Pharmacological Inhibition of the Stress-Related Deacetylase SIRT1 Enhances Eradication of CML stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 448-448
Author(s):  
Ling Li ◽  
Lisheng Wang ◽  
Liang Li ◽  
Tinisha McDonald ◽  
Yin Wei Ho ◽  
...  
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Blast Crisis ◽  
Fish Analysis ◽  
Pharmacological Inhibition ◽  
Knock Down ◽  
Cd34 Cells

Abstract Abstract 448 BCR-ABL tyrosine kinase inhibitors (TKI) are effective in inducing remissions and prolonging survival of CML patients, but fail to eradicate primitive leukemia stem cells (LSC) which remain a potential source of relapse. New strategies to enhance elimination of residual CML LSC in TKI-treated patients are required. We have previously reported that the stress-related deacetylase SIRT1 is expressed at high levels in CML stem/progenitor cells and that inhibition of SIRT1 expression using lentivirus-expressed shRNA induces apoptosis in CML progenitors and increases their sensitivity to imatinib (IM) by activating p53 signaling (Blood 2010, 116: 200A). These results support an important role for SIRT1 in CML LSC maintenance and TKI resistance, and as a potential molecular target for therapy directed against CML LSC. Tenovin-6 (TV) has been identified as a potent small molecule inhibitor of SIRT1 activity (Cancer Cell 2008, 13:454). Here we evaluated whether pharmacological inhibition of SIRT1 activity using TV could selectively inhibit CML stem/progenitor cells. As with shRNA-mediated knock-down of SIRT1, treatment with TV (0.5 μM) significantly increased apoptosis of CML CD34+ cells (TV16±7% vs. Control 3±2%, p=0.04, n=3), but not normal CD34+ cells (TV 6±2% vs. Control 4±2%, p=0.1, n=3). The combination of IM (2.5 μM) and TV induced significantly increased apoptosis in CML progenitors compared to IM alone, and to a significantly greater extent than in normal cells (CML, TV + IM 40±2% vs. IM 19±3%, p=0.009, n=3; CB, TV + IM 15±4% vs. IM 10±2%, p=0.04, n=3). TV (1 μM) increased apoptosis in both CML CD34+CD38− (TV 42±10% vs. Control 4±3%, p=0.04, n=3) and CD34+CD38+ cells (TV 35±7% vs. Control 8±2%, p=0.03, n=3). CFSE labeling indicated that treatment with TV resulted in increased apoptosis of undivided CML CD34+CD38− cells identified on the basis of high CFSE fluorescence (TV 20±7% vs. Control 2±1%, p=0.04, n=3). The combination of TV with IM resulted in a significant increase in apoptosis in CML CD34+CD38− CFSEhigh cells compared to IM alone (TV plus IM 35±5% vs. IM 10±4%, p=0.03, n=3). Treatment with TV (0.5 μM) reduced CML CFC frequency (70±9% inhibition with TV compared to untreated controls, p=0.009, n=3) without affecting normal CFC frequency. Combination of TV (0.5 μM) with IM resulted in enhanced inhibition of CML CFC compared to IM alone, but did not enhance inhibition of normal CFC (CML: TV plus IM 82±6% inhibition vs. IM 57±10%, p=0.02, n=3; CB: TV plus IM 38±7% inhibition vs. IM 36±9%, p=0.1, n=3). TV treatment effectively inhibited the growth of Baf3 cells expressing T315I-mutated BCR-ABL, and significantly enhanced apoptosis of IM-resistant CML blast crisis CD34+ cells [TV (1 μm) 30±1% vs. Control 19±5%, p=0.04, n=3], suggesting SIRT1 inhibition can also target TKI-resistant CML cells. Ex vivo treatment with TV (1 μM) significantly reduced longer-term (12 weeks) engraftment of CML CD34+ cells in NSG mice following TV treatment (TV treated 0.2*105±0.1*105 human CD45+ cells in murine BM vs. Control 1.8*105±0.6*105, p=0.009, n=5). Significant reduction in engraftment of CD33+ (p=0.008) and CD14+ myeloid cells (p=0.009) was seen. Q-PCR and FISH analysis confirmed that engrafted human cells were leukemic in origin. Interestingly, engraftment of CB CD34+ cells was not reduced after treatment with TV (TV 2.7*106±0.7*106 human CD45+ cells in murine BM, vs. Control 2.4*106±0.8*106, p=0.2, n=6). These results show that SIRT1 inhibition by TV effectively targets primitive human CML cells with in vivo multi-lineage engraftment capacity. Treatment with TV significantly enhanced acetylated p53 levels in CML CD34+ cells, indicating effective inhibition of SIRT1 activity. TV treatment also increased total p53 levels, possibly related to reduced p53 degradation. TV treatment did not increase acetylated p53 or total p53 levels in normal CD34+ cells. Importantly shRNA-mediated knock-down of p53 resulted in significant reduction of TV-induced apoptosis in CML CD34+ cells (13±6% apoptosis with p53 shRNA; 33±7% apoptosis with control shRNA, p=0.04, n=3), indicating that the effects of TV on CML CD34+ cells are related to p53 acetylation and activation. In conclusion, our studies indicate that pharmacological inhibition of SIRT1 can activate p53 and enhance eradication of CML LSC in combination with TKI treatment, and support further evaluation of targeted inhibition of SIRT1 as a therapeutic strategy in CML. Disclosures: No relevant conflicts of interest to declare.

