FG-4497, a Pharmacological Stabilizer of HIF-1α Protein, Synergistically Enhances Hematopoietic Stem Cells (HSC) Mobilization in Response to G-CSF and Plerixafor

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 216-216 ◽  
Author(s):  
Catherine E Forristal ◽  
Bianca Nowlan ◽  
Valerie Barbier ◽  
Ingrid G Winkler ◽  
Gail Walkinshaw ◽  
...  
Keyword(s):  
Hypoxia Inducible Factor ◽  
Cell Types ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Prolyl Hydroxylases ◽  
Treatment Groups ◽  
Healthy Donors ◽  
Cd34 Cells ◽  
O2 Concentration

Abstract Abstract 216 Up to 5% allogeneic healthy donors and up to 40–60% of chemotherapy-treated patients in autologous setting, fail to reach minimal threshold of 2×106 blood CD34+cells/kg in response to G-CSF, precluding transplantation. Plerixafor, a small inhibitor of the chemokine receptor CXCR4, used for 4 days in combination with G-CSF enables this minimal threshold to be reached in up to 60% patients who previously failed to mobilise in response to G-CSF alone. However, the remaining 40% of patients who failed to mobilise in response to G-CSF alone, still fail to mobilize adequately with G-CSF + Plerixafor. In an attempt to further boost HSC mobilization in response to combinations of G-CSF and Plerixafor, we have investigated the role of the hypoxia-sensing pathway in HSC mobilization. HIF-1α (Hypoxia-inducible factor-1α) controls HSC proliferation and self-renewal in poorly perfused hypoxic bone marrow (BM) niches where very quiescent HSC with highest self-renewal potential reside. When O2 concentration is above 2% in the cell microenvironment, HIF-1α protein is rapidly hydroxylated on Pro residues by prolyl hydroxylases PHD1-3. This recruits the E3 ubiquitin ligase VHL, which targets HIF-1α to rapid proteasomal degradation. When O2concentration is below 2% (hypoxia), PHDs are inactive; HIF-1α protein is stabilized, associates with its β subunit ARNT, translocates to the nucleus and activates of transcription and hypoxia-responsive genes. In this study, we have investigated the effect of pharmacological stabilization of HIF-1α protein on HSC mobilization in mice using the HIF-PHD inhibitor FG-4497. We report that FG-4497 treatment stabilizes HIF-1α protein in mouse BM. We find that FG-4497 synergizes with G-CSF and Plerixafor to enhance HSC mobilization. C57/Bl6 mice were in 4 treatment groups: (G) 250μg/kg/day G-CSF alone for 2 days; (GF) G-CSF for 2 days + 20mg/kg/day FG-4497 for 3 days; (GP) G-CSF for 2 days together with16mg/kg Plerixafor 1 hour prior harvest; (GPF) G-CSF together with Plerixafor and FG-4497 with same dosing as above. Mobilization of colony-forming cells (CFC), phenotypic Lin-CD41-Sca1+Kit+CD48-CD150+ HSC, and functional HSC in long-term competitive transplantation assays were measured. Mice in the GF group (G-CSF + FG-4497) mobilized CFC to the blood 4-fold and phenotypic HSC 3-fold more than mice mobilized with G-CSF alone (p<0.005), whereas FG-4497 alone had a poor mobilizing effect. This demonstrates synergy between G-CSF and PHD inhibition. Expectedly, Plerixafor enhanced mobilization of CFC 10-fold and phenotypic HSC 2-fold in response to G-CSF (p<0.005). Most interestingly, addition of FG-4497 boosted 4-fold mobilization of CFC and phenotypic HSC in response to G-CSF+Plerixafor (p<0.005). This was confirmed in competitive repopulation assays following transplantation of 20μL mobilized blood in competition with 2×105BM cells from congenic donors. CD45.2/CD45.1 chimerism showed that combination of G-CSF+Plerixafor+FG-4497 mobilized competitive repopulating HSC 6-fold more than G-CSF+Plerixafor (p<0.005), the best mobilizing cocktail used in the clinic currently. To determine which cell types drive HSC mobilization in a HIF-1α-dependant manner, we crossed HIF1αflox/floxmice with osxCre (HIF-1α gene deletion in osteoprogenitors), LysMCre (deletion in myeloid cells), or with SclCreER mice (tamoxifen-induced deletion in HSC). While studies in LysMCre and SclCreER mice are ongoing, we find that deletion of HIF-1α gene in osteoprogenitors (osxCre mice) decreased 2.5-fold the number of CFU/mL blood following 2 and 3 days treatment with G-CSF. This suggests that HIF-1α in osteoprogenitors and their osteoblastic progenies is necessary for optimal mobilization in response to G-CSF. In conclusion, our data highlight the importance of HIF-1α in HSC mobilization and provide a novel therapeutic strategy for increasing HSC mobilization above levels obtained with combinations of G-CSF and Plerixafor. Thus PHD inhibitors could be useful agents in patients who still fail to mobilize in response to G-CSF and plerixafor. Disclosures: Walkinshaw: Fibrogen Inc.: Employment, Equity Ownership.

