Sorting living mesenchymal stem cells using a TWIST1 RNA-based probe depends on incubation time and uptake capacity.

Bone marrow derived mesenchymal stromal cells (BMSCs) are multipotent progenitors of particular interest for cell-based tissue engineering therapies. However, one disadvantage that limit their clinical use is their heterogeneity. In the last decades a great effort was made to select BMSC subpopulations based on cell surface markers, however there is still no general consensus on which markers to use to obtain the best BMSCs for tissue regeneration. Looking for alternatives we decided to focus on a probe-based method to detect intracellular mRNA in living cells, the SmartFlare technology. This technology does not require fixation of the cells and allows us to sort living cells based on gene expression into functionally different populations. However, since the technology is available it is debated whether the probes specifically recognize their target mRNAs. We validated the TWIST1 probe and demonstrated that it specifically recognizes TWIST1 in BMSCs. However, differences in probe concentration, incubation time and cellular uptake can strongly influence signal specificity. In addition we found that TWIST1high expressing cells have an increased expansion rate compared to TWIST1low expressing cells derived from the same initial population of BMSCs. The SmartFlare probes recognize their target gene, however for each probe and cell type validation of the protocol is necessary.

Silencing of Antichondrogenic MicroRNA-221 in Human Mesenchymal Stem Cells Promotes Cartilage Repair In Vivo.

There is a growing demand for the development of experimental strategies for efficient articular cartilage repair. Current tissue engineering-based regenerative strategies make use of human mesenchymal stromal cells (hMSCs). However, when implanted in a cartilage defect, control of hMSCs differentiation toward the chondrogenic lineage remains a significant challenge. We have recently demonstrated that silencing the antichondrogenic regulator microRNA-221 (miR-221) was highly effective in promoting in vitro chondrogenesis of monolayered hMSCs in the absence of the chondrogenic induction factor TGF-ß. Here we investigated the feasibility of this approach first in conventional 3D pellet culture and then in an in vivo model. In pellet cultures, we observed that miR-221 silencing was sufficient to drive hMSCs toward chondrogenic differentiation in the absence of TGF-ß. In vivo, the potential of miR-221 silenced hMSCs was investigated by first encapsulating the cells in alginate and then by filling a cartilage defect in an osteochondral biopsy. After implanting the biopsy subcutaneously in nude mice, we found that silencing of miR-221 strongly enhanced in vivo cartilage repair compared to the control conditions (untreated hMSCs or alginate-only). Notably, miR-221 silenced hMSCs generated in vivo a cartilaginous tissue with no sign of collagen type X deposition, a marker of undesired hypertrophic maturation. Altogether our data indicate that silencing miR-221 has a prochondrogenic role in vivo, opening new possibilities for the use of hMSCs in cartilage tissue engineering. Stem Cells 2016;34:1801-1811.

Interaction Between Breast Cancer Cells and Adipose Tissue Cells Derived from Fat Grafting.

BACKGROUND: Adipose tissue transplantation has the benefit of providing both regenerative and aesthetic outcomes in breast cancer treatment. However, the transplanted tissue can stimulate the growth of residual cancer cells. OBJECTIVES: The aim of this study is to identify the interactions between adipose tissue cell subpopulations and human cancer cell lines. METHODS: Intact adipose tissue from lipofilling procedures as well as fibroblasts derived from adipose tissue, were cocultured in the presence of MDA-MB-231, MCF-7 e ZR-75-1 breast cancer cell lines. The influence on cancer cell lines of fibroblasts, induced to differentiate into specific adipocytes, was also assayed. RESULTS: All cancer cell lines displayed a significant increase in proliferation rate when cocultured in the presence of either intact adipose tissue or induced adipocytes. To a lesser extent, uninduced fibroblasts stimulate breast cancer cell proliferation. CONCLUSIONS: Recent studies have shown that the microenvironment surrounding breast cancer cells may stimulate growth and promote progression of residual cancer cells when surgery is performed on the main tumor mass. Accordingly, the graft of adipose tissue could potentially promote or accelerate the development of a subclinical tumor or support its locoregional recurrence. Our data suggest that adipocytes have a remarkable influence on the proliferation of cancer cell lines. The oncological safety of the lipofilling procedure outcome is still debated; thus, further studies and consistent follow-up examination are needed.