Mouse Hematopoietic Stem Cells, Unlike Human and Mouse Embryonic Stem Cells, Exhibit Checkpoint-Apoptosis Coupling.

Abstract Cell cycle checkpoints guarantee that cells move through the events of the cell cycle in the appropriate manner. The mitotic spindle checkpoint, also known as the spindle assembly checkpoint (SAC), helps to ensure the proper segregation of chromosomes into daughter cells during mitosis. Our lab recently reported on the condition of the SAC in both mouse and human embryonic stem cells (ESCs). We found that ESCs do not initiate apoptosis when the SAC is activated, which allowed these cells to tolerate a polyploid state resulting from the aberrant mitosis (Mantel et al. Blood.109: 4518–4527. 2007). These results lead us to conclude that the spindle checkpoint is uncoupled from apoptosis in ESCs. Knowing whether adult tissue specific stem/progenitor cells, such as hematopoietic stem cells (HSCs), have checkpoints which are uncoupled from apoptosis is extremely important information. If HSCs were to manifest such checkpoint uncoupling as that which we defined for ESCs, this might present a problem for the ex-vivo expansion and transplantation of HSCs. Using multiparametric permeablized cell flow cytometric analysis, we found the mitotic spindle checkpoint to be functional in primary murine sca 1+/c-kit+/lin- cells (LSK cells), a population highly enriched in primitive hematopoietic stem/progenitor cells. Using nocodazole, which exerts its affect by depolymerizing microtubules, we were able to activate the spindle checkpoint in low density mononuclear cells collected from murine bone marrow. Through flow cytometric analysis of the LSK cells in the mononuclear fraction, we were able to determine that spindle checkpoint activation in LSK cells resulted in a cell cycle arrest in mitosis, which was determined by DNA content of the cells, and eventually this arrest lead to cell death via apoptosis, as indicated by caspase-3 activation. This behavior is unlike that of ESCs, which exit mitosis and become polyploidy after prolonged spindle checkpoint activation. Thus the mitotic spindle checkpoint appears to be coupled to apoptosis in this particular set of tissue specific stem/progenitor cells, which lessens the possibility that ex-vivo expansion of hematopoietic stem cells will result in abnormalities to these cells that may give rise to disease initiation or progression after their transplantation.

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Malignant Myeloma Cells Impair Phenotype and Function of stem and Progenitor Cells.

Blood ◽

10.1182/blood.v114.22.1799.1799 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1799-1799

Author(s):

Ingmar Bruns ◽

Sebastian Büst ◽

Akos G. Czibere ◽

Ron-Patrick Cadeddu ◽

Ines Brückmann ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Plasma Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Healthy Donors ◽

Stem And Progenitor Cells

Abstract Abstract 1799 Poster Board I-825 Multiple myeloma (MM) patients often present with anemia at the time of initial diagnosis. This has so far only attributed to a physically marrow suppression by the invading malignant plasma cells and the overexpression of Fas-L and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by malignant plasma cells triggering the death of immature erythroblasts. Still the impact of MM on hematopoietic stem cells and their niches is scarcely established. In this study we analyzed highly purified CD34+ hematopoietic stem and progenitor cell subsets from the bone marrow of newly diagnosed MM patients in comparison to normal donors. Quantitative flowcytometric analyses revealed a significant reduction of the megakaryocyte-erythrocyte progenitor (MEP) proportion in MM patients, whereas the percentage of granulocyte-macrophage progenitors (GMP) was significantly increased. Proportions of hematopoietic stem cells (HSC) and myeloid progenitors (CMP) were not significantly altered. We then asked if this is also reflected by clonogenic assays and found a significantly decreased percentage of erythroid precursors (BFU-E and CFU-E). Using Affymetrix HU133 2.0 gene arrays, we compared the gene expression signatures of stem cells and progenitor subsets in MM patients and healthy donors. The most striking findings so far reflect reduced adhesive and migratory potential, impaired self-renewal capacity and disturbed B-cell development in HSC whereas the MEP expression profile reflects decreased in cell cycle activity and enhanced apoptosis. In line we found a decreased expression of the adhesion molecule CD44 and a reduced actin polymerization in MM HSC by immunofluorescence analysis. Accordingly, in vitro adhesion and transwell migration assays showed reduced adhesive and migratory capacities. The impaired self-renewal capacity of MM HSC was functionally corroborated by a significantly decreased long-term culture initiating cell (LTC-IC) frequency in long term culture assays. Cell cycle analyses revealed a significantly larger proportion of MM MEP in G0-phase of the cell cycle. Furthermore, the proportion of apoptotic cells in MM MEP determined by the content of cleaved caspase 3 was increased as compared to MEP from healthy donors. Taken together, our findings indicate an impact of MM on the molecular phenotype and functional properties of stem and progenitor cells. Anemia in MM seems at least partially to originate already at the stem and progenitor level. Disclosures Off Label Use: AML with multikinase inhibitor sorafenib, which is approved by EMEA + FDA for renal cell carcinoma.

