Deciphering the Mechanisms of Osteoblast-Induced Resistance of Leukemic Stem Cell (LSC) in Ph+ CML: Role of PI3-Kinase, BRD4 and MYC and Development of Strategies to Overcome Osteoblast-Induced Resistance

Abstract Chronic myeloid leukemia (CML) is a stem cell neoplasms characterized by the chromosome translocation t(9;22) and the related BCR-ABL1 fusion gene. Therapy with BCR-ABL1 kinase inhibitors is highly effective in the treatment of CML and deep molecular responses are achieved in most patients. However, not all patients respond to these drugs due to resistance of leukemic stem cells (LSC). Recent data suggest that the disease-related microenvironment, known as the stem cell niche contributes to drug resistance and relapse in CML. So far, little is known about the resistance mechanisms protecting niche cells in the bone marrow of patients with CML. We have recently shown that osteoblasts are a major site of LSC-mediated resistance against BCR-ABL1-targeting drugs in CML. In the current study, we screened for drugs that are able to suppress growth and viability of osteoblasts and/or other niche-related cells and can thereby overcome drug resistance of CML LSC. Proliferation was analyzed by determining 3H-thymidine uptake in niche-related cells and apoptosis was measured by Annexin-V/DAPI-staining and flow cytometry. We found that the dual PI3 kinase (PI3K) and mTOR inhibitor BEZ235 and the selective pan-PI3K inhibitor copanlisib suppress proliferation of primary osteoblasts (BEZ235 IC 50: 0.05 µM; copanlisib IC 50: 0.05 µM), the osteoblastic cell line CAL-72 (BEZ235 IC 50: 0.5 µM; copanlisib IC 50: 1 µM), primary human umbilical vein endothelial cells (BEZ235 IC 50: 0.5 µM; copanlisib IC 50: 0.5 µM) and the endothelial cell line HMEC-1 (BEZ235 IC 50: 1 µM; copanlisib IC 50: 1 µM), whereas no comparable effects were seen with the mTOR inhibitor rapamycin. As determined by flow cytometry, BEZ235 and copanlisib suppressed the expression of phosphorylated (p) pAKT and pS6 in endothelial cells and osteoblasts whereas rapamycin downregulated pS6 expression but did not decrease expression of pAKT. Moreover, we found that BEZ235 and copanlisib cooperate with nilotinib and ponatinib in suppressing growth and viability of osteoblasts and endothelial cells. Furthermore, BEZ235 and copanlisib were found to overcome osteoblast-induced resistance of K562, KU812 cells, and primary CD34 +/CD38 − CML LSC against nilotinib and ponatinib. This effect was also seen when CAL-72 cells were first exposed to BEZ235 or copanlisib and washed before co-cultures with CML cells and BCR-ABL1 inhibitors were prepared, suggesting that osteoblast inhibition was a crucial event capable of disrupting LSC resistance in these co-cultures. Of all other drugs tested, only the BRD4-targeting drug JQ1 was found to suppress CAL72-induced resistance in the CML cell lines KU812 and K562, suggesting that osteoblast-induced resistance of CML cells is also mediated by a BRD4-MYC pathway. In a next step, we examined the expression of resistance-mediating immune checkpoint molecules on CML cells (KU812, K562, LSC) and on osteoblasts by flow cytometry. We found that CML cells and CAL72 cells constitutively express PD-L1 and that interferon-gamma (IFN-G) promotes the expression of PD-L1 in all cell types tested. Moreover, we found that the BRD4 blocker JQ1 and the BRD4-degrader dBET6 suppress the IFN-G-induced upregulation of PD-L1 in CML LSC and osteoblasts. In conclusion, our data show that osteoblast-induced resistance of CML stem cells is mediated by a PI3K-dependent pathway and BRD4/MYC, and that BRD4-inhibition or BRD4-degradation counteracts osteoblast-induced resistance of CML (stem) cells against BCR-ABL1 inhibitors and PD-L1 expression on CML LSC and osteoblasts. We hypothesize that checkpoint inhibition may assist in drug-induced eradication of CML LSC and thus in the development of curative drug therapies in Ph + CML. Disclosures Hoermann: Novartis: Honoraria. Gleixner: Pfizer: Honoraria; Abbvie: Honoraria; BMS: Honoraria; Incyte: Honoraria; Novartis: Honoraria. Sperr: AbbVie, BMS-Celgene, Daiichi Sankyo, Deciphera, Incyte, Jazz, Novartis, Pfizer, StemLine, Thermo Fisher: Honoraria, Research Funding. Valent: Novartis: Honoraria; Pfizer: Honoraria, Research Funding; Celgene/BMS: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; OAP Orphan Pharmaceuticals: Honoraria.

