Myeloma Patient-Derived Mesenchymal Stem Cells Grown In 3-D Culture Induce Primary Myeloma Cell Proliferation and Resistance To Therapy

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1912-1912
Author(s):  
Jana Jakubikova ◽  
Richard W.J. Groen ◽  
Teru Hideshima ◽  
Danka Cholujova ◽  
Jan Sedlak ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Board Of Directors ◽  
Flow Cytometry Analysis ◽  
Advisory Committees ◽  
Bm Niche ◽  
Equity Ownership ◽  
D Model

Abstract Introduction The neoplastic bone marrow (BM) milieu or so-called myeloma niche plays an essential role in initiation, development, and progression of multiple myeloma (MM). It is known that regulatory signaling molecules and cell-cell crosstalk interactions involved in the normal hematopoietic stem cell niche have also been implicated in MM. Defining phenotypic features and molecular signatures of the neoplastic BM niche in MM will provide the framework for future development of new treatment strategies targeting MM cells and their BM niche. Methods and Results To investigate the neoplastic BM microenvironment of MM patients, we compared a special gel-based 3-dimensional (3-D) model with a 2-dimensional (2-D) system for culturing MM patient-derived mesenchymal stem cells (MM-MSCs) ex-vivo. In the gel-based 3-D model, MM-MSCs formed compact clusters with active fibrous connections between clusters to create a MM niche-like structure. Phenotypic profiling of 3-D MM-MSCs clusters revealed high expression of MSC-specific markers including CD73, CD90, CD105, CD166, and HLA-ABC. On the other hand, 3-D MM-MSCs clusters lacked expression of CD45, CD34 and HLA-DR. In order to confirm differentiation potential of 3-D MM-MSC, we cultured them with differentiation media towards either osteogenic or adipogenic lineages. The lineage differentiation capacity of the 3-D system was comparable to the 2-D MM-MSCs, with stronger calcium mineralization of clusters associated with differentiation towards the osteoblastogenic lineage. Moreover, the production of osteopontin and angiopoietin-2 was evident in 3-D MM-MSCs. In these models of the neoplastic BM microenvironment in MM, significantly higher production of IL-6 (p=0.002), IL-8, MCP-1(MCAF), RANTES, VEGF and HGF (p<0.001) was observed in 3-D vs. 2-D MM-MSCs. To define the functional myeloma niche, key hematopoietic and/or neoplastic niche molecules were investigated by analyzing 30 MM-MSCs and 5 healthy volunteers MSCs. MM-MSCs cultured in 3-D vs. 2-D model have higher expression of N-cadherin and CXCL12 and decreased expression of nestin by flow cytometry analysis, reflecting the MM BM niche. Higher pattern of cytokine production of leptin, SCF and sTie-2 was evident in 3-D MM-MSCs compared to 2-D system, as well as cancer biomarkes like IGFBP-1, sEGFR and sHER2neu performed by multiplex luminex technology. Next, we observed statistically significant higher expression of extracellular matrix (ECM) molecules including fibronectin, laminin, collagen I, and collagen IV (p<0.001) at 7 days MM-MSC culture in 3-D compared to 2-D, as determined by confocal microscopy and flow cytometry analysis. Furthermore, the activation of integrins such as VLA-2, VLA-4 and VLA-5, on the MSCs surface was also increased in 3-D MM-MSCs, as confirmed by flow cytometry analysis. To investigate the pathogenesis of MM in neoplastic BM niche, we co-cultured BM aspirates of MM cells (N=30) with MM-MSCs in vitro. Co-culturing BM aspirates with (autologous) MM-MSCs increased MM cells (CD38+/CD138+/CD19-) as well as stimulated their proliferation (determined by CFSE proliferation dye) in 3-D vs. 2-D system. MM cells in 3-D MM-MSCs were 4-fold more resistant to bortezomib and pomalidomide as well as melphalan, compared to 2-D. Finally, MM cells expressing CXCR4, as well as MM side population with “stem-like” features, were significantly increased in 3-D MM-MSCs vs. 2-D MM-MSCs cultures. Conclusions Our 3-D MM-MSCs model mimics the neoplastic BM microenvironment and myeloma niche in vitro, and may be useful to identify novel targets, validate targeted therapies and determine mechanisms of drug resistance in MM. Disclosures: Hideshima: Acetylon Pharmaceuticals: Consultancy. Richardson:Millennium: Membership on an entity’s Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity’s Board of Directors or advisory committees; Novartis: Membership on an entity’s Board of Directors or advisory committees. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.

