Skip to main content

Preclinical study for treatment of hypospadias by advanced therapy medicinal products

Abstract

Purpose

This paper explores the feasibility of a new therapy for the treatment of hypospadias patients. Hypospadias is a very common congenital malformation of male genitals, with very high rate of recurrences after surgery. The field of regenerative medicine, which offers innovative solutions for many pathologies, still does not offer reliable solution for this pathology. Here, we propose quality, safety, and clinical feasibility assessment for an oral mucosa advanced therapy medicinal product (ATMP) grown on a biocompatible scaffold for a clinical study on urethral reconstruction of hypospadias patients.

Methods

Urethral and oral mucosal epithelia from donor biopsies were cultivated between two fibrin layers, under clinical-grade conditions for cell and tissue characterization and comparison, aimed at tissue engineering. In addition, single-clone analyses were performed to analyze gene expression profiles of the two epithelia by microarray technology.

Results

Oral mucosa appeared suitable for urethral reconstruction. The resulting ATMP was proven to maintain stem cells and regenerative potency. The preclinical safety studies were performed on human tissues to assess abnormalities and tumorigenicity, and confirmed the safety of the ATMP. Finally, the patient selection and the clinical protocol for the upcoming clinical trial were defined.

Conclusions

Against this backdrop, in this paper, we are proposing a new reproducible and reliable ATMP for the treatment of hypospadias.

This is a preview of subscription content, access via your institution.

Fig. 1

Abbreviations

MO:

Human oral mucosa keratinocytes

UK:

Human urethra keratinocytes

CFE:

Colony forming efficiency

DP:

Final product

K:

Cytokeratin

ATMP:

Advanced therapy medicinal product

QFQ:

Q band by fluorescence and quinacrine

References

  1. Baskin LS, Ebbers MB (2006) Hypospadias: anatomy, etiology, and technique. J Pediatr Surg 41(3):463–472. https://doi.org/10.1016/j.jpedsurg.2005.11.059

    Article  PubMed  Google Scholar 

  2. Agrawal K, Misra A (2013) Unfavourable results in hypospadias. Indian J Plast Surg 46(2):419–427. https://doi.org/10.4103/0970-0358.118623

    Article  PubMed  PubMed Central  Google Scholar 

  3. Mann RJ, O’Brien AL, Adams NS, Girotto JA, Polley JW (2017) Repair of oropharyngeal stenosis with bilateral buccal myomucosal flaps. Ann Plast Surg 79(2):162–165. https://doi.org/10.1097/sap.0000000000001089

    CAS  Article  PubMed  Google Scholar 

  4. Yarington CT Jr (1980) Reconstruction of the base of the tongue and lateral pharyngeal wall. Laryngoscope 90(2):202–206

    Article  Google Scholar 

  5. Lin WC, Chang CY, Shen YY, Tsai HD (2003) Use of autologous buccal mucosa for vaginoplasty: a study of eight cases. Hum Reprod 18(3):604–607

    CAS  Article  Google Scholar 

  6. Wood DN, Allen SE, Andrich DE, Greenwell TJ, Mundy AR (2004) The morbidity of buccal mucosal graft harvest for urethroplasty and the effect of nonclosure of the graft harvest site on postoperative pain. J Urol 172(2):580–583. https://doi.org/10.1097/01.ju.0000132846.01144.9f

    CAS  Article  PubMed  Google Scholar 

  7. Dublin N, Stewart LH (2004) Oral complications after buccal mucosal graft harvest for urethroplasty. BJU Int 94(6):867–869. https://doi.org/10.1111/j.1464-410x.2004.05048.x

    Article  PubMed  Google Scholar 

  8. Piemonte ED, Lazos JP, Brunotto M (2010) Relationship between chronic trauma of the oral mucosa, oral potentially malignant disorders and oral cancer. J Oral Pathol Med 39(7):513–517. https://doi.org/10.1111/j.1600-0714.2010.00901.x

    Article  PubMed  Google Scholar 

  9. Barbagli G, Lazzeri M (2006) Urethral reconstruction. Curr Opin Urol 16(6):391–395. https://doi.org/10.1097/01.mou.0000250277.44990.ab

    Article  PubMed  Google Scholar 

  10. Rama P, Matuska S, Paganoni G, Spinelli A, De Luca M, Pellegrini G (2010) Limbal stem-cell therapy and long-term corneal regeneration. N Engl J Med 363(2):147–155. https://doi.org/10.1056/nejmoa0905955