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Hematopoietic stem cells differentiate into restricted myeloid progenitors before cell division

10.1101/256024 ◽

2018 ◽

Author(s):

Tatyana Grinenko ◽

Anne Eugster ◽

Lars Thielecke ◽

Beata Ramazs ◽

Anja Krueger ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Cell Division ◽

Hematopoietic Stem Cells ◽

Embryonic Stem ◽

Erythroid Progenitors ◽

Multipotent Stem Cells ◽

Hematopoietic Stem ◽

Myeloid Progenitors

SummaryHematopoietic stem cells (HSCs) continuously replenish all blood cell types through a series of differentiation steps that involve the generation of lineage-committed progenitors as well as necessary expansion due to repeated cell divisions. However, whether cell division in HSCs precedes differentiation is unclear. To this end, we used an HSC cell tracing approach and Ki67RFP knock-in mice to assess simultaneously divisional history, cell cycle progression, and differentiation of adult HSCs in vivo. Our results reveal that HSCs are able to differentiate into restricted progenitors, especially common myeloid progenitors, restricted megakaryocyte-erythroid progenitors (PreMEs) and pre-megakaryocyte progenitors (PreMegs), without undergoing cell division and even before entering the S phase of the cell cycle. Additionally, the phenotype of the undivided but differentiated progenitors correlated with expression of lineage-specific genes that manifested as functional differences between HSCs and restricted progenitors. Thus, HSC fate decisions appear to be uncoupled from physical cell division. Our results facilitate a better understanding of the mechanisms that control fate decisions in hematopoietic cells. Our data, together with separate findings from embryonic stem cells, suggest that cell division and fate choice are independent processes in pluripotent and multipotent stem cells.

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Change in SP Distribution with Age Reflects Intrinsic Age-Based Changes in Stem Cell Biology: Implications for the Mechanism of Aging.

Blood ◽

10.1182/blood.v106.11.4209.4209 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4209-4209

Author(s):

