Tissue engineering of implantable cellular constructs is an emerging cellular therapy for hepatic disease. In this study, we tested the ability of poly(ε-caprolactone) (PCL) nanofiber scaffold to support and maintain hepatic differentiation of human cord blood-derived unrestricted somatic stem cells (USSCs) in vitro. USSCs, self-renewing pluripotent cells, were isolated from human cord blood. The electrospun PCL nanofiber porous scaffold was constructed of uniform, randomly oriented nanofibers. USSCs were seeded onto PCL nanofiber scaffolds, and were induced to differentiate into hepatogenic lineages by culturing with differentiation factors for 6 weeks. RT-PCR analysis of endoderm and hepatic-specific gene expression, immunohistochemical detection of cytokeratin 18 (CK-18), α-fetoprotein, albumin, glycogen storage and indocyanine green uptake confirmed the differentiation of USSCs into endoderm and hepatocyte-like cells. In the present study, we show that hepatocyte-like cells differentiated from USSCs on the PCL nanofiber scaffold can be candidate for tissue engineering and cell therapy of hepatic tissues.

Keywords:
Cord blood, Stem cell, unrestricted somatic, Poly(ε-caprolactone), Nanofiber scaffold, Hepatocyte differentiation
1.
Anjos-Afonso, F., E.K. Siapati, D. Bonnet (2004) In vivo contribution of murine mesenchymal stem cells into multiple cell-types under minimal damage conditions. J Cell Sci 117: 5655–5664.
2.
Aurich, I., L.P. Mueller, H. Aurich, J. Luetzkendorf, K. Tisljar, M.M. Dollinger, W. Schormann, J. Walldorf, J.G. Hengstler, W.E. Fleig, B. Christ (2007) Functional integration of hepatocytes derived from human mesenchymal stem cells into mouse livers. Gut 56: 405–415.
3.
Baharvand, H., S.M. Hashemi, S. Kazemi Ashtiani, A. Farrokhi (2006) Differentiation of human embryonic stem cells into hepatocytes in 2D and 3D culture systems in vitro. Int J Dev Biol 50: 645–652.
4.
Banas, A., T. Teratani, Y. Yamamoto, M. Tokuhara, F. Takeshita, G. Quinn, H. Okochi, T. Ochiya (2007) Adipose tissue-derived mesenchymal stem cells as a source of human hepatocytes. Hepatology 46: 219–228.
5.
Boudriot, U., R. Dersch, A. Greiner, J.H. Wendorff (2006) Electrospinning approaches toward scaffold engineering – a brief overview. Artif Organs 30: 785–792.
6.
Chen, F., C.N. Lee, S.H. Teoh (2007a) Nanofibrous modification on ultra-thin poly(e-caprolactone) membrane via electrospinning. Mater Sci Eng C 27: 325–332.
7.
Chen, M., P.K. Patra, S.B. Warner, S. Bhowmick (2006) Optimization of electrospinning process parameters for tissue engineering scaffolds. Biophys Rev Lett 1: 153–178.
8.
Chen, M., P.K. Patra, S.B. Warner, S. Bhowmick (2007b) Role of fiber diameter in adhesion and proliferation of NIH 3T3 fibroblast on electrospun polycaprolactone scaffolds. Tissue Eng 13: 579–587.
9.
Dawson, E., G. Mapili, K. Erickson, S. Taqvi, K. Roy (2008) Biomaterials for stem cell differentiation. Adv Drug Deliv Rev 60: 215–228.
10.
Deitzel, J., J. Kleinmeyer, D. Harris, N. Tan (2001) The effect of processing variables on the morphology of electrospun nanofibers and textiles. Polymer 42: 261–272.
11.
Demir, M.M., I. Yilgor, E. Yilgor, B. Erman (2002) Electrospinning of polyurethane fibers. Polymer 43: 3303–3309.
12.
Du, Y., R. Han, F. Wen, S. Ng San San, L. Xia, T. Wohland, H.L. Leo, H. Yu (2008) Synthetic sandwich culture of 3D hepatocyte monolayer. Biomaterials 29: 290–301.
13.
Duan, Y., A. Catana, Y. Meng, N. Yamamoto, S. He, S. Gupta, S.S. Gambhir, M.A. Zern (2007) Differentiation and enrichment of hepatocyte-like cells from human embryonic stem cells in vitro and in vivo. Stem Cells 25: 3058–3068.
