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Human bone marrow‐derived mesenchymal stem cells (MSC) are multipotent non‐hematopoietic progenitors that have regulatory activity on immune cells. NOD‐ and Toll‐like receptors (NLR, TLR) have several roles in immunity, including those relevant to pathogen recognition and shaping the course of immune responses by controlling gene expression. We have shown that these innate immune receptors are expressed by hematopoietic CD34+ progenitors and MSC. To uncover genes critical in MSC function, first we have used microarray to screen for potential transcripts whose levels are altered in response to NOD‐1 and TLR‐2 activation, and second we validated some candidate genes using real‐time RT‐PCR, Western blots and cellular assays. Amongst the altered genes, galectin‐3 was upregulated at both mRNA and protein levels in response to TLR‐2 activation. Interestingly, MSC secreted galectin‐3, a protein known to modulate T‐cell proliferation, gene expression, cell adhesion and migration. Knockdown of galectin‐3 in MSC using small interfering RNA (siRNA) reduced the immunosuppressive effect of MSC on mixed lymphocyte cultures when compared to cells treated with an irrelevant siRNA (P < 0.05). Collectively, the data emphasize a new role of galectin‐3 in the immunomodulatory function of MSC and indicate that NOD signalling pathway is also functional in these cells.

We recently reported that human bone marrow hematopoietic CD34+ progenitors express functional Toll-like receptors (TLR) and can differentiate into myeloid cells just by stimulation with resiquimod (R848), a specific agonist for TLR7/8. However, the mechanisms by which R848 induces cell differentiation, the effects of other TLR agonists and the functionality of the differentiated cells are not known. Comparable to R848, loxoribine (a TLR7 agonist) and Pam 3 CSK 4 (a TLR2 agonist) induced cytokine production and cell differentiation along the myeloid lineage. R848 and loxoribine were more effective than Pam 3 CSK 4 at inducing the lineage-negative (CD11c + CD14 -) dendritic cells (DC), whereas Pam 3 CSK 4 was more effective at inducing CD11c + CD14 + monocytes. Both cell subsets expressed CD80/CD86 and HLA-DR molecules; however, they showed differential expression of CD1a, CD1b, CD1c, CD11b, CD206 and CD207 markers when compared with each other. Cell differentiation into DC was significantly inhibited by an anti-TNF-a nonoclonal antibody. The CD11c + CD14 -subset was isolated and shown to be more potent in stimulating an alloreaction than the CD11c + CD14 + subset. Collectively, these data highlight the differential effects of TLR agonists on human bone marow CD34 + progenitor cells and provide a new opportunity for generating functional DC that would be useful in cancer vaccination.

T he FMS-like tyrosine kinase 3 (FLT3) gene is mutated in 25-30% of patients with acute myeloid leukemia (AML). Because of the poor prognosis associated with FLT3-internal tandem duplication mutated AML, allogeneic hematopoietic stem-cell transplantation (SCT) was commonly performed in first complete remission. Remarkable progress

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Human galectins are involved in a variety of biological and pathological processes including cell adhesion, apoptosis, differentiation, immune regulation and tumour evasion. Previously, we identified galectin-3 as the first human lectin involved in the modulation of the immunosuppressive potential of mesenchymal stem cells (MSCs). In this study, we report on the expression profiles and potential activities of other galectins expressed in these cells. The data show that MSCs constitutively express galectins-1, -3 and -8 at both the mRNA and protein levels. In contrast to galectin-8, galectins-1 and -3 are secreted and found on the cell surface. MSC-mediated T cell suppression was inhibited by galectin-1-specific siRNAs but not by galectin-8-specific siRNAs. The double knockdown of galectins-1 and -3 almost abolished the immunosuppressive capacity of MSCs. The use of a competitive inhibitor for galectin binding, ß lactose, restored alloresponsiveness, implying an extracellular mechanism of action of galectins. Collectively, the data highlight the involvement of secreted galectins-1 and -3 in MSC-mediated T cell suppression. The immunosuppression by MSC-secreted galectins should facilitate the use of recombinant galectin-1 and/or -3 as a novel therapy to alleviate inflammatory reactions such as those seen in graft versus host disease (GvHD) and autoimmune disorders.

Inhibitors of B-cell lymphoma-2 (BCL-2) such as venetoclax (ABT-199) and navitoclax (ABT-263) are clinically explored in several cancer types, including acute myeloid leukemia (AML), to selectively induce apoptosis in cancer cells. To identify robust biomarkers for BCL-2 inhibitor sensitivity, we evaluated the ex vivo sensitivity of fresh leukemic cells from 73 diagnosed and relapsed/refractory AML patients, and then comprehensively assessed whether the responses correlated to specific mutations or gene expression signatures. Compared with samples from healthy donor controls (nonsensitive) and chronic lymphocytic leukemia (CLL) patients (highly sensitive), AML samples exhibited variable responses to BCL-2 inhibition. Strongest CLL-like responses were observed in 15% of the AML patient samples, whereas 32% were resistant, and the remaining exhibited intermediate responses to venetoclax. BCL-2 inhibitor sensitivity was associated with genetic aberrations in chromatin modifiers, WT1 and IDH1/IDH2. A striking selective overexpression of specific HOXA and HOXB gene transcripts were detected in highly BCL-2 inhibitor sensitive samples. Ex vivo responses to venetoclax showed significant inverse correlation to β2-microglobulin expression and to a lesser degree to BCL-XL and BAX expression. As new therapy options for AML are urgently needed, the specific HOX gene expression pattern can potentially be used as a biomarker to identify venetoclax-sensitive AML patients for clinical trials.

Lesions of hyaline cartilage do not heal spontaneously, and represent a therapeutic challenge. In vitro engineering of articular cartilage using cells and biomaterials may prove to be the best solution. Patients with osteoarthritis (OA) may require tissue engineered cartilage therapy. Chondrocytes obtained from OA joints are thought to be involved in the disease process, and thus to be of insufficient quality to be used for repair strategies. Bone marrow (BM) derived mesenchymal stem cells (MSCs) from healthy donors may represent an alternative cell source. We have isolated chondrocytes from OA joints, performed cell culture expansion and tissue engineering of cartilage using a disc-shaped alginate scaffold and chondrogenic differentiation medium. We performed real-time reverse transcriptase quantitative PCR and fluorescence immunohistochemistry to evaluate mRNA and protein expression for a range of molecules involved in chondrogenesis and OA pathogenesis. Results were compared with those obtained by using BM-MSCs in an identical tissue engineering strategy. Finally the two populations were compared using genome-wide mRNA arrays. At three weeks of chondrogenic differentiation we found high and similar levels of hyaline cartilage-specific type II collagen and fibrocartilage-specific type I collagen mRNA and protein in discs containing OA and BM-MSC derived chondrocytes. Aggrecan, the dominant proteoglycan in hyaline cartilage, was more abundantly distributed in the OA chondrocyte extracellular matrix. OA chondrocytes expressed higher mRNA levels also of other hyaline extracellular matrix components. Surprisingly BM-MSC derived chondrocytes expressed higher mRNA levels of OA markers such as COL10A1, SSP1 (osteopontin), ALPL, BMP2, VEGFA, PTGES, IHH, and WNT genes, but lower levels of MMP3 and S100A4. Based on the results presented here, OA chondrocytes may be suitable for tissue engineering of articular cartilage.