Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Generation of histocompatible tissues using nuclear transplantation

Abstract

Nuclear transplantation (therapeutic cloning) could theoretically provide a limitless source of cells for regenerative therapy. Although the cloned cells would carry the nuclear genome of the patient, the presence of mitochondria inherited from the recipient oocyte raises questions about the histocompatibility of the resulting cells. In this study, we created bioengineered tissues from cardiac, skeletal muscle, and renal cells cloned from adult bovine fibroblasts. Long-term viability was demonstrated after transplantation of the grafts into the nuclear donor animals. Reverse transcription-PCR (RT-PCR) and western blot analysis confirmed that the cloned tissues expressed tissue-specific mRNA and proteins while expressing a different mitochondrial DNA (mtDNA) haplotype. In addition to creating skeletal muscle and cardiac “patches”, nuclear transplantation was used to generate functioning renal units that produced urinelike fluid and demonstrated unidirectional secretion and concentration of urea nitrogen and creatinine. Examination of the explanted renal devices revealed formation of organized glomeruli- and tubule-like structures. Delayed-type hypersensitivity (DTH) testing in vivo and Elispot analysis in vitro suggested that there was no rejection response to the cloned renal cells. The ability to generate histocompatible cells using cloning techniques addresses one of the major challenges in transplantation medicine.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Retrieved muscle tissues.
Figure 2: RT-PCR and western blot analyses.
Figure 3: Tissue-engineered renal units.
Figure 4: Characterization of renal explants.
Figure 5: RT-PCR analyses (top panel) confirming the transcription of AQP1, AQP2, Tamm–Horsfall, and synaptopodin genes exclusively in the cloned group (Cls).
Figure 6: Elispot analyses of the frequencies of T cells that secrete IFNγ after primary and secondary stimulation with allogeneic renal cells, cloned renal cells, or nuclear donor fibroblasts.

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. Lanza, R.P. et al. The ethical reasons for stem cell research. Science 293, 1299 (2001).

  2. Atala, A. & Lanza, R.P. Methods of Tissue Engineering (Academic Press, San Diego, CA, 2001).

    Google Scholar 

  3. Atala, A. & Mooney, D. Synthetic Biodegradable Polymer Scaffolds (Birkhaüser, Boston, MA, 1997).

    Google Scholar 

  4. Machluf, M. & Atala, A. Emerging concepts for tissue and organ transplantation. Graft 1, 31–37 (1998).

    Google Scholar 

  5. Lanza, R.P., Cibelli, J.B. & West, M.D. Prospects for the use of nuclear transfer in human transplantation. Nat. Biotechnol. 17, 1171–1174 (1999).

    CAS  Article  Google Scholar 

  6. Evans, M.J. et al. Mitochondrial DNA genotypes in nuclear transfer-derived cloned sheep. Nat. Genet. 23, 90–93 (1999).

    CAS  Article  Google Scholar 

  7. Hiendleder, S., Schmutz, S.M., Erhardt, G., Green, R.D. & Plante, Y. Transmitochondrial differences and varying levels of heteroplasmy in nuclear transfer cloned cattle. Mol. Reprod. Dev. 54, 24–31 (1999).

    CAS  Article  Google Scholar 

  8. Steinborn, R. et al. Mitochondrial DNA heteroplasmy in cloned cattle produced by fetal and adult cell cloning. Nat. Genet. 25, 255–257 (2000).

    CAS  Article  Google Scholar 

  9. Vyas, J.M. et al. Biochemical specificity of H-2M3a: stereospecificity and space-filling requirement at position 1 maintains N-formyl peptide binding. J. Immunol. 149, 3605–3611 (1992).

    CAS  PubMed  Google Scholar 

  10. Morse, M. et al. The COI mitochondrial gene encodes a minor histocompatibility antigen presented by H2-M3. J. Immunol. 156, 3301–3307 (1996).

    CAS  PubMed  Google Scholar 

  11. Loveland, B., Wang, C.R., Yonekawa, H., Hermel, E. & Lindahl, K.F. Maternally transmitted histocompatibility antigens of mice: a hydrophobic peptide of a mitochondrial encoded protein. Cell 60, 971–980 (1990).

    CAS  Article  Google Scholar 

  12. Davies, J.D. et al. Generation of T cells with lytic specificity for atypical antigens. I. A mitochondrial antigen in the rat. J. Exp. Med. 173, 823–832 (1991).

    CAS  Article  Google Scholar 

  13. Lysaght, M.J. Maintenance dialysis population dynamics: current trends and long-term implications. J. Am. Soc. Nephrol. 13, S37–S40 (2002).

    PubMed  Google Scholar 

  14. Amiel, G.E. & Atala, A. Current and future modalities for functional renal replacement. Urol. Clin. 26, 235–246 (1999).

