Liquid biopsy-based monitoring of residual disease in multiple myeloma by analysis of the rearranged immunoglobulin genes-A feasibility study.

The need for sensitive monitoring of minimal/measurable residual disease (MRD) in multiple myeloma emerged as novel therapies led to deeper responses. Moreover, the potential benefits of blood-based analyses, the so-called liquid biopsy is prompting more and more studies to assess its feasibility. Considering these recent demands, we aimed to optimize a highly sensitive molecular system based on the rearranged immunoglobulin (Ig) genes to monitor MRD from peripheral blood. We analyzed a small group of myeloma patients with the high-risk t(4;14) translocation, using next-generation sequencing of Ig genes and droplet digital PCR of patient-specific Ig heavy chain (IgH) sequences. Moreover, well established monitoring methods such as multiparametric flow cytometry and RT-qPCR of the fusion transcript IgH::MMSET (IgH and multiple myeloma SET domain-containing protein) were utilized to evaluate the feasibility of these novel molecular tools. Serum measurements of M-protein and free light chains together with the clinical assessment by the treating physician served as routine clinical data. We found significant correlation between our molecular data and clinical parameters, using Spearman correlations. While the comparisons of the Ig-based methods and the other monitoring methods (flow cytometry, qPCR) were not statistically evaluable, we found common trends in their target detection. Regarding longitudinal disease monitoring, the applied methods yielded complementary information thus increasing the reliability of MRD evaluation. We also detected indications of early relapse before clinical signs, although this implication needs further verification in a larger patient cohort.

The Effect of Belantamab Mafodotin on Primary Myeloma-Stroma Co-Cultures: Asymmetrical Mitochondrial Transfer between Myeloma Cells and Autologous Bone Marrow Stromal Cells.

Belantamab mafodotin (belamaf) is an afucosylated monoclonal antibody conjugated to the microtubule disrupter monomethyl auristatin-F (MMAF) that targets B cell maturation antigen (BCMA) on the surface of malignant plasma cells. Belamaf can eliminate myeloma cells (MMs) through several mechanisms. On the one hand, in addition to inhibiting BCMA-receptor signaling and cell survival, intracellularly released MMAF disrupts tubulin polymerization and causes cell cycle arrest. On the other hand, belamaf induces effector cell-mediated tumor cell lysis via antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis. In our in vitro co-culture model, the consequences of the first mentioned mechanism can be investigated: belamaf binds to BCMA, reduces the proliferation and survival of MMs, and then enters the lysosomes of malignant cells, where MMAF is released. The MMAF payload causes a cell cycle arrest at the DNA damage checkpoint between the G2 and M phases, resulting in caspase-3-dependent apoptosis. Here, we show that primary MMs isolated from different patients can vary widely in terms of BCMA expression level, and inadequate expression is associated with extremely high resistance to belamaf according to our cytotoxicity assay. We also reveal that primary MMs respond to increasing concentrations of belamaf by enhancing the incorporation of mitochondria from autologous bone marrow stromal cells (BM-MSCs), and as a consequence, MMs become more resistant to belamaf in this way, which is similar to other medications we have analyzed previously in this regard, such as proteasome inhibitor carfilzomib or the BCL-2 inhibitor venetoclax. The remarkable resistance against belamaf observed in the case of certain primary myeloma cell cultures is a cause for concern and points towards the use of combination therapies to overcome the risk of antigen escape.

Specific Antibody and the T-Cell Response Elicited by BNT162b2 Boosting After Two ChAdOx1 nCoV-19 in Common Variable Immunodeficiency.