Ex-Vivo Expansion of Human Cord Blood CD34+ Cells by Overexpression of Bmi-1.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1329-1329
Author(s):  
Aleksandra Rizo ◽  
Edo Vellenga ◽  
Gerald de Haan ◽  
Jan Jacob Schuringa
Keyword(s):  
Cord Blood ◽  
Cell Expansion ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Human Cord Blood ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Cord Blood Cd34

Abstract Hematopoietic stem cells (HSCs) are able to self-renew and differentiate into cells of all hematopoietic lineages. Because of this unique property, they are used for HSC transplantations and could serve as a potential source of cells for future gene therapy. However, the difficulty to expand or even maintain HSCs ex vivo has been a major limitation for their clinical applications. Here, we report that overexpression of the Polycomb group gene Bmi-1 in human cord blood-derived HSCs can potentially overcome this limitation as stem/progenitor cells could be maintained in liquid culture conditions for over 16 weeks. In mouse studies, it has been reported that increased expression of Bmi-1 promotes HSC self-renewal, while loss-of-function analysis revealed that Bmi-1 is implicated in maintenance of the hematopoietic stem cells (HSC). In a clinically more relevant model, using human cord blood CD34+ cells, we have established a long-term ex-vivo expansion method by stable overexpression of the Bmi-1 gene. Bmi-1-transduced cells proliferated in liquid cultures supplemented with 20% serum, SCF, TPO, Flt3 ligand, IL3 and IL6 for more than 4 months, with a cumulative cell expansion of more then 2×105-fold. The cells remained cytokine-dependent, while about 4% continued to express CD34 for over 20 weeks of culture. The cultured cells retained their progenitor activity throughout the long-term expansion protocol. The colony-forming units (CFUs) were present at a frequency of ~ 30 colonies per 10 000 cells 16 weeks after culture and consisted of CFU-GM, BFU-E and high numbers of CFU-GEMM type progenitors. After plating the transduced cells in co-cultures with the stromal cell line MS5, Bmi-1 cells showed a proliferative advantage as compared to control cells, with a cumulative cell expansion of 44,9 fold. The non-adherent cells from the co-cultures gave rise to higher numbers of colonies of all types (~70 colonies/10.000 cells) after 4 weeks of co-culture. The LTC-IC frequencies were 5-fold higher in the Bmi-1-transduced cells compared to control cells (1/361 v.s. 1/2077, respectively). Further studies will be focused on in-vivo transplantation of the long-term cultured cells in NOD/SCID mice to test their repopulating capacity. In conclusion, our data implicate Bmi-1 as an important modulator of human HSC self-renewal and suggest that it can be a potential target for therapeutic manipulation of human HSCs.

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

Blood ◽  
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.

