Intrauterine xenotransplantation of human Wharton jelly-derived mesenchymal stem cells into the liver of rabbit fetuses: A preliminary study for in vivo expression of the human liver genes

Document Type: Original Article


1 Department of Biology, College of Science, Fars Science and Research Branch, Islamic Azad University, Fars, Iran

2 Department of Biology, College of Science, Shiraz Branch, Islamic Azad University, Shiraz, Iran

3 Stem Cells Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

4 Department of Medical Genetics, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

5 Department of Pharmacology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

6 6 Department of Clinical Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran


Objective(s): End-stage hepatic failure is a potentially life-threatening condition for which orthotopic liver transplantation is the only effective treatment. However, a shortage of available donor organs for transplantation each year results in the death of many patients waiting for liver transplantation. Xenotransplantation, or the transplantation of cells, tissues, or organs between different species, was proposed as a possible solution to the worldwide shortage of human organs and tissues for transplantation. The purpose of this preliminary study was to reconstruct human liver tissue by xenotransplantation of human Wharton jelly mesenchymal stem cells (hWJ-MSCs) into fetal rabbit.
Materials and Methods: Isolation and confirmation of hWJ-MSCs from human umbilical cord was performed. Eight rabbits at gestational day 14 were anesthetized. All rabbits carried pregnancies to term yielding 40 rabbit fetuses. Intrauterine injection of hWJ-MSCs was performed in 24 fetuses. Twenty-seven fetuses were born alive. Ten liver samples from injected fetuses were sampled, eight rabbits 3 days after birth and two rabbits 21 days after birth. The non-injected fetuses served as positive control. Fetuses of non-injected rabbits were negative controls. Using real-time polymerase chain reaction (RT-PCR), mRNA expression of albumin (ALB), α-fetoprotein (AFP), hepatic nuclear factor 4 (HNF4), and CYP2B6 (CYP) were detected in liver samples.
Results: The human ALB, AFP, HNF4, and CYP mRNAs were expressed in the injected sampled fetuses by hWJ-MSCs into fetuses of rabbits in utero.
Conclusion: Developing xenotransplantation of hWJ-MSCs into rabbit uterus can introduce an applied approach for producing human liver tissue in rabbits.


Main Subjects

1. Desmet VJ. The amazing universe of hepatic microstructure. Hepatology 2009; 50:333-344.

2. Kochanek KD, Murphy SL, Xu J, Tejada-Vera B. Deaths: final data for 2014. Natl Vital Stat Rep 2016; 65:1-122.

3. Lerut J, Porte RJ, Dutkowski P. Orthotopic liver transplantation. In: Clavien P-A, Sarr MG, Fong Y, Miyazaki M, editors. Atlas of upper gastrointestinal and hepato-pancreato-biliary surgery. Berlin, Heidelberg: Springer Berlin Heidelberg; 2016. p. 519-534.

4. Taub R. Liver regeneration: from myth to mechanism. Nat Rev Mol Cell Biol 2004; 5:836-847.

5. Hughes RD, Mitry RR, Dhawan A. Current status of hepatocyte transplantation. Transplantation 2012; 93:342-347.

6. Lv X, Miao Y, Ren X, Wu J, Zhang M, Gu Y. The study and implementation of liver volume measuring method based on 3-dimensional reconstruction technology. Optik 2015; 126:1534-1539.

7. Sakai Y, Yamanouchi K, Ohashi K, Koike M, Utoh R, Hasegawa H, et al. Vascularized subcutaneous human liver tissue from engineered hepatocyte/fibroblast sheets in mice. Biomaterials 2015; 65:66-75.

8. Takebe T, Sekine K, Enomura M, Koike H, Kimura M, Ogaeri T, et al. Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature 2013; 499:481-484.

9. Zhang W, Zhuang A, Gu P, Zhou H, Fan X. A review of the three-dimensional cell culture technique: Approaches, advantages and applications. Curr Stem Cell Res Ther 2016; 11:370-380.

10. Knosalla C, Cooper DK. Xenotransplantation and tolerance. Front Biosci 2002; 1:d1280-d1287.

11. De Los Angeles A, Ferrari F, Xi R, Fujiwara Y, Benvenisty N, Deng H, et al. Hallmarks of pluripotency. Nature 2015; 525:469-478.

