Analysis of Mesenchymal Stromal Cells (MSC) and Their Interactions with CD34 Stem and Progenitor Cells in Patients with Myelodysplastic Syndromes (MDS)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2393-2393
Author(s):  
Stefanie Geyh ◽  
Ron Patrick Cadeddu ◽  
Julia Fröbel ◽  
Ingmar Bruns ◽  
Fabian Zohren ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Myelodysplastic Syndromes ◽  
Stromal Cells ◽  
Median Number ◽  
Lower Number ◽  
Differentiation Potential ◽  
Receptor Ligand ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract Abstract 2393 Background: Myelodysplastic syndromes (MDS) represent a heterogeneous group of hematopoietic stem cell disorders and research in this field has mainly focused on hematopoietic stem and progenitor cells (HSPC). Still, recent data from mouse models indicate that the bone marrow (BM) microenvironment might be involved in the pathogenesis MDS (Raaijmakers et al., 2010). The role of mesenchymal stromal cells (MSC) in particular as a key component of the BM microenvironment remains elusive in human MDS and data so far are controversial. Design/Methods: We therefore investigated MSC and immunomagnetically enriched CD34+ HSPC from BM of 42 untreated patients (pts) with MDS (12 RCMD, 12 RAEB, 12 sAML, 3 del5q, 1 CMML-1, 1 MDS hypocellular, 1 MDS unclassifiable according to WHO) and age-matched healthy controls (HC, n=13). MSC were examined with regard to growth kinetics, morphology and differential potential after isolation and expansion according standard procedures in line with the international consensus criteria (Dominici et al., 2006). Furthermore corresponding receptor-ligand pairs on MSC and CD34+ cells (Kitlg/c-kit; CXCL12/CXCR4; Jagged1/Notch1; Angpt1-1/Tie-2; ICAM1/LFA-1) were investigated by RT-PCR. Results: In MDS, the colony forming activity (CFU-F) of MSC was significantly reduced in comparison to HC (median number of colonies per 1×107MNC in MDS: 8, range 2–74 vs. HC: 175, 10–646, p=0.003) and this was also true when looking at the different subtypes (RCMD median: 16, p=0.04; RAEB median: 8, p=0.31; sAML median: 26, p=0.02). According to this, MSC from pts with RCMD and del5q could only be maintained in culture for a lower number of passages (median number of passages: MDS 3 passages, range 1–15; HD 14 passages, range 8–15, p=0.01), had a lower number of cumulative population doublings (CPD) and needed a longer timer to reach equivalent CPD (MDS median: 18,16 CPD, HD median: 33,68 CPD, p=0,0059). All types of MDS-MSC showed an abnormal morphology, while an impaired osteogenic differentiation potential was exclusively observed in pts with RCMD. These findings of an altered morphology together with a diminished growth and differentiation potential prompted us to test, whether the interaction between MSC and CD34+ HSPC in BM of pts with MDS was also disturbed. On the MDS-MSC, we found a significant lower expression of Angpt1 in pts with RAEB (3.5-fold, p=0.01) and del5q (4.9-fold, p=0.009) compared to HD. The expression of CXCL12 (2.5-fold, p=0.057) and jagged1 was reduced in trend in MSC from pts with MDS, while no differences were observed with regard to the expression of kitlg and ICAM1. When looking on CD34+ cells, we found a significantly reduced expression of CXCR4 (RCMD 2.5-fold, p=0.02; RAEB 2.46-fold, p=0.02), notch1 (RCMD 6-fold, p=0.04) and Tie-2 (RAEB 5.91-fold, p=0.02) in pts with MDS, while LFA-1 was overexpressed in pts with RAEB (2.6-fold, p=0.036). Conclusion: Taken together, our data indicate that MSC from pts with MDS are structurally altered and that the crosstalk between CD34+ HSPC and MSC in the BM microenvironment of pts with MDS might be deregulated as a result of an abnormal expression of relevant receptor-ligand pairs. Ongoing research is required to corroborate these findings and to definitely address their functional relevance for the pathogenesis of MDS. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Aging- and Senescence-associated Changes of Mesenchymal Stromal Cells in Myelodysplastic Syndromes

