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

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ABSTRACT

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Electronic supplementary material: The online version of this article (doi:10.1186/s13287-016-0387-z) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

Spontaneous differentiation of mES cells cultured on reused nichoid substrates. Cells were cultured in the absence of a feeder layer and with LIF up to day 3, then without either a feeder layer or LIF from days 4 to 14. a Immunofluorescence for OCT4 (red), GATA4 (green), NKX2.5 (green) and DAPI (blue) at days 3, 7, and 14. The scale bar is 50 μm. b Immunofluorescence for F-actin (red) and DAPI (blue) showing a few residual embryoid bodies anchored both to the nichoid (left) and the flat glass (right) after trypsinization. The scale bar is 30 μm. c Quantification of OCT4, GATA4 and NKX2.5 expression by image processing; n = 15, *p < 0.01
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Fig6: Spontaneous differentiation of mES cells cultured on reused nichoid substrates. Cells were cultured in the absence of a feeder layer and with LIF up to day 3, then without either a feeder layer or LIF from days 4 to 14. a Immunofluorescence for OCT4 (red), GATA4 (green), NKX2.5 (green) and DAPI (blue) at days 3, 7, and 14. The scale bar is 50 μm. b Immunofluorescence for F-actin (red) and DAPI (blue) showing a few residual embryoid bodies anchored both to the nichoid (left) and the flat glass (right) after trypsinization. The scale bar is 30 μm. c Quantification of OCT4, GATA4 and NKX2.5 expression by image processing; n = 15, *p < 0.01

Mentions: Finally, our third aim was to assess the pluripotency maintenance and differentiation toward the three germ layers in feeder-free layer culture conditions in reused nichoids. We cultured mES cells in the absence of a feeder layer and with LIF conditioning up to day 3, then without either a feeder layer or LIF conditioning from day 4 to day 14. To evaluate stemness promotion and inhibition to differentiation, we stained and evaluated the co-occurrence of OCT4 and DAPI, while for the differentiation potential toward the endoderm and mesoderm germ layer, we stained and quantified the co-occurrence of GATA-4 and NKX2-5 on DAPI (Fig. 6a, c). Surprisingly, EBs were OCT4+ throughout the culture period (74.35 ± 2.70 on average), whereas both GATA4 and NKX2-5 expression were negligible. EB-GATA4+ was observed at day 3. As previously mentioned, a possible explanation could be the DMSO which was reported as an induction factor for endodermal differentiation [22]. The high OCT4 expression in the reused nichoids could depend on the protocol for cell detachment by trypsin. As shown in Fig. 6b (right), F-actin (red), and therefore fragments of plasma membranes were observed with no nuclei. Despite trypsin, cell residues (and/or other secreted proteins) may have remained anchored to the substrate, favoring cell adhesion, EB growth in nichoids (Fig. 6b, left), and pluripotency maintenance.Fig. 6


Two-photon polymerized “ nichoid ” substrates maintain function of pluripotent stem cells when expanded under feeder-free conditions
Spontaneous differentiation of mES cells cultured on reused nichoid substrates. Cells were cultured in the absence of a feeder layer and with LIF up to day 3, then without either a feeder layer or LIF from days 4 to 14. a Immunofluorescence for OCT4 (red), GATA4 (green), NKX2.5 (green) and DAPI (blue) at days 3, 7, and 14. The scale bar is 50 μm. b Immunofluorescence for F-actin (red) and DAPI (blue) showing a few residual embryoid bodies anchored both to the nichoid (left) and the flat glass (right) after trypsinization. The scale bar is 30 μm. c Quantification of OCT4, GATA4 and NKX2.5 expression by image processing; n = 15, *p < 0.01
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5016857&req=5

Fig6: Spontaneous differentiation of mES cells cultured on reused nichoid substrates. Cells were cultured in the absence of a feeder layer and with LIF up to day 3, then without either a feeder layer or LIF from days 4 to 14. a Immunofluorescence for OCT4 (red), GATA4 (green), NKX2.5 (green) and DAPI (blue) at days 3, 7, and 14. The scale bar is 50 μm. b Immunofluorescence for F-actin (red) and DAPI (blue) showing a few residual embryoid bodies anchored both to the nichoid (left) and the flat glass (right) after trypsinization. The scale bar is 30 μm. c Quantification of OCT4, GATA4 and NKX2.5 expression by image processing; n = 15, *p < 0.01
Mentions: Finally, our third aim was to assess the pluripotency maintenance and differentiation toward the three germ layers in feeder-free layer culture conditions in reused nichoids. We cultured mES cells in the absence of a feeder layer and with LIF conditioning up to day 3, then without either a feeder layer or LIF conditioning from day 4 to day 14. To evaluate stemness promotion and inhibition to differentiation, we stained and evaluated the co-occurrence of OCT4 and DAPI, while for the differentiation potential toward the endoderm and mesoderm germ layer, we stained and quantified the co-occurrence of GATA-4 and NKX2-5 on DAPI (Fig. 6a, c). Surprisingly, EBs were OCT4+ throughout the culture period (74.35 ± 2.70 on average), whereas both GATA4 and NKX2-5 expression were negligible. EB-GATA4+ was observed at day 3. As previously mentioned, a possible explanation could be the DMSO which was reported as an induction factor for endodermal differentiation [22]. The high OCT4 expression in the reused nichoids could depend on the protocol for cell detachment by trypsin. As shown in Fig. 6b (right), F-actin (red), and therefore fragments of plasma membranes were observed with no nuclei. Despite trypsin, cell residues (and/or other secreted proteins) may have remained anchored to the substrate, favoring cell adhesion, EB growth in nichoids (Fig. 6b, left), and pluripotency maintenance.Fig. 6

View Article: PubMed Central - PubMed

ABSTRACT

Background: 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.

Methods: We developed an innovative three-dimensional culture substrate made of &ldquo;nichoid&rdquo; 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&nbsp;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.

Results: 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 &alpha;-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.

Conclusions: 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.

Electronic supplementary material: The online version of this article (doi:10.1186/s13287-016-0387-z) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus