Overexpression of Nanog in amniotic fluid-derived mesenchymal stem cells accelerates dermal papilla cell activity and promotes hair follicle regeneration

Abstract

Alopecia, one of the most common chronic diseases, can seriously affect a patient's psychosocial life. Dermal papilla (DP) cells serve as essential signaling centers in the regulation of hair growth and regeneration and are associated with crosstalk between autocrine/paracrine factors and the surrounding environment. We previously demonstrated that amniotic fluid-derived mesenchymal stem cell-conditioned medium (AF-MSC-CM) accelerates hair regeneration and growth. The present study describes the effects of overexpression of a reprogramming factor, Nanog, on MSC properties, the paracrine effects on DP cells, and in vivo hair regrowth. First, we examined the in vitro proliferation and lifespan of AF-MSCs overexpressing reprogramming factors, including Oct4, Nanog, and Lin28, alone or in combination. Among these factors, Nanog was identified as a key factor in maintaining the self-renewal capability of AF-MSCs by delaying cellular senescence, increasing the endogenous expression of Oct4 and Sox2, and preserving stemness. Next, we evaluated the paracrine effects of AF-MSCs overexpressing Nanog (AF-N-MSCs) by monitoring secretory molecules related to hair regeneration and growth (IGF, PDGF, bFGF, and Wnt7a) and proliferation of DP cells. In vivo studies revealed that CM derived from AF-N-MSCs (AF-N-CM) accelerated the telogen-to-anagen transition in hair follicles (HFs) and increased HF density. The expression of DP and HF stem cell markers and genes related to hair induction were higher in AF-N-CM than in CM from AF-MSCs (AF-CM). This study suggests that the secretome from autologous MSCs overexpressing Nanog could be an excellent candidate as a powerful anagen inducer and hair growth stimulator for the treatment of alopecia.

Fig. 1. Forced expression of pluripotency-related transcription factors in amniotic fluid–derived mesenchymal stem cells (AF-MSCs).

a Schematic of the process for selecting a key transcription factor to promote stemness of AF-MSCs. b Morphological changes and c growth rate of AF-MSCs infected with retroviral vectors encoding Oct4, Nanog, and Lin28, alone or in combination. Scale bar, 1 mm. d Expression of transcription factors, alone or in combination, as determined by western blotting. e Overexpression of exogenous Nanog in AF-MSCs, detected by reverse transcriptase–polymerase chain reaction (RT-PCR) and immunofluorescence. Scale bar, 1 mm. f Nanog expression in H1 embryonic stem cells, AF-MSCs overexpressing Nanog (AF-N-MSCs), and AF-MSCs. g Expression of Nanog in AF-N (infected at 10 passages) over 40 passages. h Expression of endogenous Nanog in AF-N-MSCs, detected by quantitative real-time RT-PCR. Data are represented as the mean ± SD (n = 3). ***p < 0.001

Fig. 2. Promotion of amniotic fluid–derived mesenchymal stem cell (AF-MSC) stemness by Nanog overexpression.

a Growth rate of AF-MSCs overexpressing Nanog (AF-N-MSCs). Non-modified AF-MSCs served as a control (AF-MSCs). b β-Gal staining of AF- and AF-N-MSCs in passage 36. Scale bar, 200 µm. c mRNA (left) and protein expression (right) of senescence-related genes in AF- and AF-N-MSCs. d Quantification of p53 and p21 expression by quantitative real-time reverse transcriptase–polymerase chain reaction (qRT-PCR). e Fluorescence-activated cell sorting analysis and DHE staining to detect reactive oxygen species (ROS) accumulation in AF- and AF-N-MSCs. Scale bar, 1 mm. f Quantification of fluorescence intensity for ROS accumulation. g Colony-forming unit fibroblast analysis for evaluation of AF- and AF-N-MSC stemness. h Changes in endogenous expression of the pluripotency-related transcription factors (Oct4 and Sox2) in AF-MSCs upon Nanog overexpression. Expression levels were measured by RT-PCR and qRT-PCR. i Hierarchical clustering analysis of gene expression in AF- and AF-N-MSCs. Data are represented as the mean ± SD (n = 3). *p < 0.05; **p < 0.01