Formation of the Functional Niche in Vitro by Mimicking the Pathophysiological Features of the Bone Marrow Microenvironment in Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1812-1812
Author(s):  
Jana Jakubikova ◽  
Richard W.J. Groen ◽  
Sophia Adamia ◽  
Teru Hideshima ◽  
Danka Cholujova ◽  
...  
Keyword(s):  
Stem Cells ◽  
Multiple Myeloma ◽  
Stem Cell ◽  
Board Of Directors ◽  
Stem Cell Niche ◽  
Advisory Committees ◽  
Bm Niche ◽  
Cell Niche ◽  
D Model

Abstract Abstract 1812 The neoplastic bone marrow (BM) milieu plays important roles in the development of multiple myeloma (MM). Specifically, cell-cell crosstalk interactions and regulatory signaling molecules involved in the normal hematopoietic stem cell niche have also been implicated in initiation, development, and progression of MM. Moreover, this “myeloma niche” serves as a sanctuary site for MM stem cells. The increasing knowledge of interactions between complex of MM cells and other normal hematopoietic cells and also non-hematopoietic mesenchymal stem cells and their progeny may enhance our understanding of MM cell biology and pathogenesis as well as provide the basis for novel therapeutic strategies. In order to develop an experimental system mimicking the specialized properties of the neoplastic BM microenvironment, we used hydrogel based 3-dimensional (3-D) versus 2-dimensional (2-D) models together with MM-derived mesenchymal stem cells (MM-MSCs) to create MM niche-like structures in vitro. In this 3-D model, MM-MSCs grew with the matrix fibers and at day 7 formed stable compact clusters, with fibrous meshwork-like structure among them. Furthermore, we showed tri-lineage differentiation capacity of 3-D comparable to 2D MM-MSCs towards osteogenic, adipogenic, and chondrogenic lineages. Our 3-D MM-MSCs model also showed calcium mineralization of clusters associated with differentiation towards the osteoblastogenic lineage. The phenotypic profile of MM-MSCs grown in the 2-D vs. 3-D model using MSC-specific markers (such as CD166, HLA-ABC, CD73, CD105, and CD90) did not reveal any significant changes, however CD271 was more highly expressed in the 3-D model, while CD146 was overexpressed in 2-D cultures. Because the stem cell niche is enriched with extracellular matrix (ECM) molecules resulting in an overexpansion of the stem cell pool, we studied whether expression of these molecules was conserved in the 3-D model. Analyzing 30 MM-MSCs vs. 5 normal donor MSCs, we observed that the 3-D MM-MSCs expressed significantly high level of ECM molecules including laminin, fibronectin, collagen I, collagen IV and vitronectin (p<0.001) after 7 days of culture compared to the 2-D model, as determined by flow cytometry and confocal microscopy analysis. Furthermore, the formation of dimeric active integrins (α2β1, α4β1 and α5β1) on the MSCs surface when engaged by these ECM molecules, inducing MM proliferation and survival, was also increased in the 3-D model, associated with transcriptional up-regulation of integrin subunits as confirmed by qRT-PCR. We also investigated the key molecules that play an important role in the MM niche including N-cadherin and nestin, as well as the interactions of MM cells with MSCs in the niche via CXCL12/CXCR4 axis. MM-MSCs cultured in the 3-D vs. 2-D model have higher expression of N-cadherin and CXCL12 and decreased expression of nestin, reflecting the MM BM niche. To mimic the functional marrow niche of MM in our model, we cultured the tumor cells from MM patients (N=30) with their autologous MM-MSCs in vitro. MM plasma cells (identified as CD38/CD138+ cells) were increased in percentage and proliferation after 14 days in the 3-D vs. 2-D model. Finally, MM cells expressing CXCR4, as well as MM side population with “stem-like” features (Jakubikova J et al., Blood 2011) were also significantly increased in 3-D MM-MSCs vs. 2-D MM-MSCs cultures. Our study shows that hydrogel based 3-D MM-MSCs model forms a functional niche in vitro, and may be useful to identify novel targets and validate therapies directed at MM stem cells in the MM BM niche. Disclosures: Munshi: Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy; Oncopep: Patents & Royalties. Richardson:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees. Anderson:Onyx: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene Corp: Membership on an entity's Board of Directors or advisory committees.

Gene-Correction Rescues Reprogramming of Fanconi Anemia Fibroblasts and Enables Hematopoietic Differentiation of FA Induced Pluripotent Stem Cells in Vitro and In Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 672-672
Author(s):  
Lars Mueller ◽  
Michael D. Milsom ◽  
Chad Harris ◽  
Rutesh Vyas ◽  
Kristina Brumme ◽  
...  
Keyword(s):  
Stem Cells ◽  
Board Of Directors ◽  
Hematopoietic Differentiation ◽  
Gene Correction ◽  
Advisory Committees ◽  
Median Percentage ◽  
Equity Ownership ◽  
Induced Pluripotent

