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Hepatic differentiation from human mesenchymal stem cells on a novel nanofiber scaffold

Abstract

The emerging fields of tissue engineering and biomaterials have begun to provide potential treatment options for liver failure. The goal of the present study is to investigate the ability of a poly L-lactic acid (PLLA) nanofiber scaffold to support and enhance hepatic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs). A scaffold composed of poly L-lactic acid and collagen was fabricated by the electrospinning technique. After characterizing isolated hMSCs, they were seeded onto PLLA nanofiber scaffolds and induced to differentiate into a hepatocyte lineage. The mRNA levels and protein expression of several important hepatic genes were determined using RT-PCR, immunocytochemistry and ELISA. Flow cytometry revealed that the isolated bone marrow-derived stem cells were positive for hMSC-specific markers CD73, CD44, CD105 and CD166 and negative for hematopoietic markers CD34 and CD45. The differentiation of these stem cells into adipocytes and osteoblasts demonstrated their multipotency. Scanning electron microscopy showed adherence of cells in the nanofiber scaffold during differentiation towards hepatocytes. Our results showed that expression levels of liver-specific markers such as albumin, α-fetoprotein, and cytokeratins 8 and 18 were higher in differentiated cells on the nanofibers than when cultured on plates. Importantly, liver functioning serum proteins, albumin and α-1 antitrypsin were secreted into the culture medium at higher levels by the differentiated cells on the nanofibers than on the plates, demonstrating that our nanofibrous scaffolds promoted and enhanced hepatic differentiation under our culture conditions. Our results show that the engineered PLLA nanofibrous scaffold is a conducive matrix for the differentiation of MSCs into functional hepatocyte-like cells. This represents the first step for the use of this nanofibrous scaffold for culture and differentiation of stem cells that may be employed for tissue engineering and cell-based therapy applications.

Abbreviations

AAT:

α-1 antitrypsin

αFP:

α-fetoprotein

CK8:

cytokeratin-8

CK18:

cytokeratin-18

hMSCs:

human mesenchymal stem cells

PLLA:

poly L-lactic acid

References

  1. 1.

    Mohamadnejad, M., Alimoghaddam, K., Mohyeddin-Bonab, M., Bagheri, M., Bashtar, M., Ghanaati, H., Baharvand, H., Ghavamzadeh, A. and Malekzadeh, R. Phase 1 trial of autologous bone marrow mesenchymal stem cell transplantation in patients with decompensated liver cirrhosis. Arch. Iran Med. 10 (2007) 435–438.

    Google Scholar 

  2. 2.

    Murray, K.F. and Carithers, R.L. Jr. AASLD practice guidelines: Evaluation of the patient for liver transplantation. Hepatology 41 (2005) 1407–1432.

    PubMed  Article  Google Scholar 

  3. 3.

    Muraca, M., Gerunda, G., Neri, D., Vilei, M.T., Granato, A., Feltracco, P., Meroni, M., Giron, G. and Burlina, A.B. Hepatocyte transplantation as a treatment for glycogen storage disease type 1a. Lancet 359 (2002) 317–318.

    PubMed  Article  Google Scholar 

  4. 4.

    Khan, A.A., Parveen, N., Mahaboob, V.S., Rajendraprasad, A., Ravindraprakash, H.R., Venkateswarlu, J., Rao P, P.G., Narusu, M.L., Khaja, M.N., Pramila, R., Habeeb, A. and Habibullah, C.M. Treatment of Crigler-Najjar Syndrome type 1 by hepatic progenitor cell transplantation: a simple procedure for management of hyperbilirubinemia. Transplant. Proc. 40 (2008) 1148–1150.

    PubMed  Article  CAS  Google Scholar 

  5. 5.

    Perlmutter, D.H., Brodsky, J.L., Balistreri, W.F. and Trapnell, B.C. Molecular pathogenesis of alpha-1-antitrypsin deficiency-associated liver disease: a meeting review. Hepatology 45 (2007) 1313–1323.

    PubMed  Article  Google Scholar 

  6. 6.

    Habibullah, C.M. Stem cell in digestive diseases. Indian J. Gastroenerol. 26Suppl. 1 (2007) s23–s24.

    Google Scholar 

  7. 7.

    Jorgensen, C., Gordeladze, J. and Noel, D. Tissue engineering through autologous mesenchymal stem cells. Curr. Opin. Biotechnol. 15 (2004) 406–410.

    PubMed  Article  CAS  Google Scholar 

  8. 8.

    Stock, P., Brückner, S., Ebensing, S., Hempel, M., Dollinger, M.M. and Christ, B. The generation of hepatocytes from mesenchymal stem cells and engraftment into murine liver. Nat. Protoc. 5 (2010) 617–627.