Effective and Selective Elimination of CML Stem Cells Using Novel Ethacrynic Acid Derivatives

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4508-4508
Author(s):  
Su Chu ◽  
YinWei Ho ◽  
Guisen Zhao ◽  
Tessa L. Holyoake ◽  
Samuel Waxman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Protein Expression ◽  
Progenitor Cells ◽  
Ethacrynic Acid ◽  
Conflicts Of Interest ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Cd34 Cells ◽  
Induced Apoptosis

Abstract Tyrosine kinase inhibitors (TKI) are highly effective in the treatment of CML but do not eliminate primitive, quiescent leukemia stem cells (LSC), which persist as a potential source of leukemia relapse. Additional strategies to enhance eradication of LSC are required to increase the possibility of treatment free remissions for CML patients. Glutathione S-transferase P1-1 (GSTP1-1) is a Phase II detoxifying enzyme which is overexpressed in several cancers and causes drug resistance. The diuretic Ethacrynic acid (EA) is a GSTP1-1 activity inhibitor and also induces celldeath in malignant cells at high concentrations. We have synthesized two EAoxadiazole analogs, 6U and 6S, which demonstrate enhancedpro-apoptotic effects in CML K562 cells expressing high levels of GSTP1-1. Previously we found that 6U and 6S induced apoptosis through downregulation of anti-apoptotic protein MCL-1 in addition to their GSTP1-1 activity inhibition. We extended these observations to primary CML stem/progenitor cells. We observed increased expression of GSTP1-1 mRNA and protein, associated with increased expression of MCL-1, BCL2 and BCL-xL, in chronic phase (CP) and blast crisis (BC) CML compared to normal CD34+ cells. Treatment of CP CML CD34+ cells with 6U or 6S (1 to 6µM) for 24 to 48 hours resulted in a significant dose-dependent induction of apoptosis, inhibition of cell growth, and reduction in colony forming cell (CFC) numbers, with 6U demonstrating greater efficacy than 6S. Treatment with 6U did not induce significant apoptosis of normal (NL) CD34+ cells at doses below 4µM. 6U induced significantly less apoptosis in NL compared with CML CD34+ cells (2µM, p<0.05). We further tested the activity of 6U against purified CML and normal CD34+CD38- stem/primitive progenitors and CD34+CD38+ committed progenitors with or without the BCR-ABL TKI Dasatinib (DAS). 6U treatment induced apoptosis of CML, but not normal, CD34+CD38- and CD34+CD38+ cells (Table). Combination of 6U with DAS (50nM) selectively enhanced apoptosis of CML compared to normal cells, including quiescent, slowly dividing CML LSC that are resistant to TKI-induced apoptosis (p≤0.01). Treatment with 6U alone or with DAS, significantly increased G1, and decreased S/G2/M phase of CML, but not in normal CD34+ cells, and reduced CFC growth from CML CD34+CD38+ cells (Table). CML, but not normal CD34+ cells, treated with 6U, with or without DAS, prior to transplant, showed significantly reduced engraftment in NSG mice, indicating selective inhibition of in vivo repopulating CML LSC (Table). Treatment with 6U was also effective in inducing apoptosis and inhibiting CFC growth in BC CML progenitor cells (Table). 6U treatment resulted in down-regulation of GSTPI1-1 and MCL-1 protein expression in CP and BC CML, but not in normal CD34+ cells. Interestingly 6U treatment also reduced BCR-ABL protein expression in CP and BC CML CD34+ cells. We conclude that CML CP and BC LSC express high levels of GSTP1-1 and anti-apoptotic proteins, which can be targeted by the novel EA derivative 6U through a new mechanism. Since 6U has significantly lesser effects on normal stem cells, it may offer a promising and innovative approach to selectively target CP and BC CML LSC in combination with TKI inhibitors. Abstract 4508. Table CML CP Normal CML BC Ctrl 6U DAS DAS+ 6U Ctrl 6U DAS DAS+ 6U Ctrl 6U DAS DAS+ 6U Apoptosis (normal, CP CML: CD34+CD38-; CML BC CD34+) 3.4± 0.9 15.9±6.7 9.4± 2.6 47.4±13.6 ** 3.3± 0.9 5.1± 1.0 1.6± 0.2 7.0± 1.2 * 3.4± 0.7 30±12.7 10.6±1.8 43.3±14.1 ** CFU-GM (normal, CP CML: CD34+CD38+; CML BC CD34+) 71.3± 7.8 7± 3.2 ** 21± 7.3 ** 5 ± 2.3 ** 121±19.3 102.7±6.2 134.3±15.9 103±5.1 288.5±89.4 26.5±11.3 *** 82.7±33.1 ** 8 ± 3.6 *** NSG engraftment (CD34+) 1.8± 0.3 0.4± 0.1 *** 0.8± 0.3 ** 0.4± 0.04 *** 68.2± 4.9 61± 2.2 68.1± 2.9 64.2± 3.9 Data shown are mean ± SEM of 3-6 samples. Significance, compared to controls. *p≤0.05,**p≤0.01, ***p≤0.001 Disclosures No relevant conflicts of interest to declare.