14.
Fallahi-Sichani, M., M. Soleimani, S.M. Najafi, J. Kiani, E. Arefian, A. Atashi (2007) In vitro differentiation of cord blood unrestricted somatic stem cells expressing dopamine-associated genes into neuron-like cells. Cell Biol Int 31: 299–303.
15.
Hamazaki, T., Y. Iiboshi, M. Oka, P.J. Papst, A.M. Meacham, L.I. Zon, N. Terada (2001) Hepatic maturation in differentiating embryonic stem cells in vitro. FEBS Lett 497: 15–19.
16.
Hsu, C.H., S. Shivkumar (2004) N,N-dimethylformamide additions to the solution for the electrospinning of poly(ε-caprolactone) nanofibers. Macromol Mater Eng 28: 334–340.
17.
Imamura, T., L. Cui, R. Teng, K. Johkura, Y. Okouchi, K. Asanuma, N. Ogiwara, K. Sasaki (2004) Embryonic stem cell-derived embryoid bodies in three-dimensional culture system form hepatocyte-like cells in vitro and in vivo. Tissue Eng 10: 1716–1724.
18.
Jäger, M., O. Degistirici, A. Knipper, J. Fischer, M. Sager, R. Krauspe (2007) Bone healing and migration of cord blood-derived stem cells into a critical size femoral defect after xenotransplantation. J Bone Miner Res 22: 1224–1233.
19.
Jäger, M., M. Sager, A. Knipper, O. Degistirici, J. Fischer, G. Kögler, P. Wernet, R. Krauspe (2004) In vivo and in vitro bone regeneration from cord blood derived mesenchymal stem cells (in German). Orthopade 33: 1361–1372.
20.
Jäger, M., A. Wild, S. Lensing-Hohn, R. Krauspe (2003) Influence of different culture solutions on osteoblastic differentiation in cord blood and bone marrow derived progenitor cells. Biomed Tech (Berl) 48: 241–244.
21.
Jiang, Y., B.N. Jahagirdar, R.L. Reinhardt, R.E. Schwartz, C.D. Keene, X.R. Ortiz-Gonzalez, M. Reyes, T. Lenvik, T. Lund, M. Blackstad, J. Du, S. Aldrich, A. Lisberg, W.C. Low, D.A. Largaespada, C.M. Verfaillie (2002) Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418: 41–49.
22.
Kakinuma, S., Y. Tanaka, R. Chinzei, M. Watanabe, K. Shimizu-Saito, Y. Hara, K. Teramoto, S. Arii, C. Sato, K. Takase, T. Yasumizu, H. Teraoka (2003) Human umbilical cord blood as a source of transplantable hepatic progenitor cells. Stem Cells 21: 217–227.
23.
Khademhosseini, A., K.Y. Suh, J.M. Yang, G. Eng, J. Yeh, S. Levenberg, R. Langer (2004) Layer-by-layer deposition of hyaluronic acid and poly-L-lysine for patterned cell co-cultures. Biomaterials 25: 3583–3592.
24.
Khil, M.S., S.R. Bhattarai, H.Y. Kim, S.Z. Kim, K.H. Lee (2005) Novel fabricated matrix via electrospinning for tissue engineering. J Biomed Mater Res B Appl Biomater 72: 117–124.
25.
Kidoaki, S., I.K. Kwon, T. Matsuda (2005) Mesoscopic spatial designs of nano- and microfiber meshes for tissue-engineering matrix and scaffold based on newly devised multilayering and mixing electrospinning techniques. Biomaterials 26: 37–46.
26.
Kim, G., W. Kim (2007) Highly porous 3D nanofiber scaffold using an electrospinning technique. J Biomed Mater Res B Appl Biomater 81: 104–110.
27.
Kögler, G., T.F. Radke, A. Lefort, S. Sensken, J. Fischer, R.V. Sorg, P. Wernet (2005) Cytokine production and hematopoiesis supporting activity of cord blood-derived unrestricted somatic stem cells. Exp Hematol 33: 573–583.
28.
