Expression of HLA-DR by mesenchymal stromal cells in the platelet lysate era: an obsolete release criterion for MSCs?

BACKGROUND: According to the definition of the International Society for Cell and Gene Therapy (ISCT), mesenchymal stromal cells (MSCs) do not express HLA-DR. This phenotypic marker as a release criterion for clinical use was established at a time when MSCs were expanded in fetal bovine serum (FBS)-containing media. Replacement of FBS with platelet lysate (PLs) as a medium supplement induced a significantly higher fraction of MSCs to express MHC class II antigens. METHODS: As this raised concerns that such MSCs may play the role of antigen-presenting cells for T cells, in the current study, we studied major factors that may induce HLA-DR on MSCs by means of flow cytometry and real-time polymerase chain reaction. The immunomodulatory potential of MSCs was assessed by a mixed lymphocyte reaction. RESULTS: Our results demonstrated that a very low percentage of generated and expanded MSCs in FBS express HLA-DR (median: 1.1%, range: 0.3-22%) compared to MSCs generated and expanded in PLs (median: 28.4%, range: 3.3-73.7%). Analysis of the cytokine composition of ten PLs showed a significant positive correlation between the levels of IL-1ß, IL-4, IL-10, IL-17, bFGF and expression of HLA-DR, in contrast to no correlation with the age of MSC donors and HLA-DR (r = 0.21). Both MSCs expressing low and high levels of HLA-DR expressed class II transactivator (CIITA), a master gene coding for these molecules. Our results demonstrate for the first time that MSCs with constitutively high levels of HLA-DR also express moderate levels of indoleamine 2,3-dioxygenase (IDO). Treatment of MSCs with multiple doses of TGF-ß1 at passage 0 (P0) and passage 1 (P1) completely abrogated HLA-DR and IDO expression. In contrast, treatment of MSCs with a single dose of TGF-ß1 after P0 only partially reduced the expression of HLA-DR and CIITA. Remarkably, increased expression of HLA-DR on MSCs that constitutively express high levels of this antigen after overnight incubation with IFN-γ was rather unaffected by incubation with TGF-ß1. However, treatment of MSCs with TGF-ß1 for 24 h completely abrogated constitutive expression of IDO. CONCLUSIONS: Irrespective of HLA-DR expression at the population level, all MSC preparations significantly inhibited the proliferation of stimulated peripheral blood mononuclear cells, indicating that HLA-DR represents an obsolete release marker for the clinical use of MSCs.

Pediatric multicellular tumor spheroid models illustrate a therapeutic potential by combining BH3 mimetics with Natural Killer (NK) cell-based immunotherapy.

The induction of apoptosis is a direct way to eliminate tumor cells and improve cancer therapy. Apoptosis is tightly controlled by the balance of pro- and antiapoptotic Bcl-2 proteins. BH3 mimetics neutralize the antiapoptotic function of Bcl-2 proteins and are highly promising compounds inducing apoptosis in several cancer entities including pediatric malignancies. However, the clinical application of BH3 mimetics in solid tumors is impeded by the frequent resistance to single BH3 mimetics and the anticipated toxicity of high concentrations or combination treatments. One potential avenue to increase the potency of BH3 mimetics is the development of immune cell-based therapies to counteract the intrinsic apoptosis resistance of tumor cells and sensitize them to immune attack. Here, we describe spheroid cultures of pediatric cancer cells that can serve as models for drug testing. In these 3D models, we were able to demonstrate that activated allogeneic Natural Killer (NK) cells migrated into tumor spheroids and displayed cytotoxicity against a wide range of pediatric cancer spheroids, highlighting their potential as anti-tumor effector cells. Next, we investigated whether treatment of tumor spheroids with subtoxic concentrations of BH3 mimetics can increase the cytotoxicity of NK cells. Notably, the cytotoxic effects of NK cells were enhanced by the addition of BH3 mimetics. Treatment with either the Bcl-XL inhibitor A1331852 or the Mcl-1 inhibitor S63845 increased the cytotoxicity of NK cells and reduced spheroid size, while the Bcl-2 inhibitor ABT-199 had no effect on NK cell-mediated killing. Taken together, this is the first study to describe the combination of BH3 mimetics targeting Bcl-XL or Mcl-1 with NK cell-based immunotherapy, highlighting the potential of BH3 mimetics in immunotherapy.

