Role of p53 in Hematopoietic Recovery After Cytotoxic Treatment

Blood ◽  
1998 ◽  
Vol 91 (8) ◽  
pp. 2998-3006 ◽  
Author(s):  
Pawel Wlodarski ◽  
Mariusz Wasik ◽  
Mariusz Z. Ratajczak ◽  
Cinzia Sevignani ◽  
Grazyna Hoser ◽  
...  
Keyword(s):  
Growth Factors ◽  
Bone Marrow Cells ◽  
Positive Impact ◽  
Hematopoietic Stem ◽  
Hematopoietic Recovery ◽  
Cytotoxic Treatment

Prompt reconstitution of hematopoiesis after cytoreductive therapy is essential for patient recovery and may have a positive impact on long-term prognosis. We examined the role of the p53 tumor suppressor gene in hematopoietic recovery in vivo after treatment with the cytotoxic drug 5-fluorouracil (5-FU). We used p53 knock-out (p53−/−) and wild-type (p53+/+) mice injected with 5-FU as the experimental model. Analysis of the repopulation ability and clonogenic activity of hematopoietic stem cells (HSCs) and their lineage-committed descendants showed a greater number of HSCs responsible for reconstitution of lethally irradiated recipients in p53−/− bone marrow cells (BMCs) recovering after 5-FU treatment than in the corresponding p53+/+ BMCs. In post–5-FU recovering BMCs, the percentage of HSC-enriched Lin− Sca-1+c-Kit+ cells was about threefold higher in p53−/− than in p53+/+ cells. Although the percentage of the most primitive HSCs (Lin− Sca-1+ c-Kit+CD34low/−) did not depend on p53, the percentage of multipotential HSCs and committed progenitors (Lin−Sca-1+ c-Kit+ CD34high/+) was almost fourfold higher in post–5-FU recovering p53−/− BMCs than in their p53+/+ counterparts. The pool of HSCs from 5-FU–treated p53−/− BMCs was exhausted more slowly than that from the p53+/+ population as shown in vivo using pre–spleen colony-forming unit (CFU-S) assay and in vitro using long-term culture-initiating cells (LTC-ICs) and methylcellulose replating assays. Clonogenic activity of various lineage-specific descendants was significantly higher in post–5-FU regenerating p53−/− BMCs than in p53+/+ BMCs, probably because of their increased sensitivity to growth factors. Despite all these changes and the dramatic difference in sensitivity of p53−/− and p53+/+ BMCs to 5-FU–induced apoptosis, lineage commitment and differentiation of hematopoietic progenitors appeared to be independent of p53 status. These studies suggest that suppression of p53 function facilitates hematopoietic reconstitution after cytoreductive therapy by: (1) delaying the exhaustion of the most primitive HSC pool, (2) stimulating the production of multipotential HSCs, (3) increasing the sensitivity of hematopoietic cells to growth factors, and (4) decreasing the sensitivity to apoptosis.

Interleukin-3 supports expansion of long-term multilineage repopulating activity after multiple stem cell divisions in vitro

Blood ◽  
2000 ◽  
Vol 96 (5) ◽  
pp. 1748-1755 ◽  
Author(s):  
David Bryder ◽  
Sten E. W. Jacobsen
Keyword(s):  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Calf Serum ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Divisions ◽  
Stem Cell Divisions

Abstract Although long-term repopulating hematopoietic stem cells (HSC) can self-renew and expand extensively in vivo, most efforts at expanding HSC in vitro have proved unsuccessful and have frequently resulted in compromised rather than improved HSC grafts. This has triggered the search for the optimal combination of cytokines for HSC expansion. Through such studies, c-kit ligand (KL), flt3 ligand (FL), thrombopoietin, and IL-11 have emerged as likely positive regulators of HSC self-renewal. In contrast, numerous studies have implicated a unique and potent negative regulatory role of IL-3, suggesting perhaps distinct regulation of HSC fate by different cytokines. However, the interpretations of these findings are complicated by the fact that different cytokines might target distinct subpopulations within the HSC compartment and by the lack of evidence for HSC undergoing self-renewal. Here, in the presence of KL+FL+megakaryocyte growth and development factor (MGDF), which recruits virtually all Lin−Sca-1+kit+ bone marrow cells into proliferation and promotes their self-renewal under serum-free conditions, IL-3 and IL-11 revealed an indistinguishable ability to further enhance proliferation. Surprisingly, and similar to IL-11, IL-3 supported KL+FL+MGDF-induced expansion of multilineage, long-term reconstituting activity in primary and secondary recipients. Furthermore, high-resolution cell division tracking demonstrated that all HSC underwent a minimum of 5 cell divisions, suggesting that long-term repopulating HSC are not compromised by IL-3 stimulation after multiple cell divisions. In striking contrast, the ex vivo expansion of murine HSC in fetal calf serum-containing medium resulted in extensive loss of reconstituting activity, an effect further facilitated by the presence of IL-3.