Download Full-text

How to Overcome Myeloma Stem Cell Resistance to Therapy - Targeting the Stem Cell Niche

Blood ◽

10.1182/blood.v116.21.sci-6.sci-6 ◽

2010 ◽

Vol 116 (21) ◽

pp. SCI-6-SCI-6

Author(s):

Constantine S. Mitsiades

Keyword(s):

Stem Cells ◽

Drug Resistance ◽

Stem Cell ◽

Cancer Stem Cells ◽

Cancer Stem Cell ◽

Tumor Cells ◽

Cell Lines ◽

Cell Population ◽

Research Funding ◽

Accessory Cells

Abstract Abstract SCI-6 The concept of cancer stem cells has attracted again intense research interest, as the drug resistance attributed to this infrequent subpopulation of tumor cells could explain how patients can relapse even after prolonged complete clinical, biochemical, radiologic or even molecular remissions. In multiple myeloma (MM), several aspects of the cancer stem cell concept remain to be elucidated, including the potential heterogeneity of this cell subpopulation or whether CD138+ expression is incompatible or not with a MM stem cell. As these questions are being resolved, emerging data highlight that the drug resistance of MM cells with clonogenic/stem cell-like features is heavily influenced by interactions with non-malignant accessory cells of the local microenvironment, including bone marrow stromal cells (BMSCs). Indeed, transcriptional signatures of “stemness”, as identified in normal stem cells or cancer stem cells from other neoplasias, are detected in the bulk population of MM cell lines and are upregulated after MM cell interaction with BMSCs. MM cell lines and primary tumor cells contain subpopulations with clonogenic potential, such as the side population (SP) cells. SP cells, detected by low intracellular accumulation of Hoechst 33342 dye (in contrast to the tumor's “main population” (MP), are considered an enriched source of tumor-initiating cells in diverse neoplasias and were detected among CD138− CD20+ CD27+ clonogenic cells in primary MM samples. Interaction with BMSCs increases the viability of SP cells and their percentage within the MM cell population. While interaction with BMSCs or other accessory cells of the microenvironmental niche suppresses the anti-MM activity of glucocorticoids, conventional chemotherapeutics and certain investigational agents, other agents (e.g. immunomodulatory thalidomide derivatives (IMIDs), such as lenalidomide) have increased activity against MM SP cells in the context of this tumor-microenvironment interaction. These observations suggest that MM cells with stem cell-like features exhibit functional plasticity depending on which specific microenvironmental niche they interact with. The Hedgehog, Wnt and Notch pathways, as well as regulators of chromatin remodeling, e.g. histone demethylases, have emerged as putative links between drug resistance, “cancer stemness” and how these functional outcomes are modulated by the local microenvironment in MM. These pathways, as well as embryonic stem cell-associated antigens (e.g. SOX2), represent intriguing targets for investigational therapies. However, clinical translation of such treatments has notable challenges, as conventional criteria for response assessment may not accurately reflect the treatment's impact on clonogenic tumor cells. Progression-free survival is considered a more appropriate endpoint for cancer stem-cell targeting agents, its assessment, however, may be confounded without concomitant treatment that suppresses the bulk of the tumor. Consequently, candidate cancer stem cell-targeting agents may have to be evaluated in combination with regimens (including lenalidomide-bortezomib-Dex (RVD) or other combinations built around the therapeutic “backbone” of proteasome inhibition and IMIDs) which potently target the bulk MM cell population and induce high rates of complete/near complete responses. Further improvements of this dual targeting of clonogenic and bulk tumor cells may be facilitated by recently developed high-throughput platforms (e.g. compartment-specific bioluminescence imaging, CS-BLI) which screen, in the presence vs. absence of stroma or other accessory cells, large numbers of anti-tumor agents and combinations thereof against the bulk tumor cell population or its clonogenic compartments. These new platforms will inform the rational design of regimens that will hopefully improve the long-term outcome of MM patients by suppressing a clonogenic/stem cell-like tumor compartment and circumventing microenvironment-dependent drug resistance. Disclosures: Mitsiades: Millennium: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals: Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; Centrocor: Consultancy, Honoraria; PharmaMar: Patents & Royalties; OSI Pharmaceuticals: Research Funding; Amgen Pharmaceuticals: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis: Research Funding; Gloucester Pharmaceuticals: Research Funding.

