HaCaT‑conditioned medium supplemented with the small molecule inhibitors SB431542 and CHIR99021 and the growth factor PDGF‑AA prevents the dedifferentiation of dermal papilla cells <em>in vitro</em>

Sun,Dongjie; Huang,Zhehao; Xu,Jinying; Wang,Yiqing; Chen,Lin; Hou,Yi; Chi,Guangfan

doi:10.3892/mmr.2021.11965

May-2021 Volume 23 Issue 5

Full Size Image

Journals

International Journal of Molecular Medicine

International Journal of Molecular Medicine is an international journal devoted to molecular mechanisms of human disease.

International Journal of Oncology

International Journal of Oncology is an international journal devoted to oncology research and cancer treatment.

Molecular Medicine Reports

Covers molecular medicine topics such as pharmacology, pathology, genetics, neuroscience, infectious diseases, molecular cardiology, and molecular surgery.

Oncology Reports

Oncology Reports is an international journal devoted to fundamental and applied research in Oncology.

Experimental and Therapeutic Medicine

Experimental and Therapeutic Medicine is an international journal devoted to laboratory and clinical medicine.

Oncology Letters

Oncology Letters is an international journal devoted to Experimental and Clinical Oncology.

Biomedical Reports

Explores a wide range of biological and medical fields, including pharmacology, genetics, microbiology, neuroscience, and molecular cardiology.

Molecular and Clinical Oncology

International journal addressing all aspects of oncology research, from tumorigenesis and oncogenes to chemotherapy and metastasis.

World Academy of Sciences Journal

Multidisciplinary open-access journal spanning biochemistry, genetics, neuroscience, environmental health, and synthetic biology.

International Journal of Functional Nutrition

Open-access journal combining biochemistry, pharmacology, immunology, and genetics to advance health through functional nutrition.

International Journal of Epigenetics

Publishes open-access research on using epigenetics to advance understanding and treatment of human disease.

Medicine International

An International Open Access Journal Devoted to General Medicine.

May-2021 Volume 23 Issue 5

Full Size Image

Article Open Access

HaCaT‑conditioned medium supplemented with the small molecule inhibitors SB431542 and CHIR99021 and the growth factor PDGF‑AA prevents the dedifferentiation of dermal papilla cells in vitro

Authors:
- Dongjie Sun
- Zhehao Huang
- Jinying Xu
- Yiqing Wang
- Lin Chen
- Yi Hou
- Guangfan Chi
View Affiliations / Copyright

Affiliations: The Key Laboratory of Pathobiology, Ministry of Education, College of Basic Medical Sciences, Jilin University, Changchun, Jilin 130000, P.R. China, Department of Neurosurgery, China‑Japan Union Hospital of Jilin University, Changchun, Jilin 130000, P.R. China, Department of Genetics, Basic Medical College of Jilin University, Changchun, Jilin 130000, P.R. China, Department of Regeneration Medicine, School of Pharmaceutical Science of Jilin University, Changchun, Jilin 130000, P.R. China

Copyright: © Sun et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Article Number: 326
|
Published online on: March 8, 2021

https://doi.org/10.3892/mmr.2021.11965
Expand metrics +

Abstract

Hair loss, including alopecia, is a common and distressing problem for men and women, and as a result, there is considerable interest in developing treatments that can prevent or reverse hair loss. Dermal papillae closely interact with epidermal cells and play a key role during hair follicle induction and hair morphogenesis. As dermal papilla cells (DPCs) lose their hair‑inducing ability in monolayer cultures in vitro, it is difficult to obtain de novo hair follicle structures following DPC transplantation in vivo. The present study aimed to explore culture conditions to maintain DPC characteristics using conditioned media (CM) from the supernatant of cultured HaCaT keratinocyte cells supplemented with other components. Initially, it was observed that during passaging of in vitro monolayer DPC cultures, the Wnt/β‑catenin pathway was repressed, while the TGF‑β/Smad pathway was activated, and that HaCaT cells cultivated in 1% fetal bovine serum had higher levels of expression of Wnt3a and Wnt10b compared with normal keratinocytes. Culturing of high‑passage (P7) DPCs in CM from HaCaT cells (HaCaT‑CM) actively stimulated cell proliferation and maintained Sox2 and Versican expression levels. Supplementation of HaCaT‑CM with SB431542 (SB, a TGF‑β receptor inhibitor), CHIR99021, (CHIR, a GSK3α/β inhibitor and activator of Wnt signaling) and platelet‑derived growth factor (PDGF)‑AA further increased the expression levels of Sox2, Versican and alkaline phosphatase (ALP) in P7 DPCs. Three‑dimensional culture of P7 DPCs using hanging drop cultures in HaCaT‑CM supplemented with SB, CHIR and PDGF‑AA resulted in larger cell aggregates and a further significant upregulation of Sox2, ALP and Versican expression levels. Taken together, these findings demonstrated that HaCaT‑CM supplemented with SB, CHIR and PDGF‑AA may preserve the hair‑inducing ability of high‑passage DPCs and may therefore be useful in reconstructing new hair follicles in vivo.

