scholarly journals Microenvironmental Contribution to Dysfunctional Hematopoiesis in a Murine Model of Myelodysplastic Syndrome

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4359-4359
Author(s):  
Sophia R. Balderman ◽  
Benjamin J Frisch ◽  
Mark W LaMere ◽  
Alexandra N Goodman ◽  
Michael W. Becker ◽  
...  
Keyword(s):  
Murine Model ◽  
Bystander Effect ◽  
Flow Cytometric Analysis ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Flow Cytometric

Abstract In vitro data provide evidence of an altered bone marrow microenvironment (BMME) in the myelodysplastic syndromes (MDS). To assess the role of the BMME in MDS in vivo, we used a well-established transgenic murine model with expression of the translocation product Nup98-HOXD13 (NHD13) in hematopoietic cells that leads to development of an MDS phenotype, fully penetrant by 5 months of age. In order to assess whether the BMME contributes to diminished hematopoiesis as a feature of MDS, we transplanted marrow from 5-month-old NHD13 mice and normal competitor marrow into irradiated NHD13 mice and their wild type (WT) littermates. Serial analysis of peripheral blood (PB) indicated engraftment of NHD13 marrow was improved in WT recipients relative to NHD13 recipients (2-way ANOVA, WT vs. NHD13: p<0.0001). Flow cytometric analysis of marrow harvested at 16 weeks post-transplant revealed increased NHD13 donor contribution to the hematopoietic stem and progenitor cell (HSPC) pool in WT relative to NHD13 recipients (28.2 ± 4.3 vs. 2.4 ± 0.5 % of total Lineage-, cKit+, Sca1+ (LSK) cells, p<0.01). Surprisingly, leukopoiesis was improved after transplantation of NHD13 marrow into WT as compared to NHD13 recipients (2-way ANOVA, WT vs. NHD13: p<0.01). These data establish that the MDS BMME interferes with the ability of MDS HSPCs to function similarly to normal HSPCs. After the identification of a microenvironmental defect in adult NHD13 mice, we further investigated the NHD13 BMME support for hematopoietic progenitors. By flow cytometric analysis, there were no differences in marrow multipotent progenitors (MPPs) and long term hematopoietic stem cells (LT-HSCs) from NHD13 mice vs. WT littermates at 3 weeks of age. However, in adults there was a progressively severe decline in the NHD13 HSPC pool. HSPCs were not diminished in the spleens of NHD13 mice, suggesting a specific BMME defect. The decrease in phenotypic HSPCs in NHD13 mice was confirmed functionally by competitive repopulation assays using NHD13 or WT donor marrow transplanted into irradiated WT recipients. NHD13-derived PB cells demonstrated marked myeloid skewing relative to WT-derived cells, indicative of a differentiation defect in NHD13-associated hematopoiesis. At 16 weeks post-transplant, recipient marrow was assayed for relative NHD13 and WT donor contributions to the HSPC pool. Consistent with the decreased NHD13 donor contribution to PB counts, NHD13 donor contribution to the HSPC pool in the marrow was diminished (59.4 ± 8.7 vs. 15.5 ± 5.6, % WT donor vs. NHD13 donor contribution to total LSK cells, p<0.001). Despite robust engraftment of WT competitor marrow, cytopenias and macrocytosis were observed in the recipients of NHD13 marrow, suggesting a bystander effect by the NHD13 clone on the function of the normal competitor marrow. To determine NHD13 long-term engraftment function, secondary transplantation of marrow harvested from the primary recipients of NHD13 and WT donors was performed using WT recipients. Serial PB flow cytometric data demonstrated improved overall engraftment of the NHD13 relative to WT donor marrow with persistent and even more marked myeloid skewing of NHD13 donor derived blood cells than was seen in the primary transplant. Consistent with PB data, at 16 weeks post-transplant, the contribution of NHD13 and WT donors to the HSPC pool was similar. Improved NHD13 HSPC number and function in the secondary recipients may be related to BMME rejuvenation through serial passage into a WT BMME. Our data indicate that in this model (1) MDS hematopoietic function is improved in a normal compared to MDS microenvironment (2) the HSPC pool is defective and (3) there is suppression of non-clonal hematopoiesis via a bystander effect, possibly mediated by the MDS BMME. In aggregate our data demonstrate a contributory role of the BMME to ineffective hematopoiesis in MDS, and support a therapeutic strategy whereby manipulation of the MDS microenvironment may improve hematopoietic function. Disclosures Calvi: Fate Therapeutics: Patents & Royalties.