βig-h3, a Novel Regulator of Adhesion and Differentiation of Human Hematopoietic Stem/Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3640-3640
Author(s):  
Sofieke E Klamer ◽  
Paula B van Hennik ◽  
Daphne C Thijssen-Timmer ◽  
C. Ellen Van der Schoot ◽  
Carlijn Voermans
Keyword(s):  
Cell Surface ◽  
Mrna Expression ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Cell Types ◽  
Cell Surface Expression ◽  
Type I ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 3640 Poster Board III-576 Adult hematopoietic stem cells (HSC) reside in the bone marrow (BM) in so-called niches. Within this specialized microenvironment, the interactions of HSC with adhesion molecules on neighbouring cells and extracellular matrix (ECM) components are thought to be critical for the maintenance of the HSC population. Comparative gene-expression profiling of purified HSC in homeostatic and regenerative conditions allowed the identification of a set of differentially expressed ECM proteins. One of these proteins was the novel ECM protein βg-h3, which plays a role in cell-ECM interactions, by binding to type I, II and IV collagens and cellular integrins. We postulated that βig-h3 could have a role in HSC biology by being both a homeostatic and regenerative regulator of HSC self-renewal and differentiation. First we analyzed the mRNA expression in human CD34+ hematopoietic stem/progenitor cells (HSPC) isolated from BM, mobilized peripheral blood (MPB) and umbilical cord blood (UCB). The expression of βig-h3 was found to be significantly higher in BM-CD34+ cells as compared to MPB-CD34+ cells, suggesting a role for this ECM protein in retaining HSC in the BM. To determine expression of βig-h3 on the various subsets within the heterogeneous CD34+ population, the expression was compared between sorted sub-populations of BM-CD34+ cells: megakaryocyte-erythrocyte-progenitors (MEP: CD38+/CD110+/CD45RA−), common myeloid progenitors (CMP: CD38+/CD110−/CD45RA−), granulocyte-monocyte-progenitors (GMP: CD38+/CD110−/CD45RA+) and more immature CD34+/CD38− HSC. The purity of the sub-populations was analyzed by colony forming assays. These data indicate that at least the mRNA expression of βig-h3 was highest in GMPs. Analysis of different human cell types revealed that the highest βig-h3 mRNA expression is measured in monocytes, dendritic cells and mesenchymal stromal cells (MSC), while its expression in megakaryocytes and HUVEC is comparable to that in HSPC. In addition, cell surface expression of the βig-h3 protein was determined by flowcytometry. βig-h3 was found to be expressed on the cell surface of only a subpopulation of BM derived CD34+ cells (0.5%), monocytes (5%), MSCs (11%) and megakaryocytes (30%). Intracellular flowcytometry staining revealed that βig-h3 is expressed inside CD34+ cells derived from all sources. Since there is evidence in several other cell types that βig-h3 plays a role in enhancing cell adhesion and migration, adhesion experiments using CD34+ cells were performed. These experiments show a significant (p<0.01) two-fold increased adhesion of MPB-CD34+ cells to βig-h3 compared to a BSA coating (mean 40% (SEM ± 9.8%) and 23% (SEM ± 5.0%), respectively, (n=3)). Further experiments showed that adhesion of CD34+ cells to βig-h3 is mediated by both β1- and β2- integrins. The functional relevance of the target proteins in HSC differentiation and self-renewal was studied by lentiviral mediated overexpression. We used a βig-h3-SIN-GFP vector or a control SIN-GFP vector to transduce CD34+ cells isolated from MPB or UCB and cultured them towards a megakaryocytic lineage using TPO, SCF, Flt3 and IL6. Overexpression of βig-h3 in MPB and UCB-CD34+ cells resulted in an acceleration of the megakaryopoiesis and in an increased percentage of mature megakaryocytic cells (i.e. CD41+) two weeks after transduction. In conclusion, βig-h3 is an adhesive protein for HSPCs and GMP's express significantly more βig-h3 as compared to other CD34+ subsets. Moreover, ectopic expression of βig-h3 in CD34+ cells accelerates differentiation towards megakaryocytes. These data suggest that upregulation of βig-h3 in HSCs may be a vital element driving lineage commitment of HSCs in homeostatic or regenerative conditions. Disclosures: No relevant conflicts of interest to declare.

BMI1 Collaborates with RUNX1/AML1 Mutants in the Development of Human Myelodysplastic Syndrome (MDS) / Acute Myeloid Leukemia (AML)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 275-275
Author(s):  
Hironori Harada ◽  
Ye Ding ◽  
Jun Imagawa ◽  
Takahiko Miyama ◽  
Akiro Kimura ◽  
...  
Keyword(s):  
Clinical Features ◽  
Expression Level ◽  
Refractory Anemia ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Long Term Culture ◽  
Cd34 Cells ◽  
Impressive Result ◽  
Proliferation Ability