    CAS  Article  Google Scholar 

  15. Humes, H.D., Buffington, D.A., MacKay, S.M., Funke, A.J. & Weitzel, W.F. Replacement of renal function in uremic animals with a tissue-engineered kidney. Nat. Biotechnol. 17, 451–455 (1999).

    CAS  Article  Google Scholar 

  16. Cieslinski, D.A. & Humes, H.D. Tissue engineering of a bioartificial kidney. Biotechnol. Bioeng. 43, 781–791 (1994).

    Article  Google Scholar 

  17. MacKay, S.M., Kunke, A.J., Buffington, D.A. & Humes, H.D. Tissue engineering of a bioartificial renal tubule. ASAIO J. 44, 179–183 (1998).

    CAS  Article  Google Scholar 

  18. Aebischer, P., Ip, T.K., Panol, G. & Galletti, P.M. The bioartificial kidney: progress towards an ultrafiltration device with renal epithelial cells processing. Life Support Syst. 5, 159–168 (1987).

    CAS  PubMed  Google Scholar 

  19. Ip, T., Aebischer, P. & Galletti, P.M. Cellular control of membrane permeability. Implications for a bioartificial renal tubule. ASAIO Trans. 34, 351–355 (1988).

    CAS  PubMed  Google Scholar 

  20. Humes, H.D. Renal replacement devices. in Principles of Tissue Engineering; Edn. 2 (eds Lanza, R.P., Langer, R. & Vacanti, J.) 645–653 (Academic Press, San Diego, 2000).

    Chapter  Google Scholar 

  21. Amiel, A., Yoo, J. & Atala, A. Renal therapy using tissue engineered constructs and gene delivery. World J. Urol. 18, 71–79 (2000).

    CAS  Article  Google Scholar 

  22. Lanza, R.P., Hayes, J.L. & Chick, W.L. Encapsulated cell technology. Nat. Biotechnol. 14, 1107–1111 (1996).

    CAS  Article  Google Scholar 

  23. Kuhtreiber, W.M., Lanza, R.P. & Chick, W.L. (eds). Cell Encapsulation Technology and Therapeutics (Birkhauser, Boston, 1998).

    Google Scholar 

  24. Lanza, R.P & Chick, W.L. (eds). Immunoisolation of Pancreatic Islets (R.G. Landes, Austin, TX, 1994).

    Google Scholar 

  25. Joki, T. et al. Continuous release of endostatin from microencapsulated engineered cells for tumor therapy. Nat. Biotechnol. 19, 35–39 (2001).

    CAS  Article  Google Scholar 

  26. Qiao, J., Sakurai, H. & Nigam, S.K. Branching morphogenesis independent of mesenchymal-epithelial contact in the developing kidney. Proc. Natl. Acad. Sci. USA 96, 7330–7335 (1999).

    CAS  Article  Google Scholar 

  27. Humes, H.D., Krauss, J.C., Cieslinski, D.A. & Funke, A.J. Tubulogenesis from isolated single cells of adult mammalian kidney: clonal analysis with a recombinant retrovirus. Am. J. Physiol. 271, F42–F49 (1996).

    CAS  PubMed  Google Scholar 

  28. Lanza, R.P, Langer, R. & Vacanti, J. Principles of Tissue Engineering (Academic Press, San Diego, CA, 2000).

    Google Scholar 

  29. Atala, A. Future perspectives in reconstructive surgery using tissue engineering. Urol. Clin. 26, 157–166 (1999).

    CAS  Article  Google Scholar 

  30. Santavirta, S. et al. Immune response to polyglycolic acid implants. J. Bone Joint Surg. Br. 72, 597–600 (1990).

    CAS  Article  Google Scholar 

  31. Paivarinta, U. et al. Intraosseous cellular response to biodegradable fracture fixation screws made of polyglycolide or polylactide. Arch. Orthop. Trauma Surg. 112, 71–74 (1993).

    CAS  Article  Google Scholar 

  32. Bostman, O.M. & Pihlajamaki, H.K. Adverse tissue reactions to bioabsorbable fixation devices. Clin. Orthop. 371, 216–227 (2000).

    Article  Google Scholar 

  33. Ruuskanen, M. et al. Evaluation of self-reinforced polyglycolide membrane implanted in the subcutis of rabbits. Ann. Chir. Gynaecol. 88 308–312 (1999).

    CAS  PubMed  Google Scholar 

  34. Weiler, A., Helling, H.J., Kirch, U., Zirbes, T.K. & Rehm, K.E. Foreign-body fracture fixation: experimental study in sheep. J. Bone Joint Surg. Br. 78, 369–376 (1996).

    CAS  Article  Google Scholar 

  35. Pariente, J.L., Kim, B.S. & Atala, A. In vitro compatibility assessment of naturally-derived and synthetic biomaterials using normal human urothelial cells. J. Biomed. Mat. Res. 55, 33–39 (2001).

