Normal Bone Marrow Stromal Cells Research Articles

AML alters bone marrow stromal cell osteogenic commitment via Notch signaling.

Acute myeloid leukemia (AML) is a highly heterogeneous malignancy caused by various genetic alterations and characterized by the accumulation of immature myeloid blasts in the bone marrow (BM). This abnormal growth of AML cells disrupts normal hematopoiesis and alters the BM microenvironment components, establishing a niche supportive of leukemogenesis. Bone marrow stromal cells (BMSCs) play a pivotal role in giving rise to essential elements of the BM niche, including adipocytes and osteogenic cells. Animal models have shown that the BM microenvironment is significantly remodeled by AML cells, which skew BMSCs toward an ineffective osteogenic differentiation with an accumulation of osteoprogenitors. However, little is known about the mechanisms by which AML cells affect osteogenesis. We studied the effect of AML cells on the osteogenic commitment of normal BMSCs, using a 2D co-culture system. We found that AML cell lines and primary blasts, but not normal hematopoietic CD34+ cells, induced in BMSCs an ineffective osteogenic commitment, with an increase of the early-osteogenic marker tissue non-specific alkaline phosphatase (TNAP) in the absence of the late-osteogenic gene up-regulation. Moreover, the direct interaction of AML cells and BMSCs was indispensable in influencing osteogenic differentiation. Mechanistic studies identified a role for AML-mediated Notch activation in BMSCs contributing to their ineffective osteogenic commitment. Inhibition of Notch using a γ-secretase inhibitor strongly influenced Notch signaling in BMSCs and abrogated the AML-induced TNAP up-regulation. Together, our data support the hypothesis that AML infiltration produces a leukemia-supportive pre-osteoblast-rich niche in the BM, which can be partially ascribed to AML-induced activation of Notch signaling in BMSCs.

Lysin (K)-specific demethylase 1 inhibition enhances proteasome inhibitor response and overcomes drug resistance in multiple myeloma

BackgroundMultiple myeloma (MM) is an incurable plasma cell malignancy, accounting for approximately 1% of all cancers. Despite recent advances in the treatment of MM, due to the introduction of proteasome inhibitors (PIs) such as bortezomib (BTZ) and carfilzomib (CFZ), relapses and disease progression remain common. Therefore, a major challenge is the development of novel therapeutic approaches to overcome drug resistance, improve patient outcomes, and broaden PIs applicability to other pathologies.MethodsWe performed genetic and drug screens to identify new synthetic lethal partners to PIs, and validated candidates in PI-sensitive and -resistant MM cells. We also tested best synthetic lethal interactions in other B-cell malignancies, such as mantle cell, Burkitt’s and diffuse large B-cell lymphomas. We evaluated the toxicity of combination treatments in normal peripheral blood mononuclear cells (PBMCs) and bone marrow stromal cells (BMSCs). We confirmed the combo treatment’ synergistic effects ex vivo in primary CD138+ cells from MM patients, and in different MM xenograft models. We exploited RNA-sequencing and Reverse-Phase Protein Arrays (RPPA) to investigate the molecular mechanisms of the synergy.ResultsWe identified lysine (K)-specific demethylase 1 (LSD1) as a top candidate whose inhibition can synergize with CFZ treatment. LSD1 silencing enhanced CFZ sensitivity in both PI-resistant and -sensitive MM cells, resulting in increased tumor cell death. Several LSD1 inhibitors (SP2509, SP2577, and CC-90011) triggered synergistic cytotoxicity in combination with different PIs in MM and other B-cell neoplasms. CFZ/SP2509 treatment exhibited a favorable cytotoxicity profile toward PBMCs and BMSCs. We confirmed the clinical potential of LSD1-proteasome inhibition in primary CD138+ cells of MM patients, and in MM xenograft models, leading to the inhibition of tumor progression. DNA damage response (DDR) and proliferation machinery were the most affected pathways by CFZ/SP2509 combo treatment, responsible for the anti-tumoral effects.ConclusionsThe present study preclinically demonstrated that LSD1 inhibition could provide a valuable strategy to enhance PI sensitivity and overcome drug resistance in MM patients and that this combination might be exploited for the treatment of other B-cell malignancies, thus extending the therapeutic impact of the project.

Cytotoxicity of photoactivatable tricarbonyl Mn(I) complex with 1-(chloromethyl)-1H-benzotriazole against leukaemia

The reaction between bromo pentacarbonyl manganese(I) and two equivalents of 1-(chloromethyl)-1H-benzotriazole (BTZ-CH2Cl) afforded visible-light induced carbon monoxide releasing molecule of the general formula, fac-[MnBr(CO)3(BTZ-CH2Cl)2]. The solvatochromism, dark-stability and photolysis profile of the titled complex was examined in organic solvents with various hydrogen-bonding propensities, coordinating capabilities, and solvent polarities as well as in the presence of proteins. The number of CO equivalents released from the titled complex upon the exposure to a 468 nm LED light was determined using myoglobin assay. The titled complex displayed selective cytotoxicity towards human acute monocytic leukaemia in the dark without causing any harmful effect to the normal mice bone marrow stromal cells.

Differentiation Capacity of Human Urine-Derived Stem Cells to Retain Telomerase Activity.