The Phenotype and Functional Activity of Mesenchymal Stromal Cells in Pediatric Patients with Non-Malignant Hematological Diseases.

As the biology of mesenchymal stromal cells (MSCs) in patients with non-malignant hematological diseases (NMHD) is poorly understood, in the current study we performed a basic characterization of the phenotype and functional activity of NMHD-MSCs. Bone marrow (BM) of patients with thalassemia major (TM) possessed a significantly higher number of nucleated cells (BM-MNCs)/mL BM than healthy donors (P < 0.0001), which however did not result in a higher number of colony forming units-fibroblast (CFU-F) per milliliter BM. In contrast, from 1 × 106 BM-MNCs of patients with sickle cell disease (SCD) were generated significantly more CFU-Fs than from TM-BM-MNCs (P < 0.013) and control group (P < 0.02). In addition, NMHD-MSCs expressed significantly lower levels of CD146 molecule, demonstrated an equal proliferation potential and differentiated along three lineages (osteoblasts, chondrocytes and adipocytes) as healthy donors' MSCs, with exception of TM-MSCs which differentiated weakly in adipocytes. In contrast to other NMHD-MSCs and healthy donors' MSCs, TM-MSCs demonstrated an impaired in vitro immunosuppressive potential, either. Noteworthy, the majority of the immunosuppressive effect of NMHD-MSCs was mediated through prostaglandin-E2 (PGE2), because indomethacin (an inhibitor of PGE2 synthesis) was able to significantly reverse this effect. Our results indicate therefore that NMHD-MSCs, except TM-MSCs, may be used as an autologous cell-based therapy for post-transplant complications such as graft failure, graft-versus-host disease (GvHD) and osteonecrosis.

Tumorigenic and Antiproliferative Properties of the TALE-Transcription Factors MEIS2D and MEIS2A in Neuroblastoma.

Neuroblastoma is one of only a few human cancers that can spontaneously regress even after extensive dissemination, a poorly understood phenomenon that occurs in as many as 10% of patients. In this study, we identify the TALE-homeodomain transcription factor MEIS2 as a key contributor to this phenomenon. We identified MEIS2 as a MYCN-independent factor in neuroblastoma and showed that in this setting the alternatively spliced isoforms MEIS2A and MEIS2D exert antagonistic functions. Specifically, expression of MEIS2A was low in aggressive stage 4 neuroblastoma but high in spontaneously regressing stage 4S neuroblastoma. Moderate elevation of MEIS2A expression reduced proliferation of MYCN-amplified human neuroblastoma cells, induced neuronal differentiation and impaired the ability of these cells to form tumors in mice. In contrast, MEIS2A silencing or MEIS2D upregulation enhanced the aggressiveness of the tumor phenotype. Mechanistically, MEIS2A uncoupled a negative feedback loop that restricts accumulation of cellular retinoic acid, an effective agent in neuroblastoma treatment. Overall, our results illuminate the basis for spontaneous regression in neuroblastoma and identify an MEIS2A-specific signaling network as a potential therapeutic target in this common pediatric malignancy.Significance: This study illuminates the basis for spontaneous regressions that can occur in a common pediatric tumor, with implications for the development of new treatment strategies. Cancer Res; 78(8); 1935-47. ©2018 AACR.

In vitro migration and proliferation ("wound healing") potential of mesenchymal stromal cells generated from human CD271(+) bone marrow mononuclear cells.