Abstract Abstract 275 RUNX1/AML1 gene has been investigated in the pathogenesis of hematopoietic diseases and point mutations of RUNX1 have been frequently detected in patients with MDS or AML. We have found RUNX1 mutations in patients with MDS and MDS-related AML, including therapy-related cases. The mutations were distributed throughout the full length of the RUNX1 protein, and replacement of D171 amino acid in runt homology domain was the most frequent target of mutations in the RUNX1 gene. The D171N mutant showed a loss of normal RUNX1 trans-activation potential and dominant-negative trans-activation suppression, suggesting that this mutant may have some oncogenic potential in addition to the loss of function. In mouse transplantation systems D171N-transduced mice exhibited hyper-proliferative AML with multilineage dysplasia in collaboration with Evi1 overexpression. This impressive result indicated that RUNX1 mutations may be a cause of MDS with a leukemogenic potential. However in human, most of D171-mutated patients were MDS refractory anemia with excess blasts, and EVI1 overexpression was observed in a patient with MDS rapidly progressed to AML. Instead, most of patients with RUNX1 mutations displayed a high expression level of BMI1, suggesting that mouse phenotypes were not always meets clinical features of the patients with the mutations, partly because gene circumstances in mouse are different from those in human. Thus, biological analysis using human hematopoietic stem cells was considered to be necessary to clarify the molecular mechanisms of the RUNX1 mutations in the pathogenesis of MDS/AML. To examine the effect of RUNX1 mutants, we transduced the D171N mutant into human CD34+ cells from cord blood cells. In colony forming cell (CFC) assay, the number of burst forming unit-erythroid colonies was significantly decreased, while the number of granulocyte-macrophage colony-forming unit colonies was increased. Most of the D171N-transduced cells expressed myeloid lineage markers. The D171N cells showed replating capacity for 3 replatings, suggesting that they have self-renewal advantage. The presence of progenitors with long-term self-renewal capabilities was confirmed by long-term culture-initiating cell assay. The D171N cells showed a drastic increase in the number of colonies. However, long-term culture in complete cytokine liquid medium showed that the D171N cells grew a little, exhibiting lower proliferation ability than control. The percentage of CD34+ cells increased slightly, but gradually decreased with a maximum around day 35. At this point, although the percentage of CD34+/CD38+ cells did not increase in comparison to control cells, the percentage of CD34+/CD38− cells increased to 4 %. On day 35, a vast majority of the control cells terminally differentiated into mature myeloid cells and monocytes, whereas the D171N cells contained a large number of immature cells and displayed morphological abnormalities in all 3 hematopoietic lineages. Cell cycle analysis revealed that most of the D171N cells accumulated in G1 phase on day 53 when the cells stopped proliferating. These results indicated that the D171N cells had no proliferation ability although the mutant probably gives rise to the multi-lineage dysplasia of hematopoietic cells with increase in the number of blasts that is the main characteristic of MDS. Because the D171N cells showed low expression level of BMI1, we next performed stepwise transduction of BMI1 following D171N. In CFC assay, stepwise vector-transduced D171N cells seemed to no longer have colony forming ability, whereas stepwise BMI1-transduced D171N cells displayed an increase in both colony forming ability and replating capacity. Moreover, CD34+ cell population remained in the stepwise BMI1-transduced D171N cells. Furthermore, the cells showed long-term proliferation with a retained CD34+ cell fraction. Morphological findings showed myeloid cell dysplasia with increased blast-like cells. Taken together, the results of the D171N forced expression demonstrate that the mutation requires collaborating genes for proliferation. EVI1 and BMI1 may add distinct proliferating forces to the D171N cells, reflected in clinical features of MDS patients with their overexpression. Our results support the concept that RUNX1 mutations may become one of the genetic classification categories of myeloid neoplasms. Disclosures: No relevant conflicts of interest to declare.

STAT5-Induced Self-Renewal and Impaired Myelopoiesis of Human Hematopoietic Stem/Progenitor Cells Involves Downmodulation of C/EBPα.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 268-268
Author(s):  
Jan Jacob Schuringa ◽  
Bart-Jan Wierenga ◽  
Hein Schepers ◽  
Malcolm A.S. Moore ◽  
Edo Vellenga
Keyword(s):  
Progenitor Cells ◽  
Rt Pcr ◽  
Human Cord Blood ◽  
Self Renewal ◽  
Affymetrix Microarray ◽  
Hematopoietic Stem ◽  
Cycle Arrest ◽  
Cd34 Cells ◽  
Proliferative Advantage

Abstract Previously, we demonstrated that enforced activation of STAT5 in human cord blood (CB)-derived stem/progenitor cells results in enhanced long-term stem cell self-renewal and impaired myelopoiesis (J.J.Schuringa et al, J.Exp.Med. 2004;200:623). Now, C/EBPα was identified as a critical transcription factor that is downregulated by STAT5. Affymetrix microarray analysis on STAT5A(1*6)-transduced CD34+ cells identified C/EBPα as the most prominently downregulated gene (−3.3 fold), and these data were confirmed by RT-PCR and Western blotting. To determine the cell-biological relevance of these observations, a 4-OHT-inducible C/EBPα-ER protein was co-expressed with the STAT5A(1*6) mutant in CB CD34+ cells by using a retroviral approach. Re-expression of C/EBPα in STAT5A(1*6) cells resulted in a marked restoration of myelopoiesis as determined by morphological analyses, FACS analyses for myeloid markers such as CD11b, CD14 and CD15, and RT-PCR for myeloid-restricted genes such as g-csfr. While enforced activation of STAT5A resulted in accelerated erythropoiesis, this was blocked when C/EBPα was re-introduced into STAT5A(1*6) cells. Similarly, the proliferative advantage imposed on CD34+ cells by STAT5A(1*6) depended on the downmodulation of C/EBP as reintroduction of C/EBPα in these cells induced a quick cell cycle arrest and the onset of myeloid differentiation. At the stem/progenitor cell level, LTC-IC frequencies were elevated from 0.5% to 11% by STAT5A(1*6) as compared to controls, but these elevated LTC-IC frequencies were strongly reduced when C/EBPα was reintroduced in STAT5A(1*6) cells. Enumeration of progenitors in methylcellulose assays revealed similar results, the number of CFCs was reduced over 10-fold when C/EBPα was expressed in STAT5A(1*6) cells. Also, secondary CAFCs and long-term cultures could only be generated from STAT5A(1*6) expressing cells, but not from cells that co-expressed STAT5A(1*6) and C/EBPα. Taken together, these data indicate that STAT5-induced self-renewal and impaired myelopoiesis involves downmodulation of C/EBPα.

