Cell differentiation mediated by co-culture of human umbilical cord blood stem cells with murine hepatic cells

  • Maria StecklumEmail author
  • Annika Wulf-Goldenberg
  • Bettina Purfürst
  • Antje Siegert
  • Marlen Keil
  • Klaus Eckert
  • Iduna Fichtner


In the present study, purified human cord blood stem cells were co-cultivated with murine hepatic alpha mouse liver 12 (AML12) cells to compare the effect on endodermal stem cell differentiation by either direct cell-cell interaction or by soluble factors in conditioned hepatic cell medium. With that approach, we want to mimic in vitro the situation of preclinical transplantation experiments using human cells in mice. Cord blood stem cells, cultivated with hepatic conditioned medium, showed a low endodermal differentiation but an increased connexin 32 (Cx32) and Cx43, and cytokeratin 8 (CK8) and CK19 expression was monitored by reverse transcription polymerase chain reaction (RT-PCR). Microarray profiling indicated that in cultivated cord blood cells, 604 genes were upregulated 2-fold, with the highest expression for epithelial CK19 and epithelial cadherin (E-cadherin). On ultrastructural level, there were no major changes in the cellular morphology, except a higher presence of phago(ly)some-like structures observed. Direct co-culture of AML12 cells with cord blood cells led to less incisive differentiation with increased sex-determining region Y-box 17 (SOX17), Cx32 and Cx43, as well as epithelial CK8 and CK19 expressions. On ultrastructural level, tight cell contacts along the plasma membranes were revealed. FACS analysis in co-cultivated cells quantified dye exchange on low level, as also proved by time relapse video-imaging of labelled cells. Modulators of gap junction formation influenced dye transfer between the co-cultured cells, whereby retinoic acid increased and 3-heptanol reduced the dye transfer. The study indicated that the cell-co-cultured model of human umbilical cord blood cells and murine AML12 cells may be a suitable approach to study some aspects of endodermal/hepatic cell differentiation induction.


Co-culture Murine hepatic cells (AML12) Umbilical cord blood cells (UCBC) Cell differentiation 



The skilful technical assistance of Mrs. Vannauer is gratefully acknowledged. We thank the HELIOS-Clinics GmbH in Berlin-Buch and the Vivantes Clinics in Berlin for collecting and providing the umbilical cord blood. The work was supported by a grant from the Federal Ministry of Education and Research (BMBF; 01GN0528)