2018 ◽  
Vol 27 (5) ◽  
pp. 754-764 ◽  
Author(s):  
Domenico Mattiucci ◽  
Giulia Maurizi ◽  
Pietro Leoni ◽  
Antonella Poloni
Keyword(s):  
Progenitor Cells ◽  
Mesenchymal Stromal Cells ◽  
Myelodysplastic Syndromes ◽  
Stromal Cells ◽  
Replicative Senescence ◽  
Special Focus ◽  
Proliferative Potential ◽  
Stromal Compartment ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Hematopoietic stem and progenitor cells reside within the bone marrow (BM) microenvironment. By a well-balanced interplay between self-renewal and differentiation, they ensure a lifelong supply of mature blood cells. Physiologically, multiple different cell types contribute to the regulation of stem and progenitor cells in the BM microenvironment by cell-extrinsic and cell-intrinsic mechanisms. During the last decades, mesenchymal stromal cells (MSCs) have been identified as one of the main cellular components of the BM microenvironment holding an indispensable role for normal hematopoiesis. During aging, MSCs diminish their functional and regenerative capacities and in some cases encounter replicative senescence, promoting inflammation and cancer progression. It is now evident that alterations in specific stromal cells that comprise the BM microenvironment can contribute to hematologic malignancies, and there is growing interest regarding the contribution of MSCs to the pathogenesis of myelodysplastic syndromes (MDSs), a clonal hematological disorder, occurring mostly in the elderly, characterized by ineffective hematopoiesis and increased tendency to acute myeloid leukemia evolution. The pathogenesis of MDS has been associated with specific genetic and epigenetic events occurring both in hematopoietic stem cells (HSCs) and in the whole BM microenvironment with an aberrant cross talk between hematopoietic elements and stromal compartment. This review highlights the role of MSCs in MDS showing functional and molecular alterations such as altered cell-cycle regulation with impaired proliferative potential, dysregulated cytokine secretion, and an abnormal gene expression profile. Here, the current knowledge of impaired functional properties of both aged MSCs and MSCs in MDS have been described with a special focus on inflammation and senescence induced changes in the BM microenvironment. Furthermore, a better understanding of aberrant BM microenvironment could improve future potential therapies.

scholarly journals Roles for integrin very late activation antigen-4 in stroma-dependent erythropoiesis

Blood ◽  
1994 ◽  
Vol 83 (10) ◽  
pp. 2844-2850 ◽  
Author(s):  
N Yanai ◽  
C Sekine ◽  
H Yagita ◽  
M Obinata
Keyword(s):  
Progenitor Cells ◽  
Adhesion Molecules ◽  
Stromal Cells ◽  
Ligand Molecule ◽  
Erythroid Cells ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Erythroid Progenitor Cells ◽  
Stem And Progenitor Cells ◽  
Activation Antigen

Abstract Adhesion molecules are required for development of hematopoietic stem and progenitor cells in the respective hematopoietic microenvironments. We previously showed that development of the erythroid progenitor cells is dependent on their direct adhesion to the stroma cells established from the erythropoietic organs. In this stroma-dependent erythropoiesis, we examined the role of adhesion molecules in erythropoiesis by blocking antibodies. The development of the erythroid cells on stroma cells was inhibited by anti-very late activation antigen-4 (VLA-4 integrin) antibody, but not by anti-VLA-5 antibody, although the erythroid cells express both VLA-4 and VLA-5. Whereas high levels of expression of vascular cell adhesion molecule-1 (VCAM-1) and fibronectin, ligands for VLA-4, were detected in the stroma cells, the adhesion and development of the erythroid progenitor cells were partly inhibited by the blocking antibody against VCAM-1. VLA-5 and fibronectin could mediate adhesion of the erythroid progenitor cells to the stromal cells, but the adhesion itself may not be sufficient for the stroma-supported erythropoiesis. The stromal cells may support erythroid development by the adhesion through a new ligand molecule(s) for VLA-4 in addition to VCAM-1, and such collaborative interaction may provide adequate signaling for the erythroid progenitor cells in the erythropoietic microenvironment.

scholarly journals Directed Differentiation of Mobilized Hematopoietic Stem and Progenitor Cells into Functional NK Cells with Enhanced Antitumor Activity