    CAS  Article  PubMed  Google Scholar 

  11. Ram-Liebig G, Bednarz J, Stuerzebecher B, Fahlenkamp D, Barbagli G, Romano G, Balsmeyer U, Spiegeler ME, Liebig S, Knispel H (2015) Regulatory challenges for autologous tissue engineered products on their way from bench to bedside in Europe. Adv Drug Deliv Rev 82–83:181–191. https://doi.org/10.1016/j.addr.2014.11.009

    CAS  Article  PubMed  Google Scholar 

  12. Pellegrini G, Ardigo D, Milazzo G, Iotti G, Guatelli P, Pelosi D, De Luca M (2018) Navigating market authorization: the path holoclar took to become the first stem cell product approved in the European Union. Stem Cells Transl Med 7(1):146–154. https://doi.org/10.1002/sctm.17-0003

    Article  PubMed  Google Scholar 

  13. Barrandon Y, Green H (1987) Three clonal types of keratinocyte with different capacities for multiplication. Proc Natl Acad Sci USA 84(8):2302–2306

    CAS  Article  Google Scholar 

  14. Moll R, Divo M, Langbein L (2008) The human keratins: biology and pathology. Histochem Cell Biol 129(6):705–733. https://doi.org/10.1007/s00418-008-0435-6

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Matthias C, Mack B, Berghaus A, Gires O (2008) Keratin 8 expression in head and neck epithelia. BMC Cancer 8:267. https://doi.org/10.1186/1471-2407-8-267

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. Sun TT (2006) Altered phenotype of cultured urothelial and other stratified epithelial cells: implications for wound healing. Am J Physiol Renal Physiol 291(1):F9–F21. https://doi.org/10.1152/ajprenal.00035.2006

    CAS  Article  PubMed  Google Scholar 

  17. Corradini F, Zattoni M, Barbagli G, Bianchi G, Giovanardi M, Serafini C, Genna VG, Ribbene A, Balo S, Fidanza F, Lazzeri M, Luca M, Pellegrini G (2016) Comparative assessment of cultures from oral and urethral stem cells for urethral regeneration. Curr Stem Cell Res Ther 11(8):643–651

    CAS  Article  Google Scholar 

  18. Biehs B, Hu JK, Strauli NB, Sangiorgi E, Jung H, Heber RP, Ho S, Goodwin AF, Dasen JS, Capecchi MR, Klein OD (2013) BMI1 represses Ink4a/Arf and Hox genes to regulate stem cells in the rodent incisor. Nat Cell Biol 15(7):846–852. https://doi.org/10.1038/ncb2766

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Sheikh BN, Downer NL, Phipson B, Vanyai HK, Kueh AJ, McCarthy DJ, Smyth GK, Thomas T, Voss AK (2015) MOZ and BMI1 play opposing roles during Hox gene activation in ES cells and in body segment identity specification in vivo. Proc Natl Acad Sci USA 112(17):5437–5442. https://doi.org/10.1073/pnas.1422872112

    CAS  Article  PubMed  Google Scholar 

  20. Isono K, Mizutani-Koseki Y, Komori T, Schmidt-Zachmann MS, Koseki H (2005) Mammalian polycomb-mediated repression of Hox genes requires the essential spliceosomal protein Sf3b1. Genes Dev 19(5):536–541. https://doi.org/10.1101/gad.1284605

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. Li Z, Chen P, Su R, Hu C, Li Y, Elkahloun AG, Zuo Z, Gurbuxani S, Arnovitz S, Weng H, Wang Y, Li S, Huang H, Neilly MB, Wang GG, Jiang X, Liu PP, Jin J, Chen J (2016) PBX3 and MEIS1 cooperate in hematopoietic cells to drive acute myeloid leukemias characterized by a core transcriptome of the MLL-rearranged disease. Cancer Res 76(3):619–629. https://doi.org/10.1158/0008-5472.can-15-1566

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. Sciarra A, Salciccia S, Albanesi L, Cardi A, D’Eramo G, Di Silverio F (2005) Use of cyclooxygenase-2 inhibitor for prevention of urethral strictures secondary to transurethral resection of the prostate. Urology 66(6):1218–1222. https://doi.org/10.1016/j.urology.2005.06.090