Supplementary material


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  1. Agarwal S, Holton KL, Lanza R (2008) Efficient differentiation of functional hepatocytes from human embryonic stem cells. Stem Cells 26(5):1117–1127CrossRefPubMedGoogle Scholar
  2. Basma H, Soto-Gutierrez A, Yannam GR, Liu L, Ito R, Yamamoto T, Ellis E, Carson SD, Sato S, Chen Y, Muirhead D, Navarro-Alvarez N, Wong RJ, Roy-Chowdhury J, Platt JL, Mercer DF, Miller JD, Strom SC, Kobayashi N, Fox IJ (2009) Differentiation and transplantation of human embryonic stem cell-derived hepatocytes. Gastroenterology 136(3):990–999CrossRefPubMedCentralPubMedGoogle Scholar
  3. 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(3):833–848CrossRefPubMedGoogle Scholar
  4. Chen X, Zeng F (2011) Directed hepatic differentiation from embryonic stem cells. Prot Cell 2(3):180–188CrossRefGoogle Scholar
  5. Chen Y, Soto-Gutierrez A, Navarro-Alvarez N, Rivas-Carrillo JD, Yamatsuji T, Shirakawa Y, Tanaka N, Basma H, Fox IJ, Kobayashi N (2006) Instant hepatic differentiation of human embryonic stem cells using activin A and a deleted variant of HGF. Cell Transplant 15(10):865–871CrossRefPubMedGoogle Scholar
  6. Chen Y, Cai H, Tan W (2008) [Research on in vitro differentiation of human umbilical cord blood-derived CD34+ cells into hepatocyte-like cells]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi 22(6):747–752PubMedGoogle Scholar
  7. Chen Q, Khoury M, Limmon G, Choolani M, Chan JK, Chen J (2013) Human fetal hepatic progenitor cells are distinct from, but closely related to, hematopoietic stem/progenitor cells. Stem Cells 31(6):1160–1169CrossRefPubMedGoogle Scholar
  8. Cho CH, Parashurama N, Park EY, Suganuma K, Nahmias Y, Park J, Tilles AW, Berthiaume F, Yarmush ML (2008) Homogeneous differentiation of hepatocyte-like cells from embryonic stem cells: applications for the treatment of liver failure. FASEB J 22(3):898–909CrossRefPubMedGoogle Scholar
  9. Choi YS, Dusting GJ, Stubbs S, Arunothayaraj S, Han XL, Collas P, Morrison WA, Dilley RJ (2010) Differentiation of human adipose-derived stem cells into beating cardiomyocytes. J Cell Mol Med 14(4):878–889CrossRefPubMedGoogle Scholar
  10. Chularojmontri L, Wattanapitayakul SK (2009) Isolation and characterization of umbilical cord blood hematopoietic stem cells. J Med Assoc Thai 92(Suppl 3):S88–S94PubMedGoogle Scholar
  11. Dong XJ, Zhang GR, Zhou QJ, Pan RL, Chen Y, Xiang LX, Shao JZ (2009) Direct hepatic differentiation of mouse embryonic stem cells induced by valproic acid and cytokines. World J Gastroenterol 15(41):5165–5175CrossRefPubMedCentralPubMedGoogle Scholar
  12. Elias LA, Kriegstein AR (2008) Gap junctions: multifaceted regulators of embryonic cortical development. Trends Neurosci 31(5):243–250CrossRefPubMedCentralPubMedGoogle Scholar
  13. Esrefoglu M (2013) Role of stem cells in repair of liver injury: experimental and clinical benefit of transferred stem cells on liver failure. World J Gastroenterol 19(40):6757–6773CrossRefPubMedCentralPubMedGoogle Scholar
  14. Fiegel HC, Lioznov MV, Cortes-Dericks L, Lange C, Kluth D, Fehse B, Zander AR (2003) Liver-specific gene expression in cultured human hematopoietic stem cells. Stem Cells 21(1):98–104CrossRefPubMedGoogle Scholar
  15. Fladmark KE, Gjertsen BT, Molven A, Mellgren G, Vintermyr OK, Doskeland SO (1997) Gap junctions and growth control in liver regeneration and in isolated rat hepatocytes. Hepatology 25(4):847–855CrossRefPubMedGoogle Scholar
