Cell and Tissue Research

, Volume 366, Issue 1, pp 89–99 | Cite as

Differentiation of hepatocytes from induced pluripotent stem cells derived from human hair follicle mesenchymal stem cells

  • Xu Shi
  • Shuang Lv
  • Xia He
  • Xiaomei Liu
  • Meiyu Sun
  • Meiying Li
  • Guangfan Chi
  • Yulin LiEmail author
Regular Article


Due to the limitations of organ donors and immune rejection in severe liver diseases, stem cell-based therapy presents a promising application for tissue repair and regeneration. As a novel cell source, mesenchymal stem cells separated from human hair follicles (HF-MSCs) are convenient to obtain and have no age limit. To date, the differentiation of HF-MSCs into hepatocytes has not been reported. In this study, we explored whether HF-MSCs and HF-MSC-derived-induced pluripotent stem cells (HF-iPS) could differentiate into hepatocytes in vitro. Flow cytometry, Oil Red O stain and Alizarin Red stain were used to identify the characteristics of HF-MSCs. The expression of liver-specific gene was detected by immunofluorescence and Quantitative Polymerase Chain Reaction. Periodic Acid-Schiff stain, Indocyanine Green stain and Low-Density Lipoprotein stain were performed to evaluate the functions of induced hepatocyte-like cells (HLCs). HF-MSCs were unable to differentiate into HLCs using previously reported procedures for MSCs from other tissues. However, HF-iPS efficiently induced the generation of HLCs that expressed hepatocyte markers and drug metabolism-related genes. HF-iPS can be used as novel and alternative cellular tools for inducing hepatocytes in vitro, simultaneously benefiting from utilizing HF-MSCs as a noninvasive and convenient cell source for reprogramming.


Induced pluripotent stem cells Hair follicle mesenchymal stem cells Hepatocytes Multipotential differentiation End-stage liver disease 



We thank LetPub for its linguistic assistance during the preparation of this manuscript.

Compliance with ethical standards

Conflict of interest

None of the authors has any conflict of interest related to this manuscript.

Supplementary material

441_2016_2399_MOESM4_ESM.docx (16 kb)
ESM 1 (DOCX 15 kb)
441_2016_2399_Fig7_ESM.gif (125 kb)
Figure S1

Identification of HF-MSCs. ac Morphology characterization. A typical fibroblast-like cell shape was observed in the undifferentiated HF-MSCs. Adipogenic differentiation was detected by Oil Red O staining. The tiny red dots represent intracellular lipid droplets. Osteogenic differentiation was detected by Alizarin Red staining. A red color indicates the formation of calcium nodules. Scale bars  20 μm. dj Flow cytometric analyses of cell surface markers on HF-MSCs. A tiotal 2 × 105 HF-MSCs were incubated with primary antibodies against different biomarkers. Controls were incubated with secondary antibody only. % indicates the fraction of cells that stained positive. (GIF 124 kb)

441_2016_2399_MOESM1_ESM.tif (4.2 mb)
High resolution image (TIF 4284 kb)
441_2016_2399_Fig8_ESM.gif (139 kb)
Figure S2

Immunofluorescence analysis of DE markers in HF-MSC-derived iPS (HF-iPS). a–i Undifferentiated HF-iPS showed negative staining for DE markers—SOX17, GATA4 and FoxA2. Scale bars 50 μm. (GIF 139 kb)

441_2016_2399_MOESM2_ESM.tif (4.3 mb)
High resolution image (TIF 4416 kb)
441_2016_2399_Fig9_ESM.gif (33 kb)
Figure S3

Identification of hepatic functions in HF-MSC-derived iPS (HF-iPS). Transport and metabolic functions, as well as glycogen storage were detected in undifferentiated HF-iPS cells. ac The detection of LDL uptake in undifferentiated HF-iPS. Nuclei were stained with DAPI (blue). Scale bars 50 μm. d, e PAS staining and ICG uptake analysis in HF-iPS. Scale bars 50 μm. (GIF 33 kb)

441_2016_2399_MOESM3_ESM.tif (1.2 mb)
High resolution image (TIF 1192 kb)