The Pan-Bcl-2 Family Inhibitor 97C1 Targets Blast Crisis Chronic Myeloid Leukemia Stem Cells but Spares Normal Cord Blood Progenitor Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 516-516 ◽  
Author(s):  
Daniel Goff ◽  
Alice Shih ◽  
Angela Court Recart ◽  
Larisa Balaian ◽  
Ryan Chuang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Blast Crisis ◽  
Chemotherapy Resistance ◽  
Chronic Phase ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 516 Introduction: Several studies have demonstrated the role of leukemia stem cells (LSC) in the development and maintenance of human chronic myeloid leukemia (CML). These cells, which first develop in chronic phase CML (CP CML) with acquisition of the BCR-ABL fusion protein, are often quiescent and can be highly resistant to apoptosis induced by drugs and radiotherapy that target rapidly dividing cells. Data has also shown that CML LSC become increasingly resistant to BCR-ABL inhibition with progression to blast crisis CML (BC CML). Bcl-2 family proteins are key regulators of apoptosis and have been shown by numerous studies to regulate cancer resistance to chemotherapy. This family of proteins has also been implicated in the development of BC CML, however most studies have focused on CML cell lines and their expression of Bcl-2 family proteins in vitro. Thus, there is relatively little data on expression of Bcl-2 family proteins in primary CML LSC and on the role of these proteins in regulating chemotherapy resistance in CML LSC in vivo. As Bcl-2 family proteins are known regulators of chemotherapy resistance we hypothesized that human BC CML LSC may overexpress these proteins compared to normal hematopoietic stem cells. We analyzed Bcl-2 family mRNA and protein expression in CP CML and BC CML LSC and compared this expression to normal cord blood stem and progenitor cells. We also analyzed whether these cells were sensitive to chemotherapy treatment in vitro. Finally, we tested whether a high potency pan-Bcl-2 inhibitor, 97C1, could effectively kill CML LSC in vitro and in vivo. Methods: Bcl-2 and Mcl-1 protein expression was measured in primary CP CML, BC CML, and normal cord blood cells using intracellular FACS. We also measured Bcl-2, Mcl-1, Bcl-X, and Bfl-1 mRNA expression in FACS sorted CD34+CD38+lin− cells (LSC) from these samples. For all drug studies we used either serially transplanted CD34+ cells derived from primary BC CML patient samples or primary CD34+ normal cord blood cells. In vitro drug responses were tested by culturing CD34+ cells either alone or in co-culture with a mouse bone marrow stromal cell line (SL/M2). Effects on colony formation and replating were also tested by culturing sorted CD34+CD38+lin− cells in methylcellulose in the presence and absence of drug. For in vivo testing of 97C1 we transplanted neonatal RAG2-/-yc-/- mice with CD34+ cells from 3 different BC CML and cord blood samples. Transplanted mice were screened for peripheral blood engraftment at 6–8 weeks post-transplant and engrafted mice were then treated for 2 weeks with 97C1 by IP injection. Following the treatment period the mice were sacrificed and hemotapoietic organs were analyzed for human engraftment by FACS. Results: BC CML progenitors expressed higher levels of Bcl-2 and Mcl-1 protein compared to normal cord blood and chronic phase CML cells. mRNA expression of Mcl-1, Bcl-X, and Bfl-1 was also increased in BC CML progenitors compared to CP CML progenitors. While BC CML LSC cultured in vitro were resistant to etoposide and dasatinib-induced cell death, 97C1 treatment led to a dose-dependent increase in cell death along with a dose-dependent decrease in the frequency of CD34+CD38+lin− cells compared to vehicle treated controls. While cord blood progenitor cells were also sensitive to 97C1 treatment they had an IC50 around 10 times higher than that for the BC CML cells (100nM versus 10nM). Importantly, 97C1 treatment did not inhibit cord blood colony formation or colony replating in vitro. Mice transplanted with BC CML LSC developed CML in 6–8 weeks post-transplant with diffuse myeloid sarcomas and engraftment of human CD34+CD38+lin− cells in the peripheral blood, liver, spleen, and bone marrow. In vivo treatment with 97C1 led to a significant reduction in both total human engraftment and engraftment of CD34+CD38+lin− cells in all hematopoietic organs analyzed. Conclusion: Our results demonstrate that BC CML LSC are resistant to conventional chemotherapy but are sensitive to 97C1 in vitro and in vivo. Broad-spectrum inhibition of Bcl-2 family proteins may help to eliminate CML LSC while sparing normal hematopoietic stem and progenitor cells. Disclosures: Jamieson: CoronadoBiosciences: Research Funding; CIRM: Research Funding.

Environmental Cues Can Promote Symmetrical Division of Functional Human Hematopoietic Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1397-1397
Author(s):  
Nadim Mahmud ◽  
Kazumi Yoshinaga ◽  
Craig Beam ◽  
Hiroto Araki
Keyword(s):  
Stem Cells ◽  
Cell Division ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Absolute Number ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Differentiated Cells ◽  
Cd34 Cells ◽  
Cells Cultured