    PubMed  Article  CAS  Google Scholar 

  9. 9.

    Duan, Y., Catana, A., Meng, Y., Yamamoto, N., He, S., Gupta, S., Gambhir, S.S. and Zern, M.A. Differentiation and enrichment of hepatocyte-like cells from human embryonic stem cells in vitro and in vivo. Stem Cells 25 (2007) 3058–3068.

    PubMed  Article  CAS  Google Scholar 

  10. 10.

    Gupta, S., LaBrecque, D.R. and Shafritz, D.A. Mitogenic effect of hepatic stimulator substance on cultured nonparenchymal liver epithelial cells. Hepatology 15 (1992) 485–491.

    PubMed  Article  CAS  Google Scholar 

  11. 11.

    Newsome, P.N., Johannessen, I., Boyle, S., Dalakas, E., McAulay, K.A., Samuel, K., Rae, F., Forrester, L., Turner, M.L., Hayes, P.C., Harrison, D.J., Bickmore, W.A. and Plevris, J.N. Human cord blood-derived cells can differentiate into hepatocytes in the mouse liver with no evidence of cellular fusion. Gastroenterology 124 (2003) 1891–1900.

    PubMed  Article  Google Scholar 

  12. 12.

    Schwartz, R.E., Reyes, M., Koodie, L., Jiang, Y., Blackstad, M., Lund, T., Lenvik, T., Johnson, S., Hu, W.S. and Verfaillie, C.M. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocytelike cells. J. Clin. Invest. 109 (2002) 1291–1302.

    PubMed  CAS  Google Scholar 

  13. 13.

    Malhi, H., Irani, A.N., Gagandeep, S. and Gupta, S. Isolation of human progenitor liver epithelial cells with extensive replication capacity and differentiation into mature hepatocytes. J. Cell. Sci. 115 (2002) 2679–2688.

    PubMed  CAS  Google Scholar 

  14. 14.

    Khurana, S. and Mukhopadhyay, A. In vitro transdifferentiation of adult hematopoietic stem cells: an alternative source of engraftable hepatocytes. J. Hepatol. 49 (2008) 998–1007.

    PubMed  Article  CAS  Google Scholar 

  15. 15.

    Sato, Y., Araki, H., Kato, J., Nakamura, K., Kawano, Y., Kobune, M., Sato, T., Miyanishi, K., Takayama, T., Takahashi, M., Takimoto, R., Iyama, S., Matsunaga, T., Ohtani, S., Matsuura, A., Hamada, H. and Niitsu, Y. Human mesenchymal stem cells xenografted directly to rat liver are differentiated into human hepatocytes without fusion. Blood 106 (2005) 756–763.

    PubMed  Article  CAS  Google Scholar 

  16. 16.

    Zhao, J., Zhang, N., Prestwich, G.D. and Wen, X. Recruitment of endogenous stem cells for tissue repair. Macromol. Biosci. 8 (2008) 836–842.

    PubMed  Article  CAS  Google Scholar 

  17. 17.

    Salgado, A.J., Oliveira, J.T., Pedro, A.J and Reis, R.L. Adult stem cells in bone and cartilage tissue engineering. Curr. Stem Cell Res. Ther. 1 (2006) 345–364.

    PubMed  CAS  Google Scholar 

  18. 18.

    Mironov, V., Visconti, R.P., Kasyanov, V., Forgacs, G., Drake, C.J. and Markwald, R.R. Organ printing: Tissue spheroids as building blocks. Biomaterials 30 (2009) 2164–2174.

    PubMed  Article  CAS  Google Scholar 

  19. 19.

    Piryaei, A., Valojerdi, M.R., Shahsavandi, M., Baharvand, H. Differentiation of bone marrow-derived mesenchymal stem cells into hepatocyte-like cells on nanofibers and their transplantation into a carbon tetrachloride-induced liver fibrosis model. Stem Cell Rev. 7 (2011) 103–118.

    PubMed  Article  CAS  Google Scholar 

  20. 20.

    Shvartsman, I., Dvir, T., Harel-Adar, T. and Cohen, S. Perfusion cell seeding and cultivation induce the assembly of thick and functional hepatocellular tissue-like construct. Tissue Eng. Part A. 15 (2009) 751–760.

    PubMed  Article  CAS  Google Scholar 

  21. 21.

    Ng, S., Wu, Y.N., Zhou, Y., Toh, Y.E., Ho, Z.Z., Chia, S.M., Zhu, J.H., Mao, H.Q. and Yu, H. Optimization of 3-D hepatocyte culture by controlling the physical and chemical properties of the extra-cellular matrices. Biometerials 26 (2005) 3153–3163.