Kögler, G., S. Sensken, J.A. Airey, T. Trapp, M. Müschen, N. Feldhahn, S. Liedtke, R.V. Sorg, J. Fischer, C. Rosenbaum, S. Greschat, A. Knipper, J. Bender, O. Degistirici, J. Gao, A.I. Caplan, E.J. Colletti, G. Almeida-Porada, H.W. Müller, E. Zanjani, P. Wernet (2004) A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential. J Exp Med 200: 123–135.
29.
Kögler, G., S. Sensken, P. Wernet (2006) Comparative generation and characterization of pluripotent unrestricted somatic stem cells with mesenchymal stem cells from human cord blood. Exp Hematol 34: 1589–1595.
30.
Kollet, O., S. Shivtiel, Y.Q. Chen, J. Suriawinata, S.N. Thung, M.D. Dabeva, J. Kahn, A. Spiegel, A. Dar, S. Samira, P. Goichberg, A. Kalinkovich, F. Arenzana-Seisdedos, A. Nagler, I. Hardan, M. Revel, D.A. Shafritz, T. Lapidot (2003) HGF, SDF-1, and MMP-9 are involved in stress-induced human CD34+ stem cell recruitment to the liver. J Clin Invest 112: 160–169.
31.
Langer, R., J.P. Vacanti (1993) Tissue engineering. Science 260: 920–926.
32.
Lee, K.D., T.K. Kuo, J. Whang-Peng, Y.F. Chung, C.T. Lin, S.H. Chou, J.R. Chen, Y.P. Chen, O.K. Lee (2004) In vitro hepatic differentiation of human mesenchymal stem cells. Hepatology 40: 1275–1284.
33.
Lee, K.H., H.Y. Kim, M.S. Khil, Y.M. Ra, D.R. Lee (2003) Characterization of nano-structured poly (ε-caprolactone) nonwoven mats via electrospinning. Polymer 44: 1287–1294.
34.
Li, W.J., C.T. Laurencin, E.J. Caterson, R.S. Tuan, F.K. Ko (2002) Electrospun nanofibrous structure: a novel scaffold for tissue engineering. J Biomed Mater Res 60: 613–621.
35.
Li, W.J., R. Tuli, X. Huang, P. Laquerriere, R.S. Tuan (2005a) Multilineage differentiation of human mesenchymal stem cells in a three-dimensional nanofibrous scaffold. Biomaterials 26: 5158–5166.
36.
Li, W.J., R. Tuli, C. Okafor, A. Derfoul, K.G. Danielson, D.J. Hall, R.S. Tuan (2005b) A three-dimensional nanofibrous scaffold for cartilage tissue engineering using human mesenchymal stem cells. Biomaterials 26: 599–609.
37.
Murugan, R., S. Ramakrishna (2007) Design strategies of tissue engineering scaffolds with controlled fiber orientation. Tissue Eng 13: 1845–1866.
38.
Nair, L.S., S. Bhattacharyya, C.T. Laurencin (2004) Development of novel tissue engineering scaffolds via electrospinning. Expert Opin Biol Ther 4: 659–668.
39.
Ng, S., Y.N. Wu, Y. Zhou, Y.E. Toh, Z.Z. Ho, S.M. Chia, J.H. Zhu, H.Q. Mao, H. Yu (2005) Optimization of 3-D hepatocyte culture by controlling the physical and chemical properties of the extra-cellular matrices. Biomaterials 26: 3153–3163.
40.
Okumoto, K., T. Saito, E. Hattori, J.I. Ito, A. Suzuki, K. Misawa, R. Ishii, T. Karasawa, H. Haga, M. Sanjo, T. Takeda, K. Sugahara, K. Saito, H. Togashi, S. Kawata (2005) Differentiation of rat bone marrow cells cultured on artificial basement membrane containing extracellular matrix into a liver cell lineage. J Hepatol 43: 110–116.
41.
Pankajakshan, D., V.K. Krishnan, L.K. Krishnan (2007) Vascular tissue generation in response to signaling molecules integrated with a novel poly(epsilon-caprolactone)-fibrin hybrid scaffold. J Tissue Eng Regen Med 1: 389–397.
42.
Pham, Q.P., U. Sharma, A.G. Mikos (2006a) Electrospinning of polymeric nanofibers for tissue engineering applications: a review. Tissue Eng 12: 1197–1211.
43.
Pham, Q.P., U. Sharma, A.G. Mikos (2006b) Electrospun poly(epsilon-caprolactone) microfiber and multilayer nanofiber/microfiber scaffolds: characterization of scaffolds and measurement of cellular infiltration. Biomacromolecules 7: 2796–2805.