Daniel J. Pearce ◽

Catherine Simpson ◽

Kirsty Allen ◽

Ayad Eddaoudi ◽

Derek Davies ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Cell Biology ◽

Stem Cell Biology ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

The Absolute

Abstract It has been postulated that as we age, accumulated damage causes stem cells to die by apoptosis. This could lead to a diminished stem cell pool and consequently a reduced organ regeneration potential that contributes to somatic senescence. Hematopoietic stem cells have evolved many mechanisms to cope with their exposure to toxins during life. Cell surface transporters and anti-toxic enzymes are highly expressed in hematopoietic stem cells. If toxins do get the opportunity to damage the DNA of stem cells then the cell is more likely to die by apoptosis than attempt DNA repair and risk an error. Summarised below are our results from an investigation of the frequency, phenotype, cell cycle status and repopulation potential (in young recipients) of C57BL6 side population (SP) cells from mice with a range of ages. The absolute frequency of SP cells increases with age (Figure-A). The proportion of the lineage negative, Sca-1+, c-kit+ (KLS) cell population that is an SP stem cell increases from ~1% to over 30% during the murine lifetime (red bars in Figure-B). These SP cells from older mice have a reduced 4-month competitive repopulation potential when compared to SP cells from younger mice but contain a similarly low proportion of phenotypically-defined mature cells (blue bars in Figure-B) and have a similar cell cycle profile and progenitor cell output (2% of 3 x 96 well plates for each). SP cells from older mice contained a higher proportion of SP cells with the highest efflux ability (61 vs 414 days, p=<0.001, n=6) Engrafted cells derived from old SP cells 4 months previously still displayed an increased SP frequency when compared to engrafted cells derived from SP cells of young mice. Hence, more progenitors or committed cells have not gained the SP ability; rather this difference in SP distribution reflects an age-dependent change in hematopoietic stem cell biology that is independent of the microenvironment. Specifically, the proportion of stem and progenitor cells (KLS) that is a stem cell (SP fraction of KLS) increases with age. We hypothesize that this may be a progressive enrichment of primitive cells over time via selection. As we age, accumulative damage to hematopoietic stem and progenitor cells causes more cells to die by apoptosis. It may be that the stem/progenitor cells with the lowest hoechst efflux ability are most susceptible to damage and hence most likely to die by apoptosis. Since the HSCs with the highest efflux of hoechst are thought to be the most primitive, it may be that there is an enrichment of primitive cells. This could account for the increased SP proportion observed within KLS cells. As there may be cells with ABC/G2 activity that is undetectable via the SP technique, selection of cells with a higher pump activity could also explain the increased SP frequency we observed. This hypothetical mechanism would also be independent of microenvirinment. In summary, we surmise that HSCs have a mechanism that copes with cellular damage while compensating for the reduced cellular output of HSCs with age by increasing the absolute number of HSCs. Figure Figure

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Cyclin A2 Plays a Critical Role in Proliferation of Lymphoid Progenitors

Blood ◽

10.1182/blood.v118.21.914.914 ◽

2011 ◽

Vol 118 (21) ◽

pp. 914-914

Author(s):

Kentaro Kohno ◽

Hiromi Iwasaki ◽

Tadafumi Iino ◽

Shin-ichi Mizuno ◽

Peter Sicinski ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

B Cell ◽

Progenitor Cells ◽

Critical Role ◽

Embryonic Stem ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem ◽

M Phase ◽

Cyclin A2

Abstract Abstract 914 Cell cycle regulators could be differentially used among self–renewing stem cells, rapidly expanding progenitor cells, and terminally differentiated cells those clonally replicate. Cyclin A is a regulatory subunit for cyclin dependent kinase (Cdk) 1 and Cdk2, and it drives S phase progression as well as transition to G2/M phase in cell cycle. We have previously reported that cyclin A2 is not required for fibroblast replication but it is indispensable in maintenance of self-renewing stem cells, including embryonic stem cells and hematopoietic stem cells (HSCs) (Cell 138 2009). The question is whether cyclin A2 plays a role in proliferation of hematopoietic progenitors downstream of the HSC. Here, we further assessed the requirement of cyclin A2 in non-self-renewing hematopoietic progenitors. Quantitative RT-PCR analysis showed that cyclin A2 was expressed in hematopoietic progenitor cells as well as stem cells, and its expression level is highest in lymphoid-committed progenitor stages of both T and B cell lineages. Thus, in order to test the role of cylin A2 in early lymphopoiesis, we crossed cyclin A2 floxed mice with Rag1-Cre knock-in mice. Because recombination activating gene (RAG)-1 is essential for generation of pre-BCRs and pre-TCRs that are critical for expansion of B and T lymphoid progenitor cells, respectively, we hypothesized that the requirement of Cyclin A2 in early lymphopoiesis can be assessed in this system. As we expected, the Rag1-Cre cyclin A2 floxed/floxed mice were viable, and have normal numbers of HSCs and myeloid progenitors. They, however, displayed severe reduction of mature T and B cell numbers that were only 1/100 - 1/10 of wild-type controls. The number of common lymphoid progenitor was unchanged, but there were severely reduced preB cells in bone marrow and T cell progenitors from CD4-CD8- double negative stage in thymus. Furthermore, cell cycle analysis shows that the Cyclin A2 disrupted progenitors are unable to progress from S to G2/M phase, and in vitro culture clearly showed that those progenitors are unable to proliferate and resulted in apoptosis. These findings clearly demonstrate that cyclin A2 is indispensable not only for self-renewing HSCs, but also for proliferation of T and B cell progenitors. Disclosures: No relevant conflicts of interest to declare.

