The Science of Nature

, 103:62

Generation of functional hepatocyte-like cells from human deciduous periodontal ligament stem cells

  • Punitha Vasanthan
  • Pukana Jayaraman
  • Wijenthiran Kunasekaran
  • Anthony Lawrence
  • Nareshwaran Gnanasegaran
  • Vijayendran Govindasamy
  • Sabri Musa
  • Noor Hayaty Abu Kasim
Original Paper

Abstract

Human deciduous periodontal ligament stem cells have been introduced for as an easily accessible source of stem cells from dental origin. Although recent studies have revealed the ability of these stem cells in multipotential attribute, their efficiency of hepatic lineage differentiation has not been addressed so far. The aim of this study is to investigate hepatic lineage fate competence of periodontal ligament stem cells through direct media induction. Differentiation of periodontal ligament stem cells into hepatocyte-like cells was conducted by the exposure of two phase media induction. First phase was performed in the presence of hepatocyte growth factors to induce a definitive endoderm formation. In the subsequent phase, the cells were treated with oncostatin M and dexamethosone followed by insulin and transferrin to generate hepatocyte-like cells. Hepatic-related characters of the generated hepatocyte-like cells were determined at both mRNA and protein level followed by functional assays. Foremost changes observed in the generation of hepatocyte-like cells were the morphological features in which these cells were transformed from fibroblastic shape to polygonal shape. Temporal expression of hepatic markers ranging from early endodermal up to late markers were detected in the hepatocyte-like cells. Crucial hepatic markers such as glycogen storage, albumin, and urea secretion were also shown. These findings exhibited the ability of periodontal ligament stem cells of dental origin to be directed into hepatic lineage fate. These cells can be regarded as an alternative autologous source in the usage of stem cell-based treatment for liver diseases.

Keywords

Cell differentiation Dental stem cells Periodontal ligament 

Supplementary material

114_2016_1387_MOESM1_ESM.tif (1.8 mb)
ESM 1Supplementary Figure 1. Bona fide MSCs features in isolated PDLSCs. A) Morphologically PDLSCs culture appeared spindle shaped (Scale bar = 100μm). B and C) Growth curve and population doubling time from P1 to P5 of respective cell culture (Scale bar = 200μm). D) Formation of colonies shown for PDLSCs. E) Percentage of senescent cells in PDLSCs. F) The panels display representative of visual observation (Scale bar = 200μm) of adipocytes detected by Oil Red O staining, chondrocytes detected by Alcian Blue staining and osteocytes detected by Von Kossa staining. Representative six independent result. (TIF 1866 kb)
114_2016_1387_MOESM2_ESM.tif (857 kb)
ESM 2Supplementary Figure 2. Immunophenotyping of mesenchymal stem cells surface markers. Isolated PDLSCs shown to be positively stained the gold standard of mesenchymal stem cells surface markers and absence of hematopoetic surface markers. (TIF 857 kb)