Constitutive TGF-β1 Deficiency Induces a Defect in Hematopoietic Stem/Progenitor Cell Compartment in Fetus and Neonate.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1397-1397
Author(s):  
Claude Capron ◽  
Catherine Lacout ◽  
Yann Lecluse ◽  
Valérie Jalbert ◽  
Elisabeth Cramer Bordé ◽  
...  
Keyword(s):  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Type I ◽  
Activated T Cells ◽  
Hematopoietic Stem ◽  
Tgf Β1

Abstract TGF-β1 is a cytokine with pleiotropic effects. It has been considered that TGF-β1plays a major role on hematopoietic stem cells (HSC) based on in vitro experiment. Achieving in vivo experiments proved to be difficult because constitutive TGF-β1 knock-out (KO) in mice leads to lethality during the first 4 weeks of life from a wasting syndrome related to tissue infiltration by activated T cells and macrophages. For this reason, hematopoiesis of TGF-β1−/− mice has not been studied in details. In contrast the role of TGF-β1 has been recently extensively studied in conditional TGF-β type I receptor (TβRI) KO mice. No clear effect was observed on HSC functions, suggesting that TGF-β1 was not a key physiological regulator of hematopoiesis in the adult. However, these experiments have some limitations. They do not exclude a putative role for TGF-β1 during fetal hematopoiesis and they do not specifically address the role of TGF-β1 on hematopoiesis because KO of TGF-β receptor leads to signaling arrest for all TGF-βs. In addition, other receptors may be involved in TGF-β1 signaling. For these reasons, we have investigated the hematopoiesis of constitutive TGF-β1 KO mice with a mixed Sv129 × CF-1 genetic background allowing the birth of a high proportion of homozygotes. In 2 week-old neonate mice, we have shown a decrease of bone marrow (BM) and spleen progenitors and a decrease of immature progenitors colony forming unit of the spleen (CFU-s). Moreover this was associated with a loss in reconstitutive activity of TGF-β1−/− HSC from BM. However, although asymptomatic, these mice had an excess of activated lymphocytes and an augmentation of Sca-1 antigen on hematopoietic cells suggesting an excess of γ-interferon release. Thus we studied hematopoiesis of 7 to 10 days-old neonate mice, before phenotypic modification and inflammatory cytokine release. Similar results were observed with a decrease in the number of progenitors and in the proliferation of TGF-β1−/− BM cells along with an increased differentiation but without an augmentation in apoptosis. Moreoever, a loss of long term reconstitutive capacity of BM Lineage negative (Lin−) TGF-β1−/− cells along with a diminution of homing of TGF-β1−/− progenitors was found. These results demonstrate that TGF-β1 may play a major role on the HSC/Progenitor compartment in vivo and that this defect does not seem to be linked to the immune disease. To completely overpass the risk of the inflammatory syndrome, we analyzed hematopoiesis of fetal liver (FL) of TGF-β1−/− mice and still found a decrease in progenitors, a profound defect in the proliferative capacities, in long term reconstitutive activity and homing potential of primitive FL hematopoietic cells. Our results demonstrate that TGF-β1 plays an important role during hematopoietic embryonic development. Altogether these findings suggest that TGF-β1 is a potent positive regulator for the in vivo homeostasis of the HSC compartment.