12. Bongso A, Fong C-Y. The therapeutic potential, challenges and future clinical directions of stem cells from the Wharton's jelly of the human umbilical cord. Stem Cell Rev 2013; 9:226-240.

13. Kalaszczynska I, Ferdyn K. Wharton's jelly derived mesenchymal stem cells: future of regenerative medicine? Recent findings and clinical significance. Biomed Res Int 2015;2015:430847.

14. Lin HD, Bongso A, Gauthaman K, Biswas A, Choolani M, Fong CY. Human Wharton's jelly stem cell conditioned medium enhances freeze-thaw survival and expansion of cryopreserved CD34+ cells. Stem Cell Rev 2013; 9:172-183.

15. La Rocca G, Anzalone R, Corrao S, Magno F, Loria T, Lo Iacono M, et al. Isolation and characterization of Oct-4+/HLA-G+ mesenchymal stem cells from human umbilical cord matrix: differentiation potential and detection of new markers. Histochem Cell Biol 2009; 131:267-282.

16. Fong CY, Gauthaman K, Cheyyatraivendran S, Lin HD, Biswas A, Bongso A. Human umbilical cord Wharton's jelly stem cells and its conditioned medium support hematopoietic stem cell expansion ex vivo. J Cell Biochem 2012;113:658-668.

17. Hollweck T, Hartmann I, Eblenkamp M, Wintermantel E, Reichart B, Überfuhr P, et al. Cardiac differentiation of human Wharton’s jelly stem cells—experimental comparison of protocols. Open Tissue Eng Regen Med J 2011;4:95-102.

18. Wang L, Ott L, Seshareddy K, Weiss ML, Detamore MS. Musculoskeletal tissue engineering with human umbilical cord mesenchymal stromal cells. Regen Med 2010; 6:95-109.

19. Yan M, Sun M, Zhou Y, Wang W, He Z, Tang D, et al. Conversion of human umbilical cord mesenchymal stem cells in Wharton's jelly to dopamine neurons mediated by the Lmx1a and neurturin in vitro: potential therapeutic application for Parkinson's disease in a rhesus monkey model. PLoS One 2013; 8:e64000.

20. Wang HW, Lin LM, He HY, You F, Li WZ, Huang TH, et al. Human umbilical cord mesenchymal stem cells derived from Wharton's jelly differentiate into insulin-producing cells in vitro. Chin Med J 2011;124:1534-1539.

21. Zhang Y-N, Lie P-C, Wei X. Differentiation of mesenchymal stromal cells derived from umbilical cord Wharton's jelly into hepatocyte-like cells. Cytotherapy 2009; 11:548-558.

22. Bongso A, Fong C-Y, Gauthaman K. Taking stem cells to the clinic: Major challenges. J Cell Biochem 2008; 105:1352-1360.

23. Weiss ML, Medicetty S, Bledsoe AR, Rachakatla RS, Choi M, Merchav S, et al. Human umbilical cord matrix stem cells: preliminary characterization and effect of transplantation in a rodent model of Parkinson's disease. Stem Cells 2006; 24:781-792.

24. Fong CY, Chak LL, Biswas A, Tan JH, Gauthaman K, Chan WK, et al. Human Wharton's jelly stem cells have unique transcriptome profiles compared to human embryonic stem cells and other mesenchymal stem cells. Stem Cell Rev 2011; 7:1-16.

25. Kim D-W, Staples M, Shinozuka K, Pantcheva P, Kang S-D, Borlongan CV. Wharton’s jelly-derived mesenchymal stem cells: phenotypic characterization and optimizing their therapeutic potential for clinical applications. Int J Mol Sci 2013; 14:11692-11712.

26. Olthof PB, Heger M, van Lienden KP, de Bruin K, Bennink RJ, van Gulik TM. Comparable liver function and volume increase after portal vein embolization in rabbits and humans. Surgery 2017; 161:658-665.

