Different Histone Deacetylase Inhibitors Affect Distinct Cellular Targets within the Hierarchy of Hematopoietic Stem / Progenitor Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1721-1721
Author(s):  
Hiroto Araki ◽  
Ronald Hoffman ◽  
Nadim Mahmud
Keyword(s):  
Progenitor Cells ◽  
Histone Deacetylase ◽  
Hdac Inhibitor ◽  
Histone Deacetylase Inhibitors ◽  
Trichostatin A ◽  
Scid Mice ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Recently several laboratories have examined the in vitro effects of chromatin modifying agents on hematopoiesis. We have previously reported that the sequential addition of a hypomethylating agent, 5-aza-2′-deoxyctidine (5azaD) and a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA) to cultures of human cord blood (CB) CD34+ cells containing SCF, thrombopoietin and FLT-3 ligand (cytokines) resulted in a 10-fold expansion of SCID repopulating cells (SRC) (Araki et al. Blood2004: 104:881a). Recently others have shown that another HDAC inhibitor, valproic acid (VA) resulted in an expansion of CB CD34+ cells and murine hematopoietic stem / progenitor cells (HSPC) (DeFelice et al. Cancer Res.2005:65:1505, Beg et al. Cancer Res.2005:65:2537). In our current studies we have compared the efficacy of VA, TSA or 5azaD as single agents or in combination to promote the expansion of CB HSPC in vitro. The frequency and fold expansion of colony forming cells (CFC), cobblestone area-forming cells (CAFC) as well as SRC generated from CB CD34+ cells after 9 days of culture were examined. The addition of cytokines alone result in a 1.5-fold expansion of CD34+CD90+ cells. By contrast the addition of cytokines with VA led to a 65-fold expansion of the numbers of CD34+CD90+ cells as compared to a 1.3-fold, 5.6-fold, 4.2-fold or 12.5-fold expansion of CD34+CD90+ cells in cultures receiving cytokines with 5azaD, TSA or 5azaD/VA or 5azaD/TSA respectively. In vitro biological assays (CFC, CAFC) were performed to determine the correlation between CD34+CD90+ cell expansion and function. Cultures receiving cytokines alone or cytokines with VA had the greatest degree of expansion of CFC (14.4 and 18.6-fold respectively). By contrast cultures receiving cytokines alone contained only 70% of the numbers of CAFC as did the primary CB CD34+ cells. Cultures receiving VA or 5azaD/TSA had the greatest degree of expansion of CAFC numbers (9.6-fold and 11.5-fold respectively). The marrow repopulating potential of these various expanded cell populations were then assayed by transplanting them into NOD/SCID mice. CD34+ cells from cultures receiving cytokines alone or cytokines with 5azaD/VA were devoid of human hematopoietic cell chimerism. By contrast, all NOD/SCID mice receiving grafts from cultures treated with cytokines with 5azaD/TSA had evidence of human multilineage hematopoietic engraftment (7.5% ± 3.7%). Cells from cultures treated with cytokines with VA are capable of engraftment in 2 out of 6 mice with a barely detectable level of human cell chimerism (0.11%, 0.14%). We then assessed using western blot analysis whether the chromatin modifying agents might alter HSC function by upregulating HOXB4 protein levels. HOXB4 protein was detectable in cells cultured in the presence of cytokines with VA, cytokines with 5azaD/VA, cytokines with 5azaD/TSA but only cells treated with cytokines with 5azaD/TSA contained readily assayable SRC. These studies suggest that treatment with different chromatin modifying agents are capable of altering the differentiation program of distinct populations of HSPC. Some treatments (VA, 5azaD/VA) primarily affect CFC and CAFC but not SRC. While 5azaD/TSA targets CAFC and SRC but not CFC. In addition, although HOXB4 may participate in HSC self-renewal, additional genes are likely altered following 5azaD/TSA treatment which are required for the maintenance of SRC potential.