Telomerase activity is essential for the self-renewal and potential of embryonic, induced pluripotent, and cancer stem cells, as well as a few somatic stem cells, such as human urine-derived stem cells (USCs). However, it remains unclear how telomerase activity affects the regeneration potential of somatic stem cells. The objective of this study was to determine the regenerative significance of telomerase activity, particularly to retain cell surface marker expression, multipotent differentiation capability, chromosomal stability, and in vivo tumorigenic transformation, in each clonal population of human primary USCs. In total, 117 USC specimens from 10 healthy male adults (25–57 years of age) were obtained. Polymerase chain reaction amplification of a telomeric repeat was used to detect USCs with positive telomerase activity (USCsTA+). A total of 80 USCsTA+ (70.2%) were identified from 117 USC clones, but they were not detected in the paired normal bladder smooth muscle cell and bone marrow stromal cell specimens. In the 20–40 years age group, approximately 75% of USC clones displayed positive telomerase activity, whereas in the 50 years age group, 59.2% of the USC clones expressed positive telomerase activity. USCsTA+ extended to passage 16, underwent 62.0 ± 4.8 population doublings, produced more cells, and were superior for osteogenic, myogenic, and uroepithelial differentiation compared to USCsTA−. Importantly, USCs displayed normal chromosome and no oncological transformation after being implanted in vivo. Overall, as a safe cell source, telomerase-positive USCs have a robust regenerative potential in cell proliferation and multipotent differentiation capacity.

Effect of treadmill exercise and bone marrow stromal cell engraftment on activation of BDNF-ERK-CREB signaling pathway in the crushed sciatic nerve

The effect of combined approach of exercise training and bone marrow stromal cell (BMSC) engraftment on activation of brain-derived neurotrophic factor (BDNF)-extracellular signal-regulated kinase 1 and 2 (ERK1/2)-cyclic adenosine monophosphate response element-binding protein (CREB) signaling pathway after sciatic nerve injury (SNI) was investigated. Sixty male Sprague-Dawley rats divided into the normal control, nonexercise (NEX), exercise training (EX), BMSC transplantation (TP), and exercise training+BMSC transplantation (EX+TP) groups 4 weeks after SCI. Exercise training was carried out on the treadmill device at 5–10 m/min for 20 min for 4 weeks. Single dose of 5×106 harvested BMSC was injected into the injury area of the injured sciatic nerve. In order to evaluate induction levels of BDNF-ERK1/2-CREB signaling molecules in the whole cell and nuclear cell lysates of the injured sciatic nerve, we applied Western blot analysis. BDNF was significantly increased only in EX+TP compared to NEX, EX, and TP groups. Phosphoinositide-dependent kinase-1 was more increased in EX, TP, and EX+TP groups than NEX group, but EX+TP group showed the most upregulation of phosphorylated protein kinase B compared to other groups. In addition, in the whole cell lysate, phosphorylated ERK1/2, but not activating transcription factor-3 (ATF-3) and phosphorylated CREB, was significantly increased in TP and EX+TP groups. In the nuclear cell lysate, ATF-3 and phosphorylated CREB were strongly activated in EX+TP group compared to EX group. Regular exercise training combined with BMSC engraftment would seem to be more effective in controlling activation of regeneration-related signaling pathway after SNI.

A CREB1-miR-181a-5p loop regulates the pathophysiologic features of bone marrow stromal cells in fibrous dysplasia of bone

BackgroundFibrous dysplasia (FD) is a bone marrow stromal cell (BMSC) disease caused by activating mutations of guanine nucleotide-binding protein alpha-stimulating activity polypeptide (GNAS) and is characterized by increased proliferative activity and disrupted osteogenesis of BMSCs. However, the molecular mechanisms regulating the pathophysiologic features of BMSCs in FD remain unknown. This study aimed to identify and verify the roles of the CREB1-miR-181a-5p regulatory loop in FD pathophysiology.MethodsMicroRNA (miRNA) sequencing analysis was used to identify the possible miRNAs implicated in FD. The proliferation, apoptosis, and osteogenic differentiation of BMSCs, as well as the osteoclast-induced phenotype, were measured and compared after exogenous miR-181a-5p transfection into FD BMSCs or miR-181a-5p inhibitor transfection into normal BMSCs. Chromatin immunoprecipitation and luciferase reporter assays were performed to verify the interactions between CREB1 and miR-181a-5p and their effects on the FD pathological phenotype.ResultsCompared to normal BMSCs, FD BMSCs showed decreased miR-181a-5p levels and exhibited increased proliferative activity, decreased apoptotic capacity, and impaired osteogenesis. FD BMSCs also showed a stronger osteoclast activation effect. miR-181a-5p overexpression reversed the pathophysiologic features of FD BMSCs, whereas miR-181a-5p suppression induced an FD-like phenotype in normal BMSCs. Mechanistically, miR-181a-5p was the downstream target of CREB1, and CREB1 was posttranscriptionally regulated by miR-181a-5p.ConclusionsOur study identifies that the interaction loop between CREB1 and miR-181a-5p plays a crucial role in regulating the pathophysiologic features of FD BMSCs. MiR-181a-5p may be a potential therapeutic target for the treatment of FD.

MicroRNA-107 promotes apoptosis of acute myelocytic leukemia cells by targeting RAD51.