Download Full-text

167 ISOLATION AND COMPARATIVE PROFILING OF HUMAN ADIPOSE-DERIVED ADULT STEM CELLS

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab167 ◽

2005 ◽

Vol 17 (2) ◽

pp. 234

Author(s):

A. Boquest ◽

A. Shahdadfar ◽

K. Fronsdal ◽

J. Brinchmann ◽

P. Collas

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Flow Cytometry ◽

Endothelial Cells ◽

Adipose Tissue ◽

Stem Cell ◽

Regenerative Medicine ◽

Cell Signaling ◽

Proliferative Capacity ◽

Stromal Stem Cells

The stromal compartment of mesenchymal tissues is thought to harbor stem cells that display extensive proliferative capacity and multilineage potential. However, despite their potential impact in the field of regenerative medicine, little is known about the biology of stromal stem cells prior to culture. After removing adipocytes and erythrocytes from collagenase digested human adipose tissue, we identified two cell populations using flow cytometry which shared expression of stem cell markers SH2 and CD34, but lacked the phenotypic characteristics of leukocytes (CD45−). However, they were found to be discernible based on CD31 expression, a marker for endothelial cells. Using CD31 conjugated magnetic beads, we separated these cells (CD45-CD31− and CD45-CD31+) from three patients and compared global gene expression profiles using an Affymetrix platform. The prominant feature of CD45-CD31+ cells was the up-regulation of genes associated with endothelial cells. By contrast, CD45-CD31− cells were found to overexpress transcripts involved in cell cycle quiescence and cell signaling elements including those of the WNT pathway thought to be important for maintaining the stem cell state. Upon culture in DMEM/F12 with 20% FCS, only CD45-CD31− cells were capable of adhering to plastic and forming colonies. These cells with fibroblastic morphology met the key criterion of stem cells, the ability to proliferate while retaining the capacity to differentiate into mature tissues. Under appropriate inductive conditions, they were found to exclusively form bone, cartilage, adipose and neuronal-like tissues in vitro. Clonal cell lines generated from individually cultured CD45-CD31− cells displayed multilineage and proliferative capacity, validating our conclusion that they are true stem cells and not simply committed progenitors. We then undertook extensive comparative profiling of CD45-CD31− cells with their cultured counterparts to examine changes that stromal stem cells undergo during culture. Except for the disappearance of CD34, flow cytometry analysis using 52 antibodies revealed little change in cell surface phenotype as a result of culture. However, comparative global gene profiling revealed extensive down-regulation of many genes during culture. These included cell cycle arresting genes, as expected, and genes encoding elements involved in cell signaling including those belonging to the tumor necrosis factor, interleukin, transforming growth factor and chemokine families. The consequences of these changes remain unknown, but ultimately may affect the potential use of adipose tissue stem cells in regenerative medicine.

Download Full-text

Leukemia Stem Cells Are Characterized By CLEC12A Expression and Chemotherapy Refractoriness That Can be Overcome By Targeting with Chimeric Antigen Receptor T Cells

Blood ◽

10.1182/blood.v128.22.766.766 ◽

2016 ◽

Vol 128 (22) ◽

pp. 766-766 ◽

Cited By ~ 6

Author(s):