    Article  PubMed  Google Scholar 

  23. Barbaro V, Testa A, Di Iorio E, Mavilio F, Pellegrini G, De Luca M (2007) C/EBPdelta regulates cell cycle and self-renewal of human limbal stem cells. J Cell Biol 177(6):1037–1049. https://doi.org/10.1083/jcb.200703003

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. Molofsky AV, He S, Bydon M, Morrison SJ, Pardal R (2005) Bmi-1 promotes neural stem cell self-renewal and neural development but not mouse growth and survival by repressing the p16Ink4a and p19Arf senescence pathways. Genes Dev 19(12):1432–1437. https://doi.org/10.1101/gad.1299505

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. Priya CG, Arpitha P, Vaishali S, Prajna NV, Usha K, Sheetal K, Muthukkaruppan V (2011) Adult human buccal epithelial stem cells: identification, ex vivo expansion, and transplantation for corneal surface reconstruction. Eye (Lond) 25(12):1641–1649. https://doi.org/10.1038/eye.2011.230

    CAS  Article  Google Scholar 

  26. Gonzalez LM, Moeser AJ, Blikslager AT (2015) Porcine models of digestive disease: the future of large animal translational research. Transl Res 166(1):12–27. https://doi.org/10.1016/j.trsl.2015.01.004

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. Groenen MA, Archibald AL, Uenishi H, Tuggle CK, Takeuchi Y, Rothschild MF, Rogel- Gaillard C, Park C, Milan D, Megens HJ, Li S, Larkin DM, Kim H, Frantz LA, Caccamo M, Ahn H, Aken BL, Anselmo A, Anthon C, Auvil L, Badaoui B, Beattie CW, Bendixen C, Berman D, Blecha F, Blomberg J, Bolund L, Bosse M, Botti S, Bujie Z, Bystrom M, Capitanu B, Carvalho-Silva D, Chardon P, Chen C, Cheng R, Choi SH, Chow W, Clark RC, Clee C, Crooijmans RP, Dawson HD, Dehais P, De Sapio F, Dibbits B, Drou N, Du ZQ, Eversole K, Fadista J, Fairley S, Faraut T, Faulkner GJ, Fowler KE, Fredholm M, Fritz E, Gilbert JG, Giuffra E, Gorodkin J, Griffin DK, Harrow JL, Hayward A, Howe K, Hu ZL, Humphray SJ, Hunt T, Hornshoj H, Jeon JT, Jern P, Jones M, Jurka J, Kanamori H, Kapetanovic R, Kim J, Kim JH, Kim KW, Kim TH, Larson G, Lee K, Lee KT, Leggett R, Lewin HA, Li Y, Liu W, Loveland JE, Lu Y, Lunney JK, Ma J, Madsen O, Mann K, Matthews L, McLaren S, Morozumi T, Murtaugh MP, Narayan J, Nguyen DT, Ni P, Oh SJ, Onteru S, Panitz F, Park EW, Park HS, Pascal G, Paudel Y, Perez-Enciso M, Ramirez-Gonzalez R, Reecy JM, Rodriguez-Zas S, Rohrer GA, Rund L, Sang Y, Schachtschneider K, Schraiber JG, Schwartz J, Scobie L, Scott C, Searle S, Servin B, Southey BR, Sperber G, Stadler P, Sweedler JV, Tafer H, Thomsen B, Wali R, Wang J, Wang J, White S, Xu X, Yerle M, Zhang G, Zhang J, Zhang J, Zhao S, Rogers J, Churcher C, Schook LB (2012) Analyses of pig genomes provide insight into porcine demography and evolution. Nature 491(7424):393–398. https://doi.org/10.1038/nature11622

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. Pellegrini G, Rama P, Di Rocco A, Panaras A, De Luca M (2014) Concise review: hurdles in a successful example of limbal stem cell-based regenerative medicine. Stem Cells 32(1):26–34. https://doi.org/10.1002/stem.1517

    CAS  Article  PubMed  Google Scholar 

  29. Barbagli G, Perovic S, Djinovic R, Sansalone S, Lazzeri M (2010) Retrospective descriptive analysis of 1,176 patients with failed hypospadias repair. J Urol 183(1):207–211. https://doi.org/10.1016/j.juro.2009.08.153

    Article  PubMed  Google Scholar 

  30. Rama P, Bonini S, Lambiase A, Golisano O, Paterna P, De Luca M, Pellegrini G (2001) Autologous fibrin-cultured limbal stem cells permanently restore the corneal surface of patients with total limbal stem cell deficiency. Transplantation 72(9):1478–1485

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank Dr. Cristina Falcinelli and Dr. Olga Calabrese from laboratory of Medical Genetics of Modena for their effective collaboration.