Abstract Abstract 672 Fanconi anemia (FA) is a recessive syndrome characterized by progressive fatal bone marrow failure and chromosomal instability. FA cells have inactivating mutations in a signaling pathway that is critical for maintaining genomic integrity and repairing DNA damage caused by cross-linking agents. Transgenic expression of the implicated genes corrects the phenotype of hematopoietic cells but previous attempts at gene therapy failed largely due to inadequate numbers of hematopoietic stem cells available for gene correction and autologous engraftment. Induced pluripotent stem cells (iPSC) constitute an alternate source of autologous cells, which are amenable to ex vivo expansion and genetic correction. While fibroblasts from a limited number of FA patients have been reported to fail to undergo reprogramming (Raya et al., Nature, 2009), reproducible observations and mechanistic studies ascertained in an extended panel of patient cells and murine knock-out models are lacking to date. We undertook direct reprogramming of ten unique human FA primary fibroblast samples of the FA-A, FA-C, FA-G, and FA-D2 complementation groups. Using standard four-factor reprogramming, no human FA iPSC colonies were obtained in cells defective in the FA pathway. By contrast, reprogramming of gene-corrected patient samples, augmented by hypoxia (5%O2), yielded multiple pluripotent iPSC lines, confirming a critical cell-intrinsic role of the FA pathway in reprogramming. To determine if gene-corrected FA iPSC could be therapeutically useful, we performed karyotype analyses and evaluated in vitro hematopoietic differentiation in three FA-A iPSC lines. These FA patient iPSC lines were karyotypically normal and showed a robust multilineage hematopoietic differentiation potential, resulting in erythroid and myeloid hematopoietic colony forming units to a similar degree as compared to normal donor iPSC controls. We hypothesized that the reprogramming resistance of FA cells is due to defective DNA repair and genomic instability. To explore the mechanisms of the reprogramming defect, we transduced wild type (wt) tail-tip fibroblasts (TTF) with the reprogramming vectors. We observed significantly increased FANCD2 foci formation during reprogramming (median percentage of FANCD2 foci: mock-transduced TTF 2.5%, reprogrammed TTF 20.5%, n=8, p<0.01) indicating activation of the FA pathway. Next, we examined reprogramming in FA-deficient mouse cells. We observed a significantly higher incidence of reprogramming-induced double-strand DNA breaks and senescence in Fanca−/− TTF as compared to wt controls (γH2AX foci: wt 13%, Fanca−/− 19%; senescence: wt 47%, Fanca−/− 62%, median percentage, p<0.01). To evaluate whether these changes contribute to the reprogramming resistance of FA cells, we quantified the reprogramming efficiency of Fanca−/−, Fancc−/− and littermate wt TTF. The efficiency was 0.06% for Fanca−/− (n=8) and 0.38% for Fancc−/− (n=12) as compared to 0.55% for wt controls (n=13; p<0.01 and <0.05, respectively). To directly test the role of the FA pathway in reprogramming, TTF were transduced with retroviral vectors co-expressing FANCA and enhanced green fluorescent protein (eGFP) or encoding only eGFP as a control. Under hypoxic conditions, gene-correction of the Fanca−/− TTF with FANCA resulted in a significant reduction of senescence and rescued the reprogramming efficiency of Fanca−/− TTF to wt levels. While significant chromosomal aberrations were observed in uncorrected Fanca−/− iPCS, gene-corrected Fanca−/− iPSC did not show any significant chromosomal imbalances when analyzed by comparative genomic hybridization. To evaluate the capacity of FA iPSC to form blood cells in vivo, we injected wt, control transduced or gene-corrected Fanca−/− iPCS (CD45.1+) into wt blastocysts (CD45.2+) and analyzed the contribution of iPSC-derived hematopoietic cells in embryonic day 14.5 fetal livers. We observed 1.8–4% wt iPSC chimerism (n=15), 0.4–0.9% Fanca−/− iPSC chimerism (n=3) and 1.5 to 2.5% chimerism in gene-corrected Fanca−/− iPSC (n=11). Our data demonstrate that reprogramming activates the FA pathway. Gene-correction rescues the reprogramming block of FA cells and protects FA iPSC from genomic instability, thus yielding an expandable source of autologous stem cells with hematopoietic differentiation capacity that may be explored for future use in regenerative medicine. Disclosures: Daley: iPierian, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Epizyme, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Verastem, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Solasia, KK: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; MPM Capital, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees.

Inhibition Of Nuclear Transport Modulator Xpo1/CRM1 For The Treatment Of Acute Myeloid Leukemia (AML)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 237-237 ◽  
Author(s):  
Michael P. Rettig ◽  
Matthew Holt ◽  
Julie Prior ◽  
Sharon Shacham ◽  
Michael Kauffman ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Board Of Directors ◽  
Cell Lines ◽  
Nuclear Export ◽  
Employment Equity ◽  
Advisory Committees ◽  
Ic50 Values ◽  
Equity Ownership