IntroductionThis study aimed to investigate the role of microRNA (miRNA) that affects acute myelocytic leukemia (AML) and its potential molecular mechanism by constructing a miRNA–mRNA interaction network using bioinformatics methods.Material and methodsMicroRNA expression data of AML were retrieved from Gene Expression Omnibus (GEO) and analyzed by microarray analysis. Expression levels of miR-107 and RAD51 mRNA were detected by quantitative real time polymerase chain reaction (qRT-PCR). Protein expression of RAD51, pro-apoptotic protein Bax, apoptosis related protein CytC and anti-apoptotic protein Bcl-2 were determined by Western blot. The rate of cell apoptosis was detected by Annexin-V/PI. The predicted targeting relationship between miR-107 and the 3′UTR of RAD51 was first predicted by the online application TargetScan and then verified by dual-luciferase assay.ResultsAcute myelocytic leukemia-associated genes (n = 197) and miRNAs (n = 1701) were retrieved from the database, the interaction network of miRNA-mRNA was constructed and the core position was occupied by RAD51. miR-107 exhibited a regulatory effect on RAD51 in which the mRNA and protein expression of RAD51 were both significantly inhibited by miR-107 mimics in vitro. Additionally, down-regulated expression of miR107 as well as up-regulated expression of RAD51 were detected not only in the plasma of AML patients compared to healthy volunteers, but also in AML cell lines compared to the normal bone marrow stromal cell line. Further study found that increased expression of miR-107 and the consequent down-regulation of RAD51 could aggravate the apoptosis of AML cells in vitro.ConclusionsOur present results showed that the crucial role of RAD51 and miR-107 in the apoptosis of AML cells, i.e., miR-107 promotes the apoptosis of AML cells through down-regulating the expression of RAD51.

Expression of the RET Receptor Tyrosine Kinase and GDNFR-α in Normal and Leukemic Human Hematopoietic Cells and Stromal Cells of the Bone Marrow Microenvironment

AbstractThe RET proto-oncogene product is a receptor tyrosine kinase representing the signal-transducing molecule of a multisubunit surface receptor complex for the glial cell line-derived neurotrophic factor (GDNF ), in which a novel glycosyl-phosphatidylinositol (PI)-linked protein (termed GDNFR-α) acts as the ligand-binding component. We have analyzed expression of RET and GDNFR-α in purified normal hematolymphopoietic cells, leukemia/lymphoma cell lines, and 154 primary samples from patients with hematopoietic malignancies encompassing different lineages and differentiation stages. Relatively low amounts of RET mRNA were found in early CD34+ hematopoietic progenitors, but RET transcripts appeared to increase after myelomonocytic maturation. No expression of RET was found in peripheral blood and tissue B and T lymphocytes. Analysis of human myelomonocytic cell lines was overall consistent with results obtained on purified normal cells. Accordingly, RET expression was mainly confined to acute myeloid leukemias (AMLs) displaying either monocytic (French-American-British M4 and M5) or intermediate-mature myeloid (M2 and M3) phenotypes, being less frequently detected in early myeloid (M0 and M1) AMLs. In contrast, RET mRNA was sporadically detected in B-cell tumors, whereas, among T-cell malignancies, RET transcripts were mainly detected in cells of postthymic and mature T-cell phenotype. RET broad detection in primary tumors was not paralleled by the mutual expression of GDNFR-α, which was detected only in 2 isolated primary samples and in 3 leukemia/lymphoma cell lines. However, GDNFR-α transcripts, in the absence of RET mRNA, were found in normal bone marrow stromal cells (BMSC), in BM fibroblasts, and in two osteoblast cell lines previously described to support normal hematopoiesis. In the presence of GDNF-receptors derived from BMSC by PI-specific phospholipase C cleavage, GDNF efficiently bound RET-expressing AML blasts and was functionally active by reducing their clonogenic growth and triggering the monocytic maturation of leukemic cells.

Cell and Tissue Response to Polyethylene Terephthalate Mesh Containing Bone Allograft in Vitro and in Vivo.

Extended polyethylene terephthalate mesh (PET, Dacron) can provide containment of compressed particulate allograft and autograft. This study assessed if PET mesh would interfere with osteoprogenitor cell migration from vertebral plates through particulate graft, and its effect on osteoblast differentiation or the quality of bone forming within fusing vertebra during vertebral interbody fusion. The impact of PET mesh on the biological response of normal human osteoblasts (NHOst cells) and bone marrow stromal cells (MSCs) to particulate bone graft was examined in vitro. Cells were cultured on rat bone particles +/- mesh; proliferation and osteoblast differentiation were assessed. The interface between the vertebral endplate, PET mesh, and newly formed bone within consolidated allograft contained by mesh was examined in a sheep model via microradiographs, histology, and mechanical testing. Growth on bone particles stimulated proliferation and early differentiation of NHOst cells and MSCs, but delayed terminal differentiation. This was not negatively impacted by mesh. New bone formation in vivo was not prevented by use of a PET mesh graft containment device. Fusion was improved in sites containing allograft/demineralized bone matrix (DBM) versus autograft and was further enhanced when stabilized using pedicle screws. Only sites treated with allograft/DBM+screws exhibited greater percent bone ingrowth versus discectomy or autograft. These results were mirrored biomechanically. PET mesh does not negatively impact cell attachment to particulate bone graft, proliferation, or initial osteoblast differentiation. The results demonstrated that bone growth occurs from vertebral endplates into graft material within the PET mesh. This was enhanced by stabilization with pedicle screws leading to greater bone ingrowth and biomechanical stability across the fusion site. The use of extended PET mesh allows containment of bone graft material during vertebral interbody fusion without inhibiting migration of osteoprogenitor cells from vertebral end plates in order to achieve fusion. 5.

