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Preclinical study for treatment of hypospadias by advanced therapy medicinal products



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.


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.


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.


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

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Fig. 1



Human oral mucosa keratinocytes


Human urethra keratinocytes


Colony forming efficiency


Final product




Advanced therapy medicinal product


Q band by fluorescence and quinacrine


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


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).

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Authors and Affiliations



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.

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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.

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

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Supplementary material 1 (DOCX 26 kb)


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


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


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

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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).

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