Disruption Of The Hematopoietic Stem and Progenitor Cell Pool and Bone Marrow Microenvironment In a Murine Model Of Myelodysplastic Syndrome

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2756-2756
Author(s):  
Sophia R Balderman ◽  
Benjamin J Frisch ◽  
Mark W LaMere ◽  
Alexandra N Goodman ◽  
Michael W. Becker ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Murine Model ◽  
Bystander Effect ◽  
Conflicts Of Interest ◽  
Flow Cytometric Analysis ◽  
Bone Marrow Microenvironment ◽  
Hematopoietic Stem ◽  
Serial Blood ◽  
Post Transplant

Abstract Myelodysplastic Syndromes (MDS) are a group of clonal disorders characterized by ineffective hematopoiesis. Recently, data have emerged supporting a role of the bone marrow microenvironment (BMME ) in the initiation of MDS. We and others have previously shown that cells within the BMME play a central role in normal regulation of hematopoietic stem and progenitor cells (HSPCs). To determine if the HSPC compartment in MDS is defective and also if HSPC function in MDS is regulated by the BMME, we studied a transgenic murine model that expresses the Nup98/HOXD13 (NHD13) translocation product. As was previously reported, these mice develop ineffective hematopoiesis resulting in progressive cytopenias with dysmorphic cells, a phenotype similar to that of human MDS. We investigated the composition of the HSPC pool in these transgenic (TG) mice at 20 to 22 weeks from birth, a time when an MDS phenotype was evident but acute leukemia had not yet developed. Immunophenotypic analysis by flow cytometry on marrow cells from TG and wild type (WT) age-matched littermates demonstrated a severe defect in the TG HSPC pool, with a severe decline in Lin-Sca1+cKit+CD48-CD150+ long-term HSCs (WT vs. TG: 3.5 ± 1.2 x 104 vs 4.4 ± 3.4 x102, p =0.0025) and in Lin-Sca1+cKit+Flt3+Thy1.1- multipotent progenitors (WT vs. TG: 5.6 ± 1 x 105 vs 2.1 ± 0.4 x 104, p<0.0001), as well as in total Lin-Sca1+cKit+ cells and short-term HSCs. To determine if the numerical changes in phenotypic HSPCs corresponded with decreased HSPC function, we performed a competitive repopulation assay using whole bone marrow, and found relative loss of function of HSPCs by 9 weeks after transplantation of marrow from 22-week old TG vs littermate WT donor mice into lethally irradiated WT recipients as measured by percent of donor cells in the blood (WT vs. TG: 37.7 ± 3.4 vs 14.7 ± 1.7, p<0.0001). Serial blood cell flow cytometric analysis demonstrated myeloid skewing (marked by percent of CD11b positive cells) of HSPCs transplanted from TG mice at the expense of lymphocytes by 5 weeks (WT vs. TG: 44.0 ± 4.3 vs 64.9 ± 4.5, p=0.0047), which persisted at 9 weeks (WT vs. TG: 43.6 ± 3.6 vs 69.1 ± 5.9, p=0.0023) and 13 weeks post transplant, a feature which has been previously associated with HSPC aging. Curiously, despite robust engraftment of normal competitor marrow, serial blood counts of recipients after competitive transplant showed that mice receiving 22-week old TG marrow developed leukopenia (9 weeks, WT vs. TG: 7.3 ± 0.47 vs 4.6 ± 0.41, p=0.0008) and lymphopenia (9 weeks, WT vs. TG: 6.0 ± 0.42 vs 3.4 ± 0.37, p=0.0003), suggesting a bystander effect initiated by the TG marrow resulting in ineffective hematopoiesis in the recipients. To determine if the MDS microenvironment contributes to ineffective hematopoiesis, we transplanted NHD13 TG and normal competitor marrow into lethally irradiated TG or WT recipient mice. NHD13 TG marrow engrafted significantly better in WT compared to TG recipients as seen by 4 weeks post transplant (Percent of total cells, WT vs. TG recipient: 14.2 ± 2.3 vs 1.1 ± 0.1, p = 0.0049; Percent of CD11b positive cells, WT vs. TG recipient: 17.1 ± 4.2 vs 1.7 ± 0.1, p = 0.0208; Percent of B220 positive cells, WT vs. TG recipient: 2.7 ± 0.3 vs 0.1 ± 0.0, p = 0.0008). These aggregate results indicate (1) severe disruption of the immunophenotypic HSPC pool in this murine TG model of MDS, (2) a functional defect of HSPCs in this MDS model as evidenced by decreased engraftment and myeloid skewing, (3) contribution of the MDS BMME to ineffective hematopoiesis downstream of immature MDS cells and (4) MDS-dependent signals initiating such microenvironmental effects. Our data strongly suggest that the malignant clone in MDS initiates signals that disrupt the normal marrow microenvironment. Furthermore, these data provide support for a strategy where rejuvenation of the marrow microenvironment and/or interference with MDS-initiated signals may result in mitigation of ineffective hematopoiesis. Further understanding of the HSPC defect in this murine model of MDS and of the role of the BMME in MDS could therefore inform new therapeutic targets for this disease. Disclosures: No relevant conflicts of interest to declare.