References

  1. Alison MR, Vig P, Russo F et al (2004) Hepatic stem cells: from inside and outside liver? Cell Prolif 37:1–21. doi:10.1111/j.1365-2184.2004.00297.x CrossRefPubMedGoogle Scholar
  2. Behbahan IS, Duan Y, Lam A et al (2011) New approaches in the differentiation of human embryonic stem cells and induced pluripotent stem cells toward hepatocytes. Stem Cell Rev 7:748–759. doi:10.1007/s12015-010-9216-4 CrossRefPubMedPubMedCentralGoogle Scholar
  3. Campard D, Lysy PA, Najimi M, Sokal EM (2008) Native umbilical cord matrix stem cells express heaptic markers and differentiate into hepatocyte-like cells. Gastroenterology 134:833–848. doi:10.1053/j.gastro.2007.12.024 CrossRefPubMedGoogle Scholar
  4. Cicchini C, Amicone L, Alonzi T, Marchetti A, Mancone C, Tripodi M (2014) Molecular mechanisms controlling the phenotype and the EMT/MET dynamics of hepatocyte. Liver Int 35:302–310. doi:10.1111/liv.12577 CrossRefPubMedPubMedCentralGoogle Scholar
  5. Dhawan A, Mitry RR, Hughes RD et al (2004) Hepatocyte transplantation for inherited factor VII deficiency. Transplantation 78:1812–1814. doi:10.1097/01.TP.0000146386.77076.47 CrossRefPubMedGoogle Scholar
  6. Donato MT, Lahoz A, Montero S et al (2008) Functional assessment of the quality of human hepatocyte preparations for cell transplantation. Cell Transplant 17:1211–1219. doi:10.3727/096368908787236620 CrossRefPubMedGoogle Scholar
  7. Fawcett JK, Scott JE (1960) A rapid and precise method for the determination of urea. J Clin Pathol 13:156–159. doi:10.1136/jcp.13.2.156 CrossRefPubMedPubMedCentralGoogle Scholar
  8. Gomez-Lechon MJ, Donato MT, Castell JV, Jover R (2004) Human hepatocyte in primary culture: the choice to investigate drug metabolism in man. Curr Drug Metab 5:443–462. doi:10.2174/1389200043335414 CrossRefPubMedGoogle Scholar
  9. Govindasamy V, Ronald VS, Abdullah AN et al (2011) Differentiation of dental pulp stem cells into islet-like aggregates. J Dent Res 90:646–652. doi:10.1177/0022034510396879 CrossRefPubMedGoogle Scholar
  10. Huang L, Liang J, Geng Y et al (2013) Directing adult human periodontal ligament –derived stem cells to retinal fate. Invest Ophthalmol Vis Sci 54:3965–3974. doi:10.1167/iovs.13-11910 CrossRefPubMedGoogle Scholar
  11. Ikeda E, Yagi K, Kojima M, Yagyuu T, Ohshima A, Sobajima S, Tadokoro M, Katsube Y, Isoda K, Kondoh M, Kawase M, Go MJ, Adachi H, Yokota Y, Kirita T, Ohgushi H (2008) Multipotent cells from the human third molar: feasibility of cell-based therapy for liver disease. Differentiation 76:495–505. doi:10.1111/j.1432-0436.2007.00245.x CrossRefPubMedGoogle Scholar
  12. Ishkitiev N, Yaegaki K, Calenic B et al (2010) Deciduous and permanent dental pulp mesenchymal stem cells acquire hepatic morphology and functional features in vitro. J Endod 36:469–474. doi:10.1016/j.joen.2009.12.022 CrossRefPubMedGoogle Scholar
  13. Ishkitiev N, Yaegaki K, Imai T, Tanaka T, Nakahara T, Ishikawa H, Mitev V, Haapasalo M (2012) High-purity hepatic lineage differentiated from dental pulp stem cells in serum-free medium. J Endod 38:475–480. doi:10.1016/j.joen.2011.12.011 CrossRefPubMedGoogle Scholar
  14. Ishkitiev N, Yaegaki K, Imai T, Tanaka T, Fushimi N, Mitev V, Okada M, Tominaga N, Ono S, Ishikawa H (2015) Novel management of acute or secondary biliary liver conditions using hepatically differentiated human dental pulp cells. Tissue Eng Part A 21:586–593. doi:10.1089/ten.TEA.2014.0162 CrossRefPubMedGoogle Scholar
  15. Kabir R, Gupta M, Aggarwal A, Sharma D, Sarin A, Kola MZ (2014) Imperative role of dental pulp stem cells in regenerative therapies: a systematic review. Niger J Surg 20(1):1–8. doi:10.4103/1117-6806.127092 PubMedPubMedCentralGoogle Scholar
  16. Kalsheker N, Morley S, Morgan K (2002) Gene regulation of the serine proteinase inhibitor alpha1-antitrypsin and alpha1-antichymotrypsin. Biochem Soc Trans 30:93–98. doi:10.1042/bst0300093 CrossRefPubMedGoogle Scholar
  17. Kawanabe N, Murata S, Murakami K, Ishihara Y, Hayano S, Kurosaka H, Kamioka H, Takano-Yamamoto T, Yamashiro T (2010) Isolation of multipotent stem cells in human periodontal ligament using stage-specific embryonic antigen-4. Differentiation 79(2):74–83. doi:10.1016/j.diff.2009.10.005 CrossRefPubMedGoogle Scholar
  18. Lee JS, An SY, Kwon IK, Heo JS (2014) Transdifferentiation of human periodontal ligament stem cells into pancreatic cell lineage. Cell Biochem Funct 32:605–611. doi:10.1002/cbf.3057 CrossRefPubMedGoogle Scholar
  19. Lemaigre FP (2009) Mechanisms of liver development: concepts for understanding liver disorders and design of novel therapies. Gastroenterology 137:62–67. doi:10.1053/j.gastro.2009.03.035 CrossRefPubMedGoogle Scholar
  20. Loranger A, Duclos S (1997) Simple epithelium keratins are required for maintenance of hepatocyte integrity. Am J Pathol 151:1673–1683PubMedPubMedCentralGoogle Scholar
