Study implicated small RNAs in keeping MSCs quiescent but able to differentiate.

Researchers have fine-tuned the nanotopography of a stem cell growth substrate to generate a support that maintains mesenchymal stem cells (MSCs) in their undifferentiated, multipotent state for eight weeks. The growth support is manufactured by a straightforward injection moulding technology akin to that already used to produce Blue-ray disks. It could provide the answer to generating large-scale quantities of autologous cells for clinical use, claims the U.K.-based team.

Reporting in Nature Materials researchers at the Universities of Glasgow and Southampton say their achievement paves the way for the design of nanoscale features into tissue-engineering scaffolds that support reservoirs of progenitor cells for a range of tissue-specific stem cell types and improve the regenerative capacity of in vitro-fabricated tissue and organs. The work has also implicated upregulation of small RNAs in the mechanisms that keep MSCs quietly ticking over without losing their multipotency.

Led by Rebecca J. McMurray, Ph.D., and Matthew J. Dalby, Ph.D., at the University of Glasgow Institute of Molecular, Cell and Systems Biology’s Centre for Cell Engineering, the team describes its work in a paper titled “Nanoscale surfaces for the long-term maintenance of mesenchymal stem cell phenotype and multipotency .”

The investigators previously designed and produced a polycaprolactone (PCL)-based support with nanoscale features that allows osteogenesis from stem and progenitor mesenchymal populations cultured in osteogenic media. This osteogenesis-promoting support comprised 120 nm pits in a square arrangement with a center-center spacing of 300 nm but with a +/- 50 nm offset in pit placement in x and y axes.

What the researchers have now found is that by reducing the level of offset to as close to zero as possible (absolute square lattice symmetry, SQ), the resulting nanotopography induced a switch from osteogenic stimulation to a surface conducive to MSC growth while permitting prolonged retention of MSC markers and multipotency.

In fact, the researchers claim, MSCs cultured on SQ retained the expression of stem cell markers and, critically, displayed no markers of osteogenesis. Importantly, the SQ surface supported the growth and multipotency of a range of MSCs including commercially available skeletal- and adipose-derived MSC preparations as well as 20 different patient-derived cell populations. MSCs grown on the SQ surface for four weeks could be removed, plated onto coverslips, and treated with differentiation media to promote osteogenesis or adipogenesis.

Interestingly, MSCs grown on SQ had reduced expression of metabolic genes, supporting previous observations that stem cell populations are quiescent and relatively inactive metabolically when compared with the increased metabolomic activity found in adult stem cells undergoing active differentiation.

When the researchers looked more closely at gene expression in the SQ-supported MSCs, they found that a range of small, untranslated, RNAs were up-regulated in comparison with osteogenic MSCs. These included C/D box snoRNAs (SNORDS) that are implicated in methylation of ribosomal RNA and alternative splicing of mRNA.

This observation suggests that differential regulation of small RNAs may hold back mRNA transcription and slow growth, so reducing metabolism and repressing differentiation-related canonical signaling. Concurring with the notion that changes in metabolism and canonical signaling drive phenotypic change, the researchers found that downstream functional pathways were also repressed in MSCs grown on SQ topography.

They postulate that nanoscale modifications to surface topography alter the interaction of integrin receptors within cell adhesions, resulting in changes in intracellular tension. In support of this they found that a degree of tension is required for MSCs to retain multipotency: inhibiting actin/myosin interaction resulted in gene expression changes that promoted adipogenesis.

“Certainly, it would seem that MSCs have a direct form-function relationship, and we speculate that a surface needs to influence the adhesion/tension balance to permit self-renewal or targeted differentiation,” the authors write.

“Together, our results imply that increased self-renewal in MSCs requires the cells to be sufficiently biochemically and mechanically active to undergo proliferation, but their metabolic activity must be kept to a minimum, possibly regulated by ERK to allow proliferation but differentiation being attenuated through small RNAs.”

Critically, the production process for the support is relatively simple and reproducible, they stress. The design used for the reported studies was fabricated by electron beam lithography and processed into the thermoplastic PCL by hot embossing. The importance of the surface topography was further demonstrated by confirming that cells could be maintained when the pattern was embossed in either polycarbonate and polystyrene. The original osteogenesis-triggering surface was produced on PCA.

“The demonstrable sensitivity of MSCs to materials, with < 50nm alterations in feature placement and the role of such defined topographies on cell fate and function, offers nanoscale patterning as a powerful tool for the noninvasive manipulation of stem cells,” the team concludes.

“The data presented offers an insight into the potential role of small RNAs in mediating nanotopography-induced stem cell function and fate. Small RNAs thus provide us with potential target(s) to focus on control and modulation with materials, and such studies are continuing in our laboratories.”

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