BACKGROUND: Emerging evidence indicates that mesenchymal stromal cells (MSCs) isolated from different tissue sources may be used in vivo as tissue restorative agents. To date, there is no evidence, however, on migration and proliferation ("wound healing") potential of different subsets of MSCs. The main goal of this study was therefore to compare the in vitro "wound healing" capacity of MSCs generated from positively selected CD271(+) bone marrow mononuclear cells (CD271-MSCs) and MSCs generated by plastic adherence (PA-MSCs). METHODS: The in vitro model of wound healing (CytoSelect™ 24-Well Wound Healing Assay) was used in order to compare the migration and proliferation potential of CD271-MSCs and PA-MSCs of passage 2 and 4 cultured in presence or absence of growth factors or cytokines. RESULTS: CD271-MSCs of both passages when compared to PA-MSCs demonstrated a significantly higher potential to close the wound 12 and 24 h after initiation of the wound healing assay (P < 0.003 and P < 0.002, respectively). Noteworthy, the migration capacity of PA-MSCs of second passage was significantly improved after stimulation with FGF-2 (P < 0.02), PDGF-BB (P < 0.006), MCP-1 (P < 0.002) and IL-6 (P < 0.03), whereas only TGF-ß enhanced significantly migration process of PA-MSCs of P4 12 h after the treatment (P < 0.02). Interestingly, treatment of CD271-MSCs of both passages with growth factors or cytokines did not affect their migratory potential. CONCLUSIONS: Our in vitro data provide the first evidence that CD271-MSCs are significantly more potent in "wound healing" than their counterparts PA-MSCs.

Altered mucosal immune response after acute lung injury in a murine model of Ataxia Telangiectasia.

BACKGROUND: Ataxia telangiectasia (A-T) is a rare but devastating and progressive disorder characterized by cerebellar dysfunction, lymphoreticular malignancies and recurrent sinopulmonary infections. In A-T, disease of the respiratory system causes significant morbidity and is a frequent cause of death. METHODS: We used a self-limited murine model of hydrochloric acid-induced acute lung injury (ALI) to determine the inflammatory answer due to mucosal injury in Atm (A-T mutated)- deficient mice (Atm(-/-)). RESULTS: ATM deficiency increased peak lung inflammation as demonstrated by bronchoalveolar lavage fluid (BALF) neutrophils and lymphocytes and increased levels of BALF pro-inflammatory cytokines (e.g. IL-6, TNF). Furthermore, bronchial epithelial damage after ALI was increased in Atm(-/-) mice. ATM deficiency increased airway resistance and tissue compliance before ALI was performed. CONCLUSIONS: Together, these findings indicate that ATM plays a key role in inflammatory response after airway mucosal injury.

Smac mimetic and glucocorticoids synergize to induce apoptosis in childhood ALL by promoting ripoptosome assembly.

Apoptosis resistance contributes to poor outcome in pediatric acute lymphoblastic leukemia (ALL). Here, we identify a novel synergistic combination of Smac mimetic BV6 and glucocorticoids (GCs) (ie, dexamethasone, prednisolone) to trigger apoptosis in ALL cells. BV6 and GCs similarly cooperate to induce apoptosis in patient-derived leukemia samples, underlining the clinical relevance. Importantly, BV6/dexamethasone cotreatment is significantly more effective than monotherapy to delay leukemia growth in a patient-derived xenograft model of pediatric ALL without causing additional side effects. In contrast, BV6 does not increase cytotoxicity of dexamethasone against nonmalignant peripheral blood lymphocytes, mesenchymal stromal cells, and CD34-positive hematopoietic cells. We identify a novel mechanism by showing that BV6 and dexamethasone cooperate to deplete cIAP1, cIAP2, and XIAP, thereby promoting assembly of the ripoptosome, a RIP1/FADD/caspase-8-containing complex. This complex is critical and is required for BV6/dexamethasone-induced cell death, because RIP1 knockdown reduces caspase activation, reactive oxygen species production, and cell death. Ripoptosome formation occurs independently of autocrine/paracrine loops of death receptor ligands, because blocking antibodies for TNFα, tumor necrosis factor-related apoptosis-inducing ligand, or CD95 ligand or knockdown of death receptors fail to rescue BV6/dexamethasone-induced cell death. This is the first report showing that BV6 sensitizes for GC-triggered cell death by promoting ripoptosome formation with important implications for apoptosis-targeted therapies of ALL.

Clonal analysis of multipotent stromal cells derived from CD271+ bone marrow mononuclear cells: functional heterogeneity and different mechanisms of allosuppression.