Abstract Widespread clinical use of ex-vivo expanded human umbilical cord blood (CB) grafts has been limited by lack of proper understanding of factors regulating self-renewal type of symmetric cell divisions. The expansion of the number of functional hematopoietic stem cells (HSC) ex-vivo requires the creation of an environment which favors symmetrical division. In our current studies, addition of late acting cytokines, (GM-CSF, IL-6, Epo) with early acting cytokines (thrombopoietin, SCF, Flt-3 ligand) resulted in loss of expansion of stem/progenitor cells. These data indicate that modification of HSC fate is not fully independent of external humoral influences. We have previously demonstrated that following treatment of CD34+ cells with 5-aza-2-deoxycytidine (5azaD) and trichostatin A (TSA) there is a 10- fold increase in the number of SCID mouse repopulating cells (SRC). This increase of SRC, however, occurred concomitantly with an increase in absolute number of CD34+CD90+ cells as well as primitive progenitors which gives rise to colony forming unit Mix lineage (CFU-Mix). We hypothesized that if the primary CD34+ cells generates CFU-Mix/CFU-GM in a ratio of ‘X’, then to observe a higher rate of symmetric cell division we would expect to see the ratio increased (&gt;X) in the 5azaD/TSA treated cells in comparison to cells cultured in the absence of 5azaD/TSA (&lt; X). Interestingly, analyses of our data suggest that when 5azaD/TSA treated CD34+ cells are cultured for 5 days and assayed for colonies we observed a significant increase in the ratio of CFU-Mix/CFU-GM in contrast to cells cultured in cytokines alone, 0.373 ± 0.06 and 0.066 ± 0.032 respectively. The ratio of CFU-Mix/CFU-GM of CB CD34+ cells (day 0) was 0.262 ± 0.045. These findings indicate that 5azaD/TSA treatment promotes the ratio of CFU-Mix/CFU-GM possibly by enhancing symmetric division of CFU-Mix while in the absence of 5azaD/TSA treatment the culture condition likely induces differentiation. In addition, we have also investigated the ratio of progenitor cells/differentiated cells by assessing the ratio of human CD34+ cells/CD33+ cells in the bone marrow of immunodeficient mice following transplantation (8 weeks) of equal numbers of CD34+ cells. The ratio of CD34+ cells/CD33+ cells following transplantation of 5azaD/TSA treated cells was 0.52 ± 0.14 (n = 11) while in the absence of 5azaD/TSA the ratio dropped to 0.31± 0.16 (n = 4). The ratio following transplantation of primary CD34+ (day 0) cells was 0.62 ± 0.14 (n = 6). These data suggest that 5azaD/TSA treated cells maintain the balance of generation of CD34+ cells/CD33+ cells at a comparable rate to that of primary CD34+ cells, while the CD34+ cells generated in the absence of 5azaD/TSA promotes generation of more differentiated cells. Alternatively, it is also possible that 5azaD/TSA treatment of CD34+ cells in the culture results in inhibition of myeloid differentiation at the cost of proliferation. However, the latter possibility is unlikely, since treatment of CB cells with 5azaD/TSA results in an increase in the absolute number of progenitors including SRC possessing both myeloid and lymphoid differentiation potential. Taken together, these data support our hypothesis that chromatin modifying agents in the culture is capable of promoting self-renewal type of symmetric cell division possessing in vivo multilineage marrow repopulating potential.

Chromatin Modifying Agents Promote the Ex Vivo Production of Functional Human Erythroid Progenitor Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 340-340
Author(s):  
Pratima Chaurasia ◽  
Dmitriy Berenzon ◽  
Ronald Hoffman
Keyword(s):  
Progenitor Cells ◽  
Histone Deacetylase Inhibitors ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Significant Proportion ◽  
Erythroid Progenitor ◽  
Erythroid Precursor ◽  
Cell Product ◽  
Erythroid Progenitor Cells ◽  
Cd34 Cells

Abstract Abstract 340 Presently, blood transfusion products (TP) are composed of terminally differentiated cells with a finite life span. We attempted to develop an alternative TP which would be capable of generating additional red blood cells (RBC). Several histone deacetylase inhibitors (HDACIs) were used in vitro to reprogram cord blood (CB) CD34+ cells to differentiate to erythroid progenitor cells (EPC). We demonstrated that CB CD34+ cells in the presence of HDACIs (SAHA, VPA and TSA), and a combination of cytokines SCF, IL-3, TPO and FLT3, promoted expansion of CD34+ cells and CD34+CD90+ cells as compared to cultures containing cytokines alone. Addition of VPA resulted in the greatest expansion of CD34+ cells, CD34+CD90+cells+ (59.4 fold, p=0.01; 66.7 fold, p=0.02, respectively) as compared to SAHA and TSA. VPA also led to the generation of the greatest absolute number of EPC cells (14.9×106, p=0.002), approximately a 5500 fold in the numbers of assayable EPC, as compared to primary CB. The single cell analyses of CB CD34+ cells (Day0) and single CD34+ reisolated from ex-vivo cultures pretreated with cytokines alone or cytokines+VPA demonstrated an skewed differentiation program of CD34+ cells to EPC (>94%, p=0.003) compared to CB CD34+(50%) and cytokines alone (29%). We investigated the expression of lineage specific phenotypic markers expressed by CD34+ cells exposed to cytokines alone or cytokines plus VPA. The FACS analyses showed a significantly greater proportion of CD34+CD36+ (52.4% vs 21.0%) CD36+CD71+(44.5% vs7.6%), CD36+GPA+(12.8% Vs 4.0%) and CD71+GPA+(22.2% vs 6.3%) cells with lower numbers of CD19+(2.8% vs 13.6%) cells, CD14+(2.0% vs 8.9%), CD15+(1.8 vs 6.9%) in VPA treated CD34+ cells as compared to cytokines alone. We monitored the relative expression of a group of genes characteristic of both primitive HPC and erythroid commitment (Bmi1, Dnmt1, Ezh2, Smad5, Eklf, GATA1, GATA2, EpoR and Pu.1). Q-PCR was performed on CD34+cells reisolated from cultures treated with cytokines alone or cytokines plus VPA and compared to primary CB CD34+ cells. The expression of genes associated with retention of the biological properties of the primitive HPC (Bmi1-2.6 fold, Dnmt1-10.3 fold and Ezh2-4.8 fold) and erythroid lineage specific genes (Smad5-6.2 fold, GATA2-3.7 fold) were upregulated and Pu.1 (0.6-fold), GATA1(1.9 fold) were downregulated as compared to cytokines alone. However, expression of EpoR and Eklf were similar in the two cell populations Histone acetylation study showed that the CB CD34+ cells and VPA treated CD34+ cells had a significant proportion of acetylated H3K9 cells, 52.2% and 56.1% respectively, while this population was virtually absent in CD34+ cells exposed to cytokines alone (1.3%, p=0.001). ChIP assay demonstrated a varying degree of H3K9/14 and H3K27 acetylation within the promoters of VPA treated CD34+ cells for GATA2 (7.4 fold, 7.2 fold), Eklf (7.4 fold, 9.7 fold), Pu.1(4.5fold, 4.8 fold), EpoR (2.3 fold, 4.7 fold) and GATA1(4.7 fold, 2.9 fold). The acetylation of cytokines treated CD34+ cells were much lower than VPA treated CD34+ cells. The VPA treated cell product after 9 days (supplemented with SCF, Epo and IL-3 for 2 additional days) compared to 7 days contained a greater percentage of EPC and erythroid precursor cells CD34+CD36+(24.9% vs 23.0%), CD36+GPA+(33.9% vs 18.8%), CD36+. CD71+(55.8% vs 37.8%), CD71+GPA+(33.9% vs 20.5%) and CD34+CXCR4+(28.8% vs 21.0 %). The TP contained very limited number of CD19+(1.4%), CD14+(11.11%) or CD15+(6.8%) of cells. Approximately 50 % of the cells present in the TP expressed the chemokine receptor CXCR4. We next evaluated the behavior of ex vivo expanded cell product following transfusion into sublethally irradiated NOD/SCID mice. FACS analyses of mice peripheral blood (PB) on serial days showed evidence of circulating nucleated erythroid and enucleated red cells. The greatest number of circulating human RBC (12.4%±6.8%) was observed on day5. RT-PCR analyses on the PB of mice on day 15 revealed the presence of erythroid cells containing both human adult and fetal hemoglobin. On day 15 the mice were sacrificed and the degree of human cells engraftment in the marrow were predominately hu -CD45+ (7.4%), CD34-CD36+(1.8%), CD36 (4.5%) and GPA+(1.7%) with no evidence of CD33+, CD14+, CD19+ and CD41+ cells. The ex vivo generated EPC-TP likely represents a paradigm shift in transfusion medicine due to its continued ability to generate additional RBC. Disclosures: No relevant conflicts of interest to declare.