Common variable immunodeficiency (CVID) patients have markedly decreased immune response to vaccinations. In this study we evaluated humoral and T cell-mediated responses against severe acute respiratory syndrome coronavirus-2 (SARS-Cov-2) with additional flow cytometric changes in CVID patients receiving booster vaccination with BNT162b2 after two ChAdOx1 nCoV-19. The BNT162b2 vaccine raised the anti-spike protein S immunoglobulin G over the cut-off value from 70% to 83% in CVID, anti-neutralizing antibody had been raised over a cut-off value from 70% to 80% but levels after boosting were significantly less in both tests than in healthy controls (*p=0.02; **p=0.009 respectively). Anti-SARS-CoV-2 immunoglobulin A became less positive in CVID after boosting, but the difference was not significant. The cumulative interferon-γ positive T cell response by ELISpot was over the cut-off value in 53% of the tested individuals and raised to 83% after boosting. This and flow cytometric control of cumulative CD4+ and CD8+ virus-specific T cell absolute counts in CVID were also statistically not different from healthy individuals after boosting. Additional flow cytometric measures for CD45+ lymphocytes, CD3+, and CD19+ cells have not shown significant differences from controls except for lower CD4+T cell counts at both time points (**p=0.003; **p=0.002), in parallel CD4+ virus-specific T-cell ratio was significantly lower in CVID patients at the first time point (*p: 0.03). After boosting, in more than 33% of both CVID patients and also in their healthy controls we detected a decrease in absolute CD45+, CD3+, CD3+CD4+, and CD3+CD8+, CD19+, and CD16+56+ cell counts. CD16+CD56+ cell counts were significantly lower compared to controls before and after boosting (*p=0.02, *p=0.02). CVID patients receiving immunosuppressive therapy throughout the previous year or autologous stem cell transplantation two years before vaccination had worse responses in anti-spike, anti-neutralizing antibody, CD3+CD4+T, CD19+ B, and natural killer cell counts than the whole CVID group. Vaccinations had few side effects. Based on these data, CVID patients receiving booster vaccination with BNT162b2 after two ChadOx1 can effectively elevate the levels of protection against COVID-19 infection, but the duration of the immune response together with COVID-19 morbidity data needs further investigation among these patients.

Stromal Cells Serve Drug Resistance for Multiple Myeloma via Mitochondrial Transfer: A Study on Primary Myeloma and Stromal Cells.

Recently, it has become evident that mitochondrial transfer (MT) plays a crucial role in the acquisition of cancer drug resistance in many hematologic malignancies; however, for multiple myeloma, there is a need to generate novel data to better understand this mechanism. Here, we show that primary myeloma cells (MMs) respond to an increasing concentration of chemotherapeutic drugs with an increase in the acquisition of mitochondria from autologous bone marrow stromal cells (BM-MSCs), whereupon survival and adenosine triphosphate levels of MMs increase, while the mitochondrial superoxide levels decrease in MMs. These changes are proportional to the amount of incorporated BM-MSC-derived mitochondria and to the concentration of the used drug, but seem independent from the type and mechanism of action of chemotherapeutics. In parallel, BM-MSCs also incorporate an increasing amount of MM cell-derived mitochondria accompanied by an elevation of superoxide levels. Using the therapeutic antibodies Daratumumab, Isatuximab, or Elotuzumab, no similar effect was observed regarding the MT. Our research shows that MT occurs via tunneling nanotubes and partial cell fusion with extreme increases under the influence of chemotherapeutic drugs, but its inhibition is limited. However, the supportive effect of stromal cells can be effectively avoided by influencing the metabolism of myeloma cells with the concomitant use of chemotherapeutic agents and an inhibitor of oxidative phosphorylation.

Establishment and Characterization of a Brca1-/-, p53-/- Mouse Mammary Tumor Cell Line.