HDAC8, A Potential Therapeutic Target, Regulates Proliferation and Differentiation of Bone Marrow Stromal Cells in Fibrous Dysplasia

Fibrous dysplasia (FD) is a disease of postnatal skeletal stem cells caused by activating mutations of guanine nucleotide‐binding protein alpha‐stimulating activity polypeptide (GNAS). FD is characterized by high proliferation and osteogenesis disorder of bone marrow stromal cells (BMSCs), resulting in bone pain, deformities, and fractures. The cAMP‐CREB pathway, which is activated by GNAS mutations, is known to be closely associated with the occurrence of FD. However, so far there is no available targeted therapeutic strategy for FD, as a critical issue that remains largely unknown is how this pathway is involved in FD. Our previous study revealed that histone deacetylase 8 (HDAC8) inhibited the osteogenic differentiation of BMSCs via epigenetic regulation. Here, compared with normal BMSCs, FD BMSCs exhibited significantly high proliferation and weak osteogenic capacity in response to HDAC8 upregulation and tumor protein 53 (TP53) downregulation. Moreover, inhibition of cAMP reduced HDAC8 expression, increased TP53 expression and resulted in the improvement of FD phenotype. Importantly, HDAC8 inhibition prevented cAMP‐induced cell phenotype and promoted osteogenesis in nude mice that were implanted with FD BMSCs. Mechanistically, HDAC8 was identified as a transcriptional target gene of CREB1 and its transcription was directly activated by CREB1 in FD BMSCs. In summary, our study reveals that HDAC8 associates with FD phenotype and demonstrates the mechanisms regulated by cAMP‐CREB1‐HDAC8 pathway. These results provide insights into the molecular regulation of FD pathogenesis, and offer novel clues that small molecule inhibitors targeting HDAC8 are promising clinical treatment for FD. stem cells translational medicine 2019;8:148&14

Mobilized Peripheral Blood versus Cord Blood: Insight into the Distinct Role of Proinflammatory Cytokines on Survival, Clonogenic Ability, and Migration of CD34+Cells

Inflammation may play a role in cancer. However, the contribution of cytokine-mediated crosstalk between normal hemopoietic stem/progenitor cells (HSPCs) and their (inflammatory) microenvironment is largely elusive. Here we compared survival, phenotype, and function of neonatal (umbilical cord blood (CB)) and adult (normal G-CSF-mobilized peripheral blood (mPB)) CD34+ cells after in vitro exposure to combined crucial inflammatory factors such as interleukin- (IL-) 1β, IL-6, tumor necrosis factor- (TNF-) α, or tissue inhibitor of metalloproteinases-1 (TIMP-1). To mimic bone marrow (BM) niche, coculture experiments with normal BM stromal cells (BMSCs) were also performed. We found that combined inflammatory cytokines increased only the in vitro survival of CB-derived CD34+ cells by reducing apoptosis. Conversely, selected combinations of inflammatory cytokines (IL-1β + TNF-α, IL-6 + TNF-α, and IL-1β + TNF-α + TIMP-1) mainly enhanced the in vitro CXCR4-driven migration of mPB-derived CD34+ cells. TNF-α, alone or in combination, upregulated CD44 and CD13 expression in both sources. Finally, BMSCs alone increased survival/migration of CB- and mPB-derived CD34+ cells at the same extent of the combined inflammatory cytokines; importantly, their copresence did not show additive/synergistic effect. Taken together, these data indicate that combined proinflammatory stimuli promote distinct in vitro functional activation of neonatal or adult normal HSPCs.

Novel targets for sensitizing breast cancer cells to TRAIL-induced apoptosis with siRNA delivery.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in variety of cancer cells without affecting most normal cells, which makes it a promising agent for cancer therapy. However, TRAIL therapy is clinically not effective due to resistance induction. To identify novel regulators of TRAIL that can aid in therapy, protein targets whose silencing sensitized breast cancer cells against TRAIL were screened with an siRNA library against 446 human apoptosis-related proteins in MDA-231 cells. Using a cationic lipopolymer (PEI-αLA) for delivery of library members, 16 siRNAs were identified that sensitized the TRAIL-induced death in MDA-231 cells. The siRNAs targeting BCL2L12 and SOD1 were further evaluated based on the novelty and their ability to sensitize TRAIL induced cell death. Silencing both targets sensitized TRAIL-mediated cell death in MDA-231 cells as well as TRAIL resistant breast cancer cells, MCF-7. Combination of TRAIL and siRNA silencing BCL2L12 had no effect in normal human umbilical vein cells and human bone marrow stromal cell. The silencing of BCL2L12 and SOD1 enhanced TRAIL-mediated apoptosis in MDA-231 cells via synergistically activating capsase-3 activity. Hence, here we report siRNAs targeting BCL2L12 and SOD1 as a novel regulator of TRAIL-induced cell death in breast cancer cells, providing a new approach for enhancing TRAIL therapy for breast cancer. The combination of siRNA targeting BCL2L12 and TRAIL can be a highly effective synergistic pair in breast cancer cells with minimal effect on the non-transformed cells.