  16. Fujino H, Hiramatsu H, Tsuchiya A, Niwa A, Noma H, Shiota M, Umeda K, Yoshimoto M, Ito M, Heike T, Nakahata T (2007) Human cord blood CD34+ cells develop into hepatocytes in the livers of NOD/SCID/gamma(c)null mice through cell fusion. FASEB J 21(13):3499–3510CrossRefPubMedGoogle Scholar
  17. Hombach-Klonisch S, Panigrahi S, Rashedi I, Seifert A, Alberti E, Pocar P, Kurpisz M, Schulze-Osthoff K, Mackiewicz A, Los M (2008) Adult stem cells and their trans-differentiation potential—perspectives and therapeutic applications. J Mol Med (Berl) 86(12):1301–1314CrossRefGoogle Scholar
  18. Ishii T, Fukumitsu K, Yasuchika K, Adachi K, Kawase E, Suemori H, Nakatsuji N, Ikai I, Uemoto S (2008) Effects of extracellular matrixes and growth factors on the hepatic differentiation of human embryonic stem cells. Am J Physiol Gastrointest Liver Physiol 295(2):G313–G321CrossRefPubMedGoogle Scholar
  19. Jang YY, Collector MI, Baylin SB, Diehl AM, Sharkis SJ (2004) Hematopoietic stem cells convert into liver cells within days without fusion. Nat Cell Biol 6(6):532–539CrossRefPubMedGoogle Scholar
  20. Kakinuma S, Tanaka Y, Chinzei R, Watanabe M, Shimizu-Saito K, Hara Y, Teramoto K, Arii S, Sato C, Takase K, Yasumizu T, Teraoka H (2003) Human umbilical cord blood as a source of transplantable hepatic progenitor cells. Stem Cells 21(2):217–227CrossRefPubMedGoogle Scholar
  21. Kihara AH, Paschon V, Akamine PS, Saito KC, Leonelli M, Jiang JX, Hamassaki DE, Britto LR (2008) Differential expression of connexins during histogenesis of the chick retina. Dev Neurobiol 68(11):1287–1302CrossRefPubMedGoogle Scholar
  22. Kojima T, Yamamoto T, Murata M, Chiba H, Kokai Y, Sawada N (2003) Regulation of the blood-biliary barrier: interaction between gap and tight junctions in hepatocytes. Med Electron Microsc 36(3):157–164CrossRefPubMedGoogle Scholar
  23. Lagasse E, Connors H, Al-Dhalimy M, Reitsma M, Dohse M, Osborne L, Wang X, Finegold M, Weissman IL, Grompe M (2000) Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 6(11):1229–1234CrossRefPubMedGoogle Scholar
  24. Lavon N (2010) Generation of hepatocytes from human embryonic stem cells. Methods Mol Biol 640:237–246CrossRefPubMedGoogle Scholar
  25. Lavon N, Benvenisty N (2005) Study of hepatocyte differentiation using embryonic stem cells. J Cell Biochem 96(6):1193–1202CrossRefPubMedGoogle Scholar
  26. Lysy PA, Campard D, Smets F, Najimi M, Sokal EM (2008) Stem cells for liver tissue repair: current knowledge and perspectives. World J Gastroenterol 14(6):864–875CrossRefPubMedCentralPubMedGoogle Scholar
  27. Masson S, Harrison DJ, Plevris JN, Newsome PN (2004) Potential of hematopoietic stem cell therapy in hepatology: a critical review. Stem Cells 22(6):897–907CrossRefPubMedGoogle Scholar
  28. Matsui T, Kanai-Azuma M, Hara K, Matoba S, Hiramatsu R, Kawakami H, Kurohmaru M, Koopman P, Kanai Y (2006) Redundant roles of Sox17 and Sox18 in postnatal angiogenesis in mice. J Cell Sci 119(Pt 17):3513–3526CrossRefPubMedGoogle Scholar
  29. Minamiguchi H, Ishikawa F, Fleming PA, Yang S, Drake CJ, Wingard JR, Ogawa M (2008) Transplanted human cord blood cells generate amylase-producing pancreatic acinar cells in engrafted mice. Pancreas 36(2):e30–e35CrossRefPubMedGoogle Scholar
  30. Naiki-Ito A, Asamoto M, Naiki T, Ogawa K, Takahashi S, Sato S, Shirai T (2010) Gap junction dysfunction reduces acetaminophen hepatotoxicity with impact on apoptotic signaling and connexin 43 protein induction in rat. Toxicol Pathol 38(2):280–286CrossRefPubMedGoogle Scholar