Short Pulse of a Small Peptide Agonist of Stromal Cell-Derived Factor - 1 (SDF-1) Enhances the Engraftment of Expanded Human Cord Blood Hematopoietic Progenitor Cells in NOD/SCID Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 404-404 ◽  
Author(s):  
Karen Kwai Har Li ◽  
Carmen K.Y. Chuen ◽  
Shuk Man Lee ◽  
Donald Wong ◽  
Ahmed Merzouk ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Short Pulse ◽  
Scid Mice ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Peptide Agonist

Abstract SDF-1 is the ligand to the chemokine receptor CXCR-4. A small synthetic peptide agonist of SDF-1 (CTCE-0214) has been shown to expand human cord blood hematopoietic stem and progenitor cells. In this study, we investigated whether a brief exposure of expanded cord blood hematopoietic cells to CTCE-0214 can improve engraftment of the cells into NOD/SCID mice. Published in vivo studies demonstrated that the administration of CTCE-0214 to transplanted NOD/SCID mice mobilized human colony forming cells (CFC) and enhanced human thrombopoiesis (Exp Hematol 32, 300, 2004). Our earlier study showed that CTCE-0214 added to single factors of thrombopoietin (TPO), stem cell factor (SCF), or Flt-3 ligand (F3L) synergistically increased the survival of enriched cord blood CD34+ cells (Blood 102, 960a, 2003). In this study, we further investigated the effects of CTCE-0214 on the ex vivo expansion of CD34+ cells to multi-lineage progenitors and the homing and engraftment capacity of expanded human progenitor cells after a short in vitro exposure to the peptide prior to infusion into NOD/SCID mice. Enriched CD34+ cells (MACS) derived from cord blood were cultured for 8 days in serum-free medium QBSF-60 containing TPO (50 ng/ml), SCF (50 ng/nl) and F3L (80 ng/ml) (TSF), with or without CTCE-0214 (0.01 ng/ml) (TSF+CTCE-0214) added at day 4. Progenitor cells expanded for 8 days in the absence of CTCE-0214 were pulsed with the peptide (100 ng/ml) for 4 hours (TSFpCTCE-0214). Results are summarized in Table. CTCE-0214 significantly (N=30, p≤0.05, paired t-test) increased the fold expansion of total nucleated cells (TNC), CD34+ cells, CD34+CD38- cells, CFU-GM, CFU-E, and CFU-MK (total CFC). Expanded progenitor cells (with and without CTCE-0214) were then infused into irradiated NOD/SCID mice. After 6 weeks, enhanced engraftments of human CD45+ cells (p≤0.05, N=21) were demonstrated in the bone marrow (BM) of mice that received cells cultured in TSF+CTCE-0214. Interestingly, a short pulse of cells expanded in TSF to CTCE-0214 for 4 hours also significantly increased the NOD/SCID engraftment (N=18), although no major changes to the in vitro read-out parameters were observed. The mechanism could be associated with the increased homing capacity of progenitor cells after pulsing with CTCE-0214. In conclusion, our results showed that CTCE-0214 enhances the proliferation of early progenitor cells in culture and exposure to the peptide can enhance the engraftment potential of expanded cells in NOD/SCID mice. The SDF-1 peptide agonist could be developed for application to hematopoietic stem cell transplantation and ex vivo expansion. NOD/SCID Engraftment of Expanded Cord Blood Stem Cells TSF TSF+CTCE-0214 TSFpCTCE-0214 *Fold expansion (mean±SE); **% human CD45+ cells in BM of mice TNC* 84.6±10.4 123.5±15.3 88.5±11.2 CD34+* 8.5±1.3 14.1±2.1 9.6±1.6 CD34+CD38−* 24.6±4.8 48.7±8.6 27.5±5.3 Total CFC* 46.9±6.5 87.9±10.7 50.6±6.4 NOD/SCID** 2.8±0.9 6.7±2.5 8.3±4.0

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.

