Minimally manipulated whole human umbilical cord is a rich source of clinical-grade human mesenchymal stromal cells expanded in human platelet lysate

Abstract

Background aims. Mesenchymal stromal cells (MSC) have recently been identified as a therapeutic option in several clinical conditions. Whereas bone marrow (BM) is considered the main source of MSC (BM-MSC), the invasive technique required for collection and the decline in allogeneic donations call for alternative sources. Human umbilical cord (UC) represents an easily available source of MSC (UC-MSC). Methods. Sections of full-term UC were transferred to cell culture flasks and cultured in 5% human platelet lysate (PL)-enriched medium. Neither enzymatic digestion nor blood vessel removal was performed. After 2 weeks, the adherent cells were harvested (P1), replated at low density and expanded for two consecutive rounds (P2 and P3). Results. We isolated and expanded MSC from 9/9 UC. UC-MSC expanded with a mean fold increase (FI) of 42 735 ± 16 195 from P1 to P3 in a mean of 29 ± 2 days. By processing the entire cord unit, we theoretically could have reached a median of 9.5 × 10¹⁰ cells (ranging from 1.0 × 10¹⁰ to 29.0 × 10¹⁰). UC-MSC expressed standard surface markers; they contained more colony-forming unit (CFU)-fibroblast (F) and seemed less committed towards osteogenic, chondrogenic and adipogenic lineages than BM-MSC. They showed immunosuppressive properties both in vitro and in an in vivo chronic Graft versus Host disease (cGvHD) mouse model. Both array-Comparative Genomic Hybridization (CGH) analysis and karyotyping revealed no chromosome alterations at the end of the expansion. Animal studies revealed no tumorigenicity in vivo. Conclusions. UC constitute a convenient and very rich source of MSC for the production of third-party ‘clinical doses’ of cells under good manufacturing practice (GMP) conditions.

Key Words::

• good manufacturing practice
• mesenchymal stromal cells
• platelet lysate
• umbilical cord

Acknowledgments

The authors wish to thank the following people: Dr M. Magri for administrative and secretarial assistance; Dr E. Cattaneo (Department of Pharmacological Sciences and Centre for Stem Cell Research, Università degli Studi di Milano) and Dr P. Bellavita (Transfusion Division, Ospedali Riuniti di Bergamo) for providing cells and reagents; Dr U. Giussani (Cytogenetics Division, Ospedali Riuniti di Bergamo) for her precious advice and expertise in the cytogenetic analysis; Sig.ra B. Pasini (Obstetrics and Gynecology Division, Ospedali Riuniti di Bergamo) for providing tissues; Dr Cristina Trento and Dr Thilakavathy Karuppiah (Kennedy Institute of Rheumatology, Imperial College, London) for their expertise and precious help in the animal handling.

This work was partially supported by ‘Fondazione Italiana Ricerca Cancro (FIRC)’, by a special grant from ‘Associazione Italiana Ricerca Cancro (AIRC) 5 per mille’ to the AGIMM group (AIRC-Gruppo Italiano Malattie Mileoproliferative), by a regional grant from ‘AIRC for Molecular Imaging’ and by ‘Associazione Italiana contro le Leucemie, Linfomi e Mieloma (AIL), Bergamo-Sezione Paolo Belli’.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.