View Figures

View References

1	Xiao SE, Miao Y, Wang J, Jiang W, Fan ZX, Liu XM and Hu ZQ: As a carrier-transporter for hair follicle reconstitution, platelet-rich plasma promotes proliferation and induction of mouse dermal papilla cells. Sci Rep. 7:11252017. View Article : Google Scholar : PubMed/NCBI
2	Ohyama M and Veraitch O: Strategies to enhance epithelial-mesenchymal interactions for human hair follicle bioengineering. J Dermatol Sci. 70:78–87. 2013. View Article : Google Scholar : PubMed/NCBI
3	Biernaskie J, Paris M, Morozova O, Fagan BM, Marra M, Pevny L and Miller FD: SKPs derive from hair follicle precursors and exhibit properties of adult dermal stem cells. Cell Stem Cell. 5:610–623. 2009. View Article : Google Scholar : PubMed/NCBI
4	Wu JJ, Zhu TY, Lu YG, Liu RQ, Mai Y, Cheng B, Lu ZF, Zhong BY and Tang SQ: Hair follicle reformation induced by dermal papilla cells from human scalp skin. Arch Dermatol Res. 298:183–190. 2006. View Article : Google Scholar : PubMed/NCBI
5	Ohyama M, Kobayashi T, Sasaki T, Shimizu A and Amagai M: Restoration of the intrinsic properties of human dermal papilla in vitro. J Cell Sci. 125:4114–4125. 2012. View Article : Google Scholar : PubMed/NCBI
6	Higgins CA, Chen JC, Cerise JE, Jahoda CAB and Christiano AM: Microenvironmental reprogramming by three-dimensional culture enables dermal papilla cells to induce de novo human hair-follicle growth. Proc Natl Acad Sci USA. 110:19679–19688. 2013. View Article : Google Scholar : PubMed/NCBI
7	Driskell RR, Clavel C, Rendl M and Watt FM: Hair follicle dermal papilla cells at a glance. J Cell Sci. 124:1179–1182. 2011. View Article : Google Scholar : PubMed/NCBI
8	Yang CC and Cotsarelis G: Review of hair follicle dermal cells. J Dermatol Sci. 57:2–11. 2010. View Article : Google Scholar : PubMed/NCBI
9	Kishimoto J, Ehama R, Wu L, Jiang S, Jiang N and Burgeson RE: Selective activation of the versican promoter by epithelial- mesenchymal interactions during hair follicle development. Proc Natl Acad Sci USA. 96:7336–7341. 1999. View Article : Google Scholar : PubMed/NCBI
10	Millar SE: Molecular mechanisms regulating hair follicle development. J Invest Dermatol. 118:216–225. 2002. View Article : Google Scholar : PubMed/NCBI
11	Ouji Y, Nakamura-Uchiyama F and Yoshikawa M: Canonical Wnts, specifically Wnt-10b, show ability to maintain dermal papilla cells. Biochem Biophys Res Commun. 438:493–499. 2013. View Article : Google Scholar : PubMed/NCBI
12	Rezza A, Sennett R, Tanguy M, Clavel C and Rendl M: PDGF signalling in the dermis and in dermal condensates is dispensable for hair follicle induction and formation. Exp Dermatol. 24:468–470. 2015. View Article : Google Scholar : PubMed/NCBI
13	Kiso M, Hamazaki TS, Itoh M, Kikuchi S, Nakagawa H and Okochi H: Synergistic effect of PDGF and FGF2 for cell proliferation and hair inductive activity in murine vibrissal dermal papilla in vitro. J Dermatol Sci. 79:110–118. 2015. View Article : Google Scholar : PubMed/NCBI
14	Gemayel R and Chenette EJ: β-catenin signalling in dermal papilla cells leads to a hairy situation. FEBS J. 283:2820–2822. 2016. View Article : Google Scholar : PubMed/NCBI
15	Soma T, Fujiwara S, Shirakata Y, Hashimoto K and Kishimoto J: Hair-inducing ability of human dermal papilla cells cultured under Wnt/β-catenin signalling activation. Exp Dermatol. 21:307–309. 2012. View Article : Google Scholar : PubMed/NCBI
16	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
17	Moore GP, Du Cros DL, Isaacs K, Pisansarakit P and Wynn PC: Hair growth induction: roles of growth factors. Ann N Y Acad Sci. 642:308–325. 1991. View Article : Google Scholar : PubMed/NCBI
18	Inamatsu M, Matsuzaki T, Iwanari H and Yoshizato K: Establishment of rat dermal papilla cell lines that sustain the potency to induce hair follicles from afollicular skin. J Invest Dermatol. 111:767–775. 1998. View Article : Google Scholar : PubMed/NCBI
19	Schürer N, Köhne A, Schliep V, Barlag K and Goerz G: Lipid composition and synthesis of HaCaT cells, an immortalized human keratinocyte line, in comparison with normal human adult keratinocytes. Exp Dermatol. 2:179–185. 1993. View Article : Google Scholar : PubMed/NCBI