Gene Transfer to Repopulating Human CD34+ Cells Using Amphotropic, GALV or RD114 Pseudotyped HIV-1 Based Vectors from Stable Producer Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2106-2106
Author(s):  
Johan Richter ◽  
Maria Johansson ◽  
Thomas Relander ◽  
Karin Olsson ◽  
Yasuhiro Ikeda ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Progenitor Cells ◽  
Scid Mice ◽  
Endogenous Virus ◽  
Virus Envelope ◽  
Human Cord Blood ◽  
Packaging System ◽  
Cd34 Cells ◽  
Hiv 1

Abstract A novel, stable human immunodeficiency virus type 1 (HIV-1) vector packaging system, STAR, was tested for its ability to transduce human cord blood CD34+ progenitor cells assayed both in vitro and after transplantation to NOD/SCID mice. Vectors pseudotyped with three different gammaretrovirus envelopes were used; the amphotropic MLV envelope (MLV-A), a modified gibbon ape leukemia virus envelope (GALV+) and a modified feline endogenous virus RD114 envelope (RDpro). Titration of vectors harvested from the stable packaging cells gave the following results: MLV-A: 1–3x106 inf.u/ml, GALV+: 1x106 inf.u/ml and RDpro: 3–10x106 inf.u/ml CB CD34+ cells were transduced on Retronectin® preloaded with vector either in fresh medium (PL alone) or in vector containing medium (PL+VCM). Gene transfer to freshly thawed CD34+ in the absence of cytokines was very low. Addition of cytokines (CK; SCF, TPO and FL) increased gene transfer efficiency significantly and this was further augmented if the cells were prestimulated for 24 hours wit the same cytokines. Concentration of the vectors (15-fold) by low-speed ultracentrifugation increased gene transfer to CD34+ cells in vitro even further. More than 90% of cells were transduced with a single exposure to the RDpro vector as determined by GFP expression using flow cytometry. The two other pseudotypes transduced approximately 70% of the cells under the same conditions. Detailed in vitro transduction data is shown in table as percentage GFP positive cells: RDpro MLV-A GALV+ PL alone PL+VCM PL alone PL+VCM PL alone PL+VCM No CK 9.2±2 0.9±0.8 12.5±3.5 6.6±0.1 0.9±0.4 1.4±0.5 With CK 22.4±8.3 3.3±5.3 18.2±3.8 10.8±3.8 4.8±1.7 16.7±9.0 CK prestim f 24 hours 57.6±14.9 35.3±32.1 45.4±14.9 36.3±14.8 3.4±1.7 19.5±12.2 Concentrated vector CK prestim f 24 h 93.4±3.3 93.3±3.7 73.3±7.6 40.3±20.4 66.1±7.5 77.8±10.9 Transplantation of CD34+ cells prestimulated for 24 hours and then transduced with a single hit of concentrated vector to irradiated NOD/SCID mice revealed that the RDpro vector transduced 55.1% of NOD/SCID repopulating human cells which was significantly higher than the MLV-A (12.6%) or GALV+ (25.1%) pseudotyped vectors. Thus, the STAR packaging system, especially with the modified RD114 envelope, appears to be a new valuable tool for the genetic modification of primitive human progenitor cells.

scholarly journals Depletion of L3MBTL1 promotes the erythroid differentiation of human hematopoietic progenitor cells: possible role in 20q− polycythemia vera

Blood ◽  
2010 ◽  
Vol 116 (15) ◽  
pp. 2812-2821 ◽  
Author(s):  
Fabiana Perna ◽  
Nadia Gurvich ◽  
Ruben Hoya-Arias ◽  
Omar Abdel-Wahab ◽  
Ross L. Levine ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Progenitor Cells ◽  
Cell Fate ◽  
Myeloproliferative Neoplasms ◽  
Erythroid Differentiation ◽  
Polycomb Group ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cell Fate Decisions ◽  
Cd34 Cells