High-resolution tracking of cell division suggests similar cell cycle kinetics of hematopoietic stem cells stimulated in vitro and in vivo

Blood ◽  
2000 ◽  
Vol 95 (3) ◽  
pp. 855-862 ◽  
Author(s):  
Robert A. J. Oostendorp ◽  
Julie Audet ◽  
Connie J. Eaves
Keyword(s):  
Stem Cells ◽  
Fetal Liver ◽  
Flow Cytometric Analysis ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Flow Cytometric ◽  
Differentiation Status ◽  
Kinetics Of

The kinetics of proliferation of primitive murine bone marrow (BM) cells stimulated either in vitro with growth factors (fetal liver tyrosine kinase ligand 3 [FL], Steel factor [SF], and interleukin-11 [IL-11], or hyper–IL-6) or in vivo by factors active in myeloablated recipients were examined. Cells were first labeled with 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and then incubated overnight prior to isolating CFSE+ cells. After 2 more days in culture, more than 90% of the in vivo lymphomyeloid repopulating activity was associated with the most fluorescent CFSE+ cells (ie, cells that had not yet divided), although this accounted for only 25% of the repopulating stem cells measured in the CFSE+ “start” population. After a total of 4 days in culture (1 day later), 15-fold more stem cells were detected (ie, 4-fold more than the day 1 input number), and these had become (and thereafter remained) exclusively associated with cells that had divided at least once in vitro. Flow cytometric analysis of CFSE+ cells recovered from the BM of transplanted mice indicated that these cells proliferated slightly faster (up to 5 divisions completed within 2 days and up to 8 divisions completed within 3 days in vivo versus 5 and 7 divisions, respectively, in vitro). FL, SF, and ligands which activate gp130 are thus efficient stimulators of transplantable stem cell self-renewal divisions in vitro. The accompanying failure of these cells to accumulate rapidly indicates important changes in their engraftment potential independent of accompanying changes in their differentiation status.

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.

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.

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.

Evidence for the Involvement of CXCR4 Signaling in In Vivo Self-Renewal of Transplanted Hematopoietic Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1888-1888 ◽  
Author(s):  
Chen-YI LAI ◽  
Makoto Otsu ◽  
Motohito Okabe ◽  
Sachie Suzuki ◽  
Satoshi Yamazaki ◽  
...  
Keyword(s):  
Post Transplantation ◽  
Gain Of Function ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cxcr4 Signaling ◽  
Hematopoietic Reconstitution