Abstract Background Exportin 1 (XPO1) also called CRM1, is a widely expressed nuclear export protein, transporting a variety of molecules including tumor suppressor proteins and cell cycle regulators. Targeted inhibition of XPO1 is a new strategy to restore multiple cell death pathways in various malignant diseases. SINEs are novel, orally available, small molecule Selective Inhibitors of Nuclear Export (SINE) that specifically bind to XPO1 and inhibit its function. Methods We used WST-1 cell proliferation assays, flow cytometry, and bioluminescence imaging to evaluate the efficacy of multiple SINEs to induce apoptosis alone and in combination with cytarabine (AraC) or doxorubicin in vitro in chemotherapy sensitive and resistant murine acute promyelocytic leukemia (APL) cells. This murine model of APL was previously generated by knocking in the human PML-RARa cDNA into the 5’ regulatory sequence of the cathepsin G locus (Westervelt et al. Blood, 2003). The abnormal co-expression of the myeloid surface antigen Gr1 and the early hematopoietic markers CD34 and CD117 identify leukemic blasts. These Gr1+CD34+CD117+ APL cells partially retain the ability to terminally differentiate toward mature granulocytes (mimicking more traditional AML models) and can be adoptively transferred to secondary recipients, which develop a rapidly fatal leukemia within 3 weeks after tumor inoculation. To assess the safety and efficacy of SINEs in vivo, we injected cryopreserved APL cells intravenously via the tail vein into unconditioned genetically compatible C57BL/6 recipients and treated leukemic and non-leukemic mice (n=15/cohort) with 15 mg/kg of the oral clinical staged SINE KPT-330 (currently in Phase 1 studies in patients with solid tumors and hematological malignancies) alone or in combination with 200 mg/kg cytarabine every other day for a total of 2 weeks. Peripheral blood was obtained weekly from mice for complete blood counts and flow cytometry to screen for development of APL. Results The first generation SINE, KPT214, inhibited the proliferation of murine APL cell lines in a dose and time dependent manner with IC50 values ranging from of 95 nM to 750 nM. IC50 values decreased 2.4-fold (KPT-185) and 3.5-fold (KPT-249) with subsequent generations of the SINEs. Consistent with the WST-1 results, Annexin V/7-aminoactinomycin D flow cytometry showed a significant increase of APL apoptosis within 6 hours of KPT-249 application. Minimal toxicity against normal murine lymphocytes was observed with SINEs even up to doses of 500 nM. Additional WST-1 assays using AraC-resistant and doxorubicin-resistant APL cell lines demonstrated cell death of both chemotherapy-resistant cell lines at levels comparable to the parental chemosensitive APL cell lines. Combination therapy with low dose KPT-330 and AraC showed additive effects on inhibition of cell proliferation in vitro. This additive effect of KPT-330 and chemotherapy on APL killing was maintained in vivo. As shown in Figure 1, treatment with AraC or KPT-330 alone significantly prolonged the survival of leukemic mice from a median survival of 24 days (APL + vehicle) to 33 days or 39 days, respectively (P < 0.0001). Encouragingly, combination therapy with AraC + KPT-330 further prolonged survival compared to monotherapy (P < 0.0001), with some mice being cured of the disease. Similar in vivo studies with the AraC-resistant and doxorubicin-resistant APL cells are just being initiated. Conclusions Our data suggests that the addition of a CRM1 inhibitor to a chemotherapy regimen offers a promising avenue for treatment of AML. Disclosures: Shacham: Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Kauffman:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. McCauley:Karyopharm Therapeutics, Inc: Employment, Equity Ownership.

scholarly journals Angiogenin Is a Niche-Secreted RNase Which Regulates Hematopoietic Regeneration

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 171-171
Author(s):  
Lev Silberstein ◽  
Kevin Goncalves ◽  
Nicholas Severe ◽  
Guo-fu Hu ◽  
David T. Scadden
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Board Of Directors ◽  
Advisory Committees ◽  
Self Renewal ◽  
Cellular Source ◽  
Equity Ownership ◽  
Ng2 Cells

