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Direct induction of layered tissues from mouse embryonic stem cells: potential for differentiation into urinary tract tissue

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Abstract

The aim of this study was to induce organized layered tissues with characteristics of the urinary tract from embryonic stem (ES) cells alone. We seeded embryoid bodies (EBs) originating from mouse ES cells onto mono-layered collagen membranes and cultured them in four different media. Group 1 was grown in a mixed medium of keratinocyte serum-free medium (KSFM) and Medium 199, Group 2 in a mixed medium of KSFM and conditioned medium collected from 3T3 fibroblasts, Group 3 in an EB formation medium (control group), and Group 4 in KSFM only (control group). After 28 days, cultured tissues were transplanted into nude mice. Cultured tissues from Groups 1 and 2 formed four-layered structures comprising a stratified epithelium, a submucosal loose connective tissue layer, a smooth muscle cell layer identified immunohistochemically by α-smooth muscle actin, and a deep loose connective tissue layer identical to the adventitia. Immunohistochemistry showed that the epithelia were positive for cytokeratins. Tissues also expressed uroplakin as detected by reverse transcription/polymerase chain reaction. In contrast, specimens from Groups 3 and 4 demonstrated necrotic features. Uroplakin-positive (i.e., urothelium-like) cells developed only in Group 2 in the transplanted culture tissues in nude mice. In addition to inducing organized layered tissues from mouse ES cells directly in vitro, these findings demonstrate that tissues cultured in KSFM plus conditioned medium from 3T3 fibroblasts differentiate into luminal walls similar to those of urinary tract in vivo. These findings suggest a new approach to urinary tract regeneration.

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References

  • Baker LA, Gomez RA (1998) Embryonic development of the ureter and bladder: acquisition of smooth muscle. J Urol 160:545–550

    Article  PubMed  CAS  Google Scholar 

  • Baskin LS, Howard PS, Duckett JW, Snyder HM, Macarak EJ (1993) Bladder smooth muscle cells in culture. I. Identification and characterization. J Urol 149:190–197

    PubMed  CAS  Google Scholar 

  • Baskin LS, Hayward SW, Young PF, Cunha GR (1996) Ontogeny of the rat bladder: smooth muscle and epithelial differentiation. Acta Anat (Basel) 155:163–171

    CAS  Google Scholar 

  • Drake MJ, Fry CH, Eyden B (2006) Structural characterization of myofibroblasts in the bladder. BJU Int 97:29–32

    Article  PubMed  Google Scholar 

  • Drewa T, Sir J, Czajkowski R, Wozniak A (2006) Scaffold seeded with cells is essential in urothelium regeneration and tissue remodeling in vivo after bladder augmentation using in vitro engineered graft. Transplant Proc 38:133–135

    Article  PubMed  CAS  Google Scholar 

  • Frimberger D, Morales N, Gearhart JD, Gearhart JP, Lakshmanan Y (2006) Human embryoid body-derived stem cells in tissue engineering-enhanced migration in co-culture with bladder smooth muscle and urothelium. Urology 67:1298–1303

    Article  PubMed  Google Scholar 

  • Fujiyama C, Masaki Z, Sugihara H (1995) Reconstruction of the urinary bladder mucosa in three-dimensional collagen gel culture: fibroblast-extracellular matrix interactions on the differentiation of transitional epithelial cells. J Urol 153:2060–2067

    Article  PubMed  CAS  Google Scholar 

  • Heng BC, Cao T, Haider HK, Wang DZ, Sim EK, Ng SC (2004) An overview and synopsis of techniques for directing stem cell differentiation in vitro. Cell Tissue Res 315:291–303

    Article  PubMed  Google Scholar 

  • Hutton KA, Trejdosiewicz LK, Thomas DF, Southgate J (1993) Urothelial tissue culture for bladder reconstruction: an experimental study. J Urol 150:721–725

    PubMed  CAS  Google Scholar 

  • Invitrogen (2001) Defined keratinocyte-SFM: serum-free keratinocyte medium for the culture of human keratinocytes. Available at https://www.invitrogen.com/content/sfs/manuals/3867.pdf

  • Itskovitz-Eldor J, Schuldiner M, Karsenti D, Eden A, Yanuka O, Amit M, Soreq H, Benvenisty N (2000) Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol Med 6:88–95

    PubMed  CAS  Google Scholar 

  • Kanematsu A, Yamamoto S, Iwai-Kanai E, Kanatani I, Imamura M, Adam RM, Tabata Y, Ogawa O (2005) Induction of smooth muscle cell-like phenotype in marrow-derived cells among regenerating urinary bladder smooth muscle cells. Am J Pathol 166:565–573

    PubMed  CAS  Google Scholar 

  • Karbanova J, Mokry J (2002) Histological and histochemical analysis of embryoid bodies. Acta Histochem 104:361–365

    Article  PubMed  Google Scholar 

  • Kurosawa H, Imamura T, Koike M, Sasaki K, Amano Y (2003) A simple method for forming embryoid body from mouse embryonic stem cells. J Biosci Bioeng 96:409–411

    PubMed  CAS  Google Scholar 

  • Lewis SA (2000) Everything you wanted to know about the bladder epithelium but were afraid to ask. Am J Physiol Renal Physiol 278:F867–F874