Abstract L3MBTL1, the human homolog of the Drosophila L(3)MBT polycomb group tumor suppressor gene, is located on chromosome 20q12, within the common deleted region identified in patients with 20q deletion-associated polycythemia vera, myelodysplastic syndrome, and acute myeloid leukemia. L3MBTL1 is expressed within hematopoietic CD34+ cells; thus, it may contribute to the pathogenesis of these disorders. To define its role in hematopoiesis, we knocked down L3MBTL1 expression in primary hematopoietic stem/progenitor (ie, CD34+) cells isolated from human cord blood (using short hairpin RNAs) and observed an enhanced commitment to and acceleration of erythroid differentiation. Consistent with this effect, overexpression of L3MBTL1 in primary hematopoietic CD34+ cells as well as in 20q− cell lines restricted erythroid differentiation. Furthermore, L3MBTL1 levels decrease during hemin-induced erythroid differentiation or erythropoietin exposure, suggesting a specific role for L3MBTL1 down-regulation in enforcing cell fate decisions toward the erythroid lineage. Indeed, L3MBTL1 knockdown enhanced the sensitivity of hematopoietic stem/progenitor cells to erythropoietin (Epo), with increased Epo-induced phosphorylation of STAT5, AKT, and MAPK as well as detectable phosphorylation in the absence of Epo. Our data suggest that haploinsufficiency of L3MBTL1 contributes to some (20q−) myeloproliferative neoplasms, especially polycythemia vera, by promoting erythroid differentiation.

Long-Term Reconstitution of Transduced Rhesus CD34+ Cells Mobilized by G-CSF and Plerixafor.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1449-1449
Author(s):  
Naoya Uchida ◽  
Aylin Bonifacino ◽  
Allen E Krouse ◽  
Sandra D Price ◽  
Ross M Fasano ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Blood Cells ◽  
Transgene Expression ◽  
Hematopoietic Stem ◽  
Expression Levels ◽  
Cd34 Cells ◽  
One Year

Abstract Abstract 1449 Granulocyte colony-stimulating factor (G-CSF) in combination with plerixafor (AMD3100) produces significant mobilization of peripheral blood stem cells in the rhesus macaque model. The CD34+ cell population mobilized possesses a unique gene expression profile, suggesting a different proportion of progenitor/stem cells. To evaluate whether these CD34+ cells can stably reconstitute blood cells, we performed hematopoietic stem cell transplantation using G-CSF and plerixafor-mobilized rhesus CD34+ cells that were transduced with chimeric HIV1-based lentiviral vector including the SIV-capsid (χHIV vector). In our experiments, G-CSF and plerixafor mobilization (N=3) yielded a 2-fold higher CD34+ cell number, compared to that observed for G-CSF and stem cell factor (SCF) combination (N=5) (8.6 ± 1.8 × 107 vs. 3.6 ± 0.5 × 107, p<0.01). Transduction rates with χHIV vector, however, were 4-fold lower in G-CSF and plerixafor-mobilized CD34+ cells, compared to G-CSF and SCF (13 ± 4% vs. 57 ± 5%, p<0.01). CD123+ (IL3 receptor) rates were higher in CD34+ cells mobilized by G-CSF and plerixafor (16.4%) or plerixafor alone (21.3%), when compared to G-CSF alone (2.6%). To determine their repopulating ability, G-CSF and plerixafor-mobilized CD34+ cells were transduced with EGFP-expressing χHIV vector at MOI 50 and transplanted into lethally-irradiated rhesus macaques (N=3). Blood counts and transgene expression levels were followed for more than one year. Animals transplanted with G-CSF and plerixafor-mobilized cells showed engraftment of all lineages and earlier recovery of lymphocytes, compared to animals who received G-CSF and SCF-mobilized grafts (1200 ± 300/μl vs. 3300 ± 900/μl on day 30, p<0.05). One month after transplantation, there was a transient development of a skin rash, cold agglutinin reaction, and IgG and IgM type plasma paraproteins in one of the three animals transplanted with G-CSF and plerixafor cells. This animal had the most rapid lymphocyte recovery. These data suggested that G-CSF and plerixafor-mobilized CD34+ cells contained an increased amount of early lymphoid progenitor cells, compared to those arising from the G-CSF and SCF mobilization. One year after transplantation, transgene expression levels were 2–5% in the first animal, 2–5% in the second animal, and 5–10% in the third animal in all lineage cells. These data indicated G-CSF and plerixafor-mobilized CD34+ cells could stably reconstitute peripheral blood in the rhesus macaque. Next, we evaluated the correlation of transgene expression levels between in vitro bulk CD34+ cells and lymphocytes at one month, three months, and six months post-transplantation. At one and three months after transplantation, data from G-CSF and plerixafor mobilization showed higher ratio of %EGFP in lymphocytes to that of in vitro CD34+ cells when compared to that of G-CSF and SCF mobilization. At six months after transplantation the ratios were similar. These results again suggest that G-CSF and plerixafor-mobilized CD34+ cells might include a larger proportion of early lymphoid progenitor cells when compared to G-CSF and SCF mobilization. In summary, G-CSF and plerixafor mobilization increased CD34+ cell numbers. G-CSF and plerixafor-mobilized CD34+ cells contained an increased number of lymphoid progenitor cells and a hematopoietic stem cell population that was capable of reconstituting blood cells as demonstrated by earlier lymphoid recovery and stable multilineage transgene expression in vivo, respectively. Our findings should impact the development of new clinical mobilization protocols. Disclosures: No relevant conflicts of interest to declare.