ATG5 and ATG7 Fulfill Essential and Non-Redundant Roles in Human Hematopoietic Stem/Progenitor Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4316-4316
Author(s):  
Hendrik Folkerts ◽  
Maria Catalina Gomez Puerto ◽  
Albertus T.J. Wierenga ◽  
Koen Schepers ◽  
Jan Jacob Schuringa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Progenitor Cells ◽  
Target Genes ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Human Hscs

Abstract Macroautophagy is a catabolic process by which intracellular contents are delivered to lysosomes for degradation. ATG5 and ATG7 play an essential role in this process. Recent studies have shown that mouse hematopoietic stem cells (HSCs) lacking ATG7 were unable to survive in vivo, however, the role of macroautophagy in proliferation and survival of human HSCs has not yet been defined. Here, we demonstrate that autophagy is functional in human hematopoietic stem/progenitor cells. Robust accumulation of the autophagy markers LC3 and p62 were observed in cord blood (CB)-derived CD34+ cells treated with bafilomycin-A1 (BAF) or hydroxychloroquine (HCQ), as defined by Western blotting. When these cells were subsequently differentiated towards the myeloid or erythroid lineage, a decreased accumulation of LC3 was observed. In addition, CB CD34+CD38- cells showed enhanced accumulation of cyto-ID (a marker for autophagic vesicles) compared to CD34+CD38+ progenitor cells upon BAF or HCQ treatment. In line with these results, also more mature CB CD33+ and CD14+ myeloid cells or CD71+CD235+ erythroid cells showed reduced levels of cyto-ID accumulation upon BAF or HCQ treatment. These findings indicate that human hematopoietic stem and progenitor cells (HSPCs) have a higher basal autophagy flux compared to more differentiated cells. To study the functional consequences of autophagy in human HSCs and their progeny, ATG5 and ATG7 were downregulated in CB-derived CD34+ cells, using a lentiviral shRNA approach which resulted in 80% and 70% reduced expression, respectively. Downmodulation of ATG5 or ATG7 in CB CD34+ cells resulted in a significant reduction of erythroid progenitor frequencies, as assessed by colony forming cell (CFC) assays (shATG5 2.2 fold, p<0.05 or shATG7 1.4 fold p<0.05). Additionally, a strong reduction in expansion was observed when transduced cells were cultured under myeloid (shATG5 17.9 fold, p<0.05 or shATG7 12.3 fold, p<0.05) or erythroid permissive conditions (shATG5 6.7 fold, p<0.05 or shATG7 1.7 fold, p<0.05), whereby differentiation was not affected. The phenotype upon knockdown of ATG5 or ATG7 could not be reversed by culturing the cells on a MS5 stromal layer. In addition to progenitor cells, HSCs were also affected since long term culture-initiating cell (LTC-IC) assays in limiting dilution revealed a 3-fold reduction in stem cell frequency after ATG5 and ATG7 knockdown. The inhibitory effects of shATG5 and shATG7 in cultured CD34+ cells were at least in part due to a decline in the percentage of cells in S phase and (shATG5 1.4 fold, p<0.01 and shATG7 1.3 fold, p<0.01) and an increase of Annexin V positive cells. The changes in cell cycle and apoptosis coincided with a marked increase in expression of the cell cycle-dependent kinase inhibitor p21, an increase in p53 levels, and an increase in proapoptotic downstream target genes BAX, PUMA and PHLDA3. Additionally, ROS levels were increased after ATG5 and ATG7 knockdown. The increased apoptosis in shATG5 and shATG7 transduced cells might be triggered by elevated ROS levels. Taken together, our data demonstrate that autophagy is an important survival mechanism for human HSCs and their progeny. Disclosures No relevant conflicts of interest to declare.

scholarly journals Efficient Ex Vivo Expansion of Highly Purified Human Umbilical Cord Blood-Derived Very Small CD34+lin-CD45- Stem Cells into Functional Endothelial Cells in Vitro in Chemically Identified, Feeder Layer-Free Medium Supplemented with Nicotinamide

