Research ArticleHEALTH AND MEDICINE

Small molecule–driven direct conversion of human pluripotent stem cells into functional osteoblasts

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Science Advances  31 Aug 2016:
Vol. 2, no. 8, e1600691
DOI: 10.1126/sciadv.1600691
  • Fig. 1 Exogenous adenosine induced osteogenic differentiation of hiPSCs.

    (A) Transcription profile of 84 genes relevant to osteogenesis for hiPSCs cultured for 21 days in growth medium (GM), adenosine-supplemented growth medium (Adenosine), and conventional osteogenic induction medium (OM). Relative expressions: red (high), black (medium), and green (low). (B) Time-dependent quantitative gene expressions of hiPSCs for osteogenic markers (RUNX2, OCN, and SPP1) and pluripotent marker (NANOG) cultured in GM, adenosine, and OM. (C) Immunofluorescence staining of osteocalcin (green), F-actin (red), and nuclei (blue), as well as Alizarin Red S staining of hiPSCs cultured for 21 days in GM, adenosine, and OM. Scale bars, 100 μm. Inset shows the stained image of the entire well. Data are presented as means ± SEs (n = 3). Data are shown as fold expression of target genes after normalization to undifferentiated, pluripotent hiPSCs. For RUNX2, OCN, and SPP1, the groups with various medium conditions at the same culture time were compared by using one-way analysis of variance (ANOVA) with Tukey-Kramer post hoc test. For NANOG, all the groups were compared to undifferentiated, pluripotent hiPSCs by two-way ANOVA with Bonferroni post hoc test. Asterisks were assigned to P values with statistical significances (*P < 0.05; **P < 0.01; ***P < 0.001).

  • Fig. 2 Adenosine-induced osteogenic differentiation of hiPSCs involves A2bR.

    (A) Time-dependent quantitative gene expressions of hiPSCs for adenosine receptor subtypes (A1R, A2aR, A2bR, and A3R) cultured in growth medium (GM), adenosine-supplemented growth medium (Adenosine), and osteogenic induction medium (OM). (B) Quantitative gene expression analysis of osteogenic markers (RUNX2, OCN, and SPP1) and pluripotent marker (NANOG) for hiPSCs after 21 days of culture. The plus (+) and minus (−) symbols in gene expression denote growth medium in the presence and absence of adenosine or PSB 603 (an A2bR antagonist). (C) Immunofluorescence staining for osteocalcin (green), F-actin (red), and nuclei (blue), as well as Alizarin Red S staining of hiPSCs cultured for 21 days in the presence and absence of adenosine or PSB 603. Inset shows the stained image of the whole well. Various media include growth medium (GM), growth medium containing adenosine (Adenosine), and growth medium containing both adenosine and PSB 603 (Adenosine + PSB 603). Scale bars, 100 μm. Data are shown as means ± SEs (n = 3). Data are presented as fold expression of target genes after normalization to undifferentiated, pluripotent hiPSCs. For A1R, A2aR, A2bR, and A3R, as well as RUNX2, OCN, and SPP1, the groups with various medium conditions at the same culture time were compared by one-way ANOVA with Tukey-Kramer post hoc test. For NANOG, all the groups were compared to undifferentiated, pluripotent hiPSCs by two-way ANOVA with Bonferroni post hoc test. Asterisks indicate statistical significances according to P values (*P < 0.05; **P < 0.01; ***P < 0.001).

  • Fig. 3 In vitro bone-forming ability of hiPSC-derived cells.

    (A) Schematic of the experimental protocol used to examine the bone-forming ability of hiPSC-derived osteoblasts within the macroporous matrices. (B and C) Three-dimensional microcomputed tomography (μCT) images (B) and the corresponding bone mineral densities [bone volume per total volume (BV/TV)] (C) of cell-laden (Ad-hiPSCs and d-hiPSCs) matrices as a function of culture time. Scale bars, 2 mm. (D) H&E staining and immunohistochemical staining for osteocalcin of the macroporous matrices containing Ad-hiPSCs and d-hiPSCs cultured for 1, 2, and 3 weeks. Scale bars, 100 μm. High-magnification images are also provided. Scale bars, 50 μm. Data are displayed as means ± SEs (n = 4). Two groups at the same culture time were compared by two-tailed Student’s t test. Asterisks indicate statistical significances according to P values (***P < 0.01).