Interleukin-3 supports expansion of long-term multilineage repopulating activity after multiple stem cell divisions in vitro

Blood ◽  
2000 ◽  
Vol 96 (5) ◽  
pp. 1748-1755 ◽  
Author(s):  
David Bryder ◽  
Sten E. W. Jacobsen
Keyword(s):  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Calf Serum ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Divisions ◽  
Stem Cell Divisions

Although long-term repopulating hematopoietic stem cells (HSC) can self-renew and expand extensively in vivo, most efforts at expanding HSC in vitro have proved unsuccessful and have frequently resulted in compromised rather than improved HSC grafts. This has triggered the search for the optimal combination of cytokines for HSC expansion. Through such studies, c-kit ligand (KL), flt3 ligand (FL), thrombopoietin, and IL-11 have emerged as likely positive regulators of HSC self-renewal. In contrast, numerous studies have implicated a unique and potent negative regulatory role of IL-3, suggesting perhaps distinct regulation of HSC fate by different cytokines. However, the interpretations of these findings are complicated by the fact that different cytokines might target distinct subpopulations within the HSC compartment and by the lack of evidence for HSC undergoing self-renewal. Here, in the presence of KL+FL+megakaryocyte growth and development factor (MGDF), which recruits virtually all Lin−Sca-1+kit+ bone marrow cells into proliferation and promotes their self-renewal under serum-free conditions, IL-3 and IL-11 revealed an indistinguishable ability to further enhance proliferation. Surprisingly, and similar to IL-11, IL-3 supported KL+FL+MGDF-induced expansion of multilineage, long-term reconstituting activity in primary and secondary recipients. Furthermore, high-resolution cell division tracking demonstrated that all HSC underwent a minimum of 5 cell divisions, suggesting that long-term repopulating HSC are not compromised by IL-3 stimulation after multiple cell divisions. In striking contrast, the ex vivo expansion of murine HSC in fetal calf serum-containing medium resulted in extensive loss of reconstituting activity, an effect further facilitated by the presence of IL-3.

scholarly journals Human fetal bone marrow early progenitors for T, B, and myeloid cells are found exclusively in the population expressing high levels of CD34

Blood ◽  
1994 ◽  
Vol 84 (2) ◽  
pp. 421-432 ◽  
Author(s):  
D DiGiusto ◽  
S Chen ◽  
J Combs ◽  
S Webb ◽  
R Namikawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Cell Activity ◽  
Hematopoietic Stem ◽  
Fetal Bone ◽  
Cd34 Antigen

Experimentation on human stem cells is hampered by the relative paucity of this population and by the lack of assays identifying multilineage differentiation, particularly along the lymphoid lineages. In our current study, phenotypic analysis of low-density fetal bone marrow cells showed two distinct populations of CD34+ cells: those expressing a high density of CD34 antigen on their surface (CD34hi) and those expressing an intermediate level of CD34 antigen (CD34lo). Multiple tissues were used to characterize the in vitro and in vivo potential of these subsets and showed that only CD34hi cells support long-term B lymphopoiesis and myelopoiesis in vitro and mediate T, B, and myeloid repopulation of human tissues implanted into SCID mice. CD34lo cells repeatedly failed to provide long-term hematopoietic activity in vivo or in vitro. These results indicate that a simple fractionation based on well-defined CD34 antigen levels can be used to reproducibly isolate cells highly enriched for in vivo long-term repopulating activity and for multipotent progenitors, including T- and B-cell precursors. Additionally, given the limited variability in the results and the high correlation between in vitro and in vivo hematopoietic potential, we propose that the CD34hi population contains virtually all of the stem cell activity in fetal bone marrow and therefore is the population of choice for future studies in hematopoietic stem cell development and gene therapy.

An In Vivo Functional Screen Identifies miRNA-150 As a Regulator of Hematopoietic Regeneration Post Chemotherapeutic Injury

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2333-2333
Author(s):  
Brian D. Adams ◽  
Shangqin Guo ◽  
Haitao Bai ◽  
Changchun Xiao ◽  
E. Premkumar Reddy ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Negative Regulator ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Hematopoietic Recovery ◽  
Expression Construct