Saad S Kenderian ◽

Marco Ruella ◽

Olga Shestova ◽

Michael Klichinsky ◽

Miriam Y Kim ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

Stem Cell ◽

Single Dose ◽

Cell Line ◽

Induction Chemotherapy ◽

Chimeric Antigen Receptor ◽

Research Funding ◽

Tail Vein Injection ◽

Equity Ownership

Abstract Chemo-refractory acute myeloid leukemia (AML) is associated with poor prognosis and treatment options are extremely limited. Most of these patients are ineligible for allogeneic stem cell transplantation. Chemo-refractory AML is thought to arise due to selection pressure of resistant clones from prior use of chemotherapy or in some cases pre-exist due to properties of the leukemic stem cells (LSC). CLEC12A (also known as CLL1) has previously been described as being selectively over expressed in LSCs. Successful modalities to target CLEC12A and eradicate the LSC would overcome chemo-refractoriness in AML and would represent a vertical advance in the field. In this study, we confirm that CLEC12A is heterogenously expressed on AML blasts and over-expressed on AML LSC. We also show that CLEC12A is overexpressed on bone marrows from patients with AML that fail to achieve a complete remission after induction chemotherapy, suggesting that it could be a marker for residual disease that is refractory to chemotherapy. We then separated AML blasts into CLEC12A positive or negative cells by magnetic sorting. CLEC12A positive blasts selected from AML patients were more resistant to chemotherapy compared to CLEC12A negative blasts (20% killing of CLEC12A positive AML cells versus 43% of CLEC12A negative AML cells when cultured with cytarabine 10 µg/ml, P=0.01). This finding was confirmed by using the AML MOLM14 cell line engineered to overexpress CLEC12A. CLEC12Ahigh MOLM14 cells were more resistant to chemotherapy compared to wild type MOLM14 cells (P=0.003). We then evaluated CLEC12A resistance to chemotherapy in a patient derived AML xenograft model. We found a relative increase in CLEC12A positive cells post Ara-C induction chemotherapy in AML xenograft models (Figure 1). The observation that CLEC12A positive cells are more resistant to chemotherapy provided a solid rationale to target CLEC12A with chimeric antigen receptor T (CART) cells. We therefore developed a second generation CLEC12A directed CAR construct using CD3z and 41BB costimulatory domains and generated CLEC12A CART cells by lentiviral transduction with this construct. Upon incubation with primary AML samples or AML cell lines, CLEC12A CART cells resulted in modest effector functions, due to the heterogeneity of CLEC12A expression on AML blasts. However when CLEC12A overexpressed MOLM14 cell line or CLEC12Apos selected leukemic cells were used as targets, CLEC12A-CART cells resulted in potent cytotoxicity, proliferation and cytokine production, indicating that CLEC12A-CART cells are more specific for LSC. To test the in vivo anti-leukemic activity of CLEC12A CARTs, we used primary human AML blasts xenografted into NSG-S mice (NOD-SCID-γc-/-, additionally transgenic for human stem cell factor, IL3 and GM-CSF). Treatment with CLEC12A CART (single dose, 1x105 total T cells via tail vein injection) resulted in modest activity against AML when employed as monotherapy. To investigate the potential role of CLEC12A CART cells in eradication of MRD and LSC, mice were treated first with chemotherapy (cytarabine 60 mg/kg intraperitoneal injection daily for 5 days) followed by a single dose (1x105 total T cells via tail vein injection) of either CLEC12A CARTs or control untransduced T cells (UTD). Treatment with CLEC12A CART cells resulted in eradication of leukemia and prolonged survival in these mice (overall survival at 200 days of 100% after CLEC12A CARTs compared to 20% after UTD, p=0.01, Figure 2). In conclusion, our preclinical studies reveal that CLEC12A positive cells in leukemia are resistant to chemotherapy and can be successfully targeted with CART cells. CLEC12A CART cells can potentially be employed as a consolidation regimen after induction chemotherapy to eradicate LSC and MRD in AML. Disclosures Kenderian: Novartis: Patents & Royalties, Research Funding. Ruella:novartis: Patents & Royalties: Novartis, Research Funding. Singh:Novartis: Employment. Richardson:Novartis: Employment, Patents & Royalties, Research Funding. June:Tmunity: Equity Ownership, Other: Founder, stockholder ; Immune Design: Consultancy, Equity Ownership; Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; University of Pennsylvania: Patents & Royalties; Celldex: Consultancy, Equity Ownership; Johnson & Johnson: Research Funding; Pfizer: Honoraria. Gill:Novartis: Patents & Royalties, Research Funding.

Download Full-text

Identifying the Leukemia Stem Cell Niche Mediating Drug Resistance in Flt3-ITD Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2019-124379 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 909-909

Author(s):

Nick R Anderson ◽

Hui Li ◽

Mason W Harris ◽

Shaowei Qiu ◽

Andrew J Paterson ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Drug Resistance ◽

