Research ArticleBIOMEDICAL ENGINEERING

Bioengineering a 3D integumentary organ system from iPS cells using an in vivo transplantation model

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Science Advances  01 Apr 2016:
Vol. 2, no. 4, e1500887
DOI: 10.1126/sciadv.1500887
  • Fig. 1 Induction of epithelial tissues via the CDB transplantation method.

    (A) Schematic representation of EB cultures and the CDB transplantation method. (B) Phase-contrast images of iPS cells and the formation of EBs, which were cultured in nonadherent plastic wells for 3, 5, or 7 days. Scale bars, 100 μm. (C) mRNA expression levels of undifferentiated iPS cell markers (Nanog) and neural crest cell markers (Nestin and Pax3) during EB formation. *P < 0.001 by Student’s t test. (D) Hematoxylin and eosin (H&E) staining and immunostaining of EBs, using antibodies of epithelial (Sox2/p63 and Sox17), neural progenitor (Pax6), and neural crest markers (Snail and Twist) after 7 days. The nuclei were stained using Hoechst 33258 (white). Scale bars, 50 μm. (E) Macroscopic photographs (left panels) and microscopy (H&E staining, center and right panels) of in vivo transplants under various transplantation conditions. The in vivo transplants of 3000 dissociated iPS cells (upper), single EBs (middle), and more than 30 EBs (lower) were placed in the subrenal capsule for 30 days and then analyzed. (F) Macroscopic photographs of multiple EB in a collagen gel before transplantation. Scale bars, 1 mm. (G) Weight of the in vivo transplants. The data are presented as the median ± maximum or minimum from individual experiments; n = 8 and n = 48 per experiment. Red circles indicate the cyst, including hair follicles, in the explants. *P < 0.001 by Student’s t test. (H) The area occupancy of the cystic lumen in the whole specimens of in vivo transplants of the three types of conditions was compared. *P < 0.001 by Student’s t test. (I) Histochemical and immunohistochemical analyses of the cystic epithelia in the in vivo explants of multiple iPS cell–derived EBs. Boxed areas in the left panels show H&E staining. To identify epithelial types, such as ectodermal epithelium, integument (top panels), and endodermal epithelium, including the gastrointestinal tube (middle panels) and respiratory tract (bottom panels), we analyzed CDB transplants by immunostaining with specific antibodies for CK5, CK10, Muc2, Cdx2, villin, CC10, Tuj1, and E-cadherin. The nuclei were stained using Hoechst 33258 (blue). To identify the nonspecific fluorescence signals in these immunohistochemical analyses, we performed the experiments under the conditions without specific antibodies against antigens [negative control (NC)]. Scale bars, 1 mm (low-magnification images) and 100 μm (high-magnification images). (J) The frequency of epithelial types in CDB transplants. Epithelial types in CDB transplants were classified based on the cell morphology and number count. The data are presented as the means ± SEM from individual experiments; n = 5.

  • Fig. 2 Analysis of the bioengineered hair follicle induced from iPS cells via the CDB transplantation method.

    (A) Macroscopic (left panels) and microscopic (H&E staining; right panels) examination of the hair follicles and shafts in the CDB transplants. EBs were stimulated without Wnt10b (upper panels) or with Wnt10b (lower panel) on day 7. Scale bars, 100 μm. (B) Frequency of the epithelial tissue, including hair follicle, in CDB transplants. The data are presented as the means ± SEM from individual experiments; n = 13 (single iPS injection), n = 49 (CDB transplants without Wnt10b), and n = 15 (CDB transplants with Wnt10b). (C) Number of hair follicles in the CDB transplants. The data are presented as the means ± SEM from individual experiments; n = 13 (single iPS injection), n = 74 (CDB transplants without Wnt10b), and n = 4 (CDB transplants with Wnt10b). *P < 0.001 by Student’s t test. (D) Comparative analysis of the length of hair shafts from the hair tip to the DP in CDB explants treated with or without Wnt10b. *P < 0.05 by Student’s t test. (E) Histological analysis of the hair follicles and their surrounding tissues in iPS cell–derived bioengineered 3D IOSs. The isolated cystic structures with hair follicles were observed macroscopically (left panels). In the H&E analyses, the boxed areas in the low-magnification macroscopic views are shown at a higher magnification in other panels. CL, cystic lumen; EL, epidermal layer; DL, dermal layer; SD, subdermal tissue; SG, sebaceous gland; HO, hair opening; v, vessel. Scale bars, 500 μm (H&E; upper left) and 100 μm (others). (F) Schematic representation of stem/progenitor cells in the hair follicle and surrounding tissues on the skin at the anagen and telogen phases. APM, arrector pili muscle. (G) Immunohistochemical analyses of stem/progenitor cells in the follicles of natural pelage and enhanced green fluorescent protein (EGFP)–labeled iPS cell–derived bioengineered hair follicles. These samples were immunostained with anti-Sox9 (red), anti-CD34 (green), anti-CK15 (red), anti-Lrig1 (white), and anti-Lgr5 (red) antibodies. Arrows, epithelial cells of bulge regions; arrowheads, Lrig1-positive cells; *, background fluorescence of hair shafts. Scale bars, 200 μm (upper panels) and 50 μm (lower panels). (H) Histological and immunohistochemical analyses of the iPS cell–derived hair follicles and their surrounding tissues. Natural pelage and iPS cell–derived hair follicles were stained with calponin. Arrowheads indicate calponin-positive arrector pili muscles. Scale bars, 200 μm.