20	Dong L, Hao H, Liu J, Tong C, Ti D, Chen D, Chen L, Li M, Liu H, Fu X, et al: Wnt1a maintains characteristics of dermal papilla cells that induce mouse hair regeneration in a 3D preculture system. J Tissue Eng Regen Med. 11:1479–1489. 2017. View Article : Google Scholar : PubMed/NCBI
21	Shimizu H and Morgan BA: Wnt signaling through the β-catenin pathway is sufficient to maintain, but not restore, anagen-phase characteristics of dermal papilla cells. J Invest Dermatol. 122:239–245. 2004. View Article : Google Scholar : PubMed/NCBI
22	Kishimoto J, Burgeson RE and Morgan BA: Wnt signaling maintains the hair-inducing activity of the dermal papilla. Genes Dev. 14:1181–1185. 2000.PubMed/NCBI
23	Fujimori K, Matsumoto T, Kisa F, Hattori N, Okano H and Akamatsu W: Escape from pluripotency via inhibition of TGF-β/BMP and activation of Wnt signaling accelerates differentiation and aging in hPSC progeny cells. Stem Cell Reports. 9:1675–1691. 2017. View Article : Google Scholar : PubMed/NCBI
24	Yoshida Y, Soma T, Matsuzaki T and Kishimoto J: Wnt activator CHIR99021-stimulated human dermal papilla spheroids contribute to hair follicle formation and production of reconstituted follicle-enriched human skin. Biochem Biophys Res Commun. 516:599–605. 2019. View Article : Google Scholar : PubMed/NCBI
25	Lu J, Liu H, Huang CT, Chen H, Du Z, Liu Y, Sherafat MA and Zhang SC: Generation of integration-free and region-specific neural progenitors from primate fibroblasts. Cell Rep. 3:1580–1591. 2013. View Article : Google Scholar : PubMed/NCBI
26	Osada A, Iwabuchi T, Kishimoto J, Hamazaki TS and Okochi H: Long-term culture of mouse vibrissal dermal papilla cells and de novo hair follicle induction. Tissue Eng. 13:975–982. 2007. View Article : Google Scholar : PubMed/NCBI
27	Bin S, Li HD, Xu YB, Qi SH, Li TZ, Liu XS, Tang JM and Xie JL: BMP-7 attenuates TGF-β1-induced fibroblast-like differentiation of rat dermal papilla cells. Wound Repair Regen. 21:275–281. 2013. View Article : Google Scholar : PubMed/NCBI
28	Hou-dong L, Bin S, Ying-bin X, Yan S, Shao-hai Q, Tian-zeng L, Xu-sheng L, Jin-ming T and Ju-lin X: Differentiation of rat dermal papilla cells into fibroblast-like cells induced by transforming growth factor β1. J Cutan Med Surg. 16:400–406. 2012. View Article : Google Scholar : PubMed/NCBI
29	Zheng Y, Zhao YD, Gibbons M, Abramova T, Chu PY, Ash JD, Cunningham JM and Skapek SX: Tgfbeta signaling directly induces Arf promoter remodeling by a mechanism involving Smads 2/3 and p38 MAPK. J Biol Chem. 285:35654–35664. 2010. View Article : Google Scholar : PubMed/NCBI
30	Lebaron RG: Versican. Perspect Dev Neurobiol. 3:261–271. 1996.PubMed/NCBI
31	Huang YC, Chan CC, Lin WT, Chiu HY, Tsai RY, Tsai TH, Chan JY and Lin SJ: Scalable production of controllable dermal papilla spheroids on PVA surfaces and the effects of spheroid size on hair follicle regeneration. Biomaterials. 34:442–451. 2013. View Article : Google Scholar : PubMed/NCBI
32	Tan JJY, Common JE, Wu C, Ho PCL and Kang L: Keratinocytes maintain compartmentalization between dermal papilla and fibroblasts in 3D heterotypic tri-cultures. Cell Prolif. 52:e126682019. View Article : Google Scholar : PubMed/NCBI
33	Chi W, Wu E and Morgan BA: Dermal papilla cell number specifies hair size, shape and cycling and its reduction causes follicular decline. Development. 140:1676–1683. 2013. View Article : Google Scholar : PubMed/NCBI
34	Elliott K, Stephenson TJ and Messenger AG: Differences in hair follicle dermal papilla volume are due to extracellular matrix volume and cell number: Implications for the control of hair follicle size and androgen responses. J Invest Dermatol. 113:873–877. 1999. View Article : Google Scholar : PubMed/NCBI

Journals

International Journal of Molecular Medicine

International Journal of Oncology

Molecular Medicine Reports

Oncology Reports

Experimental and Therapeutic Medicine

Oncology Letters

Biomedical Reports

Molecular and Clinical Oncology

World Academy of Sciences Journal

International Journal of Functional Nutrition

International Journal of Epigenetics

Medicine International

HaCaT‑conditioned medium supplemented with the small molecule inhibitors SB431542 and CHIR99021 and the growth factor PDGF‑AA prevents the dedifferentiation of dermal papilla cells in vitro

This article is mentioned in:

Abstract

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Related Articles