Cadherin-11 Influences Differentiation in Human Mesenchymal Stem Cells by Regulating the Extracellular Matrix Via the TGFβ1 Pathway

For regenerative medicine, directing stem cell fate is one of the key aims. Human mesenchymal stem cells (hMSCs) are versatile adult stem cells that have been proposed for several clinical applications, making directing their fate of utmost importance. For most clinical applications, their differentiation toward the adipogenic lineage is an undesired outcome. Understanding the mechanisms that regulate hMSC commitment toward the adipogenic lineage might help open up new avenues for fine-tuning implanted hMSCs for regenerative medicine applications. We know that cadherin-11 is required for hMSC commitment to the adipogenic lineage; therefore, we sought to investigate the mechanisms through which cadherin-11 regulates adipogenic differentiation. We observed that hMSCs lacking cadherin-11 had decreased expression of type VI collagen and increased expression of fibronectin. We provide evidence of increased transforming growth factor beta 1 and the subsequent translocation of phosphorylated SMAD2/3 into the nucleus by cells that lack cadherin-11, which could be attributed to the changes in extracellular matrix composition. Taken together, our study implicates cadherin-11 in regulating extracellular matrix production and thereby helping improve cell- and material-based regenerative medicine approaches

Cell culture dimensionality influences mesenchymal stem cell fate through cadherin-2 and cadherin-11

The acquisition of a specific cell fate is one of the core aims of tissue engineering and regenerative medicine. Significant evidence shows that aggregate cultures have a positive influence on cell fate decisions, presumably through cell-cell interactions, but little is known about the specific mechanisms. To investigate the difference between cells cultured as a monolayer and as aggregates, we started by looking at cadherin expression, an important protein involved in cell adhesion, during the differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs) in aggregate and monolayer cultures. We observed that proliferating hMSCs in monolayer culture expressed lower levels of cadherin-2 and increased cadherin-11 expression at cell-cell contact sites over time, which was not evident in the aggregate cultures. By knocking down cadherin-2 and cadherin-11, we found that both cadherins were required for adipogenic differentiation in a monolayer as well as aggregate culture. However, during osteogenic differentiation, low levels of cadherin-2 were found to be favorable for cells cultured as a monolayer and as aggregates, whereas cadherin- 11 was dispensable for cells cultured as aggregates. Together, these results provide compelling evidence for the important role that cadherins play in regulating the differentiation of hMSCs and how this is affected by the dimensionality of cell culture

Computational evidence for multi-layer crosstalk between the cadherin-11 and PDGFR pathways

Various cell surface receptors play an important role in the differentiation and self-renewal of human mesenchymal stem cells (hMSCs). One example of such receptors are the cadherins, which maintain cell-cell adhesion and mechanically couple cells together. Recently, cadherin-11, which is a member of the type II classical cadherin family, has been shown to be involved in the fate commitment of hMSCs. Interestingly, cadherin-11 has no known intrinsic signaling activity and is thought to affect cell behavior via interactions with other cell surface receptors. Members of the platelet-derived growth factor receptor (PDGFR) family are hypothesized to be one of the interaction partners of cadherin-11. Experiments confirmed that PDGFR-α binding to extracellular cadherin-11 regions increases the PDGFR-α activity, whereas the interaction between PDGFR-β and cadherin-11 suppresses the activity of the growth factor receptor. Cadherin-11 knockdown experiments also decreased cell proliferation. These interactions between cadherin-11 and PDGFRs indicate a crosstalk between these receptors and their downstream signaling activities but the nature of this crosstalk is not entirely known. In this study, we used a computational model to represent the experimentally proven interactions between cadherin-11 and the two PDGFRs and we inspected whether the crosstalk also exists downstream of the signaling initiated by the two receptor families. The computational framework allowed us to monitor the relative activity levels of each protein in the network. We performed model simulations to mimic the conditions of previous cadherin-11 knockdown experiments and to predict the effect of crosstalk on cell proliferation. Overall, our predictions suggest the existence of another layer of crosstalk, namely between β-catenin (downstream to cadherin-11) and an ERK inhibitor protein (e.g. DUSP1), different than the crosstalk at the receptor level between cadherin-11 and PDGFR-α and -β. By investigating the multi-level crosstalk between cadherin and PDGFRs computationally, this study contributes to an improved understanding of the effect of cell surface receptors on hMSCs proliferation.</p

A comparative study of mesenchymal stems cells cultured as cell-only aggregates and in encapsulated hydrogels

There is increasing evidence that cells cultured in three-dimensional (3D) settings have superior performance compared to their traditional counterparts in monolayers. This has been attributed to cell-cell and cell-extracellular matrix interactions that more closely resemble the in vivo tissue architecture. The rapid adoption of 3D cell culture systems as experimental tools for diverse applications has not always been matched by an improved understanding of cell behavior in different 3D environments. Here, we studied human mesenchymal stem/stromal cells (hMSCs) as scaffold-free self-assembled aggregates of low and high cell number and compared them to cell-laden alginate hydrogels with and without arginine-glycine-aspartic acid peptides. We observed a significant decrease in the size of cell-only aggregates over 14 days in culture compared to the cells encapsulated in alginate hydrogels. Alginate hydrogels had persistently more living cells for a longer period (14 days) in culture as measured by total DNA content. Proliferation studies revealed that a weeklong culture of hMSCs in 3D culture, whether as aggregates or cell-laden alginate hydrogels, reduced their proliferation over time. Cell cycle analysis found no significant differences between days 1 and 7 for the different culture systems. The findings of this study improve our understanding of how aggregate cultures differ with or without a hydrogel carrier, and whether aggregation itself is important when it comes to the 3D culture of hMSCs

Self-Seeding Microwells to Isolate and Assess the Viability of Single Circulating Tumor Cells

The availability of viable tumor cells could significantly improve the disease management of cancer patients. Here we developed and evaluated a method using self-seeding microwells to obtain single circulating tumor cells (CTC) and assess their potential to expand. Conditions were optimized using cells from the breast cancer cell line MCF-7 and blood from healthy volunteers collected in EDTA blood collection tubes. 43% of the MCF-7 cells (nucleus+, Ethidium homodimer-1-, Calcein AM+, &#945;-EpCAM+, &#945;-CD45-) spiked into 7.5 mL of blood could be recovered with 67% viability and these could be further expanded. The same procedure tested in metastatic breast and prostate cancer patients resulted in a CTC recovery of only 0&#8315;5% as compared with CTC counts obtained with the CellSearch&#174; system. Viability of the detected CTC ranged from 0&#8315;36%. Cell losses could be mainly contributed to the smaller size and greater flexibility of CTC as compared to cultured cells from cell lines and loss during leukocyte depletion prior to cell seeding. Although CTC losses can be reduced by fixation, to obtain viable CTC no fixatives can be used and pore size in the bottom of microwells will need to be reduced, filtration conditions adapted and pre-enrichment improved to reduce CTC losses