27. Osmanski BF, Lecarpentier E, Montaldo G, Tsatsaris V, Chavatte-Palmer P, Tanter M. Discriminative imaging of maternal and fetal blood flow within the placenta using ultrafast ultrasound. Sci Rep 2015;5:13394.

28. Tsagias N, Koliakos I, Karagiannis V, Eleftheriadou M, Koliakos GG. Isolation of mesenchymal stem cells using the total length of umbilical cord for transplantation purposes. Transfus Med 2011; 21:253-261.

29. Lanning D, Zhu X, Zhai SK, Knight KL. Development of the antibody repertoire in rabbit: gut-associated lymphoid tissue, microbes, and selection. Immunol Rev 2000; 175:214-228.

30. Lopez-Tello J, Arias-Alvarez M, Jimenez-Martinez MA, Garcia-Garcia RM, Rodriguez M, Lorenzo Gonzalez PL, et al. Competition for materno-fetal Resource partitioning in a rabbit model of undernourished pregnancy. PLoS One 2017; 12:e0169194.

31. Schoeberlein A, Holzgreve W, Dudler L, Hahn S, Surbek DV. In utero transplantation of autologous and allogeneic fetal liver stem cells in ovine fetuses. Am J Obstet Gynecol 2004; 191:1030-1036.

32. Young AJ, Holzgreve W, Dudler L, Schoeberlein A, Surbek DV. Engraftment of human cord blood-derived stem cells in preimmune ovine fetuses after ultrasound-guided in utero transplantation. Am J Obstet Gynecol 2003; 189:698-701.

33. Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL. Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 2012; 13:134.

34. Bárcia RN, Santos JM, Filipe M, Teixeira M, Martins JP, Almeida J, et al. What makes umbilical cord tissue-derived mesenchymal stromal cells superior immunomodulators when compared to bone marrow derived mesenchymal stromal cells? Stem Cells Int 2015; 2015:583984.

35. Troyer DL, Weiss ML. Wharton's jelly-derived cells are a primitive stromal cell population. Stem Cells 2008; 26:591-599.

36. Abellaneda JM, Ramis G, Martínez-Alarcón L, Majado MJ, Quereda JJ, Herrero-Medrano JM, et al. Generation of human-to-pig chimerism to induce tolerance through transcutaneous in utero injection of cord blood-derived mononuclear cells or human bone marrow mesenchymals cells in a preclinical program of liver xenotransplantation: preliminary results. Transplant Proc 2012; 44:1574-1578.

37. Weiss ML, Anderson C, Medicetty S, Seshareddy KB, Weiss RJ, VanderWerff I, et al. Immune properties of human umbilical cord Wharton's jelly-derived cells. Stem Cells 2008; 26:2865-2874.

38. Campard D, Lysy PA, Najimi M, Sokal EM. Native umbilical cord matrix stem cells express hepatic markers and differentiate into hepatocyte-like cells. Gastroenterology 2008; 134:833-848.

39. Khodabandeh Z, Vojdani Z, Talaei-Khozani T, Jaberipour M, Hosseini A, Bahmanpour S. Comparison of the expression of hepatic genes by human Wharton's jelly mesenchymal stem cells cultured in 2D and 3D collagen culture systems. Iran J Med Sci 2016; 41:28-36.

40. La Rocca G, Lo Iacono M, Corsello T, Corrao S, Farina F, Anzalone R. Human Wharton's jelly mesenchymal stem cells maintain the expression of key immunomodulatory molecules when subjected to osteogenic, adipogenic and chondrogenic differentiation in vitro: new perspectives for cellular therapy. Curr Stem Cell Res Ther 2013 8:100-113.

41. Razeghian Jahromi I, Mehrabani D, Mohammadi A, Ghahremani Seno MM, Dianatpour M, Zare S, et al. Emergence of signs of neural cells after exposure of bone marrow-derived mesenchymal stem cells to fetal brain extract. Iran J Basic Med Sci 2017; 20:301-307.

42. Khajehahmadi Z, Mehrabani D, Ashraf MJ, Rahmanifar F, Tanideh N, Tamadon A, et al. Healing effect of conditioned media from bone marrow-derived stem cells in thioacetamide-induced liver fibrosis of rat. J Med Sci 2016; 16:7-15.