Breast cancer is the most commonly occurring cancer in women and the second most common cancer overall. By the age of 80, the estimated risk for breast cancer for women with germline BRCA1 or BRCA2 mutations is around 80%. Genetically engineered BRCA1-deficient mouse models offer a unique opportunity to study the pathogenesis and therapy of triple negative breast cancer. Here we present a newly established Brca1-/-, p53-/- mouse mammary tumor cell line, designated as CST. CST shows prominent features of BRCA1-mutated triple-negative breast cancers including increased motility, high proliferation rate, genome instability and sensitivity to platinum chemotherapy and PARP inhibitors (olaparib, veliparib, rucaparib and talazoparib). Genomic instability of CST cells was confirmed by whole genome sequencing, which also revealed the presence of COSMIC (Catalogue of Somatic Mutations in Cancer) mutation signatures 3 and 8 associated with homologous recombination (HR) deficiency. In vitro sensitivity of CST cells was tested against 11 chemotherapy agents. Tumors derived from orthotopically injected CST-mCherry cells in FVB-GFP mice showed sensitivity to cisplatin, providing a new model to study the cooperation of BRCA1-KO, mCherry-positive tumor cells and the GFP-expressing stromal compartment in therapy resistance and metastasis formation. In summary, we have established CST cells as a new model recapitulating major characteristics of BRCA1-negative breast cancers.

Characterization and therapeutic application of canine adipose mesenchymal stem cells to treat elbow osteoarthritis.

Visceral adipose tissue (AT) obtained from surgical waste during routine ovariectomies was used as a source for isolating canine mesenchymal stem cells (MSCs). As determined by cytofluorimetry, passage 2 cells expressed MSC markers CD44 and CD90 and were negative for lineage-specific markers CD34 and CD45. The cells differentiated toward osteogenic, adipogenic, and chondrogenic directions. With therapeutic aims, 30 dogs (39 joints) suffering from elbow dysplasia (ED) and osteoarthritis (OA) were intra-articularly transplanted with allogeneic MSCs suspended in 0.5% hyaluronic acid (HA). A highly significant improvement was achieved without any medication as demonstrated by the degree of lameness during the follow-up period of 1 y. Control arthroscopy of 1 transplanted dog indicated that the cartilage had regenerated. Histological analysis of the cartilage biopsy confirmed that the regenerated cartilage was of hyaline type. These results demonstrate that transplantation of allogeneic adipose tissue-derived mesenchymal stem cells (AT-MSCs) is a novel, noninvasive, and highly effective therapeutic tool in treating canine elbow dysplasia.

Du tissu adipeux viscéral (TA) obtenu de résidus chirurgicaux lors d'ovariectomies de routine a été utilisé comme source pour isoler des cellules souches mésenchymateuses canines (CSMs). Tel que déterminé par cytofluorométrie, les cellules du 2e passage exprimaient les marqueurs de CSM CD44 et CD90, et étaient négatives pour les marqueurs spécifiques de lignée CD34 et CD45. Les cellules se sont différenciées dans des directions ostéogéniques, adipogéniques, et chondrogéniques. À des fins thérapeutiques, 30 chiens (39 articulations) souffrant de dysplasie du coude (DC) et d'ostéoarthrite (OA) ont reçu une transplantation intra articulaire de CSMs allogéniques en suspension dans 0,5 % d'acide hyaluronique (AH). Une amélioration hautement significative a été obtenue sans aucune médication tel que démontré par le degré de boiterie durant la période de suivi de 1 an. Une arthroscopie de contrôle de un des chiens ayant reçu une transplantation montrait que le cartilage s'était régénéré. L'analyse histologique de la biopsie du cartilage a confirmé que le cartilage régénéré était de type hyalin. Ces résultats démontrent que la transplantation de cellules souches mésenchymateuses dérivées de tissu adipeux allogène est un outil thérapeutique novateur, non-invasif, et très efficace pour traiter la dysplasie du coude chez le chien.(Traduit par Docteur Serge Messier).

[Quo vadis hematology?] / Quo vadis, hematológia?