    Article  CAS  Google Scholar 

  22. 22.

    Hashemi, S.M., Soleimani, M., Zargarian, S.S., Haddadi-Asl, V., Ahmadbeigi, N., Soudi, S., Gheisari, Y., Hajarizadeh, A. and Mohammadi, Y. In vitro differentiation of human cord blood-derived unrestricted somatic stem cells into hepatocyte-like cells on poly (ɛ-Caprolactone) nanofiber scaffolds. Cells Tissues Organs 190 (2008) 135–149.

    PubMed  Article  Google Scholar 

  23. 23.

    Li, W.J., Tuli, R., Huang, X., Laquerriere, P. and Tuan, R.S. Multilineage differentiation of human mesenchymal stem cells in a three-dimensional nanofibrous scaffold. Biomaterials 26 (2005) 5185–5166.

    Google Scholar 

  24. 24.

    Li, W.J., Laurencin, C.T., Caterson, E.J., Tuan, R.S. and Ko, F.K. Electrospun nanofibrous structure: a novel scaffold for tissue engineering. J. Biomed. Mater. Res. 60 (2002) 613–621.

    PubMed  Article  CAS  Google Scholar 

  25. 25.

    Sill, T.J. and Von Recum, H.A. Electrospinning: applications in drug delivery and tissue engineering. Biomaterials 29 (2008) 1989–2006.

    PubMed  Article  CAS  Google Scholar 

  26. 26.

    Chan, G. and Mooney, D.J. New materials for tissue engineering: towards greater control over the biological response. Trends. Biotechnol. 26 (2008) 382–392.

    PubMed  Article  CAS  Google Scholar 

  27. 27.

    Hwang, do.W., Jang, S.J., Kim, Y.H., Kim, H.J., Shim, I.K., Jeong, J.M., Chung, J.K., Lee, M.C., Lee, S.J., Kim, S.U., Kim, S. and Lee, D.S. Realtime in vivo monitoring of viable stem cells implanted on biocompatible scaffolds. Eur. J. Nucl. Med. Mol. Imaging 35 (2008) 1887–1898.

    Article  CAS  Google Scholar 

  28. 28.

    Lee, J.S, Kim, S.H., Kim, Y.J., Akaike, T. and S.C.K. Hepatocyte adhesion on a poly[N-p-vinylbenzyl-4-O-beta-D-galactopyranosyl-D-glucoamide]-coated poly(L-lactic acid) surface. Biomacromolecules 6 (2005) 1906–1911.

    PubMed  Article  CAS  Google Scholar 

  29. 29.

    Li, W.J., Cooper, J.A. Jr., Mauck, R.L. and Tuan, R.S. Fabrication and characterization of six electrospun poly(alpha-hydroxy ester)-based fibrous scaffolds for tissue engineering applications. Acta. Biomater. 2 (2006) 377–385.

    PubMed  Article  Google Scholar 

  30. 30.

    Kazemnejad, S., Allameh, A., Soleimani, M., Gharehbaghian, A.,Y., Amirizadeh, N. and Jazayery, M. Biochemical and molecular characterization of hepatocyte-like cells derived from human bone marrow mesenchymal stem cells on a novel three-dimensional biocompatible nanofibrous sacffold. J. Gastroenterol. Hepatol. 24 (2008) 278–287.

    PubMed  Article  Google Scholar 

  31. 31.

    Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S. and Marshak, D.R. Multilineage potential of adult human mesenchymal stem cells. Science 284 (1999) 143–147.

    PubMed  Article  CAS  Google Scholar 

  32. 32.

    Penfornis, P. and Pochampally, R. Isolation and expansion of mesenchymal stem cells/multipotential stromal cells from human bone marrow. Methods Mol. Biol. 698 (2011) 11–21.

    PubMed  Article  CAS  Google Scholar 

  33. 33.

    Covas, D.T., Siufi, J.L., Silva, A.R. and Orellana, M.D. Isolation and culture of umbilical vein mesenchymal stem cells. Braz. J. Med. Biol. Res. 36 (2003) 1179–1183.

    PubMed  Article  CAS  Google Scholar 

  34. 34.

    Kim, J.W., Kim, S.Y., Park, S.Y., Kim, Y.M., Kim, J.M., Lee, M.H. and Ryu, H.M. Mesenchymal progenitor cells in the human umbilical cord. Ann. Hematol. 83 (2004) 733–738.

    PubMed  Article  CAS  Google Scholar 

  35. 35.