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4882-4882
Author(s):  
Alison Domingues ◽  
Kamila Bujko ◽  
Magdalena Kucia ◽  
Janina Ratajczak ◽  
Mariusz Z Ratajczak
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Cell Therapy ◽  
Progenitor Cells ◽  
Umbilical Cord Blood ◽  
Ex Vivo ◽  
Germ Layer ◽  
Differentiation Factors

Background . There is an ongoing search for multipotent stem cells from umbilical cord blood (UCB) with trans-germ layer differentiation potential that can be employed in repairing damaged organs and also expanded into transplantable hematopoietic stem cells (HSCs) and endothelial progenitor cells (EPCs). The existence of such cells in postnatal life could also revive the concept of hemangioblasts or hemangioblast-like cells in adult hematopoietic organs. Our group was the first to isolate a population of small CD34+CD133+lin-CD45- early-development stem cells from human hematopoietic tissues, including UCB. Based on the validated expression of early-development markers, these cells were named "very small embryonic-like stem cells" (VSELs, Circulation Res 2019; 124:208-210). Currently, more than 25 independent groups worldwide who have carefully followed the multicolor-staining cell-sorting strategy described by us (Current Protocols in Cytometry 2010, 9.29.1-9.29.15) have successfully isolated these cells and demonstrated their in vivo contribution to all three germ layer lineages. Thus, VSELs could be very useful in regenerative medicine in the field of angiogenesis, and UCB is an attractive source, with easy accessibility and tolerance to allogenic grafts. However, the low number of these cells in UCB and their quiescence are limiting factors. Therefore, in vitro differentiation of VSELs into endothelial progenitor cells (EPCs) would allow improvement in the ability to expand endothelial cells and could represent a clinically relevant alternative to embryonic stem cells (ESCs) and induced pluripotent stem cells (iPS) for cell therapy without ethical problems and undesirable side effects. Hypothesis. We hypothesized that UCB-purified, very small, early-developmentCD34+lin-CD45-stem cells can be ex vivo expanded into functional EPCs. Materials and Methods. VSELs highly purified by FACS were expanded into EPCs in pro-angiogenic medium supplemented with mesodermic differentiation factors and then endothelial differentiation factors in the presence of nicotinamide and UM171. In parallel, we expanded EPCs from MNCs isolated from the same UCB units by employing a classical protocol (Methods in Enzymology 2008, 445:303-29). The EPC nature of the expanded VSEL-derived cells was confirmed by the expression of typical EPC markers as well as by in vitro angiogenic assays. Results. Our differentiation cocktail allowed us to differentiate and expand VSELs into EPCs. In our expansion medium (Figure 1), the very small, round VSELs smaller than 6 mm in diameter proliferated and differentaited over time into larger and extended cells with a cobblestone morphology similar to the EPC control cells, and we confirmed their endothelial characteristics by cytometry analysis. Like EPCs, VSEL-derived EPCs were positive for CD31, CD144, KDR, and CD105 and negative for mesenchymal surface markers, such as CD90. They also performed similarly to EPCs in classical vasculogenic tests, including adhesion, proliferation, migration, and tubulogenesis assays. Conclusions. This work shows, for the first time, efficient VSEL differentiation into functional endothelial cells with vasculogenic properties without the help of co-culture over feeder-layers or viral vectors in medium supplemented with nicotinamide and UM171. These findings allow us to propose these cells as an interesting cell therapy product. These results also reopen the question of the existence of hemangioblast-like cells in postnatal tissues. We are currently testing these cells in vivo in model of hind limb ischemia. Figure 1 Disclosures No relevant conflicts of interest to declare.

Transplantation of Primary Human CD34+CD38 - Hematopoietic Stem Cells Recapitulates Idiopathic Myelofibrosis in the NOD/scid/IL2rgKO Mice.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 260-260
Author(s):  
Noriyuki Saito ◽  
Fumihiko Ishikawa ◽  
Kazuya Shimoda ◽  
Shuro Yoshida ◽  
Yoriko Saito ◽  
...  
Keyword(s):  
Stem Cells ◽  
Extramedullary Hematopoiesis ◽  
In Vivo Model ◽  
Fish Analysis ◽  
Human Stem Cells ◽  
Idiopathic Myelofibrosis ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Xenotransplantation Model

Abstract Idiopathic myelofibrosis (IMF) is characterized by clonal proliferation of abnormal myelomonocytic cells and megakaryocytes. These cells are thought to secrete various cytokines resulting in reactive fibrosis and increased collagen content in the bone marrow (BM), and the fibrotic changes in the BM leads to extramedullary hematopoiesis and increased frequency of CD34+ cells in the peripheral blood (PB). Although IMF is thought to originate from an abnormality at the level of hematopoietic stem cell (HSC), this has not been experimentally addressed using primary human IMF samples. To demonstrate the involvement of HSCs in the pathogenesis of IMF and to establish an in vivo model of IMF, we employed the newborn NOD/SCID/IL2rg-null xenotransplantation model that efficiently supports engraftment of normal and malignant human stem cells. We purified PB CD34+ cells and PB CD34+CD38- cells from six IMF patients, and intravenously transplanted the purified cells into newborn NOD/SCID/IL2rg-null recipients. In long-term observation of the recipient mice, we analyzed human CD45+ hematopoietic cell chimerism both in the PB and in the BM, suppression of murine normal hematopoiesis, and the fibrotic changes in the BM. Six out of thirteen recipients transplanted with patient HSCs exhibited human hematopoietic engraftment, and CD33+ myeloid cells accounted for 80.5+/−9.41% of all the engrafted CD45 + population (as compared with the recipients transplanted with normal HSCs). BM of all engrafted recipients demonstrated fibrotic changes associated with increased proliferation of fibroblasts and the presence of human megakaryocytes, recapitulating the clinical features of IMF. In the 7 remaining recipients, PB hCD45 chimerism was &lt; 1.5% at thirty-two weeks and decreased over time and fibroblast proliferation could not be demonstrated in the BM at forty weeks. To investigate the origin of BM fibroblast, we performed FISH analysis using human and mouse centromeric probes and immuno-staining using anti-CD45 and anti-vimentin antibodies. Of sixty fibroblasts examined, fifty-four cells were of human origin. These findings demonstrate that the IMF-initiating cells are contained within the CD34+CD38- HSC fraction and these cells possess differentiation capacity to fibroblasts. The newborn NOD/SCID/IL2rg-null xenotransplantation model provides an in vivo model of primary human IMF that may lead to better understanding of the mechanisms of IMF pathogenesis including the identification of IMF stem cells and the development of novel therapeutic agents for IMF.