For decades, developing hematopoietic cells have been strictly compartmentalized into a small population of multipotent self-renewing hematopoietic stem cells, multipotent hematopoietic progenitor cells that are undergoing commitment to myeloid or lymphoid fates, and unipotent precursor cells that mature towards peripheral blood and immune cells. Recent studies, however, have provided a battery of findings that cannot be explained by this "classical" hierarchical model for the architecture of hematopoiesis. It is emerging that heterogeneous hematopoietic stem cell populations in the bone marrow coexist, each with distinct, preprogrammed differentiation and proliferation behaviors. Three subsets can be distinguished among them: myeloid-biased (α), balanced (ß), and lymphoid-biased (γ/δ) hematopoietic stem cells. The ratio of these hematopoietic stem cell subsets is developmentally regulated in the foetal liver and hematopoietic stem cells adult bone marrow, and coordinately gives rise to hematopoiesis. Beta- and γ/δ-hematopoietic stem cells are found predominantly early in the life of an organism, whereas α-hematopoietic stem cells accumulate in aged mice and humans. In addition, new sophisticated genetic experiments in mice have identified a major role of long-lived, committed progenitor cells downstream from hematopoietic stem cells as drivers of normal adult hematopoiesis, and revealed that post-transplantation hematopoiesis differs qualitatively and quantitatively from normal steady-state hematopoiesis. These findings have important implications for understanding in situ the regulation of haematopoiesis in health and disease. Orv. Hetil., 2016, 157(46), 1819-1829.

The scaffold protein Tks4 is required for the differentiation of mesenchymal stromal cells (MSCs) into adipogenic and osteogenic lineages.

The commitment steps of mesenchymal stromal cells (MSCs) to adipogenic and other lineages have been widely studied but not fully understood. Therefore, it is critical to understand which molecules contribute to the conversion of stem cells into differentiated cells. The scaffold protein Tks4 plays a role in podosome formation, EGFR signaling and ROS production. Dysfunction of Tks4 causes a hereditary disease called Frank-ter Haar syndrome with a variety of defects concerning certain mesenchymal tissues (bone, fat and cartilage) throughout embryogenic and postnatal development. In this study, we aimed to analyze how the mutation of Tks4 affects the differentiation potential of multipotent bone marrow MSCs (BM-MSCs). We generated a Tks4 knock-out mouse strain on C57Bl/6 background, and characterized BM-MSCs isolated from wild type and Tks4-/- mice to evaluate their differentiation. Tks4-/- BM-MSCs had reduced ability to differentiate into osteogenic and adipogenic lineages compared to wild type. Studying the expression profile of a panel of lipid-regulated genes during adipogenic induction revealed that the expression of adipogenic transcription factors, genes responsible for lipid droplet formation, sterol and fatty acid metabolism was delayed or reduced in Tks4-/- BM-MSCs. Taken together, these results establish a novel function for Tks4 in the regulation of MSC differentiation.

The Role of Extracellular Vesicle and Tunneling Nanotube-Mediated Intercellular Cross-Talk Between Mesenchymal Stem Cells and Human Peripheral T Cells.

The role of extracellular vesicles (EVs) in mediating the immunosuppressory properties of mesenchymal stem cells (MSCs) has recently attracted remarkable scientific interest. The aim of this work was to analyze the transport mechanisms of membrane and cytoplasmic components between T lymphocytes and adipose tissue-derived MSCs (AD-MSCs), by focusing on the role of distinct populations of EVs, direct cell-cell contacts, and the soluble mediators per se in modulating T lymphocyte function. We found that neither murine thymocytes and human primary T cells nor Jurkat lymphoblastoid cells incorporated appreciable amounts of MSC-derived microvesicles (MVs) or exosomes (EXOs). Moreover, these particles had no effect on the proliferation and IFN-γ production of in vitro-stimulated primary T cells. In contrast, AD-MSCs incorporated large amounts of membrane components from T cells as an intensive uptake of EXOs and MVs could be observed. Interestingly, we found a bidirectional exchange of cytoplasmic components between human AD-MSCs and primary T lymphocytes, mediated by tunneling nanotubes (TNTs) derived exclusively from the T cells. In contrast, TNTs couldn't be observed between AD-MSCs and the Jurkat cells. Our results reveal a novel and efficient way of intercellular communication between MSCs and T cells, and may help a better understanding of the immunomodulatory function of MSCs.

Mesenchymal stem cells promote macrophage polarization toward M2b-like cells.