    PubMed  CAS  Google Scholar 

  • Liu W, Li Y, Cunha S, Hayward G, Baskin L (2000) Diffusable growth factors induce bladder smooth muscle differentiation. In Vitro Cell Dev Biol Anim 36:476–484

    Article  PubMed  CAS  Google Scholar 

  • Ludwikowski B, Zhang YY, Frey P (1999) The long-term culture of porcine urothelial cells and induction of urothelial stratification. BJU Int 84:507–514

    Article  PubMed  CAS  Google Scholar 

  • Marcovich R, Seifman B, Beduschi R, Wolf JS (2003) Surface modification to improve in vitro attachment and proliferation of human urinary tract cells. BJU Int 92:636–640

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Odorico JS, Kaufman DS, Thomson JA (2001) Multilineage differentiation from human embryonic stem cell lines. Stem Cells 19:193–204

    Article  PubMed  CAS  Google Scholar 

  • Olsburgh J, Harnden P, Weeks R, Smith B, Joyce A, Hall G, Poulsom R, Selby P, Southgate J (2003) Uroplakin gene expression in normal human tissues and locally advanced bladder cancer. J Pathol 199:41–49

    Article  PubMed  Google Scholar 

  • Oottamasathien S, Wang Y, Williams K, Franco OE, Wills ML, Thomas JC, Saba K, Sharif-Afshar AR, Makari JH, Bhowmick NA, Demarco RT, Hipkens S, Magnuson M, Brock JW 3rd, Hayward SW, Pope JC, Matusik RJ (2007) Directed differentiation of embryonic stem cells into bladder tissue. Dev Biol 304:556–566

    Article  PubMed  CAS  Google Scholar 

  • Owens GK (1995) Regulation of differentiation of vascular smooth muscle cells. Physiol Rev 75:487–517

    PubMed  CAS  Google Scholar 

  • Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A (2000) Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 18:399–404

    Article  PubMed  CAS  Google Scholar 

  • Sabbagh W, Masters JR, Duffy PG, Herbage D, Brown RA (1998) In vitro assessment of a collagen sponge for engineering urothelial grafts. Br J Urol 82:888–894

    PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Shamblott MJ, Axelman J, Sterneckert J, Christoforou N, Patterson ES, Siddiqi MA, Kahler H, Ifeanyi LA, Gearhart LD (2002) Stem cell culture: pluripotent stem cells. In: Atala A, Lanza RP (ed) Methods of tissue engineering. Academic Press, San Diego, pp 411–428

    Google Scholar 

  • Sun TT, Zhao H, Provet J, Aebi U, Wu XR (1996) Formation of asymmetric unit membrane during urothelial differentiation. Mol Biol Rep 23:3–11

    Article  PubMed  CAS  Google Scholar 

  • Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147

    Article  PubMed  CAS  Google Scholar 

  • Yamamoto M, Cui L, Johkura K, Asanuma K, Okouchi Y, Ogiwara N, Sasaki K (2006) Branching ducts similar to mesonephric ducts or ureteric buds in teratomas originating from mouse embryonic stem cells. Am J Physiol Renal Physiol 290:F52–F60

    Article  PubMed  CAS  Google Scholar 

  • Zhang Y, Kropp BP, Moore P, Cowan R, Furness PD, 3rd, Kolligian ME, Frey P, Cheng EY (2000) Coculture of bladder urothelial and smooth muscle cells on small intestinal submucosa: potential applications for tissue engineering technology. J Urol 164:928–934

    Article  PubMed  CAS  Google Scholar 

  • Zhang YY, Ludwikowski B, Hurst R, Frey P (2001) Expansion and long-term culture of differentiated normal rat urothelial cells in vitro. In Vitro Cell Dev Biol Anim 37:419–429

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

We thank Dr. Li Cui, Department of Anatomy and Organ Technology, Shinshu University, for instruction in the techniques for the culture of ES cells; Ms. Kayo Suzuki, Division of Instrumental Analysis, Research Center for Human and Environmental Sciences, Shinshu University, School of Medicine, for preparing tissue sections and providing expert advice on microscopic observation; and Ms. Tracey Fine of Fine Biomedical Publications for editing the manuscript.

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Authors and Affiliations

  1. Department of Urology, Shinshu University School of Medicine, 3-1-1, Asahi, Matsumoto, 390-8621, Japan

    Yoshiaki Kinebuchi, Tetsuya Imamura, Yuji Mimura & Osamu Nishizawa

  2. Department of Anatomy and Organ Technology, Shinshu University School of Medicine, Matsumoto, Japan

    Kohei Johkura & Katsunori Sasaki

Authors
  1. Yoshiaki Kinebuchi
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  2. Kohei Johkura
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  3. Katsunori Sasaki
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  4. Tetsuya Imamura
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  5. Yuji Mimura
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  6. Osamu Nishizawa
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Corresponding author

Correspondence to Yoshiaki Kinebuchi.

Additional information

This study was supported by Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science (no. 16790911).

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Kinebuchi, Y., Johkura, K., Sasaki, K. et al. Direct induction of layered tissues from mouse embryonic stem cells: potential for differentiation into urinary tract tissue. Cell Tissue Res 331, 605–615 (2008). https://doi.org/10.1007/s00441-007-0553-9

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  • DOI: https://doi.org/10.1007/s00441-007-0553-9

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