Abstract
BackgroundThe use of pluripotent cells in stem cell therapy has major limitations, mainly related to the high costs and risks of exogenous conditioning and the use of feeder layers during cell expansion passages.MethodsWe developed an innovative three-dimensional culture substrate made of “nichoid” microstructures, nanoengineered via two-photon laser polymerization. The nichoids limit the dimension of the adhering embryoid bodies during expansion, by counteracting cell migration between adjacent units of the substrate by its microarchitecture. We expanded mouse embryonic stem cells on the nichoid for 2 weeks. We compared the expression of pluripotency and differentiation markers induced in cells with that induced by flat substrates and by a culture layer made of kidney-derived extracellular matrix.ResultsThe nichoid was found to be the only substrate, among those tested, that maintained the expression of the OCT4 pluripotency marker switched on and, simultaneously, the expression of the differentiation markers GATA4 and α-SMA switched off. The nichoid promotes pluripotency maintenance of embryonic stem cells during expansion, in the absence of a feeder layer and exogenous conditioning factors, such as the leukocyte inhibitory factor.ConclusionsWe hypothesized that the nichoid microstructures induce a genetic reprogramming of cells by controlling their cytoskeletal tension. Further studies are necessary to understand the exact mechanism by which the physical constraint provided by the nichoid architecture is responsible for cell reprogramming. The nichoid may help elucidate mechanisms of pluripotency maintenance, while potentially cutting the costs and risks of both feed-conditioning and exogenous conditioning for industrial-scale expansion of stem cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0387-z) contains supplementary material, which is available to authorized users.
Highlights
The use of pluripotent cells in stem cell therapy has major limitations, mainly related to the high costs and risks of exogenous conditioning and the use of feeder layers during cell expansion passages
Upon cell seeding, a major fraction of the cells would fall by sedimentation driven by gravity inside the nichoids and that the confinement walls would prevent these cells from leaving the nichoids during culture
Our results show that the nichoid was the only substrate among those tested that maintained a pluripotency gene switched on and, simultaneously, three differentiation genes switched off in the prolonged culture of mouse embryonic stem (mES) cells
Summary
The use of pluripotent cells in stem cell therapy has major limitations, mainly related to the high costs and risks of exogenous conditioning and the use of feeder layers during cell expansion passages. Cell-based therapies represent an important strategy to restore the function of injured cells, tissues, and organs. The limited availability of functional cells has hampered the success of this strategy. ES cells do not maintain their pluripotency during expansion passages on standard plastic culture, but tend to differentiate spontaneously toward all three germ layers. Rat, and human ES cells require distinct culture conditions for the maintenance of their pluripotency state. A feeder layer or different growth factors, cytokines, and small molecules are routinely used to counteract spontaneous differentiation and to promote ES cell self-renewal
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