Advertisement

Cytotechnology

, Volume 63, Issue 3, pp 259–268 | Cite as

In vitro trans-differentiation of human umbilical cord derived hematopoietic stem cells into hepatocyte like cells using combination of growth factors for cell based therapy

  • S. Sellamuthu
  • R. Manikandan
  • R. Thiagarajan
  • G. Babu
  • D. Dinesh
  • D. Prabhu
  • C. ArulvasuEmail author
Original Research

Abstract

The aim of the study was to develop a new strategy for the differentiation of hematopoietic stem cell (HSC) derived from UCB into hepatocyte like cells and also to estimate the effects of combination of fibroblast growth factor 4 (FGF 4) and hepatocyte growth factor (HGF) on hematopoietic stem cell differentiation. HSCs were isolated and purified by magnetic activated cell sorting. HSCs were induced to hepatocyte like cells under a 2-step protocol with combination of growth factors. Reverse transcription polymerase chain reaction was performed to detect multiple genes related to hepatocyte like cells development and function. Hepatocyte like morphology was illustrated by inverted repeat microscope and the secretion of albumin and α- fetoprotein by these cells was confirmed by enzyme linked immunosorbent assay. Hepatocyte like cells was observed at the end of the protocol (days 14). These differentiated cells were observed to show high expression of genes related to hepatocytes (tryptophan 2, 3-dioxygenase [TO], glucose 6-phosphate [G6P], cytokeratin 18 [CK 18], albumin and α- fetoprotein [AFP]). The quantities of albumin and AFP at day 0 were low and upon differentiation the cells were able to produce albumin and AFP at high levels. Our results show a new strategy for differentiation in a short duration, using a combination of growth factors for the differentiation of umbilical cord blood derived HSC into hepatocyte like cells under certain in vitro conditions. After further studies this approach has the potency, for widespread cell replacement therapy for liver diseases.

Keywords

Growth factors Hematopoietic stem cell Hepatocyte Trans-differentiation Umbilical cord blood 