Myeloma Cells Induce High Level of TAF12 Expression in Bone Marrow Stromal Cells, Resulting in Increased Osteoclastogenesis and Myeloma Cell Growth in Response to 1,25(OH)2D3

1,25 dihydroxy vitamin D3 (1,25D) plays multiple roles in normal and malignant cell functions, including cell differentiation and proliferation, and is a regulator of bone metastasis. However, the role of 1,25D in multiple myeloma (MM) is poorly characterized. We previously showed that TAF12, a member of the TFIID transcription complex, acts as a co-activator for vitamin D receptor (VDR) mediated transcription to increase 1,25D responsivity. Further, we reported that IL-6 can increase TAF12 levels in bone marrow stromal cells (BMSC). Since IL-6 is upregulated in the MM microenvironment, we assessed TAF12 levels in BMSC from 13 MM patients and 10 normals. TAF12 protein levels were significantly increased (25x) in MM BMSC compared to normal. Further, co-culture of BMSC from 3 normals with primary human MM cells significantly increased TAF12 levels in the normal BMSC. Similarly, when 5TGM-1 murine MM cells were inoculated intratibially into C57BL/KaLwRij mice and bone marrow cells collected 3 weeks later, TAF12 protein levels in MM exposed BMSC were significantly increased compared to PBS injected control mice. We then assessed if increased TAF12 levels persisted in normal BMSC (SAKA-T cells) after they were co-cultured with JJN3 MM cells and subsequently cultured in the absence of MM cells. We found that increased TAF12 levels persisted for at least 2 weeks after removal of MM cells, consistent with our findings that BMSC from MM patients express elevated TAF12 levels in the absence of MM cells. To determine if IL-6 contributes to the increased TAF12 levels induced by MM cells, we co-cultured SAKA-T cells with JJN3 MM cells in the presence or absence of anti-IL-6. Anti-IL-6 blocked the increase in TAF12 levels in these co-cultures.We then tested if the increased TAF12 levels enhance the 1,25D responsivity of BMSC. Addition of 1,25D (10-10 - 10-8 M) greatly increased expression of RANKL mRNA and VCAM-1 protein in MM BMSC compared to normals. The increased RANKL and VCAM-1 in MM BMSC treated with 1,25D correlated with increased osteoclast (OCL) formation, MM cell growth and adhesion of MM cells. When human OCL precursors and MM or normal BMSC were co-cultured with 1,25D with/without osteoprotegerin (OPG), OCL numbers were significantly increased in co-cultures of MM BMSC treated with 1,25D compared to normal, and the increase was blocked by OPG. Further, co-culture of JJN3 cells with normal or MM BMSC and 1,25D for 6 hours followed by removal of non-adherent MM cells, demonstrated that the number of MM cells adherent to BMSC was significantly increased with 1,25D treatment of MM BMSC co-cultures compared to normal BMSC. In addition, when JJN3 cells were co-cultured with normal or MM BMSC and 1,25D, JJN3 cells numbers were significantly increased in co-cultures with MM BMSC compared to normal BMSC. Finally, we determined if high levels of TAF12 in MM BMSC increased their 1,25D responsivity. We knocked-down (k/d) TAF12 with a siRNA in normal and MM BMSC, and found that TAF12 k/d in MM or normal BMSC decreased the levels of VCAM 1 induced by 1,25D compared to control siRNA transduced cells. To further investigate this effect, we assessed if TAF12 levels affected the stability of VDR in the presence of 1,25D, using TAF12 heterozygous or WT mouse BMSC. VDR half-life was decreased approximately 50% in TAF12 heterozygous BMSC compared with WT.These results show that MM cells increase expression of TAF12 in BMSC that is mediated by IL-6. BMSCs expressing high TAF12 levels have greater responsivity to 1,25D that enhances RANKL and VCAM-1 expression to induce osteoclastogenesis, MM cell growth and MM cell adhesion. These results further suggest that blocking TAF12 -VDR interactions may be a potential therapeutic target for MM bone disease. DisclosuresRoodman:Amgen: Consultancy.

Cell-based Assay System for Predicting Bone Regeneration in Patient Affected by Aseptic Nonunion and Treated with Platelet Rich Fibrin

Regenerative strategies based on the use of platelet concentrates as an autologous source of growth factors (GF) has been proposed to promote the healing of long bone nonunions. However, the relatively high failure rate stimulates interest in growing knowledge and developing solutions to obtain the best results from the regenerative approach. In this study we evaluated whether a cell-based assay system could be able to recognize patients who will benefit or not from the use of autologous platelet preparations. The autologous serum was used in culture medium to promote the osteogenic differentiation of normal bone-marrow stromal cells (BMSC). Blood samples were collected from 16 patients affected by aseptic long bone nonunion who were candidates to the treatment with autologous platelet-rich fibrin. The osteoinductive effect was detected by measuring the BMSC proliferation, the mineralization activity, and the expression of bone-related genes. Serum level of basic fibroblast growth factor (bFGF) was considered as a representative marker of the delivery of osteogenic GFs from platelets. Laboratory results were related to the characteristics of the disease before the treatment and to the outcome at 12 months. Serum samples from "good responders" showed significantly higher levels of bFGF and were able to induce a significantly higher proliferation of BMSC, while no significant differences were observed in terms of osteoblast differentiation. BMSC-based assay could be a useful tool to recognize patients who have a low probability to benefit from the use of autologous platelet concentrate to promote the healing of long bone nonunion.

IL-6 Contributes to the Defective Osteogenesis of Bone Marrow Stromal Cells from the Vertebral Body of the Glucocorticoid-Induced Osteoporotic Mouse.