  31. Rolletschek A, Schroeder IS, Schulz H, Hummel O, Huebner N, Wobus AM (2010) Characterization of mouse embryonic stem cell differentiation into the pancreatic lineage in vitro by transcriptional profiling, quantitative RT-PCR and immunocytochemistry. Int J Dev Biol 54(1):41–54CrossRefPubMedGoogle Scholar
  32. Rosendaal M, Green CR, Rahman A, Morgan D (1994) Up-regulation of the connexin43+ gap junction network in haemopoietic tissue before the growth of stem cells. J Cell Sci 107(Pt 1):29–37PubMedGoogle Scholar
  33. Saez-Lara MJ, Frecha C, Martin F, Abadia F, Toscano M, Gil A, Fontana L (2006) Transplantation of human CD34+ stem cells from umbilical cord blood to rats with thioacetamide-induced liver cirrhosis. Xenotransplantation 13(6):529–535CrossRefPubMedGoogle Scholar
  34. Sautin YY, Crawford JM, Svetlov SI (2001) Enhancement of survival by LPA via Erk1/Erk2 and PI 3-kinase/Akt pathways in a murine hepatocyte cell line. Am J Physiol Cell Physiol 281(6):C2010–C2019CrossRefPubMedGoogle Scholar
  35. Simek J, Churko J, Shao Q, Laird DW (2009) Cx43 has distinct mobility within plasma-membrane domains, indicative of progressive formation of gap-junction plaques. J Cell Sci 122(Pt 4):554–562CrossRefPubMedGoogle Scholar
  36. Solan JL, Lampe PD (2009) Connexin43 phosphorylation: structural changes and biological effects. Biochem J 419(2):261–272CrossRefPubMedCentralPubMedGoogle Scholar
  37. Song L, Wang H, Gao X, Shen K, Niu W, Qin X (2010) Proliferation and differentiation potential of mouse adult hepatic progenitor cells cultured in vitro. Acta Biochim Biophys Sin (Shanghai) 42(2):122–128CrossRefGoogle Scholar
  38. Teramoto K, Asahina K, Kumashiro Y, Kakinuma S, Chinzei R, Shimizu-Saito K, Tanaka Y, Teraoka H, Arii S (2005) Hepatocyte differentiation from embryonic stem cells and umbilical cord blood cells. J Hepatobiliary Pancreat Surg 12(3):196–202CrossRefPubMedGoogle Scholar
  39. Wagers AJ, Sherwood RI, Christensen JL, Weissman IL (2002) Little evidence for developmental plasticity of adult hematopoietic stem cells. Sci 297(5590):2256–2259CrossRefGoogle Scholar
  40. Wang X, Willenbring H, Akkari Y, Torimaru Y, Foster M, Al-Dhalimy M, Lagasse E, Finegold M, Olson S, Grompe M (2003) Cell fusion is the principal source of bone-marrow-derived hepatocytes. Nat 422(6934):897–901CrossRefGoogle Scholar
  41. Wulf-Goldenberg A, Eckert K, Fichtner I (2008) Cytokine-pretreatment of CD34(+) cord blood stem cells in vitro reduces long-term cell engraftment in NOD/SCID mice. Eur J Cell Biol 87(2):69–80CrossRefPubMedGoogle Scholar
  42. Yan L, Cai H, Chen Y, Tan W (2009) [Effect of hepatocyte-like cells induced by CD34+ cells in vitro on the repair of injured hepatic tissues of mice in vivo]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi 23(10):1235–1240PubMedGoogle Scholar
  43. Zhang Y, Chai C, Jiang XS, Teoh SH, Leong KW (2006) Co-culture of umbilical cord blood CD34+ cells with human mesenchymal stem cells. Tissue Eng 12(8):2161–2170CrossRefPubMedGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2014

Authors and Affiliations

  • Maria Stecklum
    • 1
    • 3
    Email author
  • Annika Wulf-Goldenberg
    • 3
  • Bettina Purfürst
    • 1
  • Antje Siegert
    • 3
  • Marlen Keil
    • 1
    • 2
  • Klaus Eckert
    • 1
    • 3
  • Iduna Fichtner
    • 1
  1. 1.Max Delbrück Center for Molecular Medicine, Berlin-BuchBerlinGermany
  2. 2.Charite—Universitätsmedizin Berlin, Campus Virchow KlinikumBerlinGermany
  3. 3.EPO GmbHBerlinGermany

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