  21. Muraca M, Gerunda G, Neri D et al (2002) Hepatocyte transplantation as a treatment for glycogen storage disease type 1a. Lancet 359:317–318. doi:10.1016/S0140-6736(02)07529-3 CrossRefPubMedGoogle Scholar
  22. Osman A, Gnanasegaran N, Govindasamy V et al (2014) Basal expression of growth-factor-associated genes in periodontal ligament stem cells reveals multiple distinctive pathways. Int Endod J 47:639–651. doi:10.1111/iej.12200 CrossRefPubMedGoogle Scholar
  23. Peleman RR, Gavaler JS, Van Theil DH et al (1987) Orthotopic liver transplantation for acute and subacute hepatic failure in adults. Hepatology 7:484–489. doi:10.1002/hep.1840070312 CrossRefPubMedPubMedCentralGoogle Scholar
  24. Pournasr B, Mohamadnejad M, Aghdami N, Shahsavani M, Malekzadeh R, Baharvand H (2011) In vitro differentiation of human bone marrow mesenchymal stem cells into hepatocyte-like cells. Arch Iran Med 14:244–249. doi:0011144/AIM.004PubMedGoogle Scholar
  25. Rossi JM, Dunn NR, Hogan BL, Zaret KS (2001) Distinct mesodermal signals, including BMPs from septum transversum mesenchyme, are required in combination for hepatogenesis from the endoderm. Genes Dev 15:1998–2009. doi:10.1101/gad.904601 CrossRefPubMedPubMedCentralGoogle Scholar
  26. Sassa S, Sugita S, Galbraith RA, Kappas A (1987) Drug metabolism of human hepatoma cell, HepG2. Biochem Biophys Res Commun 25:52–57. doi:10.1016/0006-291X(87)90628-0 CrossRefGoogle Scholar
  27. Seo BM, Miura M, Gronthos S et al (2004) Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 364:149–155. doi:10.1016/S0140-6736(04)16627-0 CrossRefPubMedGoogle Scholar
  28. Seo BM, Miura M, Sonoyama W, Coppe C, Stanyon R, Shi S (2005) Recovery of stem cells from cryopreserved periodontal ligament. J Dent Res 84(10):907–912. doi:10.1177/154405910508401007 CrossRefPubMedGoogle Scholar
  29. Shelly LL, Tynan W, Schwid W, Schutz G, Yeoh GC (1989) Hepatocyte differentiation in vitro: initiation of tyrosine aminotransferase expression in cultured fetal rat hepatocytes. J Cell Biol 109:3403–3410. doi:10.1083/jcb.109.6.3403 CrossRefPubMedGoogle Scholar
  30. Silvério KG, Rodrigues TL, Coletta RD, Benevides L, Da Silva JS, Casati MZ, Sallum EA, Nociti FH Jr (2010) Mesenchymal stem cell properties of periodontal ligament cells from deciduous and permanent teeth. J Periodontol 81(8):1207–1215. doi:10.1902/jop.2010.090729 CrossRefPubMedGoogle Scholar
  31. Song JS, Kim SO, Kim SH, Choi HJ, Son HK, Jung HS, Kim CS, Lee JH (2012) In vitro and in vivo characteristics of stem cells derived from the periodontal ligament of human deciduous and permanent teeth. Tissue Eng Part A 18(19-20):2040–2051. doi:10.1089/ten.TEA.2011.0318 CrossRefPubMedGoogle Scholar
  32. Soto-Guiterrez A, Basma H, Navarro-Alvarez N et al (2008) Differentiating stem cells into liver. Biotechnol Genet Eng Rev 25:149–163. doi:10.5661/bger-25-149 CrossRefGoogle Scholar
  33. Stephenne X, Najimi M, Sibille C, Nassogne MC, Smets F, Sokal EM (2006) Sustained engraftment and tissue enzyme activity after liver cell transplantation for argininosuccinate lyase deficiency. Gastroenterology 130:1317–1323. doi:10.1053/j.gastro.2006.01.008 CrossRefPubMedGoogle Scholar
  34. Tomokiyo A, Maeda H, Fujii S, Wada N, Shima K, Akamine A (2008) Development of a multipotent clonal human periodontal ligament cell line. Differentiation 76:337–347. doi:10.1111/j.1432-0436.2007.00233.x CrossRefPubMedGoogle Scholar
  35. Vasanthan P, Gnanasegaran N, Govindasamy V et al (2014) Comparison of fetal bovine serum and human platelet lysate in cultivation and differentiation of dental pulp stem cells into hepatic lineage cells. Biochem Eng J 88:142–153. doi:10.1016/j.bej.2014.04.007 CrossRefGoogle Scholar
  36. Watt AJ, Garrison WD, Duncan SA (2003) HNF4: a central regulator of hepatocyte differentiation and function. Hepatology 37:1249–1253. doi:10.1053/jhep.2003.50273 CrossRefPubMedGoogle Scholar
  37. Wu XB, Tao R (2012) Hepatocyte differentiation of mesenchymal stem cells. Hepatob Pancreat Dis 11:360–371. doi:10.1016/S1499-3872(12)60193-3 CrossRefGoogle Scholar
  38. Yin L, Zhu Y, Ni Y, Zhou Z, Chen Y, Wen L (2015) Adipose tissue-derived mesenchymal stem cells differentiated into hepatocyte like cells in vivo and in vitro. Mol Med Rep 11:1722–1732. doi:10.3892/mmr.2014.2935 PubMedGoogle Scholar
  39. Zhu W, Liang M (2015) Periodontal ligament stem cells: current status, concerns, and future prospects. Stem Cells Int 2015:972313. doi:10.1155/2015/972313 PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  1. 1.Department of Peadiatric Dentistry and Orthodontics, Faculty of DentistryUniversity of MalayaKuala LumpurMalaysia
  2. 2.Department of Restorative Dentistry, Faculty of DentistryUniversity of MalayaKuala LumpurMalaysia
  3. 3.cGMP compliance Stem Cells LaboratoryHygieia Innovation Sdn Bhd, Lot 1G-2G, Lanai Complex No.2, Persiaran Seri PerdanaPutrajayaMalaysia

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