Analysis of Idiopathic Myelofibrosis Initiating Cell in NOD/SCID/IL2rgKO Mice

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3715-3715
Author(s):  
Noriyuki Saito ◽  
Fumihiko Ishikawa ◽  
Kazuya Shimoda ◽  
Shuro Yoshida ◽  
Yoriko Saito ◽  
...  
Keyword(s):  
Stem Cells ◽  
Extramedullary Hematopoiesis ◽  
In Vivo Model ◽  
Fish Analysis ◽  
Human Stem Cells ◽  
Idiopathic Myelofibrosis ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Xenotransplantation Model

Abstract Idiopathic myelofibrosis (IMF) is characterized by clonal proliferation of abnormal myelomonocytic cells and megakaryocytes. These cells are thought to secrete various cytokines resulting in reactive fibrosis and increased collagen content in the bone marrow (BM), and the fibrotic changes in the BM lead to extramedullary hematopoiesis and increased frequency of CD34+ cells in the peripheral blood (PB). Although IMF is thought to originate from an abnormality at the level of hematopoietic stem cell (HSC), this has not been experimentally addressed using primary human IMF samples. To demonstrate the involvement of HSCs in the pathogenesis of IMF and to establish an in vivo model of IMF, we employed the newborn NOD/SCID/IL2rg-null xenotransplantation model that efficiently supports engraftment of normal and malignant human stem cells. We purified PB CD34+ cells and PB CD34+CD38− cells from four IMF patients, and intravenously transplanted the purified cells into newborn NOD/SCID/IL2rg-null recipients. In long-term observation of the recipient mice, we analyzed human CD45+ hematopoietic cell chimerism both in the PB and in the BM, suppression of murine normal hematopoiesis, and the fibrotic changes in the BM. Twelve out of nineteen recipients transplanted with patient CD34+ cells or CD34+CD38− cells exhibited human hematopoietic engraftment, and the frequency of CD33+ myeloid cells (82.5+/−12.2% among the engrafted CD45+ cells) was higher than that in the recipients transplanted with normal HSCs. These CD33+ cells expressed other myelo-monocytic markers such as CD14, CD11b, CD15, and HLA-DR. BM of all engrafted recipients demonstrated fibrotic changes associated with increased proliferation of fibroblasts and the presence of human megakaryocytes, recapitulating the clinical features of IMF. In the 7 remaining recipients, PB hCD45 chimerism was < 1.5% at thirty-two weeks and decreased over time and fibroblast proliferation could not be demonstrated in the BM at forty weeks. With FISH analysis using human X probe and immuno-staining using anti-TGF-beta1 antibody, we could confirm the TGF-beta1 production of human megakaryocytes in the recipient BM. To investigate the origin of BM fibroblasts, we performed FISH analysis using human X chromosome probe and mouse centromeric probe, and immuno-staining using anti-CD45 and anti-vimentin antibodies. Of one hundred fifty fibroblasts examined, one hundred thirty six cells (90.7%) were of human origin. These findings demonstrate that the IMF-initiating cells are contained within the CD34+CD38− HSC fraction and these cells possess differentiation capacity to fibroblasts. The newborn NOD/SCID/IL2rg-null xenotransplantation model provides an in vivo model of primary human IMF that may lead to better understanding of the mechanisms of IMF pathogenesis including the identification of IMF stem cells and the development of novel therapeutic agents for IMF.

A Dendritic Cell Based Vaccination Strategy Geared towards Eradication of Leukemic Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2046-2046
Author(s):  
Hetty J Bontkes ◽  
Jurjen Ruben ◽  
Willemijn van den Ancker ◽  
Theresia M Westers ◽  
G. Ossenkoppele ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Tumor Antigens ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Vaccination Strategy ◽  
Leukemic Stem Cells ◽  
Cell Immunity