Endothelial Cells ◽

Mesenchymal Stem Cells ◽

Stem Cell ◽

Conflicts Of Interest ◽

Single Agent ◽

Fold Increase ◽

Combination Regimen

The Flt3-ITD is one of the most common mutations in AML, and defines a distinct subtype of disease with unique features and biology, and with poor prognosis related to high rates of recurrence. Although several FLT3 TKIs have been developed for clinical use, responses are limited and are not sustained. The objective of our study was to determine the contribution of bone marrow stromal populations to LSC drug resistance to Flt3-targeted TKI in Flt3-ITD AML. We utilized a newly generated Flt3-ITD TET2flox/flox Mx1-cre mouse model of AML to identify phenotypic populations with leukemia initiating capacity (LIC) in Flt3-ITD AML. Administration of pIpC leads to deletion of TET2 and development of AML-like disease characterized by leukocytosis, accumulation of blasts, anemia and thrombocytopenia. Transplantation of selected STHSC, MPP and GMP populations revealed that LIC were almost exclusively found within the phenotypic ST-HSC population (calculated stem cell frequencies: <1:180,000, 1:63,635, and 1:2,730 for GMP, MPP, and ST-HSC, respectively). We similarly found that in samples from human Flt3-ITD AML patients LIC capacity was similarly restricted to more primitive HSPC populations (Lin-CD34+CD38-) and was not seen in committed GMP (Lin-CD34+CD38+CD123+CD45RA+). We performed flow cytometry on collagenase-digested bone fragments from AML mice to characterize bone marrow stromal cells in Flt3-ITD AML mice. We also transplanted murine AML cells into CXCL12-GFP mice to assess alterations in CXCL12-expressing stromal populations in AML bone marrow. We found that several stromal populations are expanded in AML vs. WT mice, including a 3.5-fold increase in mesenchymal stem cells (CD45-Ter119-CD31-VECadherin-Sca1+CD51+), a 3.9-fold increase in endothelial cells (CD45-Ter119-CD31+), and a 1.5-fold increase in osteoprogenitors (CD45-Ter119-CD31-VECadherin-Sca1-CD51+). The expression of CXCL12, a key factor that mediates niche localization of HSC and LSC, was greater than 2-fold higher in osteoprogenitors, but not significantly different in endothelial cells, and 2-fold lower in mesenchymal stem cells in AML vs. WT mice. We also showed that Flt3-ITD AML HSPCs have nearly 2-fold higher CXCR4 expression than WT HSPCs. These data taken together supported further exploration of the role of a CXCL12-expressing osteoprogenitor niche in supporting Flt3-ITD AML LSC. We transplanted murine AML cells into CXCL12flox/flox UBC-cre or CXCL12flox/flox Osx-cre mice to assess the effect of global or osteoprogenitor-specific CXCL12-KO, respectively, on AML progression and TKI response. We found that this model of AML was resistant to single agent Flt3 TKI (AC220) treatment. Global CXCL12-KO using CXCL12flox/flox UBC-cre mice modestly improved response to TKI. We show that a combination regimen including standard-of-care "7+3" chemotherapy (cytarabine + doxorubicin) and a Flt3 TKI (AC220) results in more effective and selective targeting of leukemia cells in this model. We are currently treating osteoprogenitor-specific CXCL12-KO AML mice with the combination chemotherapy + TKI regimen to investigate the contribution of osteoprogenitors to disease progression and drug resistance in Flt3-ITD AML LSC. In conclusion, our results suggest that LSC in Flt3-ITD AML are present within a primitive phenotypic ST-HSC population more so than in MPP and GMP populations as often seen in some other types of AML. Our studies support a potential role for a CXCL12-expressing osteoprogenitor niche in supporting Flt3-ITD AML LSC growth and drug resistance, targeting of which could improve responses and outcomes in Flt3-ITD AML. Disclosures No relevant conflicts of interest to declare.

Download Full-text

High Throughput Drug Screening of Leukemia Stem Cells Reveals Resistance to Standard Therapies and Sensitivity to Other Agents in Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-180 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 180-180

Author(s):

Frances Linzee Mabrey ◽

Sylvia S Chien ◽

Timothy S Martins ◽

James Annis ◽

Taylor S Sekizaki ◽

...

Keyword(s):