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 811
Author(s):  
Pranav Oberoi ◽  
Kathrina Kamenjarin ◽  
Jose Francisco Villena Ossa ◽  
Barbara Uherek ◽  
Halvard Bönig ◽  
...  
Keyword(s):  
Nk Cells ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Cell Expansion ◽  
Nk Cell ◽  
Ex Vivo ◽  
Feeder Cells ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Obtaining sufficient numbers of functional natural killer (NK) cells is crucial for the success of NK-cell-based adoptive immunotherapies. While expansion from peripheral blood (PB) is the current method of choice, ex vivo generation of NK cells from hematopoietic stem and progenitor cells (HSCs) may constitute an attractive alternative. Thereby, HSCs mobilized into peripheral blood (PB-CD34+) represent a valuable starting material, but the rather poor and donor-dependent differentiation of isolated PB-CD34+ cells into NK cells observed in earlier studies still represents a major hurdle. Here, we report a refined approach based on ex vivo culture of PB-CD34+ cells with optimized cytokine cocktails that reliably generates functionally mature NK cells, as assessed by analyzing NK-cell-associated surface markers and cytotoxicity. To further enhance NK cell expansion, we generated K562 feeder cells co-expressing 4-1BB ligand and membrane-anchored IL-15 and IL-21. Co-culture of PB-derived NK cells and NK cells that were ex-vivo-differentiated from HSCs with these feeder cells dramatically improved NK cell expansion, and fully compensated for donor-to-donor variability observed during only cytokine-based propagation. Our findings suggest mobilized PB-CD34+ cells expanded and differentiated according to this two-step protocol as a promising source for the generation of allogeneic NK cells for adoptive cancer immunotherapy.

Enforced Expression of GATA-2 Confers Quiescence on Human Hematopoietic Stem and Progenitor Cells In Vitro and In Vivo.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 83-83
Author(s):  
Alex J. Tipping ◽  
Cristina Pina ◽  
Anders Castor ◽  
Ann Atzberger ◽  
Dengli Hong ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Progenitor Cells ◽  
Zinc Finger ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells ◽  
Colony Number

Abstract Hematopoietic stem cells (HSCs) in adults are largely quiescent, periodically entering and exiting cell cycle to replenish the progenitor pool or to self-renew, without exhausting their number. Expression profiling of quiescent HSCs in our and other laboratories suggests that high expression of the zinc finger transcription factor GATA-2 correlates with quiescence. We show here that TGFβ1-induced quiescence of wild-type human cord blood CD34+ cells in vitro correlated with induction of endogenous GATA-2 expression. To directly test if GATA-2 has a causative role in HSC quiescence we constitutively expressed GATA-2 in human cord blood stem and progenitor cells using lentiviral vectors, and assessed the functional output from these cells. In both CD34+ and CD34+ CD38− populations, enforced GATA-2 expression conferred increased quiescence as assessed by Hoechst/Pyronin Y staining. CD34+ cells with enforced GATA-2 expression showed reductions in both colony number and size when assessed in multipotential CFC assays. In CFC assays conducted with more primitive CD34+ CD38− cells, colony number and size were also reduced, with myeloid and mixed colony number more reduced than erythroid colonies. Reduced CFC activity was not due to increased apoptosis, as judged by Annexin V staining of GATA-2-transduced CD34+ or CD34+ CD38− cells. To the contrary, in vitro cultures from GATA-2-transduced CD34+ CD38− cells showed increased protection from apoptosis. In vitro, proliferation of CD34+ CD38− cells was severely impaired by constitutive expression of GATA-2. Real-time PCR analysis showed no upregulation of classic cell cycle inhibitors such as p21, p57 or p16INK4A. However GATA-2 expression did cause repression of cyclin D3, EGR2, E2F4, ANGPT1 and C/EBPα. In stem cell assays, CD34+ CD38− cells constitutively expressing GATA-2 showed little or no LTC-IC activity. In xenografted NOD/SCID mice, transduced CD34+ CD38−cells expressing high levels of GATA-2 did not contribute to hematopoiesis, although cells expressing lower levels of GATA-2 did. This threshold effect is presumably due to DNA binding by GATA-2, as a zinc-finger deletion variant of GATA-2 shows contribution to hematopoiesis from cells irrespective of expression level. These NOD/SCID data suggest that levels of GATA-2 may play a part in the in vivo control of stem and progenitor cell proliferation. Taken together, our data demonstrate that GATA-2 enforces a transcriptional program on stem and progenitor cells which suppresses their responses to proliferative stimuli with the result that they remain quiescent in vitro and in vivo.

