽  
Janis L. Abkowitz
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood Transplantation ◽  
Cell Labeling ◽  
Replication Rate ◽  
Hematopoietic Stem ◽  
Daughter Cells ◽  
Blood Transplantation ◽  
Human Hscs

Abstract Hematopoietic stem cells (HSCs) replicate (self-renew) to create 2 daughter cells with capabilities equivalent to their parent, as well as differentiate, and thus can both maintain and restore blood cell production. Cell labeling with division-sensitive markers and competitive transplantation studies have been used to estimate the replication rate of murine HSCs in vivo. However, these methods are not feasible in humans and surrogate assays are required. In this report, we analyze the changing ratio with age of maternal/paternal X-chromosome phenotypes in blood cells from females and infer that human HSCs replicate on average once every 40 weeks (range, 25-50 weeks). We then confirm this estimate with 2 independent approaches, use the estimate to simulate human hematopoiesis, and show that the simulations accurately reproduce marrow transplantation data. Our simulations also provide evidence that the number of human HSCs increases from birth until adolescence and then plateaus, and that the ratio of contributing to quiescent HSCs in humans significantly differs from mouse. In addition, they suggest that human marrow failure, such as the marrow failure that occurs after umbilical cord blood transplantation and with aplastic anemia, results from insufficient numbers of early progenitor cells, and not the absence of HSCs.

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.

scholarly journals Changes in the Proliferative Activity of Human Hematopoietic Stem Cells in NOD/SCID Mice and Enhancement of Their Transplantability after In Vivo Treatment with Cell Cycle Inhibitors

2002 ◽  
Vol 196 (9) ◽  
pp. 1141-1150 ◽  
Author(s):  
J. Cashman ◽  
B. Dykstra ◽  
I. Clark-Lewis ◽  
A. Eaves ◽  
C. Eaves
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Transforming Growth Factor ◽  
Severe Combined Immunodeficiency ◽  
High Specific Activity ◽  
Scid Mice ◽  
Hematopoietic Stem ◽  
Human Hscs

Human hematopoietic tissue contains rare stem cells with multilineage reconstituting ability demonstrable in receptive xenogeneic hosts. We now show that within 3 wk nonobese diabetic severe combined immunodeficiency (NOD/SCID) mice transplanted with human fetal liver cells regenerate near maximum levels of daughter human hematopoietic stem cells (HSCs) able to repopulate secondary NOD/SCID mice. At this time, most of the human HSCs (and other primitive progenitors) are actively proliferating as shown by their sensitivity to treatments that kill cycling cells selectively (e.g., exposure to high specific-activity [3H]thymidine in vitro or 5-fluorouracil in vivo). Interestingly, the proliferating human HSCs were rapidly forced into quiescence by in vivo administration of stromal-derived factor-1 (SDF-1) and this was accompanied by a marked increase in the numbers of human HSCs detectable. A similar result was obtained when transforming growth factor-β was injected, consistent with a reversible change in HSCs engrafting potential linked to changes in their cell cycle status. By 12 wk after transplant, most of the human HSCs had already entered Go and treatment with SDF-1 had no effect on their engrafting activity. These findings point to the existence of novel mechanisms by which inhibitors of HSC cycling can regulate the engrafting ability of human HSCs executing self-renewal divisions in vivo.

Stromal Cell Regulation of Murine Hematopoietic Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1566-1566
Author(s):  
Stefan Wohrer ◽  
Keegan Rowe ◽  
Heidi Mader ◽  
Claudia Benz ◽  
Michael R Copley ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Stromal Cell ◽  
Cultured Cells ◽  
Conflicts Of Interest ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Daughter Cells