References

  1. Bromeyer HE (1995) Questions to be answered regarding umbilical cord blood hematopoietic stem and progenitor cells and their use in transplantation. Transfusion 35:694–702CrossRefGoogle Scholar
  2. Broxmeyer HE, Douglas GW, Hangoc G et al (1989) Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells. Proc Natl Acad Sci USA 86:3828–3832CrossRefGoogle Scholar
  3. Chomaczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:196–199Google Scholar
  4. Danet GH, Luongo JL, Butler G et al (2002) C1qRp defines a new human stem cell population with hematopoietic and hepatic potential. Proc Natl Acad Sci USA 99:10441–10445CrossRefGoogle Scholar
  5. Di Campli C, Piscaglia AC, Pierelli L et al (2004) A human umbilical cord stem cell rescue therapy in a murine model of toxic liver injury. Dig Liver Dis 36:603–613CrossRefGoogle Scholar
  6. Feng G, De-Quan W, Yah-Hua H et al (2008) Extracellular matrix gel is necessary for in vitro cultivation of insulin producing cells from human umbilical cord blood derived mesenchymal stem cells. Chinese Med J 121:811–818Google Scholar
  7. Fiegel HC, Lioznov MV (2003) Cortes-Dericks liver-specific gene expression in cultured human hematopoietic stem cells. Stem Cells 21:98–104CrossRefGoogle Scholar
  8. Forbes S, Vig P, Poulsom R et al (2002) Hepatic stem cells. J Pathol 197:510–518CrossRefGoogle Scholar
  9. Fox IJ, Chowdhury JR (2004) Hepatocyte transplantation. Amer J Transplant 4:7–13CrossRefGoogle Scholar
  10. Glimm H, Tang P, Clark-Lewis I et al (2002) Ex vivo treatment of proliferating human cord blood stem cells with stroma-derived factor-1 enhances their ability to engraft NOD/SCID mice. Blood 99:3454–3457CrossRefGoogle Scholar
  11. Gluckman E, Broxmeyer HE, Auerbach AD et al (1989) Hematopoietic reconstitution in a patient with Fanconi’s anemia by means of umbilical-cord blood from an HLA-identical sibling. N Engl J Med 321:1174–1178CrossRefGoogle Scholar
  12. Ishikawa F, Drake CJ, Yang S et al (2003) Transplanted human cord blood cells give rise to hepatocytes in engrafted mice. Ann NY Sci 996:174–185CrossRefGoogle Scholar
  13. Kurtzberg J, Laughlin M, Graham ML et al (1996) Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients. N Engl J Med 335:157–166CrossRefGoogle Scholar
  14. Lazaro CA, Croager EJ, Mitchell C et al (2003) Establishment, characterization, and long-term maintenance of cultures of human fetal hepatocytes. Hepatology 38:1095–1106CrossRefGoogle Scholar
  15. Lee KD, Kuo TK, Whang-Peng J et al (2004) In vitro hepatic differentiation of human mesenchymal stem cells. Hepatology 40:1275–1284CrossRefGoogle Scholar
  16. Mayani H, Lansdorp PM (1998) Biology of human umbilical cord blood-derived hematopoietic stem/progenitor cells. Stem Cells 16:153–165CrossRefGoogle Scholar
  17. McAdams TA, Miller WM, Papoutsakis ET (1996) Hematopoietic cell culture therapies. Trends Biotechnol 14:388–396CrossRefGoogle Scholar
  18. Mossman T (1983) Rapid colorimetric assay of cellular growth and survival: a application to proliferation and cytotoxicity assays. J Immunol Method 65:55–63CrossRefGoogle Scholar
  19. Nakahata T, Ogawa M (1982) Hemopoietic colony-forming cells in umbilical cord blood with extensive capability to generate mono- and multipotential hemopoietic progenitors. J Clin Invest 70:1324–1328CrossRefGoogle Scholar
  20. Oh SH, Miyazaki M, Kouchi H et al (2000) Hepatocyte growth factor induces differentiation of adult rat bone marrow cells into a hepatocyte lineage in vitro. Biochem Biophys Res Commun 279:500–504CrossRefGoogle Scholar
  21. Qin AL, Zhou XQ, Zhang WQ et al (2004) Characteriation an enrichment of hepatic progenitor cells in adult liver. World J Gastroenterol 10:1480–1486Google Scholar
  22. Rosenberg IL, Russel CW, Giles GR (1978) Cell viability studies on the exfoliated colonic cancer cell. British J Sur 65:188–190CrossRefGoogle Scholar
  23. Sakai Y, Jiang J, Kojima N et al (2002) Enhanced in vitro maturation of fetal mouse liver cells with oncostatin M, nicotinamide, and dimethyl sulfoxide. Cell Transplant 11:435–441Google Scholar
  24. Seo MJ, Suh SY, Bae YC et al (2005) Differentiation of human adipose stromal cells into hepatic lineage in vitro and in vivo. Biochem Biophys Res Commun 328:258–264CrossRefGoogle Scholar
  25. Talens R, Bonora A, Jover R et al (2006) Hepatogenic differentiation of human mesenchymal stem cells from adipose tissue in comparison with bone marrowmesenchymal stem cells. World J Gastroenterol 12:5834–5845Google Scholar
  26. Trey C, Davidson CS (1970) The management of fulminant hepatic failure. Prog Liver Dis 3:282–298Google Scholar
  27. Visconti RT, Bonora A, Jover R et al (2006) Hepatogenic differentiation of human mesenchymal stem cells from adipose tissue in comparison with bone marrow mesenchymal stem cells. World J Gastroenterol 28:5834–5845Google Scholar
  28. Wagner JE, Kernan NA, Steinbuch M et al (1995) Allogeneic sibling umbilical-cord-blood transplantation in children with malignant and non-malignant disease. Lancet 346:214–219CrossRefGoogle Scholar
  29. Wang X, Ge S, McNamara G et al (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 101:4201–4208CrossRefGoogle Scholar
  30. Wang PP, Wang JH, Yan P et al (2004) Expression of hepatocyte-like phenotypes in bone marrow stromal cells after HGF induction. Biochem Biophys Res Commun 320:712–716CrossRefGoogle Scholar
  31. Wells JM, Melton DA (2000) Early mouse endoderm is patterned by soluble factors from adjacent germ layers. Development 127:1563–1572Google Scholar
  32. Yu S, Li C, Xin-Guo H et al (2007) Differentiation of bone marrow-derived mesenchymal stem cell from diabetic patients into insulin-producing cells in vitro. Chinese Med J 120:771–776Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • S. Sellamuthu
    • 1
  • R. Manikandan
    • 2
  • R. Thiagarajan
    • 3
  • G. Babu
    • 1
  • D. Dinesh
    • 1
  • D. Prabhu
    • 1
  • C. Arulvasu
    • 1
    Email author
  1. 1.Department of ZoologyUniversity of MadrasChennaiIndia
  2. 2.Department of Animal Health and ManagementAlagappa UniversityKaraikudiIndia
  3. 3.Department of Biotechnology, School of Chemical and BiotechnologySastra University ThanjavurIndia

Personalised recommendations