Osteoporosis is one of the most prevalent skeletal system diseases. It is characterized by a decrease in bone mass and microarchitectural changes in bone tissue that lead to an attenuation of bone resistance and susceptibility to fracture. Vertebral fracture is by far the most prevalent osteoporotic fracture. In the musculoskeletal system, osteoblasts, originated from bone marrow stromal cells (BMSC), are responsible for osteoid synthesis and mineralization. In osteoporosis, BMSC osteogenic differentiation is defective. However, to date, what leads to the defective BMSC osteogenesis in osteoporosis remains an open question. In the current study, we made attempts to answer this question. A mouse model of glucocorticoid-induced osteoporosis (GIO) was established and BMSC were isolated from vertebral body. The impairment of osteogenesis was observed in BMSC of osteoporotic vertebral body. The expression profiles of thirty-six factors, which play important roles in bone metabolisms, were compared through antibody array between normal and osteoporotic BMSC. Significantly higher secretion level of IL-6 was observed in osteoporotic BMSCs compared with normal control. We provided evidences that IL-6 over-secretion impaired osteogenesis of osteoporotic BMSC. Further, it was observed that β-catenin activity was inhibited in response to IL-6 over-secretion. More importantly, in vivo administration of IL-6 neutralizing antibody was found to be helpful to rescue the osteoporotic phenotype of mouse vertebral body. Our study provides a deeper insight into the pathophysiology of osteoporosis and identifies IL-6 as a promising target for osteoporosis therapy.

A Patient-Derived Xenograft Model for Identifying Therapies and Defining Mechanisms of TSLP-Induced CRLF2 Signals in Ph-like B-ALL

Philadelphia chromosome (Ph)-like B cell acute lymphoblastic leukemia (B-ALL) is a high-risk leukemia with a gene expression profile similar to BCR-ABL1+ B-ALL. Approximately 50% of all Ph-like B-ALL is characterized by genetic alterations leading to overexpression of CRLF2 (CRLF2 B-ALL). CRLF2 B-ALL occurs 5 times more often in Hispanic and Native American children than others and is prevalent in adolescents and young adults. The poor outcomes associated with CRLF2 B-ALL represent a major clinical challenge and an important component of pediatric cancer health disparities. Biologically, CRLF2 acts as a receptor component for the cytokine, TSLP, which induces JAK2-STAT5 and PI3/AKT/mTOR pathway activation downstream of binding to CRLF2. Activating JAK mutations are associated with CRLF2 B-ALL, but overall data indicate that JAK mutations are present in 50% or less of CRLF2 B-ALL. Our data show that normal primary human bone marrow (BM) stromal cells express TSLP, suggesting that TSLP-induced CRLF2 signals could play a role in the initiation, maintenance and progression of CRLF2 B-ALL, particularly in cases without JAK mutations. Consistent with this, TSLP has been reported to increase in vitro production of human fetal B cell precursors. However studies of TSLP in B lymphopoiesis have been conducted almost exclusively in mice which show low homology (~40%) to human TSLP and CRLF2. Further, using phospho flow cytometry we show that mouse TSLP is unable to induce increases in pSTAT5, pAKT and pS6 observed in CRLF2 B-ALL cells stimulated with human TSLP, confirming the species specificity of mouse TSLP. These findings underscore the importance and challenge of developing in vivo systems that can model human TSLP-CRLF2 interactions for evaluating therapies and studying leukemogenesis of CRRLF2 B-ALL. To address this challenge we engineered patient-derived xenograft (PDX) mice to produce human TSLP (hTSLP) by transplanting them with stromal cells transduced to express hTSLP (+T mice). Control (-T) mice were produced by transplanting with stroma transduced with a control vector. Supernatant from engineered +T stroma, but not -T stroma, induced JAK/STAT5 and PI3K/AKT/mTOR pathway activation in CRLF2 B-ALL cells. ELISA assays showed normal serum levels of hTSLP (12-32 pg/ml) in +T mice, while hTSLP was undetectable in -T mice. Since TSLP has been shown to increase in vitro production of human B cell precursors, we evaluated the in vivo functionality of our model by comparing the production of normal B cell precursors in the BM of +T and -T PDX mice generated with human umbilical cord blood CD34+ cells. Data from 3 different cord blood donors showed that production of B cell precursors is 3-5 fold increased in +T as compared to -T mice. TSLP-induced increases were specific to B lineage cells, initiated in the earliest CD19+ B cell precursors, and maintained through later stages of B cell development. Next we evaluate the in vivo functionality of our model using primary leukemia cells. +T and -T PDX mice were produced using primary CRLF2 B-ALL cells. BM was harvested and whole genome microarray was performed on isolated CRLF2 B-ALL cells. Evaluation of microarray data by Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis showed that genes downstream of mTOR pathway activation were upregulated in +T as compared to -T PDX mice, confirming hTSLP activity in the +T PDX mice. Next we tested whether +T PDX mice provide an in vivo model of B-ALL that more closely mirrors patients than -T PDX mice. +T and -T PDX mice were generated from primary high risk B-ALL. RNAseq gene expression profiles from primary patient B-ALL cells were compared to those of the same patient sample expanded in +T and -T PDX mice. The gene expression pattern in +T mice was significantly closer to the primary patient sample than those from -T mice. The +T and -T PDX mice described here provide a novel preclinical model for studying the role of TSLP in the initiation, progression and maintenance of CRLF2 B-ALL and for evaluating drug efficacy in an in vivo model that more closely mirrors the in vivo environment present in patients. DisclosuresNo relevant conflicts of interest to declare.

