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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 endodermal and mesodermal differentiation of mES cells cultured in the nichoid substrates, compared to flat glass and to kidney ECM. Cells were cultured in the absence of a feeder layer and with LIF up to day 3, then without neither a feeder layer nor LIF from day 4 to day 14. a Immunofluorescence for GATA4 (green) and DAPI (blue) in the nichoid (gray), the flat glass and the kidney ECM at day 3, 7, and 14. The scale bar is 50 μm. b Detail of a flat region surrounding a nichoid block, showing a possible paracrine effect generated by GATA4+ cells on the expression of the GATA4 marker by cells of peripheral nichoids. The scale bar is 20 μm. c Quantification of GATA4 expression by image processing; n = 15, *p < 0.01 **p < 0.05. d Immunofluorescence for α-SMA (red) and DAPI (blue) in the nichoid (gray), the flat glass and the kidney ECM at day 3, 7, and 14. The scale bar is 50 μm. e Immunofluorescence for NKX2.5 (green) and DAPI (blue) in the nichoid (gray), the flat glass and the kidney matrix substrate at day 3, 7, and 14. The scale bar is 50 μm. f Quantification of α-SMA expression by image processing; n = 15, *p < 0.01 **p < 0.05. g Quantification of NKX2.5 expression by image processing; n = 15, *p < .0.01 **p < 0.05
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Fig4: Spontaneous endodermal and mesodermal differentiation of mES cells cultured in the nichoid substrates, compared to flat glass and to kidney ECM. Cells were cultured in the absence of a feeder layer and with LIF up to day 3, then without neither a feeder layer nor LIF from day 4 to day 14. a Immunofluorescence for GATA4 (green) and DAPI (blue) in the nichoid (gray), the flat glass and the kidney ECM at day 3, 7, and 14. The scale bar is 50 μm. b Detail of a flat region surrounding a nichoid block, showing a possible paracrine effect generated by GATA4+ cells on the expression of the GATA4 marker by cells of peripheral nichoids. The scale bar is 20 μm. c Quantification of GATA4 expression by image processing; n = 15, *p < 0.01 **p < 0.05. d Immunofluorescence for α-SMA (red) and DAPI (blue) in the nichoid (gray), the flat glass and the kidney ECM at day 3, 7, and 14. The scale bar is 50 μm. e Immunofluorescence for NKX2.5 (green) and DAPI (blue) in the nichoid (gray), the flat glass and the kidney matrix substrate at day 3, 7, and 14. The scale bar is 50 μm. f Quantification of α-SMA expression by image processing; n = 15, *p < 0.01 **p < 0.05. g Quantification of NKX2.5 expression by image processing; n = 15, *p < .0.01 **p < 0.05

Mentions: We thus assessed the differentiation potential toward the endoderm germ layer by staining and quantifying the co-occurrence of GATA-4 and DAPI in feeder-free layer culture conditions (Fig. 4a, c). This endoderm marker was highly expressed in cells cultured on both EBs in the nichoids and 2-D glass substrates (75.12 ± 12.33 %, 79.57 ± 11.31 %, respectively). On the other hand, it was negligible on kidney ECM (3.42 ± 2.86 %) (Fig. 4c, n = 15, p value = 0.01) in the presence of LIF conditioning at day 3. This could be explained by the dimethyl sulfoxide (DMSO) used in cell freezing. DMSO has been reported as an induction factor for endodermal differentiation [22]. In the absence of LIF, GATA4 expression in nichoids thus slowed down significantly compared to the 2-D glass (5.24 ± 3.97 %, 24.42 ± 17.07 %, respectively. n = 15, p value = 0.05) at day 7, while it was negligible on kidney ECM up to day 14. GATA4 in the nichoids and in the 2-D glass increased at day 14 (20.39 ± 19.06 %, 42.21 ± 22.15 %, respectively. n = 15, p value = 0.05), resulting in an up-down-up expression that is well documented in the literature [7] (Fig. 4a, c). However, this behavior could also be due to a possible paracrine signaling effect (Fig. 4b). In fact, GATA4+-EB were mostly localized at the outer boundaries of the nichoids, in particular close to the GATA4+ cells grown on the 80-μm gap flat glass surface in between the nichoids. Therefore, such cells experiencing the 2-D environment could have affected the mES cell differentiation in the peripheral nichoids.Fig. 4