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The Unexpected G0/G1 Cell Cycle Status of Mobilized Hematopoietic Stem Cells From Peripheral Blood

Blood ◽

10.1182/blood.v89.2.465 ◽

1997 ◽

Vol 89 (2) ◽

pp. 465-472 ◽

Cited By ~ 138

Author(s):

Nobuko Uchida ◽

Dongping He ◽

Annabelle M. Friera ◽

Michael Reitsma ◽

Dennis Sasaki ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Healthy Volunteers ◽

Progenitor Cells ◽

Peripheral Blood ◽

Hematopoietic Stem ◽

Cell Cycling ◽

The Impact ◽

Cell Cycle Status

Abstract Treatment with a combination of cytokines and chemotherapy can effectively stimulate the release of hematopoietic stem cells (HSC) into the peripheral blood (PB), which can then be harvested for transplantation. The cell cycle status of the harvested HSC from mobilized PB (MPB) is of interest because of the impact that cell cycling may have on optimizing the conditions for ex vivo expansion, retrovirus-mediated gene transfer, and the engraftment of transplanted tissues. Therefore, we characterized the cell cycling status of mobilized HSC from mice and humans. The murine HSC, which express the phenotype c-kit+ Thy-1.1lo Lin−/lo Sca-1+, were purified from PB, bone marrow (BM), and spleen after the mice were treated with the mobilizing regimen of granulocyte colony-stimulating factor (G-CSF ) or a combination of cyclophosphamide (CTX) and G-CSF. Human HSC (CD34+ Thy-1+ Lin−) and progenitor cells (CD34+ Thy-1− Lin−) were isolated from the BM of untreated healthy volunteers and from MPB of healthy volunteers and patients treated with G-CSF or a combination of CTX and GM-CSF. Cell cycle status was determined by quantitating the amount of DNA in the purified cells after staining with the dye Hoechst 33342. Fluorescence-activated cell sorting analysis of the progenitor cells from the murine and human samples showed an unexpected finding, ie, virtually none of the cells from the MPB was cycling. The G0/G1 status of HSC from MPB was surprising, because a significant proportion of HSC from BM are actively proliferating and, after mobilization, the HSC in the spleen and BM were also actively cycling.

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Application of Umbilical Cord Blood Stem Cells in Regenerative Medicine

The Indonesian Biomedical Journal ◽

10.18585/inabj.v6i3.25 ◽

2014 ◽

Vol 6 (3) ◽

pp. 115

Author(s):

Anna Meiliana ◽

Andi Wijaya

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cord Blood ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Embryonic Stem ◽

Hematopoietic Stem ◽

Blood Stem Cells ◽

Induced Pluripotent ◽

Nerve Muscle

BACKGROUND: Since the first umbilical cord blood (UCB) transplant, performed 25 years ago, UCB banks have been established worldwide for the collection and cryopreservation of UCB for autologous and allogeneic transplants.CONTENT: Much has been learned in a relatively short time on the properties of UCB hematopoietic progenitors and their clinical application. More interestingly, non-hematopoietic stem cells have been isolated from UCB. These cells can be grown and differentiated into various tissues including bone, cartilage, liver, pancreas, nerve, muscle and so on. The non-hematopoietic stem cells have an advantage over other sources of stem cells, such as embryonic stem cells or induced pluripotent stem cells, because their supply is unlimited, they can be used in autologous or allogeneic situations, they need minimal manipulation and they raise no ethical concerns. Future studies will test the potential of UCB cells for the treatment of several diseases including, among other possibilities, diabetes, arthritis, burns, neurological disorder and myocardial infarction.SUMMARY: In addition to hematopoietic stem cells, UCB contain a large number of non-hematopoietic stem cells. In the absence of ethical concern, the unlimited supply of UCB cells explains the increasing interest of using UCB for developing regenerative medicine.KEYWORDS: UCB, transplantation, UCB bank, HSC, MSC, CD34, CD133, VSEL