A Short Pulse of Prostaglandin E2 (PGE2) Induces Long Term Chromatin Changes in Hematopoietic Stem Cells Leading to Increased Self-Renewal and Engraftment

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 246-246
Author(s):  
Eva M Fast ◽  
Ellen M Durand ◽  
Audrey Sporrij ◽  
Leslie Ojeaburu ◽  
Rebecca Maher ◽  
...  
Keyword(s):  
Gene Expression ◽  
Open Chromatin ◽  
Advisory Committees ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Equity Ownership ◽  
Induced Genes

Abstract Hematopoietic stem cells (HSCs) offer promising treatment options for many blood diseases. We have previously identified Prostaglandin E2 (PGE2), a small molecule that increased HSC numbers in the zebrafish embryo. In an adult mammalian transplantation setting a two hour treatment significantly enhanced HSC engraftment. Currently PGE2 is being tested in a phase 2 clinical trial to improve cord blood transplants. To better understand PGE2 effect on HSCs mouse multipotent progenitors (MPP), short term (ST) HSCs, and long term (LT) HSCs were isolated via FACS and given a two hour pulse of PGE2 followed by a competitive transplantation assay. Surprisingly, PGE2 treatment mainly affected ST-HSCs by dramatically prolonging their ability to contribute to peripheral blood. The effect of the two hour treatment persisted through secondary competitive transplants in which robust peripheral blood chimerism of ST-HSCs was evident even 1.5 years after having been exposed to the drug. To elucidate underlying molecular changes gene expression right after PGE2 treatment as well as in ST-HSCs after transplantation was assessed. PGE2 target genes were divided into two categories; "transiently induced" and "permanently induced" genes. Most of the transcripts upregulated two hour after PGE2 treatment were "transiently induced" meaning that they did not continue to be differentially expressed after transplantation. In contrast, a few transcripts including chemokines such as Cxcl2, Cxcl3, members of the Fos gene family as well as Nr4a1, 2 and 3 were both upregulated right after PGE2 treatment as well as in ST-HSCs after transplantation. We classified these genes as "permanently induced". ATAC (Assay for Transposase-Accessible Chromatin)-seq analysis of the transplanted PGE2 treated cells indicated that these "permanently induced" genes maintained a distinctly open chromatin profile in both promotor and enhancer regions, whereas the "transiently induced" genes did not. Gene expression in human CD34+ cells included a signature implying CREB as the main transcription factor responsible for the acute PGE2 response. Phospho-FACS in mouse ST-HSCs and Western-blot analysis in human CD34+ cells confirmed a significant increase in CREB phosphorylation after PGE2 stimulation. Chromatin immunoprecipitation (ChIP)-seq analysis of pCREB was able to identify specific genomic regions where pCREB is recruited to after PGE2 treatment. Compared to unstimulated CD34+ cells an increased binding of pCREB could be detected in promotor regions near transcription start sites. In addition over 90% of de-novo pCREB binding occurred in intergenic and intronic regions. To determine the activation state of these putative enhancers changes in the histone mark H3K27ac and open chromatin state (via ATAC-seq) were assessed after PGE2 treatment. The data suggest that PGE2-induced pCREB binding correlates with remodeling of chromatin already after two hours of drug treatment. Furthermore chromatin sites opened by PGE2 were significantly enriched for the CREB motif both in human CD34+ cells acutely after treatment as well as in mouse ST-HSCs 1.5 years after transplant. In summary this work shows that a two hour treatment with PGE2 is sufficient to confer long-term engraftment properties to ST-HSCs. PGE triggers a chromatin remodeling event through CREB that can permanently alter epigenetic state and gene expression of ST-HSCs. Understanding the self-renewal network induced by PGE2 will not only enrich current clinical applications targeted at increasing engraftable HSC numbers but also further basic understanding of HSC self-renewal. Disclosures Zon: FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

scholarly journals Estrogens Enhance Human Hematopoietic Stem Cell Engraftment in a Xenogenic Transplantation Model

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2042-2042
Author(s):  
Sara Fañanas-Baquero ◽  
Israel Orman ◽  
Federico Becerra Aparicio ◽  
Silvia Bermudez de Miguel ◽  
Jordi Garcia Merino ◽  
...  
Keyword(s):  
Stem Cell ◽  
Research Funding ◽  
Short Term ◽  
Myeloablative Conditioning ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Hematopoietic Recovery ◽  
Cd34 Cells ◽  
Equity Ownership