Abstract Abstract 2046 Poster Board II-23 Introduction: In the majority of cases, initial remission of acute myeloid leukemia (AML) is reached but unfortunately relapse rates remain high and therefore novel treatments are needed. It is thought that recurrent AML originates from chemotherapy resistant quiescent leukemic stem cells (LSC). The application of immunotherapeutic approaches to eradicate LSC remaining after first line chemotherapy may contribute to improved disease outcome. Vaccination strategies have often used dendritic cells (DC) ex vivo pulsed with tumor-derived whole lysates or peptides as modalities to present a broad range of tumor antigens to T cells to stimulate effective anti-tumor T-cell immunity in vivo. It is likely that certain proteins expressed by LSC have a distinct antigenicity as compared to more mature AML blasts and thus provide targets for specific T-cells. Even without identification of specific antigens, LSC can be a useful source of tumor antigens in DC vaccination-based immunotherapy. CD34+CD38- LSC can be identified using malignant stem cell associated cell surface markers including CLL-1 and lineage markers such as CD7, CD19 and CD56. However, the low frequency of these cells precludes the use of LSC derived apoptotic cells or lysates for DC loading. Alternatively, mRNA isolated from LSC can be amplified and subsequently transfected into DC. Materials and Methods: We have made use of the CD38- AML derived cell line MUTZ-3 which contains a subpopulation of CD34+CLL1+ cells which resembles the phenotype of a putative LSC. CLL1+CD34+ and CLL1-CD34- cells were isolated by FACS sorting and total RNA was isolated. mRNA was converted to cDNA and amplified by PCR using the SMART system. Subsequently, mRNA was in vitro transcribed from the amplified cDNA. Mature monocyte derived DC (MoDC) were generated from healthy donor blood and transfected with amplified CLL1+CD34+ derived mRNA and used to stimulate autologous CD8β+ T-cells. After three weekly re-stimulations with CLL1+CD34+ mRNA transfected DC, specificity of the T-cells was analyzed by intracellular IFNγ staining upon 5 hour stimulation with autologous immature MoDC transfected with GFP mRNA, mRNA amplified from unsorted, CLL1+CD34+ or CLL1-CD34- MUTZ-3 subpopulations. Results: Amplification of CLL1 and survivin (also expressed by MUTZ-3) transcripts was confirmed by RT-PCR. After 3 weekly re-stimulations with CLL1+CD34+ amplified RNA transfected DC, 0.04% (range 0.01-0.12%) of the T-cells were positive for IFNγ upon a 5 hr re-stimulation with GFP transfected DC. 0.44% (range 0.04-0.69%) of the T-cells responded to DC transfected with unsorted MUTZ-3 amplified mRNA (p<0.00005 versus GFP control, 2-sided student's T-test), 0.51% (range 0.24-1.35%) responded to DC transfected with CLL1+CD34+ amplified mRNA (p<0.005 versus GFP control) and 0.46% (range 0.24-0.94%) responded to DC transfected with CLL1-CD34- amplified mRNA (p<0.0001 versus GFP control). Conclusion: We show that MoDC transfected with RNA amplified from one MUTZ-3 sub-population resembling the phenotype of LCS cells are capable of inducing T-cells which recognize both cells transfected with mRNA from the LSC resembling MUTZ-3 subset as well as the CLL1-CD34- subset. We are currently testing the efficacy and feasibility of this approach in an autologous setting in vitro. CD8β+ T-cells are stimulated with autologous MoDC from AML patients transfected with amplified mRNA isolated from their own LSC enriched populations. The capacity of these T-cells to kill autologous AML blasts and LSC is subsequently analysed in a 6-colour FACS based cytotoxicity assay. Disclosures: No relevant conflicts of interest to declare.

Screen for Small Molecules Capable of Expanding Human Hematopoietic Stem Cell Ex Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1919-1919
Author(s):  
Iman Hatem Fares ◽  
Jalila Chagraoui ◽  
Jana Krosl ◽  
Denis-Claude Roy ◽  
Sandra Cohen ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Fold Increase ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 1919 Hematopoietic stem cell (HSC) transplantation is a life saving procedure whose applicability is restricted by the lack of suitable donors, by poor responsiveness to mobilization regimens in preparation of autologous transplantations, by insufficient HSC numbers in individual cord blood units, and by the inability to sufficiently amplify HSCs ex vivo. Characterization of Stemregenin (SR1), an aryl hydrocarbon receptor (AHR) antagonist that promotes HSC expansion, provided a proof of principle that low molecular weight (LMW) compounds have the ability to promote HSC expansion. To identify novel putative agonists of HSC self-renewal, we initiated a high throughput screen (HTS) of a library comprising more than 5,000 LMW molecules using the in vitro maintenance of the CD34+CD45RA- phenotype as a model system. Our study was based on the fact that mobilized peripheral blood-derived CD34+CD45RA- cells cultured in media supplemented with: stem cell factor, thrombopoietin, FLT3 ligand and interleukin 6, would promote the expansion of mononuclear cells (MNC) concomitant with a decrease in CD34+CD45RA- population and HSC depletion. LMW compounds preventing this loss could therefore act as agonists of HSC expansion. In a 384-well plate, 2000 CD34+cells were initially cultured/well in 50μl medium comprising 1μM test compounds or 0.1% DMSO (vehicle). The proportions of CD34+CD45RA− cells were determined at the initiation of experiment and after a 7-day incubation. Six of 5,280 LMW compounds (0.11%) promoted CD34+CD45RA− cell expansion, and seventeen (0.32%) enhanced differentiation as determined by the increase in proportions of CD34−CD45RA+ cells compared to control (DMSO). The 6 LMW compounds promoting expansion of the CD34+CD45RA− cell population were re-analyzed in a secondary screen. Four out of these 6 molecules suppressed the transcriptional activity of AHR, suggesting that these compounds share the same molecular pathway as SR1 in stimulating HSC expansion, thus they were not further characterized. The remaining 2 compounds promoted, similar to SR1 or better, a 10-fold and 35-fold expansion of MNC during 7 and 12-day incubations, respectively. The expanded cell populations comprised 65–75% of CD34+ cells compared to 12–30% determined for DMSO controls. During 12-day incubation with these compounds, the numbers of CD34+ cells increased ∼25-fold over their input values, or ∼ 6-fold above the values determined for controls. This expansion of CD34+ cells was associated with a ∼5-fold increase in the numbers of multilineage CFC (granulocyte, erythroid, monocyte, and megakaryocyte, or CFU-GEMM) compared to that found in DMSO control cultures. The ability of the 2 newly identified compounds to expand functional HSCs is currently being evaluated in vivo usingimmunocompromised mice. In conclusion, results of our initial screen suggest that other mechanism, besides inhibition of AhR, are at play for expansion of human HSC. Disclosures: No relevant conflicts of interest to declare.

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