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Single-cell RNA-seq reveals a concomitant delay in differentiation and cell cycle of aged hematopoietic stem cells

BMC Biology ◽

10.1186/s12915-021-00955-z ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Léonard Hérault ◽

Mathilde Poplineau ◽

Adrien Mazuel ◽

Nadine Platet ◽

Élisabeth Remy ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cell Cycle Regulators ◽

Potential Mechanism ◽

Rna Seq ◽

Hematopoietic Stem ◽

Age Related ◽

Undifferentiated State ◽

Reference Map

Abstract Background Hematopoietic stem cells (HSCs) are the guarantor of the proper functioning of hematopoiesis due to their incredible diversity of potential. During aging, heterogeneity of HSCs changes, contributing to the deterioration of the immune system. In this study, we revisited mouse HSC compartment and its transcriptional plasticity during aging at unicellular scale. Results Through the analysis of 15,000 young and aged transcriptomes, we identified 15 groups of HSCs revealing rare and new specific HSC abilities that change with age. The implantation of new trajectories complemented with the analysis of transcription factor activities pointed consecutive states of HSC differentiation that were delayed by aging and explained the bias in differentiation of older HSCs. Moreover, reassigning cell cycle phases for each HSC clearly highlighted an imbalance of the cell cycle regulators of very immature aged HSCs that may contribute to their accumulation in an undifferentiated state. Conclusions Our results establish a new reference map of HSC differentiation in young and aged mice and reveal a potential mechanism that delays the differentiation of aged HSCs and could promote the emergence of age-related hematologic diseases.

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Expression of Cell Cycle–Related Genes With Cytokine-Induced Cell Cycle Progression of Primitive Hematopoietic Stem Cells

Stem Cells and Development ◽

10.1089/scd.2009.0283 ◽

2010 ◽

Vol 19 (4) ◽

pp. 453-460 ◽

Cited By ~ 5

Author(s):

Peter J. Quesenberry ◽

Gerri J. Dooner ◽

Michael Del Tatto ◽

Gerald A. Colvin ◽

Kevin Johnson ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cell Cycle Progression ◽

Cycle Progression ◽

Hematopoietic Stem

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Isolation of Murine Hematopoietic Stem Cells and Progenitor Cells

Current Protocols in Immunology ◽

10.1002/0471142735.im22b01s67 ◽

2005 ◽

Vol 67 (1) ◽

Cited By ~ 2

Author(s):

Hideo Ema ◽

Yohei Morita ◽

Hiromitsu Nakauchi ◽

Yumi Matsuzaki

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Hematopoietic Stem

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The Ethics of Embrionic Stem Cells Research

Advances in Library and Information Science - Ethics in Research Practice and Innovation ◽

10.4018/978-1-5225-6310-5.ch013 ◽

2019 ◽

pp. 244-263

Author(s):

Alexandra Huidu

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Embryonic Stem Cell ◽

Stem Cell Research ◽

Embryonic Stem ◽

Modern Medicine ◽

Embryonic Stem Cell Research ◽

Hematopoietic Stem ◽

Research Fields

Embrionic stem cells research, as opposed to hematopoietic stem cells research, has always stirred up many controversies of ethical nature that have projected their effects in the specialized doctrine of the domain of medical bioethics and law. Some of these controversies have been transposed at the legislative level (both by international normative acts and by the national laws of the states) while others are not yet de object of consensus. All that is not transposed by law remains in the exclusive sphere of ethics, so the ethical discussion in embryonic stem cell research is not only relevant for today's modern medicine but also of the utmost importance for a category of specialists in various research fields.

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