Abstract Abstract 2333 . MicroRNAs are important regulators of many hematopoietic processes, yet little is known with regard to the role of microRNAs in controlling normal hematopoietic regeneration. The most common methodology for in vivo microRNA studies follows a hypothesis-driven candidate approach. Here, we report the establishment of an unbiased, in vivo, microRNA gain-of-function screen, and the identification of miR-150 as a negative regulator of hematopoietic recovery post chemotherapeutic challenge. Specifically, a retroviral-library consisting of 135 hematopoietic-expressed microRNAs was generated, with each expression construct containing a barcode sequence that can be specifically recognized using a novel bead-based platform. Hematopoietic-stem-and-progenitor-cell (HSPC)-enriched wild-type bone marrow was transduced with this library and transplanted into lethally-irradiated recipients. Analysis of peripheral blood samples from each recipient up to 11 weeks post transplantation revealed that 87% of the library barcodes are reliably detected. To identify microRNAs that regulate hematopoietic regeneration after chemotherapy-induced injury, we measured the change in barcode abundance for specific microRNA constructs after 5-fluorouracil (5-FU) challenge. Notably, a small number of barcodes were consistently depleted in multiple recipient mice after treatment. Among the top hits was the miR-150-associated barcode, which was selected for further experimentation. Indeed, overexpression of miR-150 in a competitive environment resulted in significantly lower recovery rates for peripheral myeloid and platelet populations after 5-FU treatment, whereas the effects on B- and T-cells were milder. Furthermore, full recovery of these cell populations did not occur until ∼12 weeks after treatment, suggesting the involvement of HSPCs and/or common lineage progenitors. Conversely, knocking out miR-150 led to an opposite phenotype, with platelets and myeloid cells displaying faster recovery in both competitive and non-competitive settings. Interestingly, we could not observe the described effects of miR-150 in bone marrow primary cell cultures, suggesting that such effects cannot be recapitulated in vitro. Overall, these data indicate that miR-150 is a novel regulator of hematopoietic recovery after chemotherapeutic-induced injury, and highlight the important role of microRNAs in the intrinsic wiring of the hematopoietic regeneration program. Our experiments also demonstrate the feasibility and power of functional in vivo screens for studying normal hematopoietic functions, which can become an important tool in the hematology field. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Human adenosine deaminase expression in mice

Blood ◽  
1990 ◽  
Vol 75 (10) ◽  
pp. 2085-2092 ◽  
Author(s):  
KA Moore ◽  
FA Fletcher ◽  
DK Villalon ◽  
AE Utter ◽  
JW Belmont
Keyword(s):  
Stem Cells ◽  
Adenosine Deaminase ◽  
Bone Marrow Cells ◽  
High Titer ◽  
Experimental Period ◽  
Hematopoietic Stem ◽  
Provirus Integration

Abstract A replication defective retroviral vector containing a human adenosine deaminase (hADA) cDNA was produced by GP + E-86 packaging cells at high titer. We report long-term expression of hADA in the hematopoietic tissues of mice transplanted with bone marrow cells infected by in vitro co-cultivation with vector producing cells. Western analysis using an hADA-specific antibody allowed detection of the protein in the peripheral blood of all 37 transplanted mice for at least 9 weeks. Sixty-eight percent of the animals continued to express hADA in one or more of their hematopoietic tissues for the experimental period, and hADA was found in both spleen colonies and tissues of secondary recipients. There was provirus integration and expression in myeloid, erythroid, and lymphoid cell lineages, indicating extensive repopulation by the progeny of infected stem cells. The vector did not contain a selectable marker, and the infected stem cells did not have a competitive in vivo advantage. Nevertheless, we observed consistent gene transfer into hematopoietic stem cells and long-term expression of a human gene product in their progeny.

Use of limiting-dilution type long-term marrow cultures in frequency analysis of marrow-repopulating and spleen colony-forming hematopoietic stem cells in the mouse

Blood ◽  
1991 ◽  
Vol 78 (10) ◽  
pp. 2527-2533 ◽  
Author(s):  
RE Ploemacher ◽  
JP van der Sluijs ◽  
CA van Beurden ◽  
MR Baert ◽  
PL Chan
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Linear Regression Analysis ◽  
Correlation Study ◽  
Hematopoietic Stem ◽  
Clonal Assay