Abstract Background. Identification of novel niche factors is critical for understanding of regulatory mechanisms which control HSPC cell fate decisions and design of novel pro-regenerative therapies. We have developed a proximity-based differential single cell analysis approach to the study of the bone marrow niche, which showed that individual osteolineage cells located in close proximity to transplanted HSPC are enriched for expression of niche factors, and have previously reported identification of IL18 and Embigin as regulators of HSPC quiescence. Here we describe the results of in vivo validation of Angiogenin (ANG) - the third molecule highlighted using the above strategy - as a potent regulator of HSPC quiescence and regeneration. Results. ANG is a secreted RNase which is known to promote angiogenesis, proliferation of cancer cells and also enhance cell survival in response to stress. Analysis of primitive cells subsets in the ANG knock-out mice (AngKO mice) revealed a 1.4-fold increase in the frequency and absolute number of long-term hematopoietic stem cells (LT-HSCs). Subsequent BrdU incorporation and cell cycle studies demonstrated increased proliferative activity in the primitive HSPC compartment indicating that ANG regulates HSPC quiescence. To confirm these findings functionally and to assess the effect of ANG on self-renewal, we exposed AngKO animals to weekly 5-FU injections and performed serial transplantation experiments of WT LT-HSCs into AngKO hosts. We noted significantly increased mortality of AngKO mice in both experimental settings; in a competitive transplant assay, we observed almost complete absence of engraftment by WT cells in the secondary hosts, in keeping with the exhaustion phenotype. Consistently, exposure of AngKO animals to a different type of hematopoietic stress, such as ageing, resulted in development of peripheral blood cytopenias and marked reduction in the number and frequency of HSPC. ANG is expressed in multiple non-hematopoietic cell types in the bone marrow, including osteoprogenitors, mature osteoblasts and nestin-positive mesenchymal stem cells and NG2-positive arteriolar sheath cells. To establish the predominant cellular source of ANG in the niche, we crossed Ang "floxed" mice with the animals in which tamoxifen-inducible Cre-recombinase was driven by the promoters targeting these cell subsets and examined the effect on hematopoiesis. We found that deletion of ANGfrom Osx+, Nes+ and NG2+ cells resulted in an increase of the number of LT-HSC and more active cycling of LT-HSC, short-term HSC (ST-HSC) and multi-potent progenitors (MPP) while ANGdeletion in mature osteoblasts had no effect on these cell populations, but was associated with an increase in number and more active cycling of common lymphoid progenitors (CLP), as was also seen upon ANGdeletion from Nes+ and NG2+ cells. These results indicate that the target cell population which is regulated by ANG depends on the cellular source. Interestingly, transplantation of WT bone marrow into the animals with Osx-specific ANG deletion resulted in development of macrocytic anemia and neutropenia at 6 months, thus indicating that Angiogenin deficiency in the niche is sufficient for the development of the hematopoietic failure. Impaired long-term reconstitution was also observed when ANG was deleted from Nestin+ mesenchymal stem cells but not col1+ mature osteoblasts in the recipient mice. Our findings that the absence of ANG negatively impacts HSPC self-renewal prompted us to investigate whether exposure of HSPC to recombinant ANG protein will have the opposite effect and enhance hematopoietic regeneration. We therefore treated mouse LT-HSCs with recombinant ANG or vehicle control ex-vivo for 2 hours and competitively transplanted them into lethally irradiated WT recipients. We found that ANG-treated LT-HSCs displayed a significantly higher long-term reconstituting ability, which persisted into the secondary transplants. Similar data were obtained following treatment of CD34+ cord blood cells with human ANG. Conclusion. Our work defines ANG as a previously unrecognized regulator of HSPC quiescence and self-renewal and suggests that it can be explored as a potential therapeutic agent to promote hematopoietic regeneration. Disclosures Scadden: Teva: Consultancy; Apotex: Consultancy; Bone Therapeutics: Consultancy; GlaxoSmithKline: Research Funding; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Fate Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Dr. Reddy's: Consultancy.

scholarly journals Modeling Diamond Blackfan Anemia and p53 Activation in the Zebrafish

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. SCI-43-SCI-43
Author(s):  
Leonard I. Zon
Keyword(s):  
Stem Cells ◽  
Board Of Directors ◽  
Advisory Committees ◽  
Diamond Blackfan Anemia ◽  
Clonal Hematopoiesis ◽  
Calmodulin Inhibitors ◽  
Human Ipscs ◽  
Equity Ownership ◽  
Insight Into

Marrow dysplasias are pre-leukemic conditions that include ribosomopathies, a group of rare genetic diseases, or myelodysplasia preceded by clonal expansion in older adults with somatic mutations. To discover novel therapies for ribosomopathies, we performed two chemical suppressor screens using a ribosomal protein mutant zebrafish and human induced pluripotent stem cells (iPSCs) derived from ribosomopathy patients. In the zebrafish screen, we found calmodulin inhibitors rescued the anemia in zebrafish and also rescued the erythroid defect of the mouse (in vivo) and human (in vitro) models of marrow failure. The screen in RPS19 mutant human iPSCs identified a compound, SMER28, which enhanced erythropoiesis in anemia models through an autophagy-dependent mechanism. The compounds identified in our chemicals screens have provided new insight into the etiology of marrow failure, and calmodulin inhibitors will soon be studied in a clinical trial with adult Diamond-Blackfan anemia patients. To study the abnormal clonal hematopoiesis associated with myelodysplasia, we have generated a new system using the zebrafish in which single stem cells and their progeny express unique combinations of fluorescent proteins, allowing them to be tracked over time by their specific color. Mosaic mutagenesis induced by clustered regularly interspaced short palindromic repeats /cas9 and/or gene overexpression in this system led to expansion of specifically colored blood, modeling the clonal hematopoiesis of human myelodysplasia. Our studies should provide new insight into the ribosomopathies and myelodysplasia. Disclosures Zon: Marauder Therapeutics: Equity Ownership, Other: Founder; Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Fate, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

Flow Cytometry Analysis of Type IIB Procollagen as Quality Control of Chondrogenic Commitment of MSCs

2021 ◽  
Author(s):  
Emeline Perrier-Groult ◽  
Elisabeth Aubert-Foucher ◽  
Marielle Pasdeloup ◽  
Jérôme Lafont ◽  
Hugo Fabre ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Splice Variants ◽  
Type Ii ◽  
Flow Cytometry Analysis ◽  
Gene Encoding ◽  
Marrow Mesenchymal Stem Cells ◽  
Reliable Marker