Malignant transformation of bone marrow stromal cells induced by the brain glioma niche in rats.

Normal human embryonic stem cells (hESCs) can develop neoplastic cancer stem cell (CSC) properties after coculture with transformed hESCs in vitro. In the present study, the influence of the tumor microenvironment on malignant transformation of bone marrow stromal cells (BMSCs) was studied after allografting a mixture of enhanced green fluorescent protein (EGFP)-labeled BMSCs and C6 glioma cells into the rat brain to understand the influence of the cellular environment, especially the tumor environment, on the transformation of grafted BMSCs in the rat brain. We performed intracerebral transplantation in the rat brain using EGFP-labeled BMSCs coinjected with C6 tumor cells. After transplantation, the EGFP-labeled cells were isolated from the tumor using fluorescence-activated cell sorting, and the characteristics of the recovered cells were investigated. Glioma-specific biomarkers of the sorted cells and the biological characteristics of the tumors were analyzed. The BMSCs isolated from the cografts were transformed into glioma CSCs, as indicated by the marked expression of the glioma marker GFAP in glioma cells, and of Nestin and CD133 in neural stem cells and CSCs, as well as rapid cell growth, decreased level of the tumor suppressor gene p53, increased level of the oncogene murine double minute gene 2 (MDM2), and recapitulation of glioma tissues in the brain. These data suggest that BMSCs can be transformed into CSCs, which can be further directed toward glioma formation under certain conditions, supporting the notion that the tumor microenvironment is involved in transforming normal BMSCs into glial CSCs.

Abstract 2425: An in vitro platform to dissect drug responsiveness in refractory anemia with ringed sideroblasts (RARS)

Abstract Background: The pathophysiology of low-risk myelodysplastic syndrome (LR-MDS) remains unknown and therapeutic options are limited. In a phase-II trial, with oral rigosertib, a small molecule inhibitor of PI3K and PLK pathways, was shown to have a 39% response rate, in patients with LR-MDS. Rigosertib caused an increase in hemoglobin, a decrease in transfusion requirements, and, in some patients who were previously refractory, “re-sensitization” to erythropoietin (EPO) therapy. Thus far, the mechanism of this responsiveness remains unknown. Whole exome sequencing revealed broad spectrum mutations in these patients, including mutations in SF3B1, SRSF2 and TET2. However, there was no correlation between mutational spectrum and responsiveness. Methods: CD34+ cells were isolated from bone marrow (BM) mono nuclear cells derived from BM aspirates. Bone marrow stromal cells (BMSC) were isolated from bone biopsies of patients with MDS. Normal CD34+ cells and BMSC were obtained commercially. CD34+ cells were co-cultured with stromal cells plated a day before and stimulated with concentrations of EPO and rigosertib. Differentiation was assessed by staining cells with various markers of erythroid differentiation and analyzed by FACS. We show that co-culture of CD34+ stem cells from the bone marrow of patients with their own BMSC recapitulates key features of the MDS phenotype and rigosertib responsiveness. Results: Normal (non-MDS) CD34+ cells co-cultured with normal BMSC showed striking responsiveness to EPO stimulation, as evidenced by the increased production of CD45- low/GPA+/ band 3+/Integrin-Alpha4+ erythroid cells (2.47 fold increase compared to no EPO). In contrast, RARS CD34+ cells co-cultured with MDS stromal cells showed no erythroid differentiation with EPO stimulation (1.09 fold change compared to EPO). Notably, LR-MDS co-cultures showed increased erythroid differentiation with the addition of 20 nm and 100 nm rigosertib in the presence of EPO (1.28 and 1.4 fold increase, respectively, compared to EPO alone). Moreover, co-cultures obtained from 3/3 patients that showed responsiveness in vivo were responsive to combined EPO/rigosertib stimulation in vitro, while 2/2 non-responsive patients were not responsive in vitro. Conclusions: We have created an in vitro platform to dissect the mechanism of rigosertib responsiveness in RARS patients. This is a novel co-culture-based system that recapitulates key features of the RARS phenotype. Without reliable animal models for this disease, this platform may offer a viable method to characterize drug responsiveness, dissect mechanisms, and offer patient-specific drug responsiveness information, since individual CD34+ cells are co-cultured with a patient's own BMSC. We wish to use this in vitro co- culture test to prospectively predict responsiveness to experimental drugs in patients, thereby focusing the drug on only selected patients. Citation Format: Daniela Georgieva, Sheherzad Preisler, Michael Churchill, Abdullah Mahmood Ali, Azra Raza, Siddhartha Mukherjee. An in vitro platform to dissect drug responsiveness in refractory anemia with ringed sideroblasts (RARS). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2425. doi:10.1158/1538-7445.AM2015-2425

SHH Inhibitor PF-0449913 in Vitro Remodels Leukemic Niche into HSC Supportive Niche

