Skip to main content

Advertisement

Log in

Side Population Cells Derived from Adult Human Liver Generate Hepatocyte-like Cells In Vitro

  • Published:
Digestive Diseases and Sciences Aims and scope Submit manuscript

Abstract

We sought to determine whether hepatic side population (SP) cells derived from adult human liver possess the potential of a novel candidate hepatic stem cell. Human cadaveric donor liver was subjected to collagenase perfusion and hepatocytes were separated from nonparenchymal cells by differential centrifugation. SP cells were isolated from the nonparenchymal portion after Hoechst 33342 staining. Since CD45 is a panleukocyte antigen, CD45-negative SP cells were separated from the vast majority of CD45-positive SP cells (90%), and hepatic growth medium was used to culture both groups. Both CD45-negative and CD45-positive hepatic SP cells generated colonies in the hepatic growth medium in 2–3 weeks. The colonies yielded large cells morphologically consistent with human hepatocytes, demonstrating granule-rich cytoplasm, dense, often double nuclei, and intracellular lipofuscin pigment. The cultured cells from both sources were positive for markers of human hepatocytes: HepPar, cytokeratin 8 (CK8), and human albumin. Reverse transcriptase–polymerase chain reaction (RT-PCR) performed on both groups demonstrated positivity for additional liver markers including human albumin, CK18, α-1 anti-trypsin, and the human cytochrome P450 enzyme CYP2B6. Double immunostaining (CD45 and HepPar) and RT-PCR confirmed that the hepatocyte-like cells derived from the CD45-negative SP cells acquired HepPar positivity but had no detectable CD45 antigen expression. In contrast, the cultured cells derived from the CD45-positive SP cells also acquired HepPar positivity, but only a minimal fraction expressed the CD45 antigen. We conclude that hepatic SP cells derived from the nonparenchymal portion of human liver are a potential source of human hepatocytes irrespective of their CD45 status, and further animal studies will be required to assess their regenerative potential.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC: Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 183:1797–1806, 1996

    Article  PubMed  Google Scholar 

  2. Goodell MA, Rosenzweig M, Kim H, Marks DF, DeMaria M, Paradis G, Grupp SA, Sieff CA, Mulligan RC, Johnson RP: Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat Med 3:1337–1345, 1997

    Article  PubMed  Google Scholar 

  3. Michalopoulos GK, DeFrances MC: Liver regeneration. Science 276:60–66, 1997

    Article  PubMed  Google Scholar 

  4. Forbes S, Vig P, Poulsom R, Thomas H, Alison M: Hepatic stem cells. J Pathol 197:510–518, 2002

    Article  PubMed  Google Scholar 

  5. Oh SH, Hatch HM, Petersen BE: Hepatic oval ‘stem’ cell in liver regeneration. Semin Cell Dev Biol 13:405–409, 2002

    Article  PubMed  Google Scholar 

  6. Petersen BE: Hepatic “stem” cells: coming full circle. Blood Cells Mol Dis 27:590–600, 2001

    Article  PubMed  Google Scholar 

  7. Suzuki A, Nakauchi H: Identification and propagation of liver stem cells. Semin Cell Dev Biol 13:455–461, 2002

    Article  PubMed  Google Scholar 

  8. Uchida N, Fujisaki T, Eaves AC, Eaves CJ: Transplantable hematopoietic stem cells in human fetal liver have a CD34(+) side population (SP)phenotype. J Clin Invest 108:1071–1077, 2001

    Article  PubMed  Google Scholar 

  9. Asakura A, Rudnicki MA: Side population cells from diverse adult tissues are capable of in vitro hematopoietic differentiation. Exp Hematol 30:1339–1345, 2002

    Article  PubMed  Google Scholar 

  10. Zhou S, Schuetz JD, Bunting KD, Colapietro AM, Sampath J, Morris JJ, Lagutina I, Grosveld GC, Osawa M, Nakauchi H, Sorrentino BP: The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat Med 7:1028–1034, 2001

    Article  PubMed  Google Scholar 

  11. Zhou S, Morris JJ, Barnes Y, Lan L, Schuetz JD, Sorrentino BP: Bcrp1 gene expression is required for normal numbers of side population stem cells in mice, and confers relative protection to mitoxantrone in hematopoietic cells in vivo. Proc Natl Acad Sci USA 99:12339–12344, 2002

    Article  PubMed  Google Scholar 

  12. Wulf GG, Luo KL, Jackson KA, Brenner MK, Goodell MA: Cells of the hepatic side population contribute to liver regeneration and can be replenished with bone marrow stem cells. Haematologica 88:368–378, 2003

    PubMed  Google Scholar 

  13. Knapp W, Dorken B, Rieber P, Schmidt RE, Stein H, von dem Borne AE: CD antigens 1989. Blood 74:1448–1450, 1989

    PubMed  Google Scholar 

  14. Strom SC, Pisarov LA, Dorko K, Thompson MT, Schuetz JD, Schuetz EG: Use of human hepatocytes to study P450 gene induction. Methods Enzymol 272:388–401, 1996

    PubMed  Google Scholar 

  15. 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: Human umbilical cord blood as a source of transplantable hepatic progenitor cells. Stem Cells 21:217–227, 2002