Type II collagen is the major collagen protein in cartilage, synthesized as precursor forms (procollagens). Several splice variants of the gene encoding type II procollagen have been identified such as IIA and IIB isoforms. Interestingly, a shift from IIA to IIB transcripts has been reported to occur during cartilage development and during chondrogenic differentiation of mesenchymal stem cells in vitro. Thus, type IIB procollagen represents a reliable marker of chondrocyte differentiation. We characterized previously the first antibody (referred as anti-pNIIB52) able to selectively detect the IIB form of human type II procollagen in Western-blot or immunohistochemistry analysis. More recently, we used anti-pNIIB52 in flow cytometry to quantify chondrogenic induction of bone marrow-mesenchymal stem cells cultivated in agarose hydrogel, after release of the cells from the gel. Here, we use imaging flow cytometry and anti-pNIIB52 to visualize directly intracellular accumulation of type IIB procollagen in cells undergoing chondrogenesis. Our data together show that flow cytometry analysis using anti-pNIIB52 represents an efficient and rapid diagnostic tool of good chondrogenic conversion, at the cellular level.

Neonatal Recipients Offer Permissive Hematopoietic Microenvironment for Engraftment of Embryonic Murine Hematopoietic Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2344-2344
Author(s):  
Natasha Arora ◽  
Shannon McKinney-Freeman ◽  
Garrett C Heffner ◽  
Il-Ho Jang ◽  
Pamela L. Wenzel ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Board Of Directors ◽  
Early Embryo ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Hematopoietic Microenvironment ◽  
Equity Ownership

Abstract Abstract 2344 The first hematopoietic stem cells (HSC) that give rise to robust, long-term, multi-lineage reconstitution in irradiated adult recipients arise in the murine embryo at embryonic day 11.5 (E11.5). However, long-term multi-lineage engraftment in neonatal recipients has been observed from E9.0 yolk sac, suggesting that the neonatal hematopoietic microenvironment is more permissive for engraftment of embryonic HSCs. To resolve the apparent discrepancy between the numbers of candidate HSCs detected by direct visualization in the early embryo, relative to the numbers that can be measured by limiting dilution, we sought to characterize engraftment of neonatal recipients versus adult recipients with hematopoietic populations dissected from the aorta-gonad-mesonephros (AGM) region of the early embryo, the first putative site of intraembryonic origin of definitive HSCs. We dissected whole AGM from E11.5 embryos and injected cell dilutions from 2 embryo equivalents (ee) to 0.25 ee into the facial vein of day 1–2 neonatal recipients that had received sublethal conditioning with 350 rad irradiation. In neonatal recipients we detected robust, long-term, multi-lineage hematopoietic engraftment from as little as 0.25 ee. From less than 1 ee of whole AGM, the engraftment chimerism ranged from 5–20%. With 2 ee, chimerism was as high as 70%. Most animals showed balanced donor derived myeloid and lymphoid contribution by 10 weeks post-transplant. Interestingly, some animals had predominantly myeloid reconstitution for as long as 18 weeks, suggesting the presence of a novel long-term, myeloid-restricted, embryonic HSC. We also explored the neonatal engraftment potential of VE-cadherin+ CD45+ and VE-cadherin+ CD45− populations. As expected from the literature, only the VE-cadherin+ CD45+ population engrafted the neonatal recipients. Our data indicate that the neonate harbors a more permissive hematopoietic microenvironment that enables more robust engraftment of early embryonic hematopoietic populations, thereby allowing us to identify potentially novel classes of embryonic hematopoietic progenitors. We are currently exploring the neonatal engraftment potential of E9.5 and E10.5 embryonic populations, additional FACS-purified populations, and hematopoietic populations derived from pluripotent stem cells in vitro. Disclosures: Daley: iPierian, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Epizyme, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Verastem, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Solasia, KK: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; MPM Capital, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Multiparametric Flow-Cytometry Is a Reliable Tool for Measurable Residual Disease Assessment and Risk-Stratification of FLT3-Mutated AML Patients

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5083-5083
Author(s):  
Raffaele Palmieri ◽  
Luca Maurillo ◽  
Alfonso Piciocchi ◽  
Maria Ilaria Del Principe ◽  
Valentina Arena ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Board Of Directors ◽  
Residual Disease ◽  
Cell Transplant ◽  
Advisory Committees ◽  
Npm1 Mutation ◽  
Multiparametric Flow Cytometry ◽  
Reliable Tool ◽  
Measurable Residual Disease