Mesenchymal stem or stromal cells (MSCs) act on different components of the immune response including macrophages (MΦs). Therefore this study has been committed to explore how MSCs may modify the effect of MΦ polarization upon an inductive environment using mouse bone marrow (BM)-derived "naïve", unpolarized MΦs. Phagocytosis of various MΦ subtypes was different since M1 and M2b showed poorer, while M2a higher rate of phagocytosis. MSCs significantly promoted yeast ingestion by M1 and M2b and diminished it by M2a cells. Under polarizing conditions, MSCs profoundly affected the TNFα production of MΦ subtypes since M1 and M2b MΦs produced less and M2a produced higher amount of TNFα while the amount of IL-10 was not affected. The most striking effect of MSCs was registered on M2b cells since the inflammatory TNFα dominance remarkably shifted to the immunosuppressive IL-10. Prepolarized M1 cells readily converted to M2a and M2b states when polarizing conditions changed from M1 to M2a or M2b induction, respectively. Repolarizing from M1 to M2a resulted in the decline of IL-10 and TNFα and defined elevation of Ym1 similar to levels characteristic to M2a primarily polarized from naïve BM-MΦs. Similarly, polarization of M1 to M2b MΦs was successful showing increase in IL-10 and reduction in TNFα levels characteristic to M2b cells. However, when co-culturing with MSCs, M1-M2a or M1-M2b transition was not affected. Crosstalk between MΦs and MSCs depended on PGE-2 since COX-2 inhibition reduced the effect of MSCs to establish an IL-10-dominant cytokine production by MΦs.

Galectin-1 is a local but not systemic immunomodulatory factor in mesenchymal stromal cells.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) have powerful immunosuppressive activity. This function of MSCs is attributed to plethora of the expressed immunosuppressive factors, such as galectin-1 (Gal-1), a pleiotropic lectin with robust anti-inflammatory effect. Nevertheless, whether Gal-1 renders or contributes to the immunosuppressive effect of MSCs has not been clearly established. Therefore, this question was the focus of a complex study. METHODS: MSCs were isolated from bone marrows of wild-type and Gal-1 knockout mice and their in vitro anti-proliferative and apoptosis-inducing effects on activated T cells were examined. The in vivo immunosuppressive activity was tested in murine models of type I diabetes and delayed-type hypersensitivity. RESULTS: Both Gal-1-expressing and -deficient MSCs inhibited T-cell proliferation. Inhibition of T-cell proliferation by MSCs was mediated by nitric oxide but not PD-L1 or Gal-1. In contrast, MSC-derived Gal-1 triggered apoptosis in activated T cells that were directly coupled to MSCs, representing a low proportion of the T-cell population. Furthermore, absence of Gal-1 in MSCs did not affect their in vivo immunosuppressive effect. CONCLUSIONS: These results serve as evidence that Gal-1 does not play a role in the systemic immunosuppressive effect of MSCs. However, a local contribution of Gal-1 to modulation of T-cell response by direct cell-to-cell interaction cannot be excluded. Notably, this study serves a good model to understand how the specificity of a pleiotropic protein depends on the type and localization of the producing effector cell and its target.

Licensing by Inflammatory Cytokines Abolishes Heterogeneity of Immunosuppressive Function of Mesenchymal Stem Cell Population.