44.
Rosso, F., A. Giordano, M. Barbarisi, A. Barbarisi (2004) From cell-ECM interactions to tissue engineering. J Cell Physiol 199: 174–180.
45.
Sato, Y., H. Araki, J. Kato, K. Nakamura, Y. Kawano, M. Kobune, T. Sato, K. Miyanishi, T. Takayama, M. Takahashi, R. Takimoto, S. Iyama, T. Matsunaga, S. Ohtani, A. Matsuura, H. Hamada, Y. Niitsu (2005) Human mesenchymal stem cells xenografted directly to rat liver are differentiated into human hepatocytes without fusion. Blood 106: 756–763.
46.
Schwartz, R.E., M. Reyes, L. Koodie, Y. Jiang, M. Blackstad, T. Lund, T. Lenvik, S. Johnson, W.S. Hu, C.M. Verfaillie (2002) Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J Clin Invest 109: 1291–1302.
47.
Semler, E.J., C.S. Ranucci, P.V. Moghe (2000) Mechanochemical manipulation of hepatocyte aggregation can selectively induce or repress liver-specific function. Biotechnol Bioeng 69: 359–369.
48.
Sensken, S., S. Waclawczyk, A.S. Knaupp, T. Trapp, J. Enczmann, P. Wernet, G. Kögler (2007) In vitro differentiation of human cord blood-derived unrestricted somatic stem cells towards an endodermal pathway. Cytotherapy 9: 362–378.
49.
Serban, M.A., L. Yanchun, G.D. Prestwich (2008) Effects of extracellular matrix analogues on primary human fibroblast behavior. Acta Biomater 4: 67–75.
50.
Sgodda, M., H. Aurich, S. Kleist, I. Aurich, S. Konig, M.M. Dollinger, W.E. Fleig, B. Christ (2007) Hepatocyte differentiation of mesenchymal stem cells from rat peritoneal adipose tissue in vitro and in vivo. Exp Cell Res 313: 2875–2886.
51.
Shields, K.J., M.J. Beckman, G.L. Bowlin, J.S. Wayne (2004) Mechanical properties and cellular proliferation of electrospun collagen type II. Tissue Eng 10: 1510–1517.
52.
Sun, T., S. Mai, D. Norton, J.W. Haycock, A.J. Ryan, S. MacNeil (2005) Self-organization of skin cells in three-dimensional electrospun polystyrene scaffolds. Tissue Eng 11: 1023–1033.
53.
Talens-Visconti, R., A. Bonora, R. Jover, V. Mirabet, F. Carbonell, J.V. Castell, M.J. Gomez-Lechon (2006) Hepatogenic differentiation of human mesenchymal stem cells from adipose tissue in comparison with bone marrow mesenchymal stem cells. World J Gastroenterol 12: 5834–5845.
54.
Tan, P.S., S.H. Teoh (2007) Effect of stiffness of polycaprolactone (PCL) membrane on cell proliferation. Mater Sci Eng C 27: 304–308.
55.
Wang, X., S. Ge, G. McNamara, Q.L. Hao, G.M. Crooks, J.A. Nolta (2003) Albumin-expressing hepatocyte-like cells develop in the livers of immune-deficient mice that received transplants of highly purified human hematopoietic stem cells. Blood 101: 4201–4208.
56.
Yamada, T., M. Yoshikawa, S. Kanda, Y. Kato, Y. Nakajima, S. Ishizaka, Y. Tsunoda (2002) In vitro differentiation of embryonic stem cells into hepatocyte-like cells identified by cellular uptake of indocyanine green. Stem Cells 20: 146–154.
57.
Yang, F., R. Murugan, S. Wang, S. Ramakrishna (2005) Electrospinning of nano/micro scale poly(L-lactic acid) aligned fibers and their potential in neural tissue engineering. Biomaterials 26: 2603–2610.
58.
Zavan, B., P. Brun, V. Vindigni, A. Amadori, W. Habeler, P. Pontisso, D. Montemurro, G. Abatangelo, R. Cortivo (2005) Extracellular matrix-enriched polymeric scaffolds as a substrate for hepatocyte cultures: in vitro and in vivo studies. Biomaterials 26: 7038–7045.
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