Muc1 Expression Identifies Human Self-Renewing Stem/Progenitor Populations and Is Elevated in AML CD34+ Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4040-4040
Author(s):  
Szabolcs Fatrai ◽  
Simon M.G.J. Daenen ◽  
Edo Vellenga ◽  
Jan J. Schuringa
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Fold Increase ◽  
Marrow Stromal Cells ◽  
Muc1 Expression ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Mucin1 (Muc1) is a membrane glycoprotein which is expressed on most of the normal secretory epithelial cells as well as on hematopoietic cells. It is involved in migration, adhesion and intracellular signalling. Muc1 can be cleaved close to the membrane-proximal region, resulting in an intracellular Muc1 that can associate with or activate various signalling pathway components such as b-catenin, p53 and HIF1a. Based on these properties, Muc1 expression was analysed in human hematopoietic stem/progenitor cells. Muc1 mRNA expression was highest in the immature CD34+/CD38− cells and was reduced upon maturation towards the progenitor stage. Cord blood (CB) CD34+ cells were sorted into Muc1+ and Muc1− populations followed by CFC and LTC-IC assays and these experiments revealed that the stem and progenitor cells reside predominantly in the CD34+/Muc1+ fraction. Importantly, we observed strongly increased Muc1 expression in the CD34+ subfraction of AML mononuclear cells. These results tempted us to further study the role of Muc1 overexpression in human CD34+ stem/progenitor cells. Full-length Muc1 (Muc1F) and a Muc1 isoform with a deleted extracellular domain (DTR) were stably expressed in CB CD34+ cells using a retroviral approach. Upon coculture with MS5 bone marrow stromal cells, a two-fold increase in expansion of suspension cells was observed in both Muc1F and DTR cultures. In line with these results, we observed an increase in progenitor counts in the Muc1F and DTR group as determined by CFC assays in methylcellulose. Upon replating of CFC cultures, Muc1F and DTR were giving rise to secondary colonies in contrast to empty vector control groups, indicating that self-renewal was imposed on progenitors by expression of Muc1. A 3-fold and 2-fold increase in stem cell frequencies was observed in the DTR and Muc1F groups, respectively, as determined by LTC-IC assays. To determine whether the above mentioned phenotypes in MS5 co-cultures were stroma-dependent, we expanded Muc1F and DTR-transduced cells in cytokine-driven liquid cultures. However, no proliferative advantage or increase in CFC frequencies was observed suggesting that Muc1 requires bone marrow stromal cells. In conclusion, our data indicate that HSCs as well as AML cells are enriched for Muc1 expression, and that overexpression of Muc1 in CB cells is sufficient to increase both progenitor and stem cell frequencies.

Mesenchymal Stem and Progenitor Cells In the Mouse Bone Marrow Lack Expression of CD44.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2581-2581
Author(s):  
Hong Qian ◽  
Mikael Sigvardsson
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Colony Forming Unit ◽  
Stem And Progenitor Cells ◽  
Cell Fractions