Abstract Hematopoietic Stem Cells (HSCs) is a rare cell population that sits atop a hierarchy of progenitors that become progressively restricted to several or a single blood lineage. HSCs are capable of self-renewal and multipotent differentiation to all blood cell lineages. HSCs are crucial in the maintenance of lifelong production of all blood cells. HSCs are highly regulated to maintain homeostasis through a delicate balance between quiescence, self-renewal and differentiation. However, this balance is altered during the hematopoietic recovery after Hematopoietic Stem Cell Transplantation (HSCT). HSCT is routinely used to reconstitute hematopoiesis after myeloablation, being the most commonly-used cell therapy. HSCT efficacy and multilineage reconstitution can be limited by inadequate HSC number, poor homing, engraftment, or limited self-renewal. Recent evidence indicates that estrogens are involved in regulating the hematopoietic system homeostasis. Estrogens are the primary female sex hormones and are responsible for controlling many cellular processes including growth, differentiation and function of the reproductive system. However, estrogens have also been proposed to regulate HSCs. b-Estradiol (E2) was shown to promote the cell cycle of HSCs and multipotent progenitors (MPPs) and increase erythroid differentiation in females (1). On the other hand, tamoxifen reduces the number of MPPs and short-term HSCs but activates proliferation of long-term HSCs (2). The potential clinical application of estrogens in HSCT mainly derives from the possibility that these drugs may enhance the engraftment of transplanted HSCs, thus reducing side effects associated to myeloablative conditioning. Here, we show that a short-term treatment of immunodeficient mice transplanted with hCD34+ cells with estrogens such as E2 and estetrol (E4) improves human hematopoietic engraftment. Fifty-thousand cord blood CD34+ cells (CB-CD34+) were transplanted into sublethally irradiated immunodeficient NSG mice. Three days after transplantation, mice were treated for four days with daily subcutaneous doses of E2, E4 or vehicle. Human hematopoietic engraftment was evaluated in the BM of transplanted mice at four months later. E2 and E4 estrogens increased the proportion of hCD45+ cells 1.8-fold and 2.4-fold as compared to values determined in control mice, without modifying the proportion of myeloid and lymphoid lineages. Significantly, animals treated with either estrogen had significantly higher levels of human hematopoietic progenitors (hCD45+CD34+). To study the engraftment of long-term engraftment HSCs in transplanted mice, human CD45+ cells from primary recipients were sorted and transplanted in secondary NSG recipients. Three months after transplants, the proportion of human hematopoietic cells in secondary recipients was also higher when primary recipients were treated with E2 or E4 than in vehicle-treated animals. Improved engraftments associated to the administration of E2 or E4 estrogens were confirmed when very low doses of CB-CD34+ cells were transplanted (5x103 hCD34+/mouse) in recipients of either sex. Collectively, our data support a new application of estrogens to improve the hematopoietic recovery after HSCT. This application may have particular relevance to enhance the hematopoietic recovery after myeloablative conditioning and when limiting numbers of HSCs are available. Disclosures Bueren: Rocket Pharmaceuticals Inc: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Segovia:Rocket Pharmaceuticals Inc: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

scholarly journals Hepatoma-Derived Growth Factor Is a Novel Factor to Promote the Proliferation of Hematopoietic Stem Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1471-1471
Author(s):  
Munetada Haruyama ◽  
Kozo Yamaichi ◽  
Akira Niwa ◽  
Megumu K Saito ◽  
Tatsutoshi Nakahata
Keyword(s):  
Bone Marrow ◽  
Growth Factor ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Absolute Number ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
The Mean