The Tetraspanin CD9 Regulates Engraftment and Mobilization of Human CD34+ Hematopoietic Stem/Progenitor Cells and Modulates VLA-4 Activity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1169-1169
Author(s):  
Kam Tong Leung ◽  
Karen Li ◽  
Yorky Tsin Sik Wong ◽  
Kathy Yuen Yee Chan ◽  
Xiao-Bing Zhang ◽  
...  
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Scid Mouse ◽  
Conflicts Of Interest ◽  
Chimeric Mice ◽  
Hematopoietic Stem ◽  
Cell Engraftment ◽  
Cd34 Cells

Abstract Migration, homing and engraftment of hematopoietic stem/progenitor cells depend critically on the SDF-1/CXCR4 axis. We previously identified the tetraspanin CD9 as a downstream signal of this axis, and it regulates short-term homing of cord blood (CB) CD34+ cells (Leung et al, Blood, 2011). However, its roles in stem cell engraftment, mobilization and the underlying mechanisms have not been described. Here, we provided evidence that CD9 blockade profoundly reduced long-term bone marrow (BM; 70.9% inhibition; P = .0089) and splenic engraftment (87.8% inhibition; P = .0179) of CB CD34+ cells (n = 6) in the NOD/SCID mouse xenotransplantation model, without biasing specific lineage commitment. Interestingly, significant increase in the CD34+CD9+ subsets were observed in the BM (9.6-fold; P < .0001) and spleens (9.8-fold; P = .0014) of engrafted animals (n = 3-4), indicating that CD9 expression on CD34+ cells is up-regulated during engraftment in the SDF-1-rich hematopoietic niches. Analysis of paired BM and peripheral blood (PB) samples from healthy donors revealed higher CD9 expressions in BM-resident CD34+ cells (46.0% CD9+ cells in BM vs 26.5% in PB; n = 13, P = .0035). Consistently, CD34+ cells in granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood (MPB) expressed lower levels of CD9 (32.3% CD9+ cells; n = 25), when compared with those in BM (47.7% CD9+ cells; n = 16, P = .0030). In vitro exposure of MPB CD34+ cells to SDF-1 significantly enhanced CD9 expression (1.5-fold increase; n = 4, P = .0060). Treatment of NOD/SCID chimeric mice with G-CSF decreased the CD34+CD9+ subsets in the BM from 79.2% to 62.4% (n = 8, P = .0179). These data indicate that CD9 expression is down-regulated during egress or mobilization of CD34+ cells. To investigate the possible mechanisms, we performed a VCAM-1 (counter receptor of the VLA-4 integrin) binding assay on BM CD34+ cells. Our results demonstrated that CD34+CD9+ cells preferentially bound to soluble VCAM-1 (17.2%-51.4% VCAM-1-bound cells in CD9+ cells vs 12.8%-25.9% in CD9- cells; n = 10, P ≤ .0003), suggesting that CD9+ cells possess higher VLA-4 activity. Concomitant with decreased CD9 expression, MPB CD34+ cells exhibited lower VCAM-1 binding ability (2.8%-4.0% VCAM-1-bound cells; n = 3), when compared to BM CD34+ cells (15.5%-37.7%; n = 10, P < .0130). In vivo treatment of NOD/SCID chimeric mice with G-CSF reduced VCAM-1 binding of CD34+ cells in the BM by 49.0% (n = 5, P = .0010). Importantly, overexpression of CD9 in CB CD34+ cells promoted VCAM-1 binding by 39.5% (n = 3, P = .0391), thus providing evidence that CD9 regulates VLA-4 activity. Preliminary results also indicated that enforcing CD9 expression in CB CD34+ cells could enhance their homing and engraftment in the NOD/SCID mouse model. Our findings collectively established that CD9 expression and associated integrin VLA-4 activity are dynamically regulated in the BM microenvironment, which may represent important events in governing stem cell engraftment and mobilization. Strategies to modify CD9 expression could be developed to enhance engraftment or mobilization of CD34+ 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.