When mesenchymal stem cells (MSCs) are used for therapy of immunological pathologies, they get into an inflammatory environment, altering the effectiveness of the treatment. To establish the impact of environmental inflammatory factors on MSCs' immunofunction in the mirror of intrinsic heterogeneity of mouse MSC population, individual MSC clones were generated and characterized. Adipogenic but not osteogenic differentiation and pro-angiogenic activity of five independent MSC cell lines were similar. Regarding osteogenic differentiation, clones MSC3 and MSC6 exhibited poorer capacity than MSC2, MSC4, and MSC5. To study the immunosuppressive heterogeneity, in vitro and in vivo experiments have been carried out using T-cell proliferation assay and delayed-type hypersensitivity (DTH) response, respectively. A remarkable difference was found between the clones in their ability to inhibit T-cell proliferation in the following order: MSC2≥MSC5>MSC4>MSC3 >> MSC6. Nevertheless, the differences between the immunosuppressive activities of the individual clones disappeared on pretreatment of the cells with pro-inflammatory cytokines, a procedure called licensing. Stimulation of all clones with IFN-γ and TNF-α resulted in elevation of their inhibitory capability to a similar level. Nitric oxide (NO) and prostaglandin E2 (PGE2) were identified as major mediators of immunofunction of the MSC clones. The earlier findings were also supported by in vivo results. Without licensing, MSC2 inhibited DTH response, while MSC6 did not affect DTH response. In contrast, prestimulation of MSC6 with inflammatory cytokines resulted in strong suppression by this clone as well. Here, we have showed that MSC population is functionally heterogeneous in terms of immunosuppressive function; however, this variability is largely reduced under pro-inflammatory conditions.

ABCG2 is a selectable marker for enhanced multilineage differentiation potential in periodontal ligament stem cells.

Periodontal ligament stem cells (PDLSCs) provide an important source for tissue regeneration and may become especially useful in the formation of osteogenic seeds. PDLSCs can be cultured, expanded, and differentiated in vitro; thus, they may be applied in the long-term treatment of the defects in the dental regions. Here we studied numerous potential markers allowing the selection of human PDLSCs with a maximum differentiation potential. We followed the expression of the ATP-binding cassette subfamily G member 2 (ABCG2) membrane transporter protein and isolated ABCG2-expressing cells by using a monoclonal antibody, recognizing the transporter at the cell surface in intact cells. The expression of the ABCG2 protein, corresponding to the so-called side-population phenotype in various tissue-derived stem cells, was found to be a useful marker for the selection of PDLSCs with enhanced osteogenic, chondrogenic, and adipogenic differentiation. These findings may have important applications in achieving efficient dental tissue regeneration by using stem cells from extracted teeth.

Regulation of mouse microglia activation and effector functions by bone marrow-derived mesenchymal stem cells.

Mesenchymal stems or stromal cells (MSCs) are rare multipotent cells with potent regenerative and immunomodulatory properties. Microglial cells (MGs) are specialized tissue macrophages of the central nervous system (CNS) that continuously survey their environment with highly motile extensions. Recently, several studies have shown that MSCs are capable of reprogramming microglia into an "M2-like" phenotype characterized by increased phagocytic activity and upregulated expression of anti-inflammatory mediators in vitro. However, the precise polarization states of microglia in the presence of MSCs under physiological or under inflammatory conditions remain largely unknown. In this study, we found that MSCs induce a mixed microglia phenotype defined as Arg1-high, CD86-high, CD206-high, IL-10-high, PGE2-high, MCP-1/CCL2-high, IL-1ß-moderate, NALP-3-low, and TNF-α-low cells. These MSC-elicited MGs have high phagocytic activity and antigen-presenting ability. Lipopolysaccharide is able to shape this microglia phenotype quantitatively, but not qualitatively in the presence of MSCs. This unique polarization state resembles a novel regulatory microglia phenotype, which might contribute to the resolution of inflammation and to tissue repair in the CNS.

A novel cyclic RGD-containing peptide polymer improves serum-free adhesion of adipose tissue-derived mesenchymal stem cells to bone implant surfaces.

Seeding of bone implants with mesenchymal stem cells (MSCs) may promote osseointegration and bone regeneration. However, implant material surfaces, such as titanium or bovine bone mineral, fail to support rapid and efficient attachment of MSCs, especially under serum-free conditions that may be desirable when human applications or tightly controlled experiments are envisioned. Here we demonstrate that a branched poly[Lys(Ser(i)-DL-Ala(m))] polymer functionalized with cyclic arginyl-glycyl-aspartate, when immobilized by simple adsorption to tissue culture plastic, surgical titanium alloy (Ti6Al4V), or Bio-Oss(®) bovine bone substitute, significantly accelerates serum-free adhesion and enhances seeding efficiency of human adipose tissue-derived MSCs. Moreover, when exposed to serum-containing osteogenic medium, MSCs survived and differentiated on the peptide-coated scaffolds. In summary, the presented novel polypeptide conjugate can be conveniently used for coating various surfaces, and may find applications whenever quick and efficient seeding of MSCs is required to various scaffolds in the absence of serum.