Abstract Abstract 2581 The bone marrow (BM) microenvironment consists of a heterogeneous population including mesenchymal stem cells and as well as more differentiated cells like osteoblast and adipocytes. These cells are believed to be crucial regulators of hematopoetic cell development, however, so far, their identity and specific functions has not been well defined. We have by using Ebf2 reporter transgenic Tg(Ebf2-Gfp) mice found that CD45−TER119−EBF2+ cells are selectively expressed in non-hematopoietic cells in mouse BM and highly enriched with MSCs whereas the EBF2− stromal cells are very heterogenous (Qian, et al., manuscript, 2010). In the present study, we have subfractionated the EBF2− stromal cells by fluorescent activated cell sorter (FACS) using CD44. On contrary to previous findings on cultured MSCs, we found that the freshly isolated CD45−TER119−EBF2+ MSCs were absent for CD44 whereas around 40% of the CD45−TER119−EBF2− cells express CD44. Colony forming unit-fibroblast (CFU-F) assay revealed that among the CD45−LIN−EBF2− cells, CD44− cells contained generated 20-fold more CFU-Fs (1/140) than the CD44+ cells. The EBF2−CD44− cells could be grown sustainably in vitro while the CD44+ cells could not, suggesting that Cd44− cells represents a more primitive cell population. In agreement with this, global gene expression analysis revealed that the CD44− cells, but not in the CD44+ cells expressed a set of genes including connective tissue growth factor (Ctgf), collagen type I (Col1a1), NOV and Runx2 and Necdin(Ndn) known to mark MSCs (Djouad et al., 2007) (Tanabe et al., 2008). Furthermore, microarray data and Q-PCR analysis from two independent experiments revealed a dramatic downregulation of cell cycle genes including Cdc6, Cdca7,-8 and Ki67, Cdk4-6) and up-regulation of Cdkis such as p57 and p21 in the EBF2−CD44− cells, compared to the CD44+ cells indicating a relatively quiescent state of the CD44− cells ex vivo. This was confirmed by FACS analysis of KI67 staining. Furthermore, our microarray analysis suggested high expression of a set of hematopoietic growth factors and cytokines genes including Angiopoietin like 1, Kit ligand, Cxcl12 and Jag-1 in the EBF2−CD44− stromal cells in comparison with that in the EBF2+ or EBF2−CD44+ cell fractions, indicating a potential role of the EBF2− cells in hematopoiesis. The hematopoiesis supporting activity of the different stromal cell fractions were tested by in vitro hematopoietic stem and progenitor assays- cobblestone area forming cells (CAFC) and colony forming unit in culture (CFU-C). We found an increased numbers of CAFCs and CFU-Cs from hematopoietic stem and progenitor cells (Lineage−SCA1+KIT+) in culture with feeder layer of the EBF2−CD44− cells, compared to that in culture with previously defined EBF2+ MSCs (Qian, et al., manuscript, 2010), confirming a high capacity of the EBF2−CD44− cells to support hematopoietic stem and progenitor cell activities. Since the EBF2+ cells display a much higher CFU-F cloning frequency (1/6) than the CD44−EBF2− cells, this would also indicate that MSCs might not be the most critical regulators of HSC activity. Taken together, we have identified three functionally and molecularly distinct cell populations by using CD44 and transgenic EBF2 expression and provided clear evidence of that primary mesenchymal stem and progenitor cells reside in the CD44− cell fraction in mouse BM. The findings provide new evidence for biological and molecular features of primary stromal cell subsets and important basis for future identification of stage-specific cellular and molecular interaction pathways between hematopoietic cells and their cellular niche components. Disclosures: No relevant conflicts of interest to declare.

Long-Term Production System of Red Blood Cells Using Human Cord Blood and Stromal Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3211-3211
Author(s):  
Masayoshi Kobune ◽  
Shohei Kikuchi ◽  
Kazuyuki Murase ◽  
Satoshi Iyama ◽  
Tsutomu Sato ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Production System ◽  
Ex Vivo ◽  
Erythroid Progenitor ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Human Stromal Cells

Abstract Abstract 3211 We have previously shown that primary human stromal cells and hTERT-transduced human stromal cells (hTERT-stromal cells) support cord blood (CB) hematopoietic stem/progenitor cells. However, it is unclear whether human stromal cells maintain the expansion of erythroid progenitor cells without losing erythroid differentiation potential for a long-term ex vivo culture. In an attempt to evaluate the efficacy of human stromal cells, erythroid induction was conducted by SCF, EPO and IGF-1, 2-week after expansion of CB CD34+ cells with or without human stromal cells. The maturation of erythroid cells were evaluated by morphological findings, transferrin receptor (TfR)/glycophorin A (GPA) expression and hemoglobin (Hb) synthesis (MCH, pg/cells). The number of BFU-E upon 2-week coculture with the hTERT-stromal cells was significantly higher than those without hTERT-stromal cells (BFU-E, 639±102 vs. 4078±1935, the initial cell number of BFU-E was 513±10). Hb concentration of erythroblasts that had been derived from coculture with stromal cells, was significantly higher than that derived from stroma-free condition 14 days after erythroid induction (MCH, 0.78±0.11 vs. 2.62±0.12; p<0.05). Moreover, cobblestone area (CA)-forming cells existed beneath stromal layer weekly produced the large number of BFU-E from 4th week to at least 8th week (the total number of BFU-E, 57246±1288)(Figure A). Notably, these BFU-Es derived from CA could simultaneously differentiate into orthophilic erythroblasts with nearly normal Hb synthesis (MHC, 24.5±6.4 pg/cell)(Figure B) and GPA expression. Furthermore, most of these erythroblasts derived from CA underwent enucleation spontaneously after further 7 days culture. Thus, using hTERT-stromal cells, the long-term ex vivo erythroid production could be attained from CB cells. These findings contribute to constructing long-term of ex vivo erythroid production system using human stromal cells. Disclosures: No relevant conflicts of interest to declare.