Background: Mutations of the gene encoding Fms Related Tyrosine Kinase 3 (FLT3), at the juxta-membrane level (ITD), represent the most common lesions found in Acute Myeloid Leukemia (AML), identifying a subgroup of patients (pts) with unfavorable prognosis. FLT3-ITD mutations are considered an unreliable tool for measurable residual disease (MRD) monitoring, due to their intraclonal heterogeneity and instability during the course of disease. Instead, multiparametric flow cytometry (MFC) may represent an alternative to monitor MRD in this molecular subset. In fact, through the recognition and monitoring of leukemia associated immunophenotypes, MFC is applicable to > 90% of AML patients with a sensitivity of 10-4. Aims: The aim of our study was to investigate the reliability of MFC in MRD assessment of 72 FLT3-ITD positive pts whose treatment allocation was prospectively decided according to the genetic/cytogenetic profile at diagnosis and post consolidation MRD. FLT3-ITD pts were to receive, after induction and consolidation, allogeneic stem cell transplant (ASCT), whatever the source of stem cells. In this subgroup analysis, we investigated if FLT3-ITD mutated pts have a different propensity to achieve high quality (e.g. MRD negative) complete remission as compared to FLT3 wildtype ones. Furthermore, we seek for a correlation between different levels of MRD and overall (OS) and disease-free survival (DFS). Methods: We included in the analysis 72 pts with de novo AML carrying FLT3-ITD mutations whose MRD assessment at the post-consolidation timepoint was available. Pts were defined as MRD-negative, when obtaining a residual leukemic cells count below the threshold of 3.5x10-4 (0.035%). MRD positive pts (with MRD ≥ 3.5x10-4 RLC) were stratified into 3 classes according to the levels of MRD (0.035%-0.1%; >0.1%-1%; >1%). We compared the MRD status and clinical outcome with a matched group of FLT3 wildtype AML (n = 203) treated in the same protocol. Results: Overall median age was 49 (range 18-60.9). The 2 cohorts were balanced in terms of age and sex distribution. In the FLT3-ITD group, 80/126 (64%) cases carried a concomitant NPM1 mutation vs 107/374 (28.6%) of FLT3 wildtype ones (p <0.001). Furthermore, FLT3 mutated pts had a median WBC count of 35x109/L vs 9.5x109/L of those FLT3 wildtype (p < 0.001). MRD determination after consolidation cycle was available in 72/126 FLT3-ITD pts (57%) and in 203/374 FLT3 wildtypeones (54.3%), respectively. After having received induction and consolidation course, 47/72 FLT3-ITD pts (65,2%) were submitted to allogenic stem cells transplantation (ASCT). At the post-consolidation time-point, MRD negativity rate was significantly lower in FTL3-ITD pts (27/72, 37.5%) as compared to those FLT3 wildtype (94/203, 46.3%). Furthermore, 38/72 (52.8%) and 10/72 (13.9%) FLT3-ITD pts had a level of MRD > 0.1% and > 1%, respectively as compared to 65/203 (33.0%) and 15/203 (7.4%) of FLT3 wildtypeones, respectively (p=0.017). When considering the different MRD stratification levels of FLT3-ITD pts, OS probability at 24 months was 57.2% (27 pts), 71.4% (7 pts), 53.6% (28 pts) and 20% (10 pts), for the MRD categories <0.035%, 0.035%-0.1%, >0.1%-1%, >1%, respectively (p=0.028). DFS probability at 24 months was 53.8% (27 pts), 71.4% (7 pts), 34.9% (27 pts) and 20% (10 pts), for the MRD categories <0.035%, 0.035%-0.1%, >0.1%-1%, >1%, respectively (p=0.038). Summary/Conclusion: We demonstrated that MRD determination by MFC is a reliable tool to assess remission quality and prognosis in FLT-ITD positive patients. This subpopulation shows a lower propensity to obtain a MRD negative CR, with the majority of pts maintaining an amount of MRD > 0.1% after standard treatment. Even though most of these pts were addressed to ASCT, post-consolidation MRD maintained its negative impact on OS and DFS, particularly for those pts with MRD >1%. In the attempt to improve the quality of response, prevent leukemia recurrence and pursue a durable remission, delivery of FLT3 inhibitors as a maintenance after transplant may represent a promising option. Disclosures Venditti: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy; Astellas: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees. Buccisano:Janssen: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

scholarly journals Effect of Silicon, Titanium, and Zirconium Ion Implantation on NiTi Biocompatibility

2012 ◽  
Vol 2012 ◽  
pp. 1-16 ◽  
Author(s):  
L. L. Meisner ◽  
A. I. Lotkov ◽  
V. A. Matveeva ◽  
L. V. Artemieva ◽  
S. N. Meisner ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Mesenchymal Stem Cells ◽  
Ion Implantation ◽  
Cell Counting ◽  
High Dose ◽  
Surface Layers ◽  
Test Flow ◽  
Ion Implanted

The objective of the work was to study the effect of high-dose ion implantation (HDII) of NiTi surface layers with Si Ti, or Zr, on the NiTi biocompatibility. The biocompatibility was judged from the intensity and peculiarities of proliferation of mesenchymal stem cells (MSCs) on the NiTi specimen surfaces treated by special mechanical, electrochemical, and HDII methods and differing in chemical composition, morphology, and roughness. It is shown that the ion-implanted NiTi specimens are nontoxic to rat MSCs. When cultivated with the test materials or on their surfaces, the MSCs retain the viability, adhesion, morphology, and capability for proliferationin vitro, as evidenced by cell counting in a Goryaev chamber, MTT test, flow cytometry, and light and fluorescence microscopy. The unimplanted NiTi specimens fail to stimulate MSC proliferation, and this allows the assumption of bioinertness of their surface layers. Conversely, the ion-implanted NiTi specimens reveal properties favorable for MSC proliferation on their surface.