Two-photon polymerized “ nichoid ” substrates maintain function of pluripotent stem cells when expanded under feeder-free conditions
Spontaneous endodermal and mesodermal differentiation of mES cells cultured in the nichoid substrates, compared to flat glass and to kidney ECM. Cells were cultured in the absence of a feeder layer and with LIF up to day 3, then without neither a feeder layer nor LIF from day 4 to day 14. a Immunofluorescence for GATA4 (green) and DAPI (blue) in the nichoid (gray), the flat glass and the kidney ECM at day 3, 7, and 14. The scale bar is 50 μm. b Detail of a flat region surrounding a nichoid block, showing a possible paracrine effect generated by GATA4+ cells on the expression of the GATA4 marker by cells of peripheral nichoids. The scale bar is 20 μm. c Quantification of GATA4 expression by image processing; n = 15, *p < 0.01 **p < 0.05. d Immunofluorescence for α-SMA (red) and DAPI (blue) in the nichoid (gray), the flat glass and the kidney ECM at day 3, 7, and 14. The scale bar is 50 μm. e Immunofluorescence for NKX2.5 (green) and DAPI (blue) in the nichoid (gray), the flat glass and the kidney matrix substrate at day 3, 7, and 14. The scale bar is 50 μm. f Quantification of α-SMA expression by image processing; n = 15, *p < 0.01 **p < 0.05. g Quantification of NKX2.5 expression by image processing; n = 15, *p < .0.01 **p < 0.05
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Fig4: Spontaneous endodermal and mesodermal differentiation of mES cells cultured in the nichoid substrates, compared to flat glass and to kidney ECM. Cells were cultured in the absence of a feeder layer and with LIF up to day 3, then without neither a feeder layer nor LIF from day 4 to day 14. a Immunofluorescence for GATA4 (green) and DAPI (blue) in the nichoid (gray), the flat glass and the kidney ECM at day 3, 7, and 14. The scale bar is 50 μm. b Detail of a flat region surrounding a nichoid block, showing a possible paracrine effect generated by GATA4+ cells on the expression of the GATA4 marker by cells of peripheral nichoids. The scale bar is 20 μm. c Quantification of GATA4 expression by image processing; n = 15, *p < 0.01 **p < 0.05. d Immunofluorescence for α-SMA (red) and DAPI (blue) in the nichoid (gray), the flat glass and the kidney ECM at day 3, 7, and 14. The scale bar is 50 μm. e Immunofluorescence for NKX2.5 (green) and DAPI (blue) in the nichoid (gray), the flat glass and the kidney matrix substrate at day 3, 7, and 14. The scale bar is 50 μm. f Quantification of α-SMA expression by image processing; n = 15, *p < 0.01 **p < 0.05. g Quantification of NKX2.5 expression by image processing; n = 15, *p < .0.01 **p < 0.05
Mentions: We thus assessed the differentiation potential toward the endoderm germ layer by staining and quantifying the co-occurrence of GATA-4 and DAPI in feeder-free layer culture conditions (Fig. 4a, c). This endoderm marker was highly expressed in cells cultured on both EBs in the nichoids and 2-D glass substrates (75.12 ± 12.33 %, 79.57 ± 11.31 %, respectively). On the other hand, it was negligible on kidney ECM (3.42 ± 2.86 %) (Fig. 4c, n = 15, p value = 0.01) in the presence of LIF conditioning at day 3. This could be explained by the dimethyl sulfoxide (DMSO) used in cell freezing. DMSO has been reported as an induction factor for endodermal differentiation [22]. In the absence of LIF, GATA4 expression in nichoids thus slowed down significantly compared to the 2-D glass (5.24 ± 3.97 %, 24.42 ± 17.07 %, respectively. n = 15, p value = 0.05) at day 7, while it was negligible on kidney ECM up to day 14. GATA4 in the nichoids and in the 2-D glass increased at day 14 (20.39 ± 19.06 %, 42.21 ± 22.15 %, respectively. n = 15, p value = 0.05), resulting in an up-down-up expression that is well documented in the literature [7] (Fig. 4a, c). However, this behavior could also be due to a possible paracrine signaling effect (Fig. 4b). In fact, GATA4+-EB were mostly localized at the outer boundaries of the nichoids, in particular close to the GATA4+ cells grown on the 80-μm gap flat glass surface in between the nichoids. Therefore, such cells experiencing the 2-D environment could have affected the mES cell differentiation in the peripheral nichoids.Fig. 4

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