BackgroundAcute myelogenous leukemia (AML) is a highly lethal disease with only 20% of 5 years survival. Most AML patients experience relapse usually leading to death. Progression to therapy resistant AML is driven by leukemia stem cells (LSC) harboring enhanced survival, dormancy and self-renewal capacity in supportive niches. Growing evidence indicates that in the development of myeloid malignancies deregulation of stem cells activity is as important as deregulation of the BM microenvironment. Sonic hedgehog (SHH) pathway molecules in general and SMO in particular are well known to maintain the growth of cancer cells. However, it’s role in leukemia niche-stem cell interaction is not well defined. Therefore, here we investigate: 1/ how the small molecule, SMO antagonist, PF-0449913 impacts the AML BM microenvironment and 2/ how in turn, changes in the activity of AML BM niche cells contribute to its remodeling into HSC supportive niche.MethodsCD34- cell from bone marrow patients undergoing hip replacement surgery (normal BM) as well as AML bone marrow were utilized for the development of the primary human stromal monolayers. We investigated stromal cultures of primary normal BM (NBM, n=6) and AML (n=6). As a control human normal bone marrow stromal cell line HS-5 was used. Then human CD34+ cells were selected from AML primary samples (n=6). As a normal control, CD34+ cells from cord blood (CB, n=5), or NBM (n=5) were utilized for the co-culture experiments for up to 9 weeks and then plated in survival and self-renewal assays. PF-0449913 was added to stromal co-cultures or, as a pre-treatment, directly to stromal monolayers.ResultsIn order to examine the role of SMO regulation in LSC generation and maintenance, primary AML (n=6) or cord blood (n=3) CD34+ cells were co-cultures with normal BM (n=3) or AML (n=3) stroma in the presence of novel small molecule inhibitor (PF-0449913) for 2 weeks. It significantly reduced LSC survival and self-renewal, while spared HSC. These effects were not observed in the absence of stroma. Importantly, pre-treatment of the AML stroma with PF-0449913 for 1 week prior- to co-culture also resulted in LSC’s inhibition of survival and self-renewal.AML- and normal BM-derived stroma differ in their ability to support HSC and LSC: LSC (n=6) were capable to self-renew after 9 weeks of co-culture with both normal and AML stroma, while cord blood (n=4) as well as normal BM (n=5) HSC lost their self-renewal potential after only 2 weeks of co-culture with the AML stroma. PF-0449913 induced changes in AML stroma. which was previously unsupportive to HSC survival, it became compliant : self-renewal of HSC increased more than 3 times. It reversed also the inhibitory effect of AML stroma-derived conditioned media (CM).Pre-treatment of the HS-5 cells with CM from AML stroma for 4 weeks prior to co-culture experiments led to significant inhibition of the cord blood (n=3) and NBM (n=3) HSC survival and self-renewal. However, in CM obtained from AML stroma treated with PF-0449913 that effect was reversed.Combined treatment of AML cells (n=6) with PF-0449913 and de-methylating agent Vidaza (5-azacytodine) or CXCR4 antagonist AMD3100 resulted in even greater inhibition of LSC survival and self-renewal. However, Vidaza did not mediate changes in AML stroma.ConclusionsTogether these data indicate that, while both conditioned media and co-culture with AML stroma impaired HSC survival and self-renewal, co-culture conditions resulted in a greater reduction in survival and self-renewal capacity, implicating that cell-cell contact or unstable secreted factors exacerbate the effects. That suggests that microenvironmental cues play a key role in regulating normal HSC versus LSC survival and maintenance, and leukemic stroma exibit severely compromised ability to maintain normal HSCs, but effectively support LSCs. PF-0449913treatment of AML induces not only eradication of LSC, but also mediate changes in AML stroma, supporting HSC generation and maintenance.Simultaneous targeting of both AML niche and LSC could represent a novel avenue for treatment of AML patients. DisclosuresNo relevant conflicts of interest to declare.

In Vitro Osteogenic Induction of Bone Marrow Stromal Cells with Encapsulated Gene-Modified Bone Marrow Stromal Cells and In Vivo Implantation for Orbital Bone Repair

Osteogenic induction with either growth factors or genetic modification has limitations due to the short half-life and cost of the former, or safety concerns regarding the latter. The objective of this study was to employ a microcapsulation technique to separate genetically modified and nonmodified bone marrow stromal cells (BMSCs) to establish a cost-effective and biosafe osteogenic induction methodology with functional evaluation in vitro and in vivo in a canine model. Autologous BMSCs were isolated and transduced with adenoviral vectors containing either BMP-2 or vascular endothelial growth factor (VEGF) or were dual transduced followed by encapsulation in alginate microcapsules using an electrostatic bead generator. After cocultured with encapsulated cells, normal autologous BMSCs were analyzed for osteogenic differentiation and seeded onto tricalcium phosphate (TCP) scaffolds for in vivo implantation to repair orbital wall bone defects (12 mm in diameter) in a canine model. In vitro assays showed that the expression of the transduced genes was significantly upregulated, with significantly more transduced proteins released from the transduced cells compared with control cells. Importantly, examination of the BMSCs induced by soluble factors released from the encapsulated cells revealed a significant upregulation of expression of osteogenic markers Runx2, BSP, OPN, and OCN in dual-transduction or induction groups. In addition, dual transduction and induction resulted in the highest increase of alkaline phosphatase activity and mineralization compared with other experimental groups. In vivo assays using CT, micro-CT, and histology further supported the qPCR and western blot findings. In conclusion, encapsulation of genetically modified BMSCs was able to release a sufficient amount of BMP-2 and VEGF, which effectively induced osteogenic differentiation of normal-cultured BMSCs and demonstrated bone repair of the orbital wall defect after implantation with β-TCP in vivo.