  • Fig. 4 hiPSC-derived osteoblasts (Ad-hiPSCs) contribute to the healing of critical-sized bone defects through the formation of vascularized neobone tissue.

    (A) Three-dimensional μCT images of cranial bone defects with no implantation (Sham), as well as defects treated with acellular and Ad-hiPSC–laden matrices [Ad-hiPSCs; hiPSC derivatives generated by culturing in growth medium supplemented with adenosine (30 μg/ml) for 21 days] after 16 weeks. Scale bars, 1 mm. (B) Bone mineral densities [bone volume per total volume (BV/TV)] of defects treated with acellular and Ad-hiPSC–laden matrices, as well as sham group following 4 and 16 weeks of implantation. Native mouse cranial bone was used as a control. (C) H&E staining and immunohistochemical staining for osteocalcin of cranial bone defects treated with acellular and Ad-hiPSCs–laden matrices, and sham group after 16 weeks. Scale bars, 500 μm. High-magnification images show the center and edge of cranial bone defects. White and black dashed lines mark the interface between newly formed tissue and the native bone tissue. Yellow arrows in H&E staining denote microvessels containing red blood cells. Scale bars, 100 μm. (D) Histomorphometric analysis for neobone density within cranial bone defects (Bone area/defect area) determined from H&E staining images for Sham, Acellular, and Ad-hiPSCs groups at 4 and 16 weeks after treatment. Data are shown as means ± SEs (n = 6). Multiple groups at the same post-implantation time were compared by one-way ANOVA with Tukey-Kramer post hoc test. Asterisks were assigned to P values with statistical significances (*P < 0.05; ***P < 0.001).

  • Fig. 5 hiPSC-derived osteoblasts contributed to the repair of critically sized cranial defects through the formation of vascularized neobone tissue.

    (A and B) Immunofluorescence staining for osteocalcin (red) and human-specific lamin A/C (green) (A) and CD31 (red) along with nuclei (blue; Hoechst), as well as TRAP staining (B) of cranial bone defects treated with Ad-hiPSC–laden (Ad-hiPSCs) and acellular matrices, and sham group after 16 weeks of implantation. Red arrows indicate TRAP-positive stains. Scale bars, 50 μm.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/8/e1600691/DC1

    fig. S1. Attachment and growth of hPSCs cultured under various medium conditions.

    fig. S2. Exogenous adenosine induced osteogenic differentiation of hESCs.

    fig. S3. Exogenous adenosine induced expressions of osteoblastic markers for hiPSCs.

    fig. S4. Adenosine-induced osteogenic differentiation of hESCs uses A2bR.

    fig. S5. In vitro hard tissue–forming ability of hiPSC-derived cells.

    fig. S6. Minimal hard tissue formation for Ad-hiPSC–laden matrices before implantation.

    fig. S7. hiPSC-derived osteoblasts (Ad-hiPSCs) facilitate the repair of critical-sized bone defects.

    fig. S8. Donor hiPSC–derived cells (Ad-hiPSCs) contribute to the regeneration of vascularized neobone tissue.

    table S1. List of primer sequences used for qPCR analysis.

    movie S1. Minimal calcification from d-hiPSCs in vitro.

    movie S2. Dense and homogeneous calcified tissue formation from Ad-hiPSCs in vitro.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Attachment and growth of hPSCs cultured under various medium conditions.
    • fig. S2. Exogenous adenosine induced osteogenic differentiation of hESCs.
    • fig. S3. Exogenous adenosine induced expressions of osteoblastic markers for hiPSCs.
    • fig. S4. Adenosine-induced osteogenic differentiation of hESCs uses A2bR.
    • fig. S5. In vitro hard tissue–forming ability of hiPSC-derived cells.
    • fig. S6. Minimal hard tissue formation for Ad-hiPSC–laden matrices before implantation.
    • fig. S7. hiPSC-derived osteoblasts (Ad-hiPSCs) facilitate the repair of criticalsized bone defects.
    • fig. S8. Donor hiPSC–derived cells (Ad-hiPSCs) contribute to the regeneration of vascularized neobone tissue.
    • table S1. List of primer sequences used for qPCR analysis.
    • Legends for movies S1 and S2

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    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.avi format). Minimal calcification from d-hiPSCs in vitro.
    • movie S2 (.avi format). Dense and homogeneous calcified tissue formation from Ad-hiPSCs in vitro.

    Files in this Data Supplement:

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