  1. Banas A, Teratani T, Yamamoto Y, Tokuhara M, Takeshita F, Quinn G, Okochi H, Ochiya T (2007) Adipose tissue-derived mesenchymal stem cells as a source of human hepatocytes. Hepatology 46:219–228CrossRefPubMedGoogle Scholar
  2. Becerra J, Santos-Ruiz L, Andrades JA, Marí-Beffa M (2011) The stem cell niche should be a key issue for cell therapy in regenerative medicine. Stem Cell Rev 7(2):248–255CrossRefPubMedGoogle Scholar
  3. Bernal W, Auzinger G, Dhawan A, Wendon J (2010) Acute liver failure. Lancet 376:190–201CrossRefPubMedGoogle Scholar
  4. Bernuau J, Rueff B, Benhamou JP (1986) Fulminant and subfulminant liver failure: definitions and causes. Semin Liver Dis 6:97–106CrossRefPubMedGoogle Scholar
  5. Beuers U, Trauner M, Jansen P, Poupon R (2015) New paradigms in the treatment of hepatic cholestasis: from UDCA to FXR, PXR and beyond. J Hepatol 62:S25–37CrossRefPubMedGoogle Scholar
  6. Bigildeev AE, Zezina EA, Shipounova IN, Drize NJ (2015) Interleukin-1 beta enhances human multipotent mesenchymal stromal cell proliferative potential and their ability to maintain hematopoietic precursor cells. Cytokine 71(2):246–254CrossRefPubMedGoogle Scholar
  7. Campard D, Lysy PA, Najimi M, Sokal EM (2008) Native umbilical cord matrix stem cells express hepatic markers and differentiate into hepatocyte-like cells. Gastroenterology 134:833–848CrossRefPubMedGoogle Scholar
  8. Chen YF, Tseng CY, Wang HW, Kuo HC, Yang VW, Lee OK (2012) Rapid generation of mature hepatocyte-like cells from human induced pluripotent stem cells by an efficient three-step protocol. Hepatology 55(4):1193–1203CrossRefPubMedPubMedCentralGoogle Scholar
  9. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop DJ, Horwitz E (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8(4):315–317CrossRefPubMedGoogle Scholar
  10. Eminli S, Utikal J, Arnold K, Jaenisch R, Hochedlinger K (2008) Reprogramming of neural progenitor cells into induced pluripotent stem cells in the absence of exogenous Sox2 expression. Stem Cells 26:2467–2474CrossRefPubMedGoogle Scholar
  11. Farci P, Alter HJ, Shimoda A, Govindarajan S, Cheung LC, Melpolder JC, Sacher RA, Shih JW, Purcell RH (1996) Hepatitis C virus-associated fulminant hepatic failure. N Engl J Med 335:631–634CrossRefPubMedGoogle Scholar
  12. Gao Y, Liu F, Zhang L, Su X, Liu JY, Li Y (2014) Acellular blood vessels combined human hair follicle mesenchymal stem cells for engineering of functional arterial grafts. Ann Biomed Eng 42(10):2177–2189CrossRefPubMedGoogle Scholar
  13. Hashimoto H, Toide K, Kitamura R, Fujita M, Tagawa S, Itoh S, Kamataki T (1993) Gene structure of CYP3A4, an adult-specific form of cytochrome P450 in human livers, and its transcriptional control. Eur J Biochem 218:585–595CrossRefPubMedGoogle Scholar
  14. Hayashi S, Watanabe J, Kawajiri K (1991) Genetic polymorphisms in the 5′-flanking region change transcriptional regulation of the human cytochrome P450IIE1 gene. J Biochem 110(4):559–565PubMedGoogle Scholar
  15. Herencia C, Martínez-Moreno JM, Herrera C, Corrales F, Santiago-Mora R, Espejo I, Barco M, Almadén Y, de la Mata M, Rodríguez-Ariza A, Muñoz-Castañeda JR (2012) Nuclear translocation of β-catenin during mesenchymal stem cells differentiation into hepatocytes is associated with a tumoral phenotype. PLoS ONE 7(4), e34656. doi: 10.1371/journal.pone.0034656 CrossRefPubMedPubMedCentralGoogle Scholar
