Two-photon polymerized "nichoid" substrates maintain function of pluripotent stem cells when expanded under feeder-free conditions.
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.



We 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.



The 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.



We 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.
Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Politecnico di Milano, 32, piazza Leonardo da Vinci, 20133, Milan, Italy. michele.nava@polimi.it.
Michele M
MM
Nava
Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Politecnico di Milano, 32, piazza Leonardo da Vinci, 20133, Milan, Italy.
Alessio
A
Piuma
IRCCS Istituto di Ricerche Farmacologiche "Mario Negri", Bergamo, Italy.
Marina
M
Figliuzzi
IRCCS Istituto di Ricerche Farmacologiche "Mario Negri", Bergamo, Italy.
Irene
I
Cattaneo
IRCCS Istituto di Ricerche Farmacologiche "Mario Negri", Bergamo, Italy.
Barbara
B
Bonandrini
Istituto di Fotonica e Nanotecnologie (IFN) - CNR and Department of Physics, Politecnico di Milano, Milan, Italy.
Tommaso
T
Zandrini
Istituto di Fotonica e Nanotecnologie (IFN) - CNR and Department of Physics, Politecnico di Milano, Milan, Italy.
Giulio
G
Cerullo
Istituto di Fotonica e Nanotecnologie (IFN) - CNR and Department of Physics, Politecnico di Milano, Milan, Italy.
Roberto
R
Osellame
Department of Management, Information and Production Engineering, University of Bergamo, Dalmine, Italy.
Andrea
A
Remuzzi
Department of Chemistry, Materials and Chemical Engineering "Giulio Natta", Politecnico di Milano, 32, piazza Leonardo da Vinci, 20133, Milan, Italy.
Manuela T
MT
Raimondi
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000502:physiology; D002465:Cell Movement / Q000502:physiology; D002478:Cells, Cultured; D065150:Cellular Reprogramming / Q000502:physiology; D058732:Embryoid Bodies / Q000166:cytology / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D061252:Feeder Cells / Q000166:cytology* / Q000378:metabolism; D007834:Lasers; D008297:Male; D051379:Mice; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D017207:Rats, Sprague-Dawley
D015415:Biomarkers
Embryonic stem cells; Mechanobiology; Nanofabrication; Pluripotency; Scaffold; Two-photon polymerization