Stem Cells ◽

Drug Resistance ◽

Stem Cell ◽

High Throughput ◽

Research Funding ◽

Drug Susceptibility ◽

Patient Specific ◽

Leukemia Stem Cells ◽

Leukemia Stem Cell ◽

Susceptibility Patterns

Abstract Background: Leukemia stem cells (LSCs) play a critical role in AML propagation and relapse. Other investigators have also highlighted unique gene expression profiles for the leukemia stem cell population. Here we compared the results of in vitro drug sensitivity testing against a custom panel of drugs and drug combinations for blast populations vs. leukemia stem cell populations derived from the same patients, as well as mutation analysis for a panel of 194 recurrently mutated genes in AML. Patients and Methods: Patient AML samples were obtained with IRB approval. LSCs were isolated by fluorescence-activated cell sorting (FACS) and the blast population enriched to >90% using immunomagnetic beads from blood samples from 5 patients with AML. A sixth AML patient sample was used for NOD/SCID IL2R γc−/− engraftment, in order to compare characteristics of pre- and post-engraftment subclones. Our CLIA approved custom assay includes 153 drugs and targeted agents, both FDA approved and investigational, with additional drug combinations. High throughput screens (HTS) were conducted with enriched cells adherent to matrix protein in 384 well plates with 8 concentrations of each drug spanning 4 logs. Viability was assessed with CellTiter-Glo (Promega). HTS were performed on LSCs, blasts and pre- as well as post-engraftment AML subclones from the xenograft. Dose-response curves were generated to calibrate drug resistance patterns. Mutation analysis by NGS for a panel of 194 recurrently mutated genes in AML (MyAML®) including 37 translocations was also conducted for the LSC and blast populations. Results: AML blasts and LSCs exhibited divergent drug susceptibility patterns (see volcano plot in Figure). Of 11 drugs commonly used in AML, 8 were typical chemotherapy drugs. Five of these compounds were effective against blasts, but none were effective against LSCs (p-value: 0.0256), suggesting a possible mechanism for post-treatment relapse or primary refractoriness. LSCs were also resistant to mitomycin-C, an agent that induces DNA interstrand crosslinks and DNA breaks, in contrast to blasts that were variably sensitive. Of note, we identified 12 drugs from 8 classes defined by mechanism of action that may target LSCs, in some cases preferentially, when compared with blasts. Drugs effective in preferentially targeting LSCs included tyrosine kinase inhibitors, histone deacetylase inhibitors, 1 cyclin-dependent kinase inhibitor, 1 proteasome inhibitor and 1 microtubule assembly inhibitor. Several of the drugs that efficiently killed LSCs have been studied clinically in AML, while others have theoretical or established efficacy against LSCs by drug class. Only one commonly used drug in AML, sorafenib, a multikinase inhibitor used in FLT3+ disease that may improve survival in younger patients, was effective against LSCs. Blast specific drugs include romidepsin, dinaciclib, alvocidib, ganetespib, selinexor, dorsomorphin, vinblastine, cladribine, dabrafenib, selumetinib, etoposide, torkinib and those in Figure. Blast and LSC drug susceptibility patterns were distinct for each patient. Further, the engrafted xenograft subclone grew very rapidly, was resistant to standard chemotherapy, and possessed three new deleterious mutations in KMT2C (2), SF3B1 and 1 possibly damaging mutation in NUP214, suggesting possible genetic contributions to chemotherapy resistance. We also compared mutation profiles for LSCs vs. blasts in 5 patients, and identified LSC specific mutations in WNK3, WNK4 and BUB1, each in 2 of the 5, and there were also other mutations that were LSC or blast specific. Of note, Bub1 is a mitotic checkpoint serine/threonine kinase that controls mitosis in cancer stem cells (Venere et al Cancer Discov. 2013). WNK3 and WNK4 also both encode serine/threonine protein kinases. Conclusions: The distinct drug susceptibility patterns of patient-specific LSC and blast populations highlight the potential of an individualized approach to treat AML. LSCs are resistant to S-phase agents used in standard-of-care chemotherapy. Genetically distinct minority resistant LSC subclones present at diagnosis may grow rapidly under some conditions, and contribute to drug resistance and relapse. Incorporating the results of functional drug screening focused on LSC subclones may allow more individualized treatment of AML patients and identify patient-specific therapies that lead to improved outcomes. Figure Figure. Disclosures Carson: Invivoscribe Inc.: Employment. Patay:Invivoscribe Inc.: Consultancy, Equity Ownership, Patents & Royalties. Becker:Novartis: Research Funding; Trovagene: Research Funding; CVS Caremark: Consultancy; JW Pharmaceuticals: Research Funding; Rocket Pharmaceuticals: Research Funding; Pfizer: Consultancy; Amgen: Research Funding; BMS: Research Funding; Abbvie: Research Funding; GlycoMimetics: Research Funding.

Download Full-text

Comparison Of Autologous Microparticles and Fucosyltransferase-7 For Adhesion Improvement Of Hematopoietic Stem Cells To Bone Marrow Endothelial Cells In a Microfluidic Flow Chamber

Blood ◽

10.1182/blood.v122.21.2000.2000 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2000-2000

Author(s):