Funding

This work funded by Esperia Bank Group (“Regenerative Medicine of urethra” from Esperia Trust), Regione Lombardia (“Lombardia è Ricerca” Milano, November 8, 2018) and Ministry of Education, University and Research; IRMI (CTN01_00177_888744). Associazione Italiana per la Ricerca sul Cancro (AIRC), IG project number #19818; AIRC 5 per 1000 project #21267; Italian Ministry of Health (project number #RF-2016-02362930).

Author information

Authors and Affiliations

Authors

Contributions

VS protocol development, data collection and management, data analysis, manuscript writing/editing. EA protocol development, data collection and management, data analysis, manuscript writing/editing. EB protocol development, data collection and management, data analysis, manuscript editing. GG data collection, data analysis. MM protocol development, data collection and management, data analysis, manuscript editing. FC protocol development, data collection and management. MF data collection, manuscript editing. AR protocol development. LL protocol development, data analysis. SB data collection. ML project development, data collection and management. CT data collection and management. MR data collection and management. RM data analysis, Manuscript editing. GB project development, data collection and management. GP protocol/project development, data collection and management, data analysis, manuscript writing/editing.

Corresponding author

Correspondence to G. Pellegrini.

Ethics declarations

Conflict of interest

G. Pellegrini is member of the Board of Directors, R&D Director of Holostem Terapie Avanzate and J-TEC consultant. V. Sceberras is employee of Holostem Terapie Avanzate. E. Attico, M. Melonari, G. Galaverni, M. Fantacci, F. Corradini, G. Barbagli, S. Balò, C. Trombetta, M. Rizzo, L. Losi, E. Bianchi, R. Manfredini, A. Ribbene and M. Lazzeri declare that they have no conflict of interest.

Research involving human participants

The study will be submitted to AIFA (Italian Drug Agency) for clinical trial approval.

Informed consent

Informed consent was obtained from all donors and will be obtained by individual participants included in the study.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 26 kb)

345_2019_2864_MOESM2_ESM.tif

Supplementary material 2 (TIFF 3645 kb) S1 Representative life spans of healthy urethra and oral mucosa samples. Oral mucosa (N = 5) and healthy urethra (N = 4) samples showed a mean of 88 and 167 cell doublings, respectively

345_2019_2864_MOESM3_ESM.tiff

Supplementary material 3 (TIFF 30661 kb) S2 Hierarchical clustering and heat map for the transcripts differentially expressed (i.e. upregulated, Fold Change ≥ 2, FDR ≤ 0.05 or downregulated, Fold Change ≤ -2, FDR ≤ 0.05) in oral mucosa (n = 15) versus urothelial (n = 4) holoclones. For each transcript, red bars indicate higher signal intensity, while green bars represent lower signal intensity and black intermediate. The clustering of the samples is indicated by the dendrogram on the left side

345_2019_2864_MOESM4_ESM.tif

Supplementary material 4 (TIFF 9683 kb) S3 Example of safety assays (a) adhesion dependence: growth curve of feeder cells (3T3-J2) and cultured oral mucosa cells (MO) vs cells from carcinomas (UM-SCC-14C and MCF7) maintained in soft agar. The first two were unable to grow in the absence of adhesion, whereas the second group produced a standard amplification. b Growth factor dependence: cultured oral mucosa (MO), compared with carcinoma cell line (UM-SCC-14C) in culture with growth factor stimulation (CTR), in the presence of feeder layer and without growth factors (+FL−GF), without feeder layer and in the presence of growth factor (−FL/+GF) and without feeder layer and growth factor (−FL/−GF). The carcinoma cell line UM-SCC-14C maintained colonies in the absence of growth factor, whereas the cultured oral mucosa was unable to develop colonies

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Sceberras, V., Attico, E., Bianchi, E. et al. Preclinical study for treatment of hypospadias by advanced therapy medicinal products. World J Urol 38, 2115–2122 (2020). https://doi.org/10.1007/s00345-019-02864-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00345-019-02864-x

Keywords

  • Hypospadias
  • Regenerative medicine
  • Oral mucosa
  • Clinical trial
  • Tissue engineering
  • Stem cells