    CAS  Article  Google Scholar 

  36. Rosenberger, G. Clinical Examination of Cattle (Verlag Paul Parey, Berlin, 1979), pp.275–281.

    Google Scholar 

  37. Smith, B.P. Large Animal Internal Medicine: Diseases of Horses, Cattle, Sheep and Goats, Edn. 2 pp. 467–469 (Mosby, St. Louis, 1996).

    Google Scholar 

  38. Lanza, R.P. et al. Cloning of an endangered species (Bos gaurus) using interspecies nuclear transfer. Cloning 2, 79–90 (2000).

    CAS  Article  Google Scholar 

  39. Fischer Lindahl, K., Hermel, E., Loveland, B.E. & Wang, C.R. Maternally transmitted antigen of mice. Ann. Rev. Immunol. 9, 351–372 (1991).

    CAS  Article  Google Scholar 

  40. Hadley, G.A., Linders, B. & Mohanakumar, T. Immunogenicity of MHC class I alloantigens expressed on parenchymal cells in the human kidney. Transplantation 54, 537–542 (1992).

    CAS  Article  Google Scholar 

  41. Yard, B.A. et al. Analysis of T cell lines from rejecting renal allografts. Kidney Int. 43, S133–S138 (1993).

    Google Scholar 

  42. Bailey, D.W. Genetics of histocompatibility in mice. I. New loci and congenic lines. Immunogenetics 2, 249–256 (1975).

    Article  Google Scholar 

  43. Mohanakumar, T. The Role of MHC and Non-MHC Antigens in Allograft Immunity pp. 1–115 (R.G. Landes Company, Austin, TX, 1994).

    Google Scholar 

  44. Lanza, R.P., Cibelli, J.B. & West, M.D. Human therapeutic cloning. Nat. Med. 5, 975–977 (1999).

    CAS  Article  Google Scholar 

  45. Cibelli, J.B. et al. Somatic cell nuclear transfer in humans: pronuclear and early embryonic development. e-biomed: J. Regen. Med. 2, 25–31 (2001).

    Article  Google Scholar 

  46. Lanza, R.P. et al. The ethical validity of using nuclear transfer in human transplantation. JAMA 284, 3175–3179 (2000).

    CAS  Article  Google Scholar 

  47. Itskovitz-Eldor, J. et al. Differentiation of human embryonic stem cells into embryoid bodies comprising the three embryonic germ layers. Mol. Med. 5, 88–95 (2000).

    Article  Google Scholar 

  48. Schuldiner, M., Yanuka, O., Itskovitz-Eldor, J., Melton, D.A. & Benvenisty, N. Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci USA 97, 11307–11312 (2000).

    CAS  Article  Google Scholar 

  49. Kaufman, D.S. et al. Directed differentiation of human embryonic stem cells into hematopoietic colony forming cells. Blood 94 (Suppl. 1, part 1 of 2), 34a (1999).

    Google Scholar 

  50. Reubinoff, B.E. et al. Neural progenitors from human embryonic stem cells. Nat. Biotechnol. 19, 1134–1140 (2001).

    CAS  Article  Google Scholar 

  51. Reubinoff, B.E., Pera, M.F., Fong, C.Y., Trounson, A. & Bongso, A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat. Biotechnol. 18, 399–404 (2000).

    CAS  Article  Google Scholar 

  52. Cibelli, J.B. et al. Parthenogenetic stem cells in nonhuman primates. Science 295, 819 (2002).

  53. Oberpenning, F.O., Meng, J., Yoo, J. & Atala, A. De novo reconstitution of a functional urinary bladder by tissue engineering. Nat. Biotechnol. 17, 149–155 (1999).

    CAS  Article  Google Scholar 

  54. Kaushal, S. et al. Circulating endothelial cells for tissue engineering of small diameter vessels. Nat. Med. 7, 1035–1040 (2001).

    CAS  Article  Google Scholar 

  55. Presicce, G.A. & Yang, X. Parthenogenetic development of bovine oocytes matured in vitro for 24 hr and activated by ethanol and cycloheximide. Mol. Reprod. Dev. 38, 380–385 (1994).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank Jose B. Cibelli, Frederick F. Hess (DVM), R. T. Duby, and the Department of Veterinary Sciences, University of Massachusetts, Amherst. We also thank Kyung-Ha Kang (Brigham & Women's Hospital), Wendy Nevala (Mayo Clinic), and Maria P. Bayona-Bafaluy (University of Miami) for their help with the histologic, Elispot, and molecular analyses. This research was supported in part by National Institutes of Health grants AI-16052 (to P.J.W.) and RO1DK57260 (to A.A.).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Anthony Atala.

Ethics declarations

Competing interests

R.P.L., C.B., and M.D.W. are employed by Advanced Cell Technology, which is pursuing therapeutic cloning.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Lanza, R., Chung, H., Yoo, J. et al. Generation of histocompatible tissues using nuclear transplantation. Nat Biotechnol 20, 689–696 (2002). https://doi.org/10.1038/nbt703

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt703

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing