Induced pluripotent stem cells from human hair follicle mesenchymal stem cells

Abstract

Reprogramming of somatic cells into inducible pluripotent stem cells (iPSCs) provides an alternative to using embryonic stem cells (ESCs). Mesenchymal stem cells derived from human hair follicles (hHF-MSCs) are easily accessible, reproducible by direct plucking of human hairs. Whether these hHF-MSCs can be reprogrammed has not been previously reported. Here we report the generation of iPSCs from hHF-MSCs obtained by plucking several hairs. hHF-MSCs were isolated from hair follicle tissues and their mesenchymal nature confirmed by detecting cell surface antigens and multilineage differentiation potential towards adipocytes and osteoblasts. They were then reprogrammed into iPSCs by lentiviral transduction with Oct4, Sox2, c-Myc and Klf4. hHF-MSC-derived iPSCs appeared indistinguishable from human embryonic stem cells (hESCs) in colony morphology, expression of alkaline phosphotase, and expression of specific hESCs surface markers, SSEA-3, SSEA-4, Tra-1-60, Tra-1-81, Nanog, Oct4, E-Cadherin and endogenous pluripotent genes. When injected into immunocompromised mice, hHF-MSC-derived iPSCs formed teratomas containing representatives of all three germ layers. This is the first study to report reprogramming of hHF-MSCs into iPSCs.

Fig. 1

Isolation and characterization of human hair follicle mesenchymal stem cells (hHF-MSCs). The hHF-MSCs, resembling typical fibroblast-like cells, migrated out from the hair follicles (a Bars = 500 μm; b Bars = 100 μm). Keratinocytes also migrated out from the hair follicles (c Bars = 500 μm). hHF-MSCs from passage 2 (d), passage 8 (e) to passage 12 (F Bars = 200 μm). Flow cytometric analysis of cell surface markers on hHF-derived fibroblast-like cells. 2 × 10⁵ cells were incubated with primary antibodies against CD29, CD73, CD105, CD90, HLA-DR, CD31, CK15 or CD45,respectively, followed by incubation with a secondary FITC-labeled antibody. Controls were incubated with secondary antibody only. Percentages indicate the fraction of cells that stained positive (g). Adipogenic differentiation of hHF-MSCs. Compared to non-induced control (h Bars = 100 μm), induction after 3 weeks, the number of intracellular lipid droplets was further increased (i Bars = 100 μm) and was detected by Oil-red O staining (j Bars = 100 μm). Osteogenic differentiation of hHF-MSCs. Compared to non-induced control (k Bars = 200 μm), calcium nodules were formed after induction for 4 weeks and was demonstrated by Alizarin red staining (l Bars = 200 μm) and Alkaline phosphatase staining (m Bars = 200 μm)

Fig. 2

Generation of induced pluripotent stem cells from hHF-MSCs. (a) Schematic diagram of the reprogramming protocol used. (b) hHF-MSCs were transduced with four factors and seeded onto mouse embryonic fibroblasts until hESCs-like colonies emerged (representative colonies). (a) Typical non-hESCs-like colony. (b) hESCs colony, X01 at passage 24. (c) High magnification of the hESCs at passage 24. (d) Typical iPS colony, hHF-MSC-derived iPSCs 10-1 at passage 2. (e) High magnification of the hHF-MSC-derived iPSCs 10-1 at passage 2. (f) hHF-MSC-derived iPSCs 10-1 at passage 16. (g) Arrow indicates area of differentiation in the center of hHF-MSC-derived iPSCs 10-1 at passage 16. (h) hHF-MSC-derived iPSCs 20-1 at passage 3. (i) High magnification of hHF-MSC-derived iPSCs. at passage 3. (j) hHF-MSC-derived iPSCs 20-1 at passage 17. (k) High magnification of hHF-MSC-derived iPSCs 20-1 at passage 17. Bars = 200 μm

Fig. 3

hESCs pluripotency marker expression by these iPSCs. a hESCs colonies, X01 at passage 25 were fixed and stained with antibodies against hESCs associated pluripotent proteins and examined under the fluorescence microscopy. b hHF-MSC-derived iPSCs 10-1 at passage 5. c hHF-MSC-derived iPSCs 20-1 at passage 6. Expressed proteins appeared red in the fluorescence microscope. Bars = 200 μm

Fig. 4

a Quantitative reverse transcription-polymerase chain reaction (PCR) analyses of endogenous Oct4, Sox2, Nanog and other pluripotency genes expression in hESCs and hHF-MSC-derived iPSCs relative to parental somatic cell populations. b Quantitative RT-PCR analyses of exogenous gene Oct4 and Sox2 expression

Fig. 5

Karyotype analysis. hHF-MSCs at passage 5 (a), hHF-MSC-derived iPSCs 10-1 at passage 10 (b) and hHF-MSC-derived iPSCs 20-1 at passage 11(c) all showed a normal 46XY karyotype

Fig. 6

Hematoxylin-eosin staining of teratomas derived from hHF-MSC-derived iPSCs. Teratomas is composed of various type of tissues: (a) Pigmented epithelium (ectoderm). b Rosettes of the neural epithelium (ectoderm). c squamous epithelium (ectoderm). d cartilage tissue (mesoderm). e Respiratory epithelium (endoderm). f Gland-like structures (endoderm). Bars = 500 μm