Abstract Abstract 1888 Hematopoietic stem cells (HSCs) represent the unique cell population capable of self-renewal and multi-lineage differentiation, thereby lifelong sustainment of the hematopoiesis. HSC transplantation has proven beneficial for various diseases, it is therefore important to elucidate the molecular determinants for successful HSC engraftment. Signaling through the chemokine receptor CXCR4 has been implicated in HSC engraftment by the observation that transplantation of HSCs lacking this molecule results in poor hematopoietic reconstitution. Because this impairment, however, can be attributed to the defects in any of the post-transplantation processes that include bone marrow (BM)-homing, -repopulation, or –retention, it is still unclear whether CXCR4 plays an essential role in HSC self-renewal upon transplantation. To elucidate the role of CXCR4 signaling in HSC self-renewal in conjunction with transplantation, we used a purified CD34neg/low c-Kit+ Sca-1+ Lineage-markerneg population as the defined stem cell source. As a loss-of-function study, CXCR4 was conditionally deleted in HSCs before transplantation. As a gain-of-function study, we generated the HSC populations overexpressing either wild-type (wt)- or C-terminal truncated (δC)-CXCR4 (OE-HSCs), the latter of which is known to exhibit enhancement in the SDF-1 signaling, by gene transfer and subsequent cell sorting. We compared these cells with control HSCs in in vitro assays with regard to the biological characteristics including chemotaxis, proliferation, colony formation, and cobblestone-area (CA) forming ability. To dissect in vivo post-transplantation processes, we investigated hematopoietic repopulation kinetics in the recipient BM at the homing/lodging phase (within 1 wk) and the early repopulation phase (2–3 wks) for the above test HSCs. The self-renewal potential of each HSC population was estimated by competitive repopulation assay. In vitro studies: OE-HSCs with wt- or δC-CXCR4 exhibited enhanced chemotaxis and proliferation in response to SDF1, confirming the gain-of-function effects of these modifications. CA forming ability was greater in OE-HSCs with δC-CXCR4 than control counterparts and absent in CXCR4-KO HSCs, suggesting the critical role of CXCR4-signaling in HSC proliferation in the presence of stromal support. In vivo studies: 1) the homing/lodging phase. Unexpectedly, we did not find significant alteration in the numbers of early progenies detectable in recipient BM 3 days after transplantation of HSCs receiving either loss- or gain-of-function modification to CXCR4, indicating that this signaling is indispensable in HSC homing. 2) the early repopulation phase. Impairment of hematopoietic repopulation in BM became evident for CXCR4-KO HSCs through 2–3 wks. On the other hand, OE-HSCs with CXCR4, more remarkably of ΔC-mutation, showed enhanced BM repopulation kinetics at ∼3 wks post transplantation, suggesting the importance of CXCR4 signaling in HSC amplification at this post-transplantation phase. 3) long-term hematopoiesis. CXCR4-KO-HSCs showed poor hematopoietic reconstitution potentials, consistent with previous observations. Interestingly, impaired peripheral repopulation was also observed with OE-HSCs with wt- or ΔC-CXCR4. Further characterization revealed that the recipients of CXCR4-overexpressing HSCs did retain their progenies, which showed multilineage differentiation, but exhibited impaired release of mature leukocytes from the BM to the peripheral blood. Most importantly, however, test-cell chimerism in the long-term HSC fraction was significantly higher in the mice receiving OE-HSCs with CXCR4, especially of ΔC-type, than those transplanted with control HSCs, indicating that the augmentation of CXCR4 signaling enhanced competitive repopulation ability of HSCs. These modified HSCs demonstrated repopulation abilities also in secondary recipients. We demonstrated that CXCR4 signaling is indispensible for HSC homing and that continuous overexpression of CXCR4 cannot benefit the peripheral reconstitution in contrary to the expectation. More importantly, our studies showed that augmentation of CXCR4 signaling leads to HSC expansion in vivo upon transplantation. We thus conclude that CXCR4 signaling has a role in HSC self-renewal and that its regulation may find the approach that will improve HSC transplantation outcomes. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Gli1 regulates the proliferation and differentiation of HSCs and myeloid progenitors

Blood ◽  
2010 ◽  
Vol 115 (12) ◽  
pp. 2391-2396 ◽  
Author(s):  
Akil Merchant ◽  
Giselle Joseph ◽  
Qiuju Wang ◽  
Sarah Brennan ◽  
William Matsui
Keyword(s):  
Homozygous Deletion ◽  
Hematopoietic Stem ◽  
Downstream Effectors ◽  
Gli Transcription Factors ◽  
Proliferation And Differentiation ◽  
Myeloid Development

The Hedgehog (Hh) pathway is essential for normal embryonic development and tissue repair. The role of Hh signaling in hematopoiesis has been studied primarily by modulating the activity of Patched and Smoothened, but results have been conflicting. Some studies demonstrate a requirement for pathway activity in hematopoiesis, whereas others report that it is dispensable. Hh activity converges on the Gli transcription factors, but the specific role of these downstream effectors in hematopoiesis has not been reported. We have analyzed hematopoietic stem cell (HSC) and progenitor function in mice with a homozygous deletion of Gli1 (Gli1null). Gli1null mice have more long-term HSCs that are more quiescent and show increased engraftment after transplantation. In contrast, myeloid development is adversely affected with decreased in vitro colony formation, decreased in vivo response to granulocyte colony-stimulating factor (G-CSF), and impaired leukocyte recovery after chemotherapy. Levels of the proto-oncogene Cyclin D1 are reduced in Gli1null mice and may explain the loss of proliferation seen in HSCs and progenitor cells. These data demonstrate that Gli1 regulates normal and stress hematopoiesis. Moreover, they suggest that Gli1 and Smoothened may not be functionally redundant, and direct GLI1 inhibitors may be needed to effectively block HH/GLI1 activity in human disease.