Loss of ASXL1 Triggers an Apoptotic Response in Human Hematopoietic Stem and Progenitor Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4107-4107
Author(s):  
Susan Hilgendorf ◽  
Hendrik Folkerts ◽  
Jan Jacob Schuringa ◽  
Edo Vellenga
Keyword(s):  
Progenitor Cells ◽  
Colony Formation ◽  
Erythroid Differentiation ◽  
Lentiviral Vectors ◽  
Apoptotic Response ◽  
Double Positive ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract In recent clinical studies, it has been shown that ASXL1 is frequently mutated in myelodysplastic syndrome (MDS), in particular in high-risk MDS patients who have a significant chance to progress to acute myeloid leukemia (AML). The majority of ASXL1 mutations leads to truncation of the protein and thereby to loss of its chromatin interacting and modifying domain, possibly facilitating malignant transformation. However, the functions of ASXL1 in human hematopoietic stem and progenitor cells are not well understood. In this study, we addressed whether manipulation of ASXL1-expression in the hematopoietic system in vitro mimics the changes observed in MDS-patients. We downregulated ASXL1 in CD34+ cord blood (CB) cells using lentiviral vectors containing several independent shRNAs and obtained a 40-50% reduction of ASXL1 expression. Colony Forming Cell (CFC) assays revealed that erythroid colony formation was significantly impaired (p<0.01) and, to some extent, granulocytic and macrophage colony formation as well (p<0.09, p<0.05 respectively). In myeloid suspension culture assays, we observed a modest reduction in expansion (two-fold at week 1) upon ASXL1 knockdown under myeloid conditions. In erythroid conditions, shASXL1 CB CD34+ cells showed a strong four-fold growth disadvantage, with a more than two-fold delay in erythroid differentiation. The reduced expansion was partly due to a significant increase in apoptosis (5.9% in controls vs. 14.0% shASXL1, p<0.02). The increase in cell death was restricted to differentiating cells, defined as CD71 bright- and CD71/GPA-double positive. In addition, we tested whether HSCs were affected by ASXL1 loss. Long-term culture-initiating cell (LTC-IC) assays revealed a two-fold decrease in stem cell frequency. To test dependency of shASXL1 CB 34+ cells on the microenvironment, transduced cells were cultured on MS5 bone marrow stromal cells with or without additional cytokines. shASXL1 CB CD34+ cells cultured on MS5 showed a modest two-fold reduction in cell growth at week 4. In the presence of EPO and SCF, we detected a growth disadvantage (three-fold at week 2) and a delay in erythroid differentiation, similar to what was observed in liquid culture. ASXL1 has been proposed to be an epigenetic modifier by recruiting/stabilizing the polycomb repressive complex 2 (PRC2). Active PRC2 can lead to trimethylation of H3K27 and silencing of certain loci. It has been proposed that perturbed ASXL1 activity may disturb PRC2 function, leading to reduced H3K27me3 and increased gene expression. Using an erythroid leukemic cell line, we downregulated ASXL1 and as a positive control EZH2, one of the core subunits of PRC2. We then performed ChIP and did PCR for several loci. Upon knockdown of ASXL1, we did not observe changes in H3K27me3 on any of he investigated loci. However, upon knockdown of EZH2 we observed more than 50% loss of the H3k27m3 mark for many of the loci. This implies that our observed phenotypes may not be conveyed via the PRC2 complex but maybe via an alternative pathway. Preliminary data revealed an increase in H2AK119ub, suggesting that the BAP1-ASXL1 complex may be involved. In patients, mutations in ASXL1 are frequently accompanied by a mutation of TP53. Possibly, this additional mutation is necessary to allow ASXL1-mutant induced transformation thereby bypassing the apoptotic response. Therefore, we modeled simultaneous loss of ASXL1 and TP53 using shRNA lentiviral vectors. Our data showed that while in primary CFC cultures shASXL1/shTP53 did not give rise to more colonies, an increase in colony-forming activity was observed upon replating of the cells. Furthermore, shASXL1/shTP53 transduced cells grown in erythroid liquid conditions revealed a decrease in apoptosis compared to the ASXL1 single mutation and an outgrowth of these double positive cells. Nevertheless, no transformation occurred in vitro. We therefore injected shASXL/TP53 transduced CB CD34+ in a humanized scaffold model in mice to determine whether transformation can occur in vivo. In conclusion, our data indicate that mutations in ASXL1 trigger an apoptotic response in CB CD34+ cells with a delay in differentiation, which leads to reduced stem and progenitor output in vitro without affecting H3K27me3. Disclosures No relevant conflicts of interest to declare.

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