Identification of galectin-1 as a critical factor in function of mouse mesenchymal stromal cell-mediated tumor promotion.

Bone marrow derived mesenchymal stromal cells (MSCs) have recently been implicated as one source of the tumor-associated stroma, which plays essential role in regulating tumor progression. In spite of the intensive research, the individual factors in MSCs controlling tumor progression have not been adequately defined. In the present study we have examined the role of galectin-1 (Gal-1), a protein highly expressed in tumors with poor prognosis, in MSCs in the course of tumor development. Co-transplantation of wild type MSCs with 4T1 mouse breast carcinoma cells enhances the incidence of palpable tumors, growth, vascularization and metastasis. It also reduces survival compared to animals treated with tumor cells alone or in combination with Gal-1 knockout MSCs. In vitro studies show that the absence of Gal-1 in MSCs does not affect the number of migrating MSCs toward the tumor cells, which is supported by the in vivo migration of intravenously injected MSCs into the tumor. Moreover, differentiation of endothelial cells into blood vessel-like structures strongly depends on the expression of Gal-1 in MSCs. Vital role of Gal-1 in MSCs has been further verified in Gal-1 knockout mice. By administering B16F10 melanoma cells into Gal-1 deficient animals, tumor growth is highly reduced compared to wild type animals. Nevertheless, co-injection of wild type but not Gal-1 deficient MSCs results in dramatic tumor growth and development.These results confirm that galectin-1 is one of the critical factors in MSCs regulating tumor progression.

Combined introduction of Bmi-1 and hTERT immortalizes human adipose tissue-derived stromal cells with low risk of transformation.

Adipose tissue-derived stromal cells (ASCs) are increasingly being studied for their usefulness in regenerative medicine. However, limited life span and donor-dependent variation of primary cells such as ASCs present major hurdles to controlled and reproducible experiments. We therefore aimed to establish immortalized ASC cell lines that provide steady supply of homogeneous cells for in vitro work while retain essential features of primary cells. To this end, combinations of human telomerase reverse transcriptase (hTERT), murine Bmi-1, and SV40 large T antigen (SV40T) were introduced by lentiviral transduction into ASCs. The resulting cell lines ASC(hTERT), ASC(Bmi-1), ASC(Bmi-1+hTERT) and ASC(SV40T+hTERT) were tested for transgene expression, telomerase activity, surface immunomarkers, proliferation, osteogenic and adipogenic differentiation, karyotype, tumorigenicity, and cellular senescence. All cell lines have maintained expression of characteristic surface immunomarkers, and none was tumorigenic. However, ASC(Bmi-1) had limited replicative potential, while the rapidly proliferating ASC(SV40T+hTERT) acquired chromosomal aberrations, departed from MSC phenotype, and lost differentiation capacity. ASC(hTERT) and ASC(hTERT+Bmi-1), on the other hand, preserved all essential MSC features and did not senesce after 100 population doublings. Notably, a subpopulation of ASC(hTERT) also acquired aberrant karyotype and showed signs of transformation after long-term culture. In conclusion, hTERT alone was sufficient to extend the life span of human ASC, but ASC(hTERT) are prone to transformation during extensive subculturing. The combination of Bmi-1 and hTERT successfully immortalized human ASCs without significantly perturbing their phenotype or biological behavior.

[Beyond genetics--the emerging role of epigenetics and its clinical aspects]. / A genetikán is túl - Az epigenetika eloretörése és orvosi vonatkozásai.

