Research ArticleCELL BIOLOGY

Three-dimensional system enabling the maintenance and directed differentiation of pluripotent stem cells under defined conditions

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Science Advances  12 May 2017:
Vol. 3, no. 5, e1602875
DOI: 10.1126/sciadv.1602875
  • Fig. 1 Characterization of mESCs cultured under 2D (plates) or 3D (scaffolds) conditions.

    (A) Nuclei 4′,6-diamidino-2-phenylindole (DAPI) staining on sections of scaffolds seeded with mESCs. The staining was performed on frozen sections. Scale bars, 100 μm. (B) Cell proliferation of mESCs cultured under the 2iLIF condition. Calorimetric assay was performed on the indicated days. The absorbance (bars) and the absorbance relative to day 1 (lines) are means ± SD from three independent experiments. *P < 0.05 in 3D-cultured mESCs versus 2D-cultured mESCs. (C) mRNA expression determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis in mESCs cultured under 2D or 3D conditions in the presence (+) or absence (−) of 2iLIF. The Sox17, T, and Sox1 expression data are shown in the rectangular box with the smaller scale of the y axis. *P < 0.05, **P < 0.01, and ***P < 0.001 as indicated or versus NIH3T3 (Fibro) or EB. Data are means ± SD from four independent experiments. (D) Protein expressions of pluripotency markers (NANOG and SSEA1) in mESCs cultured under the 3D condition in scaffolds in the presence (+) or absence (−) of 2iLIF. Immunostaining was performed on frozen sections. Nuclei were stained with DAPI. Scale bars, 100 μm.

  • Fig. 2 Differentiation of mESCs cultured under 2D (plates) or 3D (scaffolds) conditions.

    (A) Schematic representation of the in vivo implantation procedure and gross appearance of a teratoma in a nude mouse. (B) Representative pictures of teratomas derived from mESCs cultured under 2D and 3D conditions (upper) and the table showing the occurrence of teratomas (lower). Sections were stained with hematoxylin and eosin (H&E). Scale bars, 100 μm. (C) A schematic of the protocols used for in vitro early differentiation of mESCs. RA, retinoic acid; Cyc, cyclopamine. (D) mRNA expression determined by RT-qPCR analysis in the 2D or 3D differentiation cultures of mESCs; EBs and spontaneously differentiated mESCs (Spont) in 2D cultures were used for controls. Data are means ± SD from four independent experiments. *P < 0.05 in Endo-induced mESCs versus Meso- and Ecto-induced mESCs, Spont, and EB; **P < 0.01 in Ecto versus Endo, Meso, Spont, and EB; and ***P < 0.001 in Meso versus Endo, Ecto, Spont, and EB.

  • Fig. 3 Osteogenic differentiation of mESCs cultured under 2D (plates) or 3D (scaffolds) conditions.

    (A) A schematic of the strategy for inducing osteogenic differentiation of mPSCs. SAG, smoothened agonist. (B) mRNA expression determined by RT-qPCR analysis in the osteogenic 2D or 3D cultures of mESCs. Data are means ± SD from six independent experiments. **P < 0.01 and ***P < 0.001 in 3D-cultured mESCs versus 2D-cultured mESCs. (C) SP7 protein expression in the osteogenic 3D culture of mESCs (mESC-OB, day 26) and NIH3T3 cultured on scaffolds (negative control). Immunostaining was performed for SP7 on frozen sections. Nuclei were stained with DAPI. Scale bars, 50 μm. (D) Green fluorescent protein (GFP) expression in the osteogenic 3D culture of 2.3-kb Col1a1-GFP mESCs (day 26). GFP signal was assessed in the whole scaffold by fluorescence microscopy. Scale bars, 100 μm. (E) Cross-sectional view of the surface of the scaffold in the osteogenic 3D culture of 2.3-kb Col1a1-GFP mESCs (day 26). Immunostaining was performed for GFP on frozen sections. Nuclei were stained with DAPI. Scale bars, 250 μm.

  • Fig. 4 Characterization of the mESC-derived cell-scaffold complex after osteogenic differentiation (day 26) and the coculture with BM cells (day 26 + 7).

    (A) Representative pictures of H&E staining, von Kossa staining with nuclear fast red, and SEM in the osteogenic 3D culture of mESCs. Insets show fivefold magnification views of regions marked by rectangular boxes. (B) Representative pictures of H&E staining and SEM in 3D cocultures of the mESC-derived osteoblast/osteocyte population and BM cells. (C) TRAP staining in cocultures of the 3D mESC– or 2D mESC–derived osteoblasts (OB) and BM cells. Coculture of PO and BM cells in 2D is shown as a positive control.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/5/e1602875/DC1

    fig. S1. Pluripotency maintenance of mESCs cultured under 3D (scaffolds) conditions in the presence or absence of 2iLIF, related to Fig. 1.

    fig. S2. Representative pictures of H&E staining in the osteogenic 3D culture of mESCs on day 26.

    fig. S3. Representative SEM pictures in the osteogenic 3D culture of mESCs at day 26.

    fig. S4. Osteogenic differentiation of miPSCs cultured under 2D (plates) or 3D (scaffolds) conditions.

    table S1. Primer sequences (forward and reverse, 5′-3′) used for RT-qPCR.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Pluripotency maintenance of mESCs cultured under 3D (scaffolds) conditions in the presence or absence of 2iLIF, related to Fig. 1.
    • fig. S2. Representative pictures of H&E staining in the osteogenic 3D culture of mESCs on day 26.
    • fig. S3. Representative SEM pictures in the osteogenic 3D culture of mESCs at day 26.
    • fig. S4. Osteogenic differentiation of miPSCs cultured under 2D (plates) or 3D (scaffolds) conditions.
    • table S1. Primer sequences (forward and reverse, 5′-3′) used for RT-qPCR.

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