  • Fig. 3 Transplantation of the bioengineered 3D IOS.

    (A) Schematic representation of the methods used for the generation and transplantation of iPS cell–derived hair follicles. Cystic tissue with hair follicles was isolated and divided into small pieces containing 10 to 20 hair follicles (lower left panels). The small pieces were transplanted into the back skin of nude mice using a follicular unit transplantation (FUT) method developed in humans (lower right panels). Scale bars, 500 μm (lower left panels) and 1 mm (lower right panels). (B) Macromorphological observations of two independent engraftments into the dorsoventral skin of nude mice showing the eruption and growth of iPS cell–derived hair follicles. Scale bars, 1 mm. (C) Y-FISH analysis of the CDB transplants using male mouse–specific DNA probes. H&E and differential interference contrast (DIC) images are shown in the left panels. FISH images are shown in the upper left panel. Green and blue signals indicate Y-chromosome–positive cells and nuclei, respectively. Boxed areas in the FISH image are shown at a higher magnification in the right panels. Boxed areas U1 and U2 indicate the skin epithelium and upper region of the hair follicle. U3 indicates the bulge region. Boxed area sU3 indicates the Y-FISH–positive sebaceous gland isolated from fig. S5. Boxed areas L1 and L2 indicate the lower hair bulb region. Boxed area L3 indicates the intracutaneous adipose tissue. Broken lines in the differential interference contrast image indicate the outermost limit of the dermis. Scale bars, 200 μm (whole images) and 100 μm (high-magnification images). (D) Analysis of the integration with surrounding tissues, such as the arrector pili muscles and nerves, in the cervical skin of nude mice. The differential interference contrast images show black hair shafts derived from the bioengineered 3D IOS (origin: C57BL/6 mice) among the white hairs of nude mice. The arrector pili muscles and nerve fibers were analyzed by immunohistochemical staining using specific antibodies against calponin (red) for smooth muscle and neurofilament H (NF-H; white). The nuclei (Nuc) were stained using Hoechst 33258 (blue). The boxed areas in the left panels are shown at a higher magnification in the right panels. The arrows and arrowheads indicate nerve fibers and muscles connected with hair follicles, respectively. Broken lines indicate the outermost limit of each hair follicle. Scale bars, 200 μm (low-magnification photographs) and 100 μm (high-magnification photographs).

  • Fig. 4 Analysis of iPS cell–derived hair types and hair cycle.

    (A) Microscopic observation of the iPS cell–derived bioengineered hair shafts showing zigzag, awl/auchene, and guard hairs. The hair shafts were analyzed using light microscopy. Whole (left) and high-magnification (right) views are shown. Scale bars, 2 mm (low magnification) and 20 μm (high magnification). (B) Analysis of hair types, such as zigzag (Z), awl/auchene (A/Au), and guard (G), of natural mouse pelage and iPS cell–derived hair shafts. The hair types were determined by microscopic observations. The data are presented as the means ± SEM from six individual experiments; n = 104 (natural skin) and n = 248 (iPS cell–derived transplants). (C) Distance between hair shafts in natural pelage and iPS cell–derived bioengineered hair shafts. The distances were calculated by microscopic observations of H&E-stained sections. The data are presented as the means ± SEM from individual experiments; n = 6 (natural skin) and n = 6 (iPS cell–derived transplants). (D) Macromorphological observations at the anagen phase of the hair cycles in iPS cell–derived bioengineered hair. Scale bars, 1 mm. (E) Assessment of the hair growth (closed circles) and regression (open circles) phases of the bioengineered hair, including zigzag and awl/auchene and guard hair types. The data are presented as the means ± SEM; n = 5 (zigzag) and n = 10 (awl/auchene and guard).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/4/e1500887/DC1

    Fig. S1. Analysis of the area of the cystic lumen.

    Fig. S2. Histochemical and immunohistochemical analyses of the cystic epithelia in the in vivo explants of multiple iPS cell–derived EBs.

    Fig. S3. Culture of iPS cells for EB formation.

    Fig. S4. Gene expression in iPS cell–derived bioengineered hair follicle germs in a 3D IOS generated via the CDB transplantation method.

    Fig. S5. Y-FISH analysis of the distribution of iPS cell–derived cells among the 3D IOSs grafted to the natural murine skin.

    Fig. S6. Distribution of hair species of iPS cell–derived hairs.

    Table S1. Primer sequences used in real-time PCR.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Analysis of the area of the cystic lumen.
    • Fig. S2. Histochemical and immunohistochemical analyses of the cystic epithelia in the in vivo explants of multiple iPS cell–derived EBs.
    • Fig. S3. Culture of iPS cells for EB formation.
    • Fig. S4. Gene expression in iPS cell–derived bioengineered hair follicle germs in a 3D IOS generated via the CDB transplantation method.
    • Fig. S5. Y-FISH analysis of the distribution of iPS cell–derived cells among the 3D IOSs grafted to the natural murine skin.
    • Fig. S6. Distribution of hair species of iPS cell–derived hairs.
    • Table S1. Primer sequences used in real-time PCR.

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