Previous reports demonstrated a relationship between proliferation potential and trilineage differentiation in mesenchymal stromal cell-derived clones generated using plastic adherence (PA-MSCs). However, there are no reports presenting a clonal analysis of the proliferative potential, differentiation potential and allosuppressive effects of human mesenchymal stromal cell subsets. In this study, we performed a clonal analysis of mesenchymal stromal cells generated from human CD271(+) bone marrow mononuclear cells (CD271-MSCs). After transfection with the gene encoding green fluorescent protein, the cells were single-cell sorted and cultured for 2-4 weeks. A population doubling analysis demonstrated that 25% of CD271-MSC clones are fast-proliferating clones compared to only 10% of PA-MSC clones. Evaluation of the allosuppressive potential demonstrated that 81.8% of CD271-MSC clones were highly allosuppressive compared to only 58% of PA-MSC clones. However, no consistent correlation was observed between allosuppression and proliferative potential. Prostaglandin E2 levels were positively correlated with the allosuppressive activity of individual clones, suggesting that this molecule may be a useful predictive biomarker for the allosuppressive potential of mesenchymal stromal cells. In contrast, inhibitory studies of indoleamine 2,3 dioxygenase indicated that none of the clones used this enzyme to mediate their allosuppressive effect. Differentiation studies revealed the presence of tripotent, bipotent and unipotent CD271-MSC and PA-MSC clones which suppressed the allogeneic reaction to differing extents in vitro. In conclusion, our findings demonstrate differences between CD271-MSCs and PA-MSCs and indicate that neither proliferation potential nor differentiation potential represents a consistent predictive parameter for the immunomodulatory effects of either type of mesenchymal stromal cells.

Cytotoxic effects of treosulfan and busulfan against leukemic cells of pediatric patients.

PURPOSE: The alkylating agent treosulfan exerts a high cytotoxic activity against various malignant cells. Due to limited non-hematological toxicity, treosulfan might be a promising compound in myeloablative therapy for hematopoietic transplantation in children. Since in vitro data regarding the activity of treosulfan against childhood leukemic cells are limited, we compared the effect of treosulfan and busulfan against pediatric leukemic and non-malignant cells. EXPERIMENTAL DESIGN: Both agents were tested alone and in combination with fludarabine by means of the MTT and/or a five color-flow cytometric assay. Moreover, the induction of apoptosis by treosulfan was investigated via regulation of the proteinase caspase 3. RESULTS: Treosulfan was more active against leukemic cells of 20 children as well as against 3 leukemia-derived cell lines than busulfan, with increasing IC50 values from initial diagnosis to relapse. Overall purified stem cells were most sensitive, followed by CD56+CD3- NK and CD3+ T cells. The combination of treosulfan with fludarabine resulted in a synergistic effect against leukemic cells. In malignant cells, treosulfan induced rapid cell apoptosis measured by the activation of the centrally proteinase caspase 3. CONCLUSION: Our results indicate that treosulfan has activity against pediatric leukemic cells, myeloablative potential and immunosuppressive properties suitable for conditioning regimen in childhood malignancies.

Phylogeny of three parapatric species of desert ants, Cataglyphis bicolor, C. viatica, and C. savignyi: a comparison of mitochondrial DNA, nuclear DNA, and morphological data.

Due to morphological comparisons the Tunisian desert ant species Cataglyphis bicolor has been divided into three parapatric species: C. bicolor, C. viatica, and C. savignyi. The species status of the latter is supported by sequence analyses of the mitochondrial CO1 and CO2 region, while analyses of the same mitochondrial region lacked resolution for the separation of C. bicolor and C. viatica. However, the geographic distribution of mtDNA haplotypes points to different population viscosities with C. bicolor queens having longer migration distances than queens of C. viatica. Furthermore, by the use of microsatellites we excluded ongoing gene flow between geographically overlapping populations of C. bicolor and C. viatica, and hence support the morphology-based three-species hypothesis. Concerning the ongoing discussion on the future roles of morphology and molecular biology in systematics we call for a combination of both whenever possible.