43. Rahmanifar F, Tamadon A, Mehrabani D, Zare S, Abasi S, Keshavarz S, et al. Histomorphometric evaluation of treatment of rat azoospermic seminiferous tubules by allotransplantation of bone marrow-derived mesenchymal stem cells. Iran J Basic Med Sci 2016; 19:653-661.

44. Aliborzi G, Vahdati A, Mehrabani D, Ebrahim Hosseini S, Tamadon A. Isolation, characterization and growth kinetic comparison of bone marrow and adipose tissue mesenchymal stem cells of Guinea pig. Int J Stem Cells 2015; 9:115-123.

45. Razeghian Jahromi I, Mehrabani D, Mohammadi A, Dianatpour M, Tamadon A, Zare S, et al. The effect of fetal rat brain extract on morphology of bone marrow-derived mesenchymal stem cells. Comp Clin Pathol 2016; 25:343-349.

46. Tamadon A, Mehrabani D, Rahmanifar F, Raayat Jahromi A, Panahi M, Zare S, et al. Induction of spermatogenesis by bone marrow-derived mesenchymal stem cells in busulfan-induced azoospermia in hamster. Int J Stem Cells 2015; 8:134-145.

47. Mehrabani D, Khodakaram-Tafti A, Asadi-Yousefabad SL, Dianatpour M, Zare S, Tamadon A, et al. Effect of age and passage on canine bone marrow derived mesenchymal stem cells. Online J Vet Res 2015; 19:663-671.

48. Asadi-Yousefabad S-L, Khodakaram-Tafti A, Dianatpour M, Mehrabani D, Zare S, Tamadon A, et al. Genetic evaluation of bone marrow-derived mesenchymal stem cells by a modified karyotyping method. Comp Clin Pathol 2015; 24:1361-1366.

49. Hajihoseini M, Vahdati A, Ebrahim Hosseini S, Mehrabani D, Tamadon A. Induction of spermatogenesis after stem cell therapy of azoospermic guinea pigs. Vet Arh 2017; 87:333-350.

50. Mehrabani D, Rabiee M, Tamadon A, Zare S, Jahromi IR, Dianatpour M, et al. The growth kinetic, differentiation properties, karyotyping, and characterization of adipose tissue-derived stem cells in hamster. Comp Clin Pathol 2016; 25:1017-1022.

51. Shaterzadeh-Yazdi H, Mehrabani D, Khodakaram-Tafti A, Dianatpour M, Zare SH, Tamadon A, et al. Osteogenic potential of subcutaneous adipose-derived stem cells in a rabbit model. Online J Vet Res 2015; 19:436-445.

52. Mehrabani D, Hassanshahi MA, Tamadon A, Zare S, Keshavarz S, Rahmanifar F, et al. Adipose tissue-derived mesenchymal stem cells repair germinal cells of seminiferous tubules of busulfan-induced azoospermic rats. J Hum Reprod Sci 2015; 8:103-110.

53. Tamadon A, Mehrabani D, Zarezadeh Y, Rahmanifar F, Dianatpour M, Zare S. Caprine endometrial mesenchymal stromal stem cell: multi-lineage potential, characterization and growth kinetics in breeding and anestrous stages. Vet Med Int 2017; 2017:5052801.

54. Mehrabani D, Rahmanifar F, Mellinejad M, Tamadon A, Dianatpour M, Zare S, et al. Isolation, culture, characterization, and adipogenic differentiation of heifer endometrial mesenchymal stem cells. Comp Clin Pathol 2015; 24:1159-1164.

55. Mehrabani D, Mahdiyar P, Torabi K, Robati R, Zare S, Dianatpour M, et al. Growth kinetics and characterization of human dental pulp stem cells: Comparison between third molar and first premolar teeth. J Clin Exp Dent 2017;9:e172-e177.

56. Mehrabani D, Bahrami Nazarabadi R, Dianatpour M, Vahdati A, Tamadon A, Kasraeian M, et al. Growth kinetics, characterization and plasticity of human menstrual blood stem cells. Iran J Med Sci 2015; 41:132-139.