Abstract Abstract 1566 Recent advances in purifying murine hematopoietic stem cells (HSCs) to near homogeneity (>20%) have made it possible to analyze their in vivo clonal growth, self-renewal and differentiation properties over prolonged periods and the effects of various manipulations on these key functional parameters. However, conditions that allow genetically unaltered HSCs to maintain their original functional properties over equivalent periods of prolonged proliferation in vitro have not yet been identified. Since initial studies showed that the UG26 stromal cell could support murine HSC maintenance for limited periods, we first asked whether the addition of cytokines that also maintain HSCs for short periods might synergize with UG26 cells to enable HSC expansion to occur. Limiting dilution transplants that used a 6-month read-out of reconstituted blood elements (>1%) showed that the addition of 100 ng/ml Steel Factor (SF) and 20 ng/ml IL-11 to cultures containing UG26 cells and single purified (50%) HSCs (EPCR+CD150+CD48-, ESLAM cells) consistently stimulated a 3–5 fold HSC expansion after 7 days (3 expts). Furthermore, the effect of the UG26 cells could be replaced by UG26 conditioned medium (CM) and, in the presence of the CM+SF/IL-11 cocktail, the HSCs showed sustained longterm in vivo lympho-myeloid reconstituting activity in both primary and secondary recipients. Under these conditions, every ESLAM cell isolated proliferated several times within 7 days, but individual analysis of paired daughter cells showed that most first divisions (13/42) were, nevertheless, asymmetrical in terms of the numbers and types of different lineages produced by each of the 2 daughter cells for at least 4 months, although occasional evidence of symmetry was obtained (2/42 divisions). Interestingly, these first divisions showed a biphasic curve with 75% of the cells dividing before and 25% after 48 hours - the late dividers being more highly enriched for HSCs (95% vs 20%). We next asked whether TGF-β might be an important factor in UG26 CM, since UG26 cells exert a strong cell cycle inhibitory effect, and produce abundant TGF-beta. Accordingly, we next analyzed the effect of adding a neutralizing anti-TGF-β antibody or replacing the CM with TGF-β in the same type of single HSC cultures by tracking the survival and division kinetics of the cells as well as measuring the repopulating activity of their in vitro progeny present after 7 days. Strikingly, the addition of anti-TGF-β to the CM+SF/IL-11 supplemented HSC cultures eliminated the late wave of first cell divisions and caused an accompanying loss of myeloid reconstituting ability in recipients transplanted with the cultured cells. Conversely, replacement of the CM with TGF-β restored a biphasic division kinetics curve to cultures supplemented with SF/IL-11 but no CM. However, this did not protect against the early 50% loss of cells by apoptosis. These findings provide evidence of a new role of TGF-β in preserving the integrity of HSC functionality in vitro, but suggest a requirement for other types of factors released by certain stromal cells to achieve sustained symmetrical HSC self-renewal in vitro. Disclosures: No relevant conflicts of interest to declare.

scholarly journals CCND1–CDK4–mediated cell cycle progression provides a competitive advantage for human hematopoietic stem cells in vivo

2015 ◽  
Vol 212 (8) ◽  
pp. 1171-1183 ◽  
Author(s):  
Nicole Mende ◽  
Erika E. Kuchen ◽  
Mathias Lesche ◽  
Tatyana Grinenko ◽  
Konstantinos D. Kokkaliaris ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Competitive Advantage ◽  
Cell Cycle Progression ◽  
Cycle Phase ◽  
Cycle Progression ◽  
Hematopoietic Stem ◽  
Human Hscs

Maintenance of stem cell properties is associated with reduced proliferation. However, in mouse hematopoietic stem cells (HSCs), loss of quiescence results in a wide range of phenotypes, ranging from functional failure to extensive self-renewal. It remains unknown whether the function of human HSCs is controlled by the kinetics of cell cycle progression. Using human HSCs and human progenitor cells (HSPCs), we report here that elevated levels of CCND1–CDK4 complexes promoted the transit from G0 to G1 and shortened the G1 cell cycle phase, resulting in protection from differentiation-inducing signals in vitro and increasing human leukocyte engraftment in vivo. Further, CCND1–CDK4 overexpression conferred a competitive advantage without impacting HSPC numbers. In contrast, accelerated cell cycle progression mediated by elevated levels of CCNE1–CDK2 led to the loss of functional HSPCs in vivo. Collectively, these data suggest that the transition kinetics through the early cell cycle phases are key regulators of human HSPC function and important for lifelong hematopoiesis.

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.

scholarly journals Identification of Potent Quiescent Human Hematopoietic Stem Cells Using Mitochondrial Profile

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5602-5602
Author(s):  
Jiajing Qiu ◽  
Jana Gjini ◽  
Miao Lin ◽  
Tasleem Arif ◽  
Saghi Ghaffari
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Live Cells ◽  
Mitochondrial Activity ◽  
Hematopoietic Stem ◽  
Human Hscs ◽  
Time Point