    Lee, K.D., Kuo, T.K., Whang-Peng, J., Chung, Y.F., Lin, C.T., Chou, S.H., Chen, J.R., Chen, Y.P. and Lee, O.K. In vitro hepatic differentiation of human mesenchymal stem cells. Hepatology 40 (2004) 1275–1284.

    PubMed  Article  CAS  Google Scholar 

  36. 36.

    Nichols, J.E. and Cortiella, J. Engineering of a complex organ: progress toward development of a tissue-engineered lung. Proc. Am. Thorac. Soc. 5 (2008) 723–730.

    PubMed  Article  Google Scholar 

  37. 37.

    Li, W.J., Cooper, J.A. Jr, Mauck, R.L. and Tuan, R.S. Fabrication and characterization of six electrospun poly(alpha-hydroxy ester)-based fibrous scaffolds for tissue engineering applications. Acta Biomater. 2 (2006) 377–385.

    PubMed  Article  Google Scholar 

  38. 38.

    Vaquette, C., Fawzi-Grancher, S., Lavalle, P., Frochot, C., Viriot, M.L., Muller, S. and Wang, X. In vitro biocompatibility of different polyester membranes. Biomed. Mater. Eng. 16 (2006) S131–136.

    PubMed  CAS  Google Scholar 

  39. 39.

    Tuzlakoglu, K. and Reis, R.L. Biodegradable polymeric fiber structures in tissue engineering. Tissue Eng. Part B. Rev. 15 (2009) 17–27.

    PubMed  Article  CAS  Google Scholar 

  40. 40.

    Smith, L.A, Liu, X. and Ma, P.X. Tissue engineering with nano-fibrous scaffolds. Soft. Matter. 4 (2008) 2144–2149.

    PubMed  Article  CAS  Google Scholar 

  41. 41.

    Howard, D., Buttery, L.D. Shakesheff, K.M. and Roberts, S.J. Tissue engineering: strategies, stem cells and scaffolds. J. Anat. 213 (2008) 66–72.

    PubMed  Article  CAS  Google Scholar 

  42. 42.

    Yang, J., Bei, J. and Wang, S. Enhanced cell affinity of poly (D,L-lactide) by combining plasma treatment with collagen anchorage. Biomaterials 23 (2002) 2607–2614.

    PubMed  Article  CAS  Google Scholar 

  43. 43.

    Duan, Y., Wang, Z., Yan, W., Wang, S., Zhang, S. and Jia, J. Preparation of collagen-coated electrospun nanofibers by remote plasma treatment and their biological properties. J. Biomater. Sci. Polym. Ed. 18 (2007) 1153–1164.

    PubMed  Article  CAS  Google Scholar 

  44. 44.

    Zhu, Y., Gao, C. and Shen, J. Surface modification of polycaprolactone with poly(methacrylic acid) and gelatin covalent immobilization for promoting its cytocompatibility. Biomaterials 23 (2002) 4889–4895.

    PubMed  Article  CAS  Google Scholar 

  45. 45.

    Zhu, Y., Gao, C., Liu, X., He, T. and Shen, J. Immobilization of biomacromolecules onto aminolyzed poly(L-lactic acid) toward acceleration of endothelium regeneration. Tissue Eng. 10 (2004) 53–61.

    PubMed  Article  CAS  Google Scholar 

  46. 46.

    Ma, Z., Kotaki, M., Yong, T., He, W. and Ramakrishna, S. Surface engineering of electrospun polyethylene terephthalate (PET) nanofibers towards development of a new material for blood vessel engineering. Biomaterials 26 (2005) 2527–2536.

    PubMed  Article  CAS  Google Scholar 

  47. 47.

    Zhao, J.H., Wang, J., Tu, M., Luo, B.H. and Zhou, C.R. Improving the cell affinity of a poly(D,L-lactide) film modified by grafting collagen via a plasma technique. Biomed. Mater. 1 (2006) 247–252.

    PubMed  Article  CAS  Google Scholar 

  48. 48.

    Ma, Z., Gao, C., Gong, Y. and Shen, J. Cartilage tissue engineering PLLA scaffold with surface immobilized collagen and basic fibroblast growth factor. Biomaterials 26 (2005) 1253–1259.

    PubMed  Article  CAS  Google Scholar 

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Correspondence to Yuyou Duan or Abbas S. Lotfi.

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Ghaedi, M., Soleimani, M., Shabani, I. et al. Hepatic differentiation from human mesenchymal stem cells on a novel nanofiber scaffold. Cell Mol Biol Lett 17, 89–106 (2012). https://doi.org/10.2478/s11658-011-0040-x

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Key words

  • Poly L-lactic acid
  • Nanofiber
  • Hepatic differentiation
  • Mesenchymal stem cells
  • Electrospinning