Abstract We have developed an in vitro clonal assay of murine hematopoietic precursor cells that form spleen colonies (CFU-S day 12) or produce in vitro clonable progenitors in the marrow (MRA cells) of lethally irradiated mice. The assay is essentially a long-term bone marrow culture in microtiter wells containing marrow-derived stromal “feeders” depleted for hematopoietic activity by irradiation. To test the validity of the assay as a quantitative in vitro stem cell assay, a series of unsorted and physically sorted bone marrow cells were simultaneously assayed in vivo and overlaid on the feeders in a range of concentrations, while frequencies of cells forming hematopoietic clones (cobblestone area forming cells, CAFC) were calculated by means of Poisson statistics. Linear regression analysis of the data showed high correlations between the frequency of CFU-S day 12 and CAFC day 10, and between MRA cells and CAFC day 28. A majority of MRA activity and CAFC day 28 was separable from CFU-S day 12 and CAFC day 10. This correlation study validates the CAFC system as a clonal assay facilitation both the quantitative assessment of a series of subsets in the hematopoietic stem cell hierarchy and the study of single long-term repopulating cells in vitro.

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.

scholarly journals Murine hematopoietic stem and progenitor cells: I. Enrichment and biologic characterization

Blood ◽  
1995 ◽  
Vol 85 (6) ◽  
pp. 1472-1479 ◽  
Author(s):  
CL Li ◽  
GR Johnson
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Cell Populations ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Stem And Progenitor Cells

Murine bone marrow cells were fractionated by fluorescence-activated cell sorting into Rh123lo Lin- c-kit+ Ly6A+, Rh123hi Lin-c-kit+ Ly6A+, and Lin- c-kit+ Ly6A- populations within which most, if not all, of the hematopoietic activities of the marrow resided. The Rh123lo Lin- c- kit+Ly6A+ cells, which consist exclusively of small- or medium-sized lymphocyte-like cells, are highly enriched for long-term hematopoietic in vivo repopulating cells. The enrichment factor for these cells from the marrow was estimated as 2,000-fold. The Rh123hi Lin- c-kit+ Ly6A+ cells, although also highly enriched for day-12 spleen colony-forming units, were relatively depleted of long-term in vivo repopulation capacity. Most, if not all Lin- c-kit+ Ly6A- cells were Rb123hi. In contrast to both Rh123lo and Rh123hi Lin- c-kit+ Ly6A+ stem cell populations, the Lin- c-kit+ Ly6A- cells can be stimulated to proliferate in vitro in the presence of single cytokines, which is a characteristic of committed progenitor cells. No marked synergistic interactions between individual cytokines were observed with this cell population. Both Rh123hi Lin- c-kit+ Ly6A+ mature stem cell and Lin- c- kit+ Ly6A- progenitor cell populations displayed in vivo repopulation kinetics resembling those of the putative short-term hematopoietic repopulating cells.

scholarly journals Use of limiting-dilution type long-term marrow cultures in frequency analysis of marrow-repopulating and spleen colony-forming hematopoietic stem cells in the mouse

Blood ◽  
1991 ◽  
Vol 78 (10) ◽  
pp. 2527-2533 ◽  
Author(s):  
RE Ploemacher ◽  
JP van der Sluijs ◽  
CA van Beurden ◽  
MR Baert ◽  
PL Chan
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Linear Regression Analysis ◽  
Correlation Study ◽  
Hematopoietic Stem ◽  
Clonal Assay

We have developed an in vitro clonal assay of murine hematopoietic precursor cells that form spleen colonies (CFU-S day 12) or produce in vitro clonable progenitors in the marrow (MRA cells) of lethally irradiated mice. The assay is essentially a long-term bone marrow culture in microtiter wells containing marrow-derived stromal “feeders” depleted for hematopoietic activity by irradiation. To test the validity of the assay as a quantitative in vitro stem cell assay, a series of unsorted and physically sorted bone marrow cells were simultaneously assayed in vivo and overlaid on the feeders in a range of concentrations, while frequencies of cells forming hematopoietic clones (cobblestone area forming cells, CAFC) were calculated by means of Poisson statistics. Linear regression analysis of the data showed high correlations between the frequency of CFU-S day 12 and CAFC day 10, and between MRA cells and CAFC day 28. A majority of MRA activity and CAFC day 28 was separable from CFU-S day 12 and CAFC day 10. This correlation study validates the CAFC system as a clonal assay facilitation both the quantitative assessment of a series of subsets in the hematopoietic stem cell hierarchy and the study of single long-term repopulating cells in vitro.

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