Figure 1.
Directed differentiation of iPSCs into fibroblasts.
(A) Schematic representation of the differentiation strategy for generating fibroblasts from iPSCs. Cells growing out from attached embryoid bodies made in medium supplemented with AA and TGFβ2 were repeatedly split to obtain a fibroblast-like cell population. (B) Morphology of iPSC-derived fibroblasts. After 5 passages, fibroblast-like spindle-shape cells were observed. (C) Gene expression in iPSC-derived fibroblasts. Expression of stem cell markers (OCT4 and SOX2) disappeared, and markers of fibroblasts (CD10, CD73, type I and III collagen) were increased in iPSC-derived fibroblasts compared with iPSCs.
Figure 2.
Characteristics of iPSC-derived fibroblasts.
(A) Cell surface marker expression in iPSC-derived fibroblasts. FACS analysis showed that the cell surface marker profile of iPSC-derived fibroblasts was very similar to normal human fibroblasts, with over 90% expression of each marker. (B) Protein expression of iPSC-derived fibroblasts. Immunostaining demonstrated that iPSC-derived fibroblasts express fibroblast markers vimentin (VIM) and prolyl-4-hydroxylase beta (P4HB), and produced multiple types of collagen as well as normal human fibroblasts. (C) Detection of mature form of type VII collagen. Western blotting analysis detected 290kD bands in each lane, indicating that type VII collagen was synthesized from iPSC-derived fibroblasts, as well as from normal fibroblasts. The lower band of 250kD may be from either degradation or an immature form of type VII collagen, since it appears to be more abundant in the cells (lanes 3 and 4) than in the culture media (lanes 1 and 2).
Figure 3.
Generation of reconstituted skin using iPSC-derived fibroblasts.
(A) iPSC-derived fibroblasts can contribute to forming normal skin structures in vivo, comparable to normal human fibroblasts. Staining with an anti-human nuclear antibody (green signal) clearly shows the presence of human cells on the back skin of the mice. The white arrow indicates the border of the human cells on the back skin of mouse. Red: keratin 14. Green: human nuclei. Blue: DAPI (both human and mouse nuclei). (B) Desmoglein 3 is expressed though the epidermis. Green: human desmoglein 3 (does not recognize mouse desmoglein 3). Blue: DAPI. Dotted line: BMZ. (C) Human type VII collagen (using LH7.2 antibody which is specific for human type VII collagen) is observed at the BMZ of reconstituted skin using iPSC-derived fibroblasts. Red: keratin 14. Green: human type VII collagen (not mouse). Blue: DAPI. (D) Keratin 1 is present only in the suprabasal layer of epidermis. Green: human keratin 1 (does not recognize mouse keratin 1). Blue: DAPI. (E) Loricrin, a terminal differentiation marker of keratinocytes, is observed in the upper layer of epidermis. Red: loricrin. Green: human nuclei. Blue: DAPI.
Figure 4.
Generation of 3D skin equivalent.
3D skin equivalents were successfully generated using iPSC-derived keratinocytes and fibroblasts, and are histologically comparable to those generated using normal human keratinocytes and fibroblasts. H & E staining revealed normal epidermal and dermal morphology, as well as stratification and terminal differentiation.