Analysis of genomic sequences has clearly shown that the genomic differences among species do not explain the diversity of life. The genetic code itself serves as only a part of the dynamic complexity that results in the temporal and spatial changes in cell phenotypes during development. It has been concluded that the phenotype of a cell and of the organism as a whole is more influenced by environmentally-induced changes in gene activity than had been previously thought. The emerging field of epigenetics focuses on molecular marks on chromatin; called the epigenome, which serve as transmitters between the genome and the environment. These changes not only persist through multiple cell division cycles, but may also endure for multiple generations. Irregular alterations of the epigenome; called epimutations, may have a decisive role in the etiology of human pathologies such as malignancies and other complex human diseases. Epigenetics can provide the missing link between genetics, disease and the environment. Therefore, this field may have an increasing impact on future drug design and serve as a basis for new therapeutic/preventative approaches.

Activated T-cells and pro-inflammatory cytokines differentially regulate prostaglandin E2 secretion by mesenchymal stem cells.

In recent years it has become clear that mesenchymal stem or stromal cells (MSCs) are capable of modulating inflammatory and immune responses through interaction with a wide variety of cells. Whereas several studies indicated that PGE2 is one of the chief soluble mediators involved in these processes, here we investigated prostaglandin E2 (PGE2) production of murine bone marrow- (BM-) and adipose tissue- (Ad-) derived MSCs stimulated with pro-inflammatory cytokines TNF-α and IFN-γ, or co-cultured with ConA-induced T-cell blasts. We found that both MSC populations are able to produce high amounts of PGE2 in MSC/activated T-cell co-cultures. This effect was markedly attenuated when direct cell-cell contact was prevented in transwell system, indicating that the elicitation of the PGE2 secretion of MSCs is contact-dependent in this experimental setting. In contrast, when soluble recombinant pro-inflammatory cytokines were added to the MSC cultures, TNF-α and IFN-γ act synergistically to induce PGE2 production, whereas only high amount of TNF-α but not IFN-γ was able to do so alone. Although the PGE2 secretion by MSCs was completely abrogated by addition of indomethacin under all culture conditions tested, L-NMA, a NOS inhibitor could only partially inhibit it when the cells were elicited in the concomitant presence of TNF-α and IFN-γ. These results, combined with others, suggest that NO acts downstream of IFN-γ but upstream of COX2. Taken together, our findings demonstrate that the induction of PGE2 secretion by BM- and Ad-MSCs is not mediated by a single or unique, nonredundant molecular mechanism under different experimental conditions.

Positional identity of murine mesenchymal stem cells resident in different organs is determined in the postsegmentation mesoderm.

Although mesenchymal stem cells (MSCs) of distinct tissue origin have a large number of similarities and differences, it has not been determined so far whether tissue-resident MSCs are the progenies of one ancestor cell lineage or the results of parallel cell developmental events. Here we compared the expression levels of 177 genes in murine MSCs derived from adult and juvenile bone marrow and adult adipose tissue, as well as juvenile spleen, thymus, and aorta wall by quantitative real-time polymerase chain reaction and the results were partially validated at protein level. All MSC lines uniformly expressed a large set of genes including well-known mesenchymal markers, such as α-smooth muscle actin, collagen type I α-chain, GATA6, Mohawk, and vimentin. In contrast, pluripotency genes and the early mesodermal marker T-gene were not expressed. On the other hand, different MSC lines consistently expressed distinct patterns of Hox genes determining the positional identity of a given cell population. Moreover, MSCs of different origin expressed a few other transcription factors also reflecting their topological identity and so the body segment or organ to which they normally contributed in vivo: (1) thymus-derived cells specifically expressed Tbx5 and Pitx2; (2) spleen-derived MSCs were characterized with Tlx1 and Nkx2.5; (3) Pitx1 designated femoral bone marrow cells and (4) En2 appeared in aorta wall-derived MSCs. Thus, MSCs exhibited topographic identity and memory even after long-term cultivation in vitro. On the basis of these results, we suggest that postnatal MSCs isolated from different anatomical sites descend from precursor cells developing in the postsegmentation mesoderm.