Quiescence is the main property of the most potent hematopoietic stem cells (HSCs). Recent evidence suggests that mitochondrial activity may be implicated in the maintenance of stem cell quiescence. However, the potential function of mitochondria in the regulation of human HSCs remains largely unknown. To address this, we measured mitochondrial membrane potential (MMP) in subpopulations of human HSCs, using Tetramethylrhodamine, ethyl ester (TMRE) a cationic fluorescent dye sequestered by active mitochondria. We found that MMP profiles progressively shift towards lower levels in subpopulations with phenotypes of higher hematopoietic hierarchy. Subpopulation of CD34+CD38-CD45- cells within the 10% lowest MMP enriched for highly primitive CD90+ HSCs from 17.5% to 49.0% as compared to those within the 10% highest MMP (p=0.0049, n=5). These results are consistent with the murine HSC finding in our laboratory. To analyze the functional activity of HSCs with various MMP levels, subpopulations of CD90+ HSCs (CD34+CD38-CD45-CD90+) with the lowest and the highest 25% MMP were FACS-sorted as MMP-low and MMP-high HSCs respectively and subjected to long-term culture initiating cell (LTC-IC) limiting dilution assay. The results after 5 weeks revealed that the frequency of LTC-IC is much greater in MMP-low HSCs (1 in 7.85 cells) than in MMP-high HSCs (1 in 59.3 cells) (n=2). These results suggest that MMP-low HSCs maintain higher functional potential than MMP-high HSCs. The in vivo analysis of the MMP-low and MMP-high HSCs transplanted into NSG mice is ongoing (will be performed at 5 months). FACS-sorted MMP-low and high HSCs were subjected to cell cycle analysis by Pyronin Y/ Hoechst-33342 staining. We found that above 90% of both MMP-low and MMP-high HSCs were in a quiescence state (MMP-low: 4.48 %, MMP-high: 9.38% in G1; 0% in S/G2/M). Additionally, the expression of CDK6 that is associated with HSC activation was not detectable by confocal microscopy in either MMP-low or MMP-high HSCs (low: n=31, high: n=19). To identify potential distinct kinetic of cell cycle entry, FACS-sorted single HSCs were cultured in serum free medium (SFM), STEM SPAN, supplemented with cytokines (SCF 100ng/ml, TPO 50ng/ml, Flt3 50ng/ml). The occurrence of cell division in each well was monitored under microscopy every 12 hours for 6 days. At each time point the percentage of divided HSCs among total initial seeding HSCs were plotted and curve fitted to calculate the kinetics of cumulative first division (low: n=58, R2=0.9981; high: n=58, R2=0.9973). These analyses showed that MMP-low HSCs were delayed by 1.9 hrs as compared to MMP-high HSCs for cumulative 50% cells to complete the first division. The percentage of newly divided cells of first division at each time point was also plotted. Two waves of first division were revealed in both MMP-low and MMP-high HSCs. The peak of the first wave was delayed by 7 hours in MMP-low HSCs, while the second wave was delayed by 14 hours as compared to MMP-high HSCs. These results indicate that even though both MMP-low and MMP-high CD90+ HSCs are mostly in a quiescence state, upon cytokine exposure in vitro, MMP-high HSCs exist G0 phase more rapidly than MMP-low HSCs. In agreement with this interpretation, MMP-high HSCs express significantly higher level of CDK6 when cultured for 34 hours in the presence of the same cytokines as described above (low: n=14, high n=35; p=0.006). We also investigated whether MMP-low and MMP-high CD90+ HSCs can be maintained in vitro in the absence of cytokines. Both populations were cultured in 96 well plates for 7 days in SFM without cytokines. The percentage of live cells was significantly higher in MMP-low HSCs cultured for 7 days (64.9%) as compared to MMP-high HSCs (44.2%) (p=0.03). Furthermore, morphological analysis by mitochondrial specific probe TOM20 showed that MMP-low contained more fragmented mitochondria as compared to MMP-high HSCs. Our findings suggest that mitochondrial activity may be implicated in the regulation of HSC quiescence. Altogether these results support the notion that human MMP-low CD90+ HSCs are molecularly distinct from MMP-high CD90+ HSCs and maintain quiescence in vitro to a higher degree than MMP-high CD90+ HSCs which are more primed for activation. Disclosures Ghaffari: Rubius Therapeutics: Consultancy.

scholarly journals High-level correction of the sickle mutation amplified in vivo during erythroid differentiation

2018 ◽  
Author(s):  
Wendy Magis ◽  
Mark A. DeWitt ◽  
Stacia K. Wyman ◽  
Jonathan T. Vu ◽  
Seok-Jin Heo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Genetic Disorders ◽  
Erythroid Differentiation ◽  
Hematopoietic Stem ◽  
Curative Therapy ◽  
Human Hscs ◽  
High Level

ABSTRACTSickle Cell Disease (SCD), one of the world’s most common genetic disorders, causes anemia and progressive multiorgan damage that typically shortens lifespan by decades; currently there is no broadly applicable curative therapy. Here we show that Cas9 RNP-mediated gene editing with an ssDNA oligonucleotide donor yields markerless correction of the sickle mutation in more than 30% of long-term engrafting human hematopoietic stem cells (HSCs), using a selection-free protocol that is directly applicable to a clinical setting. We further find that in vivo erythroid differentiation markedly enriches for corrected ß-globin alleles. Adoption of a high-fidelity Cas9 variant demonstrates that this approach can yield efficient editing with almost no off-target events. These findings indicate that the sickle mutation can be corrected in human HSCs at curative levels with a streamlined protocol that is ready to be translated into a therapy.ONE SENTENCE SUMMARYCas9-mediated correction of the sickle mutation in human hematopoietic stem cells can be accomplished at curative levels.

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