  16. Higgins CA, Itoh M, Inoue K, Richardson GD, Jahoda CA, Christiano AM (2012) Reprogramming of human hair follicle dermal papilla cells into induced pluripotent stem cells. J Invest Dermatol 132:1725–1727CrossRefPubMedGoogle Scholar
  17. Ishkitiev N, Yaegaki K, Calenic B, Nakahara T, Ishikawa H, Mitiev V, Haapasalo M (2010) Deciduous and permanent dental pulp mesenchymal cells acquire hepatic morphologic and functional features in vitro. J Endod 36:469–474CrossRefPubMedGoogle Scholar
  18. Kfoury Y, Scadden DT (2015) Mesenchymal cell contributions to the stem cell niche. Cell Stem Cell 16(3):239–253CrossRefPubMedGoogle Scholar
  19. Kim JB, Zaehres H, Wu G, Gentile L, Ko K, Sebastiano V, Araúzo-Bravo MJ, Ruau D, Han DW, Zenke M, Schöler HR (2008) Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors. Nature 454:646–650CrossRefPubMedGoogle Scholar
  20. Kim JB, Sebastiano V, Wu G, Araúzo-Bravo MJ, Sasse P, Gentile L, Ko K, Ruau D, Ehrich M, van den Boom D, Meyer J, Hübner K, Bernemann C, Ortmeier C, Zenke M, Fleischmann BK, Zaehres H, Schöler HR (2009) Oct4-induced pluripotency in adult neural stem cells. Cell 136:411–419CrossRefPubMedGoogle Scholar
  21. Kir S, Zhang Y, Gerard RD, Kliewer SA, Mangelsdorf DJ (2012) Nuclear receptors HNF4α and LRH-1 cooperate in regulating Cyp7a1 in vivo. J Biol Chem 287(49):41334–41341CrossRefPubMedPubMedCentralGoogle Scholar
  22. Kunisato A, Wakatsuki M, Kodama Y, Shinba H, Ishida I, Nagao K (2010) Generation of induced pluripotent stem cells by efficient reprogramming of adult bone marrow cells. Stem Cells Dev 19(2):229–238CrossRefPubMedGoogle Scholar
  23. Lee KD, Kuo TK, Whang-Peng J, Chung YF, Lin CT, Chou SH, Chen JR, Chen YP, Lee OK (2004) In vitro hepatic differentiation of human mesenchymal stem cells. Hepatology 40:1275–1284CrossRefPubMedGoogle Scholar
  24. Ling L, Ni Y, Wang Q, Wang H, Hao S, Hu Y, Jiang W, Hou Y (2008) Transdifferentiation of mesenchymal stem cells derived from human fetal lung to hepatocyte-like cells. Cell Biol Int 32:1091–1098CrossRefPubMedGoogle Scholar
  25. Liu X, Song L, Liu J, Wang S, Tan X, Bai X, Bai T, Wang Y, Li M, Song Y, Li Y (2013) miR-18b inhibits TGF-β1-induced differentiation of hair follicle stem cells into smooth muscle cellsby targeting SMAD2. Biochem Biophys Res Commun 438(3):551–556CrossRefPubMedGoogle Scholar
  26. Liu H, Lou G, Li C, Wang X, Cederbaum AI, Gan L, Xie B (2014) HBx inhibits CYP2E1 gene expression via downregulating HNF4α in human hepatoma cells. PLoS ONE 9(9), e107913CrossRefPubMedPubMedCentralGoogle Scholar
  27. Liu Z, Lu SJ, Lu Y, Tan X, Zhang X, Yang M, Zhang F, Li Y, Quan C (2015) Transdifferentiation of human hair follicle mesenchymal stem cells into red blood cells by OCT4. Stem Cells Int 2015:389628PubMedPubMedCentralGoogle Scholar
  28. Ma X, Duan Y, Tschudy-Seney B, Roll G, Behbahan IS, Ahuja TP, Tolstikov V, Wang C, McGee J, Khoobyari S, Nolta JA, Willenbring H, Zern MA (2014) Highly efficient differentiation of functional hepatocytes from human induced pluripotent stem cells. Stem Cells Transl Med 2(6):409–419CrossRefGoogle Scholar