    Article  Google Scholar 

  16. Selden C, Chalmers SA, Jones C, Standish R, Quaglia A, Rolando N, Burroughs AK, Rolles K, Dhillon A, Hodgson HJ: Epithelial colonies cultured from human explanted liver in subacute hepatic failure exhibit hepatocyte, biliary epithelial, and stem cell phenotypic markers. Stem Cells 21:624–631, 2003

    Article  PubMed  Google Scholar 

  17. Schwartz RE, Reyes M, Koodie L, Jiang Y, Blackstad M, Lund T, Lenvik T, Johnson S, Hu WS, Verfaillie CM: Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J Clin Invest 109:1291–1302, 2002

    Article  PubMed  Google Scholar 

  18. Avital I, Feraresso C, Aoki T, Hui T, Rozga J, Demetriou A, Muraca M: Bone marrow–derived liver stem cell and mature hepatocyte engraftment in livers undergoing rejection. Surgery 132:384–390, 2002

    Article  PubMed  Google Scholar 

  19. Lagasse E, Connors H, Al-Dhalimy M, Reitsma M, Dohse M, Osborne L, Wang X, Finegold M, Weissman IL, Grompe M: Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 6:1229–1234, 2000

    Article  PubMed  Google Scholar 

  20. Petersen BE, Goff JP, Greenberger JS, Michalopoulos GK: Hepatic oval cells express the hematopoietic stem cell marker Thy-1 in the rat. Hepatology 27:433–445, 1998

    Article  PubMed  Google Scholar 

  21. Jones EA, Tosh D, Wilson DI, Lindsay S, Forrester LM: Hepatic differentiation of murine embryonic stem cells. Exp Cell Res 272:15–22, 2002

    Article  PubMed  Google Scholar 

  22. Oh SH, Miyazaki M, Kouchi H, Inoue Y, Sakaguchi M, Tsuji T, Shima N, Higashio K, Namba M: Hepatocyte growth factor induces differentiation of adult rat bone marrow cells into a hepatocyte lineage in vitro. Biochem Biophys Res Commun 279:500–504, 2000

    Article  PubMed  Google Scholar 

  23. Avital I, Inderbitzin D, Aoki T, Tyan DB, Cohen AH, Ferraresso C, Rozga J, Arnaout WS, Demetriou AA: Isolation, characterization, and transplantation of bone marrow-derived hepatocyte stem cells. Biochem Biophys Res Commun 288:156–164, 2001

    Article  PubMed  Google Scholar 

  24. Fiegel HC, Lioznov MV, Cortes-Dericks L, Lange C, Kluth D, Fehse B, Zander AR: Liver-specific gene expression in cultured human hematopoietic stem cells. Stem Cells 21:98–104, 2003

    Article  PubMed  Google Scholar 

  25. Danet GH, Luongo JL, Butler G, Lu MM, Tenner AJ, Simon MC, Bonnet DA: C1qRp defines a new human stem cell population with hematopoietic and hepatic potential. Proc Natl Acad Sci USA 99:10441–10445, 2002

    Article  PubMed  Google Scholar 

  26. Wang X, Willenbring H, Akkari Y, Torimaru Y, Foster M, Al-Dhalimy M, Lagasse E, Finegold M, Olson S, Grompe M: Cell fusion is the principal source of bone-marrow-derived hepatocytes. Nature 422:897–901, 2003

    Article  PubMed  Google Scholar 

  27. Vassilopoulos G, Wang PR, Russell DW: Transplanted bone marrow regenerates liver by cell fusion. Nature 422:901–904, 2003

    Article  PubMed  Google Scholar 

  28. Newsome PN, Johannessen I, Boyle S, Dalakas E, McAulay KA, Samuel K, Rae F, Forrester L, Turner ML, Hayes PC, Harrison DJ, Bickmore WA, Plevris JN. Human cord blood-derived cells can differentiate into hepatocytes in the mouse liver with no evidence of cellular fusion. Gastroenterology 124:1891–1900, 2003

    Article  PubMed  Google Scholar 

  29. Crosby HA, Kelly DA, Strain AJ: Human hepatic stem-like cells isolated using c-kit or CD34 can differentiate into biliary epithelium. Gastroenterology. 120:534–544, 2001

    Article  PubMed  Google Scholar 

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

    PubMed  Google Scholar 

  31. Minervini MI, Demetris AJ, Lee RG, Carr BI, Madariaga J, Nalesnik MA: Utilization of hepatocyte-specific antibody in the immunocytochemical evaluation of liver tumors. Mod Pathol 10:686–692, 1997

    PubMed  Google Scholar 

  32. Moll R, Franke WW, Schiller DL, Geiger B, Krepler R: The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell 31:11–24, 1982

    Article  PubMed  Google Scholar 

  33. Jenkins DD, Streetz K, Tataria M, Sahar D, Kurobe M, Longaker MT, Kay MA, Sylvester KG: Donor-derived, liver-specific protein expression after bone marrow transplantation. Transplantation 78:530–536, 2004

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to John F. Tisdale MD.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hussain, S.Z., Strom, S.C., Kirby, M.R. et al. Side Population Cells Derived from Adult Human Liver Generate Hepatocyte-like Cells In Vitro. Dig Dis Sci 50, 1755–1763 (2005). https://doi.org/10.1007/s10620-005-2933-x

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10620-005-2933-x

Key Words

Navigation