ABT-737 Targets Leukemic Stem Cells In Mouse Models of Mutant NRASD12/hBCL-2- Mediated Acute Myeloid Leukemia Progression with Increased Survival

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3308-3308
Author(s):  
Rose Ann Padua ◽  
Stephanie Beurlet ◽  
Patricia Krief ◽  
Nader Omidvar ◽  
Carole Le Pogam ◽  
...  
Keyword(s):  
Stem Cells ◽  
Disease Progression ◽  
Board Of Directors ◽  
Research Funding ◽  
Leukemic Stem Cells ◽  
Spleen Cells ◽  
Employment Equity ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Bh3 Mimetic

Abstract Abstract 3308 Background: Animal models enable us to understand disease progression and provide us with reagents to test various therapeutic strategies. We have previously developed a mouse model of myelodysplasia/acute myelogenous leukemia (MDS/AML) progression using mutant NRASD12 and overexpression of human hBCL-2 (Omidvar et al Cancer Res 67:11657-67, 2007). Expanded leukemic stem cells (LSC) were identified as Lin-/Sca1+/KIT+ (LSK) populations, with increased myeloid colony growth and were transplantable. Increased hBCL-2 and RAS-GTP complex were observed in both MDS/AML diseases. The MDS-like disease had increased apoptosis, whilst the AML-like mice had liver apoptosis patterns similar to wild type. The single NRASD12 line also had increased apoptosis. In this present study using a BCL-2 homology domain 3 (BH3) mimetic ABT-737 (Abbott), we have evaluated the effects of targeting BCL-2 in our preclinical models. Methods & Results: Treatment with the inhibitor shows a reduction of LSK cells, reduced progenitor numbers in colony assays and clearance of the liver infiltrations in both MDS and AML models. Gene expression profiling of the MDS mice shows regulation of 399 genes upon treatment including 58 genes expressed by the single mutant RAS mice and not expressed in the untreated AML mice. 78 genes were shared between single NRASD12 and diseased mice and not the treated mice. These studies potentially identify the contribution of NRASD12 genes to disease progression. By confocal microscopy we observed that in the MDS mice the majority of the RAS and BCL-2 co-localized to the plasma membrane, where active pro-apoptotic RAS is normally located, whereas in the AML disease RAS and BCL-2 co-localized in the mitochondria, where BCL-2 is normally found (Omidvar et al 2007). After treatment with the inhibitor the AML co-localization of RAS and BCL-2 shifted to the plasma membrane where single NRASD12 is normally localized. Furthermore, increased RAS-GTP levels was detected in both Sca1+ and Mac1+ enriched spleen cells and interestingly an increase in BCL-2 expression was observed in peripheral blood and in spleen cells after treatment; this increase in BCL-2 was associated with a decrease in the phosphorylation of serine 70 and an increase in phosphorylation of threonine 56 of BCL-2. ABT-737 treatment led to increased phosphorylated ERK resembling RAS and reduced MEK and AKT phosphorylation, changes detected by western blots and the nanoimmunoassay (NIA, NanoPro, Cell Biosciences) that might account for the increased apoptosis, measured by TUNEL and In vivo imaging by single-photon emission computed tomography (SPECT) using Tc-99m-labelled AnnexinV (SPECT). In contrast, although treated MDS mice had increased apoptosis they did not have an increase in overall expression of BCL-2 or in RAS-GTP levels. Treatment of both MDS and AML models with this inhibitor significantly extended lifespan from diagnosis with mean survival of 28 days untreated vs 80 days treated (p=0.0003) and mean survival from birth of 39 untreated vs 85 days treated (p<0.0001) respectively Conclusions: Genomics, proteomics and imaging have been employed in the MDS/AML models to characterize disease progression and follow response to treatment to the BH3 mimetic ABT-737 in order to gain molecular insights in the evaluation of the efficacy. ABT-737 appears to target LSCs, induce apoptosis, regulating RAS and BCL-2 signalling pathways, which translated into significantly increased survival. Disclosures: Padua: Vivavacs SAS: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Auboeuf:GenoSplice technology: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. de la Grange:GenoSplice technology: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Fenaux:Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen Cilag: Honoraria, Research Funding; ROCHE: Honoraria, Research Funding; AMGEN: Honoraria, Research Funding; GSK: Honoraria, Research Funding; Merck: Honoraria, Research Funding; Cephalon: Honoraria, Research Funding. Tu:Cell Biosciences Inc;: Employment. Yang:Cell Biosciences Inc;: Employment. Weissman:Amgen, Systemix, Stem cells Inc, Cellerant: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Felsher:Cell Bioscience:. Chomienne:Vivavacs SAS: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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