  29. Majumdar MK, Keane-Moore M, Buyaner D, Hardy WB, Moorman MA, McIntosh KR, Mosca JD (2003) Characterization and functionality of cell surface molecules on human mesenchy mal stem cells. J Biomed Sci 10(2):228–241CrossRefPubMedGoogle Scholar
  30. Navarro VJ, Senior JR (2006) Drug-related hepatotoxicity. N Engl J Med 354:731–739CrossRefPubMedGoogle Scholar
  31. Obermajer N, Popp FC, Johnson CL, Benseler V, Dahlke MH (2014) Rationale and prospects of mesenchymal stem cell therapy for liver transplantation. Curr Opin Organ Transplant 19(1):60–64CrossRefPubMedGoogle Scholar
  32. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284:143–147CrossRefPubMedGoogle Scholar
  33. Si-Tayeb K, Noto FK, Nagaoka M, Li J, Battle MA, Duris C, North PE, Dalton S, Duncan SA (2010) Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology 51(1):297–305CrossRefPubMedPubMedCentralGoogle Scholar
  34. Takayama K, Inamura M, Kawabata K, Sugawara M, Kikuchi K, Higuchi M, Nagamoto Y, Watanabe H, Tashiro K, Sakurai F, Hayakawa T, Furue MK, Mizuguchi H (2012) Generation of metabolically functioning hepatocytes from human pluripotent stem cells by FOXA2 and HNF1α transduction. J Hepatol 57(3):628–636CrossRefPubMedGoogle Scholar
  35. Tsai SY, Clavel C, Kim S, Ang YS, Grisanti L, Lee DF, Kelley K, Rendl M (2010) Oct4 and klf4 reprogram dermal papilla cells into induced pluripotent stem cells. Stem Cells 28:221–228PubMedGoogle Scholar
  36. Wan YJ, An D, Cai Y, Repa JJ, Hung-Po Chen T, Flores M, Postic C, Magnuson MA, Chen J, Chien KR, French S, Mangelsdorf DJ, Sucov HM (2000) Hepatocyte-specific mutation establishes retinoid X receptor alpha as a heterodimeric integrator of multiple physiological processes in the liver. Mol Cell Biol 20(12):4436–4444CrossRefPubMedPubMedCentralGoogle Scholar
  37. Wang Y, Liu J, Tan X, Li G, Gao Y, Liu X, Zhang L, Li Y (2013) Induced pluripotent stem cells from human hair follicle mesenchymal stem cells. Stem Cell Rev 9(4):451–460CrossRefPubMedGoogle Scholar
  38. Wu C, Liu F, Li P, Zhao G, Lan S, Jiang W, Meng X, Tian L, Li G, Li Y, Liu JY (2015) Engineered hair follicle mesenchymal stem cells overexpressing controlled-release insulin reverse hyperglycemia in mice with type I diabetes. Cell Transplant 24(5):891–907CrossRefPubMedGoogle Scholar
  39. Zheng YB, Gao ZL, Xie C, Zhu HP, Peng L, Chen JH, Chong YT (2008) Characterization and hepatogenic differentiation of mesenchymal stem cells from human amniotic fluid and human bone marrow: a comparative study. Cell Biol Int 32:1439–1448CrossRefPubMedGoogle Scholar
  40. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH (2001) Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 7:211–228CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Xu Shi
    • 1
    • 2
  • Shuang Lv
    • 1
  • Xia He
    • 1
  • Xiaomei Liu
    • 1
  • Meiyu Sun
    • 1
  • Meiying Li
    • 1
  • Guangfan Chi
    • 1
  • Yulin Li
    • 1
    Email author
  1. 1.The Key Laboratory of Pathobiology, Ministry of Education, College of Basic Medical SciencesJilin UniversityChangchunChina
  2. 2.Genetic Diagnosis Center, Central LaboratoryThe First Hospital of Jilin UniversityChangchunChina

Personalised recommendations