Fig. 3. Retention of the mesenchymal stem cell (MSC) phenotype of amniotic fluid–derived MSCs (AF-MSCs).

a Expression of mesenchymal lineage markers (fibronectin, MMP1, Snail, Slug) in AF- and AF-MSCs overexpressing Nanog (AF-N-MSCs). b Adipogenic, osteogenic, and chondrogenic differentiation of AF-N-MSCs at passage 20, evaluated by Oil Red O, von Kossa staining, and Alcian Blue staining, respectively. Scale bar, 200 µm. c mRNA expression analysis of adipogenic differentiation (aP2, PPARγ). d mRNA expression analysis of osteogenic differentiation (Osteopontin, Osteocalcin). e mRNA expression analysis of chondrogenic differentiation (Collagen II, Aggrecan). Data are represented as the mean ± SD (n = 3). *p < 0.05

Fig. 4. Increased expression of paracrine factors related to hair regeneration and growth in amniotic fluid–derived mesenchymal stem cells overexpressing Nanog (AF-N-MSCs).

a Schematic describing the identification of the main paracrine factors from AF-N-MSCs. b Comparison of cytokine expression between AF-MSCs and AF-N-MSCs. c Relative mRNA expression levels of hair growth–related markers (bFGF, IGF, Wnt7a, PDGF-AA) in AF- and AF-N-MSCs. d Protein expression of the paracrine factors related to hair growth in AF- and AF-N-MSCs, as determined by western blot. e Cytokines related to hair growth secreted from AF- and AF-N-MSCs. Cytokine concentration was measured by enzyme-linked immunosorbent assays. f Paracrine effect of AF-N-MSC–derived conditioned media (AF-N-CM) on the growth of hair follicle dermal papilla cells. Minoxidil (1 µM) served as a positive control. Data are represented as the mean ± SD (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 5. Increased hair growth in mice by amniotic fluid–derived mesenchymal stem cell overexpressing Nanog (AF-N-MSC)–derived conditioned media (AF-N-CM).

a Schematic of a process for assessing the effect of AF-N-CM on hair regeneration. b Digital images of the dorsal skin of telogen C57BL/6 mice treated with AF-CM, AF-N-CM, or minoxidil. Stages of the hair cycle were determined by comparing dorsal skin colors. c Representative hematoxylin–eosin (H&E) images of dorsal skin sections for determination of hair follicle (HF) number and cycle. Scale bar, 200 µm. d HFs in 0.36 cm². e H&E-stained dorsal skin section 5 days post-depilation. Scale bar, 600 µm. Data are represented as the mean ± SD (n = 3). **p < 0.01

Fig. 6. Histological analysis of amniotic fluid–derived mesenchymal stem cell overexpressing Nanog (AF-N-MSC)–derived conditioned media (AF-N-CM)-treated dorsal skin.

a Expression patterns of the structural indicators at 5 days post-treatment: AP for hair follicle dermal papilla cell population (top) and CK15 for bulge stem cells (bottom). Quantification of the AP-positive dermal papilla region (right). Scale bar, 200 µm. b Quantification of the hair follicle dermal papilla region area (μm²) determined by ImageJ. c Expression of hair growth markers (ALP, LEF1, and Versican) in AF-CM, AF-N-CM, and minoxidil treatments, as determined by quantitative real-time reverse transcriptase–polymerase chain reaction. d Protein levels of ALP, LEF1, and Versican in AF-CM, AF-N-CM, and minoxidil treatments, as determined by western blot. Data are represented as the mean ± SD (n = 3). *p < 0.05, **p < 0.01