The use of nanofibrillar cellulose hydrogel as a flexible three-dimensional model to culture human pluripotent stem cells

Abstract

Human embryonic stem cells and induced pluripotent stem cells have great potential in research and therapies. The current in vitro culture systems for human pluripotent stem cells (hPSCs) do not mimic the three-dimensional (3D) in vivo stem cell niche that transiently supports stem cell proliferation and is subject to changes which facilitate subsequent differentiation during development. Here, we demonstrate, for the first time, that a novel plant-derived nanofibrillar cellulose (NFC) hydrogel creates a flexible 3D environment for hPSC culture. The pluripotency of hPSCs cultured in the NFC hydrogel was maintained for 26 days as evidenced by the expression of OCT4, NANOG, and SSEA-4, in vitro embryoid body formation and in vivo teratoma formation. The use of a cellulose enzyme, cellulase, enables easy cell propagation in 3D culture as well as a shift between 3D and two-dimensional cultures. More importantly, the removal of the NFC hydrogel facilitates differentiation while retaining 3D cell organization. Thus, the NFC hydrogel represents a flexible, xeno-free 3D culture system that supports pluripotency and will be useful in hPSC-based drug research and regenerative medicine.

FIG. 1.

Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) form three-dimensional (3D) spheroids in the nanofibrillar cellulose (NFC) hydrogel. (A, B) A time course shows that WA07 (A) and iPS(IMR90)-4 (B) cells formed 3D spheroids in 0.5 wt.% NFC hydrogel. (C, D) Live/dead staining images show living WA07 (C) and iPS(IMR90)-4 (D) cells in the spheroids on day 9. Scale bars=200 μm. Color images available online at www.liebertpub.com/scd

FIG. 2.

Enzymatic removal of the NFC hydrogel. (A, B) Mitochondrial metabolic activity of WA07 and iPS(IMR90)-4 cells (A) and H9-GFP cells (B) as a function of the concentration of cellulase. Cells cultured on a standard Matrigel platform were treated with cellulase at various concentrations (μg cellulase/mg cellulose). The mitochondrial metabolic activity was determined by an AlamarBlue^® assay, and the increase in fluorescence intensity after a 24-h treatment with cellulase is presented. Four (A) or six (B) biological samples for each condition were prepared. The results are expressed as mean±standard deviation (SD). (C) Removal of cellulose by 0, 50, 200, and 500 μg cellulase/mg cellulose is visualized by calcofluor white staining of cellulose, which is represented in blue. H9-GFP cells are seen in green. (D) A pluripotency marker OCT4 is expressed in H9-GFP cells that are cultured in 0.5 wt.% NFC hydrogel after treatment with cellulase at 50, 200, and 500 μg/mg cellulose. Scale bars=100 μm. **P<0.01 and ***P<0.001. Color images available online at www.liebertpub.com/scd

FIG. 3.

OCT4 expression in the 3D human pluripotent stem cell (hPSC) spheroids. The pluripotency marker OCT4 is expressed in WA07 cells that are cultured in 0.5% NFC hydrogel for 7 days [(A), 5 μm paraffin section], for 9 days (B), for 16 days after one subculture (C), and for 26 days after three subcultures [(D), 5 μm paraffin section]. OCT4 is also expressed in iPS(IMR90)-4 cells that are cultured in 0.5 wt.% NFC hydrogel for 9 days (E). Scale bars=200 μm (A) and 100 μm (B–E). Color images available online at www.liebertpub.com/scd

FIG. 4.

Gene expression in hPSCs cultured in 3D and two-dimensional (2D) platforms. Real-time quantitative reverse transcription–polymerase chain reaction (RT-PCR) analyses of OCT4 (A) and NANOG (B) mRNA expression in WA07 cells cultured in the NFC hydrogel for 9 days and iPS(IMR90)-4 cells cultured in the hydrogel for 12 days and then transferred to 2D platforms [3D to M, 3D to laminin-511 (LN-511), 3D to LN-521, and 3D to vitronectin (VN)] as well as WA07 cells in 3D spheroids at days 7 (3D day 7) and 26 (3D day 26). Relative mRNA expression was normalized to the control gene RPLP0, and fold inductions were calculated with reference to the cells cultured on the standard Matrigel platform (M ctrl). n=3 biological samples. Error bars are SD. ***P<0.001.