Abstract Ex vivo expansion of hematopoietic stem cells (HSCs) is an attractive therapeutic strategy for many hematologic diseases and genetic disorders. Therefore, a variety of ex vivo expansion techniques have been developed, however these systems were not well done to get long term HSCs (LT-HSCs) which have a long term hematopoietic reconstitution ability. As the reasons, it is considered that the factors associating with the proliferation and self-renewal of LT-HSCs have not been clear yet. To obtain the factors to stimulate the proliferation and self-renewal of LT-HSCs, various conditioned media were evaluated. The supernatants of COS-1 cells transfected with cDNA cording for RelA (one of nuclear factor kappa B subunits) stimulated the proliferation of human CD34+ cells derived from umbilical cord blood (UCB) and increased the number of CFU-Mix strongest of all evaluated conditioned media. 60 liters of the supernatants of COS-1 cells transfected RelA genes were separated by column chromatography purifications. LC-MS/MS analysis of the final active fraction provided the information of hepatoma-derived growth factor (HDGF) as a growth factor. HDGF is a 24kD heparin-binding protein and has reported to stimulate the proliferation in various types of cells including fibroblasts, endothelial cells and hepatoma cells, its receptor(s) and signaling remain unclear, moreover, has no known function in hematopoiesis. The recombinant human HDGF indicated the ability to enhance the proliferation of CD34+ cells dose-dependently and increased the number of CFU-Mix in combination with cytokines compared to cytokines alone, especially HDGF showed the strongest synergy effect in a combination with TPO in all combinations of cytokines. Next, uncultured (UC) CD34+ cells, the cells of an equal initial number of CD34+ cells after the serum-free condition cultures in the presence of TPO alone (T), HDGF alone (H) and HDGF+TPO (HT) were transplanted into sublethally irradiated NOG (NOD/Shi-scid,IL-2RγKO) mice. HT increased the number of CD34+CD38- cells compared to UC, T and H. Analysis of CD34+CD38- cells in bone marrow cells of NOG mice 24 weeks after transplantation revealed that the mean of absolute number of CD34+CD38- cells in HT group showed about 4-fold, that in H group showed about 3-fold compared to that in UC group, however, that in T group were not detected.These results indicated that HT increased HSCs including short term and long term HSCs. In order to investigate whether HDGF could increase the number of LT-HSCs, serial transplantation experiment was carried out. Uncultured CD34+ cells and the CD34+ cells cultured with HT were transplanted into sublethally irradiated NOG mice. At 24 weeks after transplantation, the mean of absolute number of CD34+CD38- cells in HT group showed 6-fold compared to that in UC group, a half of total number of bone marrow cells from each mouse in both groups were transplanted into one secondary sublethally irradiated NOG mouse. Analysis of human hematopoietic cells in both group 20 weeks after transplantation revealed that multi-lineage human hematopoietic cells, such as CD3+ cells, CD19+ cells, CD33+ cells, CD235a+ cells, erythrocytes and platelets, were detected in all mice in HT group, but were not detected in all mice in UC group. The mean of absolute number of CD34+CD38- cells in bone marrow of HT group showed 30-fold compared to that of UC group. These results indicated that HDGF could increase the number of LT-HSCs. We showed here that the CD34+ cells cultured with HDGF can be transplanted to secondary hosts to give rise to long-term multilineage repopulation. Thus, HDGF is a novel factor to promote the proliferation of HSCs and plays an important role in hematopoiesis. HDGF will contribute the new HSCs expansion system development by using UCB for hematopoietic stem cell transplantation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Administration of Granulocyte Colony-Stimulating Factor Post-Transplant Impedes Engraftment of CRISPR-Cas9 Edited Long-Term Repopulating Hematopoietic Stem and Progenitor Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2936-2936
Author(s):  
Daisuke Araki ◽  
Christi Salisbury-Ruf ◽  
Waleed Hakami ◽  
Keyvan Keyvanfar ◽  
Richard H. Smith ◽  
...  
Keyword(s):  
Gene Editing ◽  
Treated Group ◽  
Untreated Group ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Cell Engraftment ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract Transplantation of genetically modified autologous hematopoietic stem and progenitor cells (HSPCs) holds a curative potential for subjects with inherited blood disorders. In recent years, transfer of a therapeutic gene to HSPCs has been successfully achieved using replication-incompetent integrating lentiviral vectors. More recently, advances have emerged to more precisely edit cellular genomes by specific correction of mutations or targeted gene addition at endogenous genomic loci. However, cellular processes triggered in HSPCs by the programmable nucleases utilized in these gene editing approaches may negatively impact their ability to reconstitute and maintain hematopoiesis long-term in recipient hosts. Granulocyte colony-stimulating factor (G-CSF) use after autologous HSPC transplantation is generally recommended to shorten the duration of severe neutropenia. However, little is known about the safety and efficacy of G-CSF use after transplantation of genetically modified autologous HSPCs. G-CSF is the principal cytokine regulating granulopoiesis, but also plays an important role in regulating hematopoietic stem cell (HSC) function (Schuttpelz, Leukemia 2014). Studies have suggested that G-CSF can exacerbate HSC damage caused by chemotherapeutic agents and irradiation by promoting differentiation at the expense of self-renewal and by inducing cellular senescence (van Os, Stem Cells 2000; Li, Cell Biosci 2015). Here, we asked whether G-CSF use after transplantation of gene edited HSPCs may negatively affect their long-term repopulating (LTR) and self-renewal capacities. To assess the effect of G-CSF use post-transplant on HSPC repopulating function after gene editing, mobilized human CD34+ cells were stimulated for 2 days, electroporated with AAVS1-specific sgRNA/Cas9 ribonucleoprotein complexes, and subsequently transplanted into NSG mice following busulfan conditioning. We subcutaneously injected G-CSF (125 mcg/kg/day) or PBS from post-transplant day 1 to 14 and compared hematopoietic reconstitution between both groups. The use of G-CSF initially increased human CD45+ cells in peripheral blood (PB) at 2 weeks post-transplant by enhancing CD13+ myeloid cell proliferation from committed progenitors (Fig. A). However, starting at 10 weeks post-transplant when hematopoiesis begins to emerge from the most primitive HSPCs, administration of G-CSF resulted in a 3 to 4-fold reduction in PB human cell engraftment compared to untreated mice (Fig. A). Similarly, G-CSF treated mice had significantly lower bone marrow (BM) and splenic engraftment at the endpoint (22 weeks) analysis, with comparable editing efficiency and lineage composition detected within human CD45+ cells (Fig. B, C). Importantly, percentages of immunophenotypic HSCs were 2-fold lower within the BM of G-CSF treated mice relative to the untreated group (Fig. D). To determine whether the negative effect of G-CSF post-transplant is specific to CRISPR-Cas9 gene editing, similar experiments were conducted using unmanipulated CD34+ cells or CD34+ cells transduced with a lentivirus vector expressing GFP. Interestingly, we found no differences in engraftment levels or immunophenotypic HSC frequencies between G-CSF treated and untreated mice. To assess the self-renewal capacity and quantify the frequency of gene edited LTR-HSCs, human CD45+ cells obtained from the BM of primary mice were serially transplanted into secondary recipient (NBSGW) mice at limiting dilution and BM engraftment was analyzed at 20 weeks post-transplant (total period of engraftment was 42 weeks). Notably, the secondary mice in the untreated group showed significantly superior human CD45+ cell engraftment compared with those in G-CSF treated group at the highest dose tested (Fig. E). The extreme limiting dilution analysis indicated that the frequency of LTR-HSCs was 5.1-fold higher (p = 0.011) in the untreated group compared with G-CSF treated group (Fig. F, G). Considering total engraftment in primary mice and the frequency of edited LTR-HSCs in secondary mice, we estimated the frequency of edited LTR-HSCs was reduced by 10-fold with G-CSF administration post-transplant. Collectively, our data suggest that G-CSF use post-transplant significantly reduces LTR and self-renewal capacities of CRISPR-Cas9 gene edited HSPCs. This understanding could have important clinical implications in HSPC gene therapy protocol. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