scholarly journals Identification of Lymphomyeloid Primitive Progenitor Cells in Fresh Human Cord Blood and in the Marrow of Nonobese Diabetic–Severe Combined Immunodeficient (NOD-SCID) Mice Transplanted with Human CD34+ Cord Blood Cells

1999 ◽  
Vol 189 (10) ◽  
pp. 1601-1610 ◽  
Author(s):  
Catherine Robin ◽  
Françoise Pflumio ◽  
William Vainchenker ◽  
Laure Coulombel
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Scid Mice ◽  
Human Cord Blood ◽  
Human Cd34 ◽  
Nonobese Diabetic ◽  
Direct Demonstration ◽  
Cd34 Cells ◽  
Cell Assays ◽  
Cord Blood Cells

Transplantation of genetically marked donor cells in mice have unambiguously identified individual clones with full differentiative potential in all lymphoid and myeloid pathways. Such evidence has been lacking in humans because of limitations inherent to clonal stem cell assays. In this work, we used single cell cultures to show that human cord blood (CB) contains totipotent CD34+ cells capable of T, B, natural killer, and granulocytic cell differentiation. Single CD34+ CD19−Thy1+ (or CD38−) cells from fresh CB were first induced to proliferate and their progeny separately studied in mouse fetal thymic organotypic cultures (FTOCs) and cocultures on murine stromal feeder layers. 10% of the clones individually analyzed produced CD19+, CD56+, and CD15+ cells in stromal cocultures and CD4+CD8+ T cells in FTOCs, identifying totipotent progenitor cells. Furthermore, we showed that totipotent clones with similar lymphomyeloid potential are detected in the bone marrow of nonobese diabetic severe combined immunodeficient (NOD-SCID) mice transplanted 4 mo earlier with human CB CD34+ cells. These results provide the first direct demonstration that human CB contains totipotent lymphomyeloid progenitors and transplantable CD34+ cells with the ability to reconstitute, in the marrow of recipient mice, the hierarchy of hematopoietic compartments, including a compartment of functional totipotent cells. These experimental approaches can now be exploited to analyze mechanisms controlling the decisions of such primitive human progenitors and to design conditions for their ampification that can be helpful for therapeutic purposes.

scholarly journals Activation of OCT4 Enhances Ex Vivo Expansion of Phenotypically Defined and Functionally Engraftable Human Cord Blood Hematopoietic Stem and Progenitor Cells By Regulating HOXB4 Expression