Arne Trummer ◽

Dennis Rataj ◽

Sonja Werwitzke ◽

Andreas Tiede

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Flow Cytometry ◽

Endothelial Cells ◽

Stem Cell ◽

Flow Chamber ◽

Sialyl Lewis X ◽

Study Phase ◽

Hematopoietic Stem ◽

Microfluidic Flow

Abstract The improvement of graft function and time to engraftment might help to reduce infection-related mortality in stem cell transplantation (SCT). While the concept of stem cells fucosylation for accelerated engraftment has already reached clinical study phase (for cord blood transplantation; NCT01471067), own previous work has shown an association between engraftment time and circulating microparticles bearing P-Selectin and P-Selectin glycoprotein ligand 1 (PSGL-1). PSGL-1 contains the sialyl Lewis x (CD15s) antigen that requires fucosylation for optimal binding of P- and E-Selectin on endothelial cells. We therefore hypothesized that addition of microparticles (MP) might enhance adhesion of human stem cells (HSC) to bone marrow endothelial cells and that MP might have synergistic effects in combination with stem cell fucosylation. HSC were obtained from apheresis products of allogeneic donors, purified by Ficoll and magnetic bead separation for CD34, stained with calcein AM and perfused through an automated microfluidic flow chamber (Bioflux 200, Fluxionbio, USA) covered with a confluent layer of an immortalized human bone marrow endothelial cell line (HBMEC). Photos (and videos) were taken using a fluorescence microscope at start, 5 min and 10 min and analyzed for adherent HSC across the whole chamber (about 1.5 sqmm) using ImageJ software. Autologous MP were generated by addition of calcimycin to apheresis and isolation of MP by centrifugation. For control experiments, one part of the MP solution was passed through a 0,2µm-filter to remove MP. MP concentration (mean: 1362/µl) was assessed by detection of Annexin V binding in flow cytometry, using TrucountBeads® for quantification. Fucosylation was performed by 1h incubation of isolated CD34+ stem cells with GDP-fucose and fucosyltransferase 7 (FUT7). Successful fucosylation was controlled by CD15s staining of HSC in flow cytometry. Results of seven experiments (in duplicate) demonstrated the highest number of adherent HSC in the MPpositiv/FUT7negativ preparation (median: 32 HSC/sqmm; range: 15-78), followed by MPpositiv/FUT7positiv (30 HSC/sqmm; range: 16-38), MPnegativ/FUT7positiv (median: 25/sqmm; range: 11-27) and MPnegativ/FUT7negativ (20 HSC/sqmm; range: 0-22). Comparison of the MPpositiv/FUT7negativ and MPnegativ/FUT7negativ as well as the MPpositiv/FUT7positiv and MPpositiv/FUT7negativ preparations showed statistically significant differences in Wilcoxon rank test (p<.05) while comparison of MPpositiv/FUT7positiv vs. MPnegativ/FUT7positiv and MPnegativ/FUT7positiv vs. MPnegativ/FUT7negativ preparations did not. In summary, these results demonstrate that MP can improve HSC adhesion to bone marrow endothelial cells similar to fucosylation. The effect of fucosylation on HSC adhesion appears to be mediated by MP. However, there is not a synergistic effect between MP and fucosylation. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

In vitro Three Dimensional Scaffold-free Construct of Human Adipose-derived Stem Cells in Coculture with Endothelial Cells and Fibroblasts

Revista de Chimie ◽

10.37358/rc.17.6.5670 ◽

2017 ◽

Vol 68 (6) ◽

pp. 1341-1344

Author(s):

Grigore Berea ◽

Gheorghe Gh. Balan ◽

Vasile Sandru ◽

Paul Dan Sirbu

Keyword(s):

Tissue Engineering ◽

Stem Cells ◽

Endothelial Cells ◽

Single Cell ◽

Cell Line ◽

Bone Repair ◽

Umbilical Vein ◽

Human Fibroblasts ◽

Three Dimensional ◽

Adipose Derived Stem Cells

Complex interactions between stem cells, vascular cells and fibroblasts represent the substrate of building microenvironment-embedded 3D structures that can be grafted or added to bone substitute scaffolds in tissue engineering or clinical bone repair. Human Adipose-derived Stem Cells (hASCs), human umbilical vein endothelial cells (HUVECs) and normal dermal human fibroblasts (NDHF) can be mixed together in three dimensional scaffold free constructs and their behaviour will emphasize their potential use as seeding points in bone tissue engineering. Various combinations of the aforementioned cell lines were compared to single cell line culture in terms of size, viability and cell proliferation. At 5 weeks, viability dropped for single cell line spheroids while addition of NDHF to hASC maintained the viability at the same level at 5 weeks Fibroblasts addition to the 3D construct of stem cells and endothelial cells improves viability and reduces proliferation as a marker of cell differentiation toward osteogenic line.