STAT5-induced self-renewal and impaired myelopoiesis of human hematopoietic stem/progenitor cells involves down-modulation of C/EBPα

Blood ◽  
2006 ◽  
Vol 107 (11) ◽  
pp. 4326-4333 ◽  
Author(s):  
Albertus T. J. Wierenga ◽  
Hein Schepers ◽  
Malcolm A. S. Moore ◽  
Edo Vellenga ◽  
Jan Jacob Schuringa
Keyword(s):  
Progenitor Cells ◽  
Pcr Analysis ◽  
Human Cord Blood ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cycle Arrest ◽  
Cd34 Cells ◽  
Polymerase Chain ◽  
Proliferative Advantage

AbstractPreviously, we demonstrated that enforced activation of signal transducer and activator of transcription 5 (STAT5A) in human cord blood (CB)–derived stem/progenitor cells results in enhanced self-renewal and impaired myelopoiesis. The present study identifies C/EBPα as a critical component that is down-regulated by STAT5. Microarray and reverse transcriptase–polymerase chain reaction (RT-PCR) analysis on STAT5A1*6-transduced CD34+ cells identified C/EBPα as the most prominently down-regulated gene. To determine the cell-biological relevance of these observations, a 4-OHT-inducible C/EBPα-ER protein was co-expressed with the STAT5A1*6 mutant in CB CD34+ cells using a retroviral approach. Re-expression of C/EBPα in STAT5A1*6 cells resulted in a marked restoration of myelopoiesis. The proliferative advantage imposed on CD34+ cells by STAT5A1*6 depended on the down-modulation of C/EBPα, as reintroduction of C/EBPα induced a quick cell-cycle arrest and the onset of myeloid differentiation. Long-term culture–initiating cell (LTC-IC) frequencies were elevated from 0.8% ± 0.6% to 7.8% ± 1.9% by STAT5A1*6 as compared with controls, but these elevated LTC-IC frequencies were strongly reduced upon re-introduction of C/EBPα in STAT5A1*6 cells, and no second cobble-stone area–forming cells (CAFCs) could be generated from double-transduced cells. Enumeration of progenitors revealed that the number of colony-forming cells (CFCs) was reduced more than 20-fold when C/EBPα was co-expressed in STAT5A1*6 cells. Our data indicate that down-modulation of C/EBPα is a prerequisite for STAT5-induced effects on self-renewal and myelopoiesis.

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