FIG. 5.

Transfer of WA07 3D spheroids to 2D platforms. (A–D) WA07 cells were first cultured in 0.5 wt.% NFC hydrogel for 9 days and then transferred to 2D platforms after a 24-h-treatment with 300 μg cellulase/mg cellulose at 37°C. The cells exhibited typical hPSC morphology and expressed the pluripotency markers OCT4 and SSEA-4 on Matrigel-coated dishes (A), LN-511-coated dishes (B), LN-521-coated dishes (C), and VN-coated dishes (D). Scale bars=100 μm. Color images available online at www.liebertpub.com/scd

FIG. 6.

Transfer of iPS(IMR90)-4 3D spheroids to 2D platforms. iPS(IMR90)-4 cells were first cultured in 0.5% NFC hydrogel for 12 days and then transferred to 2D platforms after a 24-h-treatment with 300 μg cellulase/mg cellulose at 37°C. The cells exhibited typical hPSC morphology and expressed the pluripotency markers OCT4 and SSEA-4 on Matrigel-coated dishes (A), LN-511-coated dishes (B), LN-521-coated dishes (C), and VN-coated dishes (D). Scale bars, 100 μm. Color images available online at www.liebertpub.com/scd

FIG. 7.

In vitro differentiation of iPS(IMR90)-4 cells. (A) Embryoid bodies (EBs) were derived from iPS(IMR90)-4 cells that had previously been cultured in 0.5% NFC hydrogel for 8 days followed by a 24-h-treatment with 300 μg cellulase/mg cellulose at 37°C. Cells dissociated from 1-week-old EBs were cultured on gelatin-coated dishes for 1 week and were then detected to express three germ layer markers, β-tubulin type III (b-tub), muscle actin (MA), and α-fetoprotein (AFP). Scale bars=100 μm. (B) Real-time quantitative RT-PCR analyses of PAX6, CDX2, and BRACHYURY mRNA expression in 4-week-old EBs derived from iPS(IMR90)-4 cells that had previously been cultured in 0.5 wt.% NFC hydrogel for 9 days. Relative mRNA expression was normalized to the control gene RPLP0, and fold inductions were calculated with reference to the cells cultured on the standard Matrigel platform (M ctrl). n=3 biological samples. Error bars are SD. Color images available online at www.liebertpub.com/scd

FIG. 8.

In vivo differentiation of WA07 cells. (A–J) Hematoxylin and eosin staining of 6-week-old teratoma sections (5 μm) of WA07 cells that had previously been cultured in 0.5 wt.% NFC hydrogel for 26 days. (A) Cartilage (1), bone (2), epithelial tissue (3), and pigmented cells (4). (B) Neuronal rosettes (ectoderm). (C) Pigmented cells (ectoderm). (D) Blood vessels (indicated by asterisks, mesoderm). (E, F) Muscle (indicated by arrows, mesoderm). (G) Cartilage (mesoderm). (H–J) Endodermal epithelia (indicated by arrows). Scale bars=100 μm. (K) Real-time quantitative RT-PCR analyses of PAX6, CDX2, and BRACHYURY mRNA expression in 6-week-old teratoma formed from WA07 cells that had previously been cultured in 0.5 wt.% NFC hydrogel for 26 days. Relative mRNA expression was normalized to the control gene RPLP0, and fold inductions were calculated with reference to the cells cultured on the standard Matrigel platform (M ctrl). Color images available online at www.liebertpub.com/scd

FIG. 9.

The karyotypes of human ESCs and iPSCs. WA07 and iPS(IMR90)-4 cells were first cultured in 0.5% NFC hydrogel and then transferred to Matrigel-coated dishes after a 24-h treatment with 300 μg cellulase/mg cellulose at 37°C. G-banding chromosome analysis showed normal chromosomes in WA07, p41 (A) and iPS(IMR90)-4, and p18+45(20) (B) cells.