Download Full-text

Synergy of molecularly mobile polyrotaxane surfaces with endothelial cell co-culture for mesenchymal stem cell mineralization

RSC Advances ◽

10.1039/d1ra01296g ◽

2021 ◽

Vol 11 (30) ◽

pp. 18685-18692

Author(s):

Hiroki Masuda ◽

Yoshinori Arisaka ◽

Masahiro Hakariya ◽

Takanori Iwata ◽

Tetsuya Yoda ◽

...

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Mesenchymal Stem Cells ◽

Stem Cell ◽

Endothelial Cell ◽

Mesenchymal Stem Cell ◽

Molecular Mobility ◽

Culture System

Molecular mobility of polyrotaxane surfaces promoted mineralization in a co-culture system of mesenchymal stem cells and endothelial cells.

Download Full-text

A selective cytotoxic adenovirus vector for concentration of pluripotent stem cells in human pluripotent stem cell-derived neural progenitor cells

Scientific Reports ◽

10.1038/s41598-021-90928-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Takamasa Hirai ◽

Ken Kono ◽

Rumi Sawada ◽

Takuya Kuroda ◽

Satoshi Yasuda ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Stem Cell ◽

Progenitor Cells ◽

Pluripotent Stem Cells ◽

Pluripotent Stem Cell ◽

Neural Progenitor Cells ◽

Neural Progenitor ◽

Sensitive Detection ◽

Quality And Safety

AbstractHighly sensitive detection of residual undifferentiated pluripotent stem cells is essential for the quality and safety of cell-processed therapeutic products derived from human induced pluripotent stem cells (hiPSCs). We previously reported the generation of an adenovirus (Ad) vector and adeno-associated virus vectors that possess a suicide gene, inducible Caspase 9 (iCasp9), which makes it possible to sensitively detect undifferentiated hiPSCs in cultures of hiPSC-derived cardiomyocytes. In this study, we investigated whether these vectors also allow for detection of undifferentiated hiPSCs in preparations of hiPSC-derived neural progenitor cells (hiPSC-NPCs), which have been expected to treat neurological disorders. To detect undifferentiated hiPSCs, the expression of pluripotent stem cell markers was determined by immunostaining and flow cytometry. Using immortalized NPCs as a model, the Ad vector was identified to be the most efficient among the vectors tested in detecting undifferentiated hiPSCs. Moreover, we found that the Ad vector killed most hiPSC-NPCs in an iCasp9-dependent manner, enabling flow cytometry to detect undifferentiated hiPSCs intermingled at a lower concentration (0.002%) than reported previously (0.1%). These data indicate that the Ad vector selectively eliminates hiPSC-NPCs, thus allowing for sensitive detection of hiPSCs. This cytotoxic viral vector could contribute to ensuring the quality and safety of hiPSCs-NPCs for therapeutic use.

Download Full-text

Effect of Placental Extract on Omentum Mesenchymal Stem Cells Differentiation in NMRI Mice

Journal of Molecular Biology Research ◽

10.5539/jmbr.v7n1p176 ◽

2017 ◽

Vol 7 (1) ◽

pp. 176

Author(s):

Maryam Sadat Nezhadfazel ◽

Kazem Parivar ◽

Nasim Hayati Roodbari ◽

Mitra Heydari Nasrabadi

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Flow Cytometry ◽

Endothelial Cells ◽

Mesenchymal Stem Cells ◽

Control Group ◽

Fat Cell ◽

Nmri Mice ◽

Differentiated Cells ◽

Placental Extract

Omentum mesenchymal stem cells (OMSCs) could be induced to differentiate into cell varieties under certain conditions. We studied differentiation of OMSCs induced by using placenta extract in NMRI mice. Mesenchymal stem cells (MSCs) were isolated from omentum and cultured with mice placenta extract. MSCs, were assessed after three passages by flow cytometry for CD90, CD44, CD73, CD105, CD34 markers and were recognized their ability to differentiate into bone and fat cell lines. Placenta extract dose was determined with IC50 test then OMSCs were cultured in DMEM and 20% placenta extract.The cell cycle was checked. OMSCs were assayed on 21 days after culture and differentiated cells were determined by flow cytometry and again processed for flow cytometry. CD90, CD44, CD73, CD105 markers were not expressed, only CD34 was their marker. OMSCs were morphologically observed. Differentiated cells are similar to the endothelial cells. Therefore, to identify differentiated cells, CD31 and FLK1 expression were measured. This was confirmed by its expression. G1 phase of the cell cycle shows that OMSCs compared to the control group, were in the differentiation phase. The reason for the differentiation of MSCs into endothelial cells was the sign of presence of VEGF factor in the medium too high value of as a VEGF secreting source.

Download Full-text