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4332-4332
Author(s):  
Xinxin Huang ◽  
Scott Cooper ◽  
Hal E. Broxmeyer
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Fold Increase ◽  
Lentiviral Vectors ◽  
Ex Vivo Expansion ◽  
Transcriptional Factor ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Allogeneic hematopoietic cell transplantation (HCT) is well established as a clinical means to treat patients with hematologic disorders and cancer. Human cord blood (CB) is a viable source of hematopoietic stem cells (HSC) for transplantation. However, numbers of nucleated cells retrieved, as well as limited numbers of HSC/progenitor cells (HPC) present, during collection may be problematic for treatment of adult patients with single CB HCT. One means to address the problem of limiting numbers of HSC/HPC is to ex vivo expand these cells for potential clinical use. While progress has been made in this endeavor, there is still a clinically relevant need for additional means to ex vivo expansion of human HSC and HPC. OCT4, a transcriptional factor, plays an essential role in pluripotency and somatic cell reprogramming, however, the functions of OCT4 in HSC are largely unexplored. We hypothesized that OCT4 is involved in HSC function and expansion, and thus we first evaluated the effects of OAC1 (Oct4-activating compound 1) on ex vivo culture of CB CD34+ cells in the presence of a cocktail of cytokines (SCF, TPO and Flt3L) known to ex vivo expand human HSC. We found that CB CD34+ cells treated with OAC1 for 4 days showed a significant increase (2.8 fold increase, p<0.01) above that of cytokine cocktail in the numbers of rigorously defined HSC by phenotype (Lin-CD34+CD38-CD45RA-CD90+CD49f+) and in vivo repopulating capacity in both primary (3.1 fold increase, p<0.01) and secondary (1.9 fold increase, p<0.01) recipient NSG mice. OAC1 also significantly increased numbers of granulocyte/macrophage (CFU-GM, 2.7 fold increase, p<0.01), erythroid (BFU-E, 2.2 fold increase, p<0.01), and granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM, 2.6 fold increase, p<0.01) progenitors above that of cytokine combinations as determined by colony assays. To further confirm the role of OCT4 in human HSC, we performed OCT4 overexpression in CB CD34+ cells using lentiviral vectors and found that overexpression of OCT4 also resulted in significant increase (2.6 fold increase, p<0.01) in the number of phenotypic HSC compared to control vectors. Together, our data indicate that activation of OCT4 by OAC1 or lentiviral vectors enhances ex vivo expansion of cytokine stimulated human CB HSC. HOXB4 is a homeobox transcriptional factor that appears to be an essential regulator of HSC self-renewal. Overexpression of HOXB4 results in high-level ex vivo HSC expansion. It is reported that OCT4 can bind to the promoter region of HOXB4 at the site of 2952 bp from the transcription start point. We hypothesized that activation of OCT4 might work through upregulation of HOXB4 expression to ex vivo expand HSC. We observed that the expression of HOXB4 was largely increased (2.3 fold increase, p<0.01) after culture of CB CD34+ cells with OAC1 compared to vehicle control. siRNA mediated inhibition of OCT4 resulted in the marked reduction of HOXB4 expression (p<0.01) in OAC1-treated cells indicating that OAC1 treatment lead to OCT4-mediated upregulation of HOXB4 expression in HSC. Consistently, siRNA-mediated knockdown of HOXB4 expression led to a significant reduction in the number of Lin-CD34+CD38-CD45RA-CD90+CD49f+ HSC in OAC1-treated cells (p<0.05), suggesting HOXB4 is essential for the generation of primitive HSC in OAC1-treated cells. Our study has identified the OCT4-HOXB4 axis in ex vivo expansion of human CB HSC and sheds light on the potential clinical application of using OAC1 treatment to enhance ex vivo expansion of cytokine stimulated human HSC. Disclosures Broxmeyer: CordUse: Membership on an entity's Board of Directors or advisory committees.

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