THE ROLE OF POLYETHYLENIMINE IN ENHANCING PERFORMANCE OF GLUTAMATE BIOSENSORS

2018 ◽  
Vol 8 (3) ◽  
pp. 36-41
Author(s):  
Diep Do Thi Hong ◽  
Duong Le Phuoc ◽  
Hoai Nguyen Thi ◽  
Serra Pier Andrea ◽  
Rocchitta Gaia
Keyword(s):  
First Generation ◽  
Good Reproducibility ◽  
Complex Matrices ◽  
Long Term Stability ◽  
In Vitro Characterization ◽  
Glutamate Oxidase

Background: The first biosensor was constructed more than fifty years ago. It was composed of the biorecognition element and transducer. The first-generation enzyme biosensors play important role in monitoring neurotransmitter and determine small quantities of substances in complex matrices of the samples Glutamate is important biochemicals involved in energetic metabolism and neurotransmission. Therefore, biosensors requires the development a new approach exhibiting high sensibility, good reproducibility and longterm stability. The first-generation enzyme biosensors play important role in monitoring neurotransmitter and determine small quantities of substances in complex matrices of the samples. The aims of this work: To find out which concentration of polyethylenimine (PEI) exhibiting the most high sensibility, good reproducibility and long-term stability. Methods: We designed and developed glutamate biosensor using different concentration of PEI ranging from 0% to 5% at Day 1 and Day 8. Results: After Glutamate biosensors in-vitro characterization, several PEI concentrations, ranging from 0.5% to 1% seem to be the best in terms of VMAX, the KM; while PEI content ranging from 0.5% to 1% resulted stable, PEI 1% displayed an excellent stability. Conclusions: In the result, PEI 1% perfomed high sensibility, good stability and blocking interference. Furthermore, we expect to develop and characterize an implantable biosensor capable of detecting glutamate, glucose in vivo. Key words: Glutamate biosensors, PEi (Polyethylenimine) enhances glutamate oxidase, glutamate oxidase biosensors

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

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

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

scholarly journals The role of the PZP domain of AF10 in acute leukemia driven by AF10 translocations

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Brianna J. Klein ◽  
Anagha Deshpande ◽  
Khan L. Cox ◽  
Fan Xuan ◽  
Mohamad Zandian ◽  
...  
Keyword(s):  
Critical Role ◽  
Cellular Transformation ◽  
Acute Leukemias ◽  
Hematopoietic Stem ◽  
Nucleosome Core ◽  
Stem And Progenitor Cells ◽  
Transforming Activity

AbstractChromosomal translocations of the AF10 (or MLLT10) gene are frequently found in acute leukemias. Here, we show that the PZP domain of AF10 (AF10PZP), which is consistently impaired or deleted in leukemogenic AF10 translocations, plays a critical role in blocking malignant transformation. Incorporation of functional AF10PZP into the leukemogenic CALM-AF10 fusion prevents the transforming activity of the fusion in bone marrow-derived hematopoietic stem and progenitor cells in vitro and in vivo and abrogates CALM-AF10-mediated leukemogenesis in vivo. Crystallographic, biochemical and mutagenesis studies reveal that AF10PZP binds to the nucleosome core particle through multivalent contacts with the histone H3 tail and DNA and associates with chromatin in cells, colocalizing with active methylation marks and discriminating against the repressive H3K27me3 mark. AF10PZP promotes nuclear localization of CALM-AF10 and is required for association with chromatin. Our data indicate that the disruption of AF10PZP function in the CALM-AF10 fusion directly leads to transformation, whereas the inclusion of AF10PZP downregulates Hoxa genes and reverses cellular transformation. Our findings highlight the molecular mechanism by which AF10 targets chromatin and suggest a model for the AF10PZP-dependent CALM-AF10-mediated leukemogenesis.

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