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.

Introduction

As defined by the International Society for Cellular Therapy (ISCT), mesenchymal stromal cells (MSC) are plastic-adherent cells with a specific combination of surface markers that have the capacity to self-renew and differentiate into various lineages, including bone, cartilage and adipose tissue (1). These cells can be derived from several different sources, such as bone, adipose tissue, synovium skeletal muscle, dermis, pericytes, cord blood and bone marrow (BM) (2).

MSC derived from BM (BM-MSC) have been studied extensively and nowadays are considered good candidates for cell-based therapies (3., 4., 5., 6., 7.). It is estimated that more than 100 clinical trials are on-going using BM-MSC for a variety of indications (www.clinicaltrials.gov [Accessed: January 2010]). Whereas BM is considered the main source of MSC, the invasive and painful procedure for BM collection as well as the concomitant decline in allogeneic BM donations have raised an increasing interest in investigating the availability of MSC from alternative sources. In this context, human umbilical cord (UC) is already considered to be a useful alternative to BM (8). The UC is a tissue of extra-embryonic origin lying between the mother and the fetus, consisting of two arteries, one vein, inter-vessels connective tissue and umbilical epithelium. The connective tissue, namely the Wharton's jelly, is made up of a sponge-like structure waved by collagen fibers, proteoglycan and embedded stromal cells (9). The UC is normally discarded after birth and its collection does not require an invasive procedure with ethical concerns. Moreover, UC-derived MSC (UC-MSC) possess many advantageous features, namely high frequency, pluripotency, high proliferation capacity, immunomodulatory properties and no age donor-dependent variations (10., 11., 12., 13.).

MSC have been isolated from different compartments of UC, i.e. from Wharton's jelly (14), perivascular areas (15,16) and subendothelium of the umbilical vein (17). Furthermore, UC-MSC have been transplanted safely in mice without inducing tumors or death and have ameliorated symptoms in several models of regenerative medicine (7,14,18., 19., 20.).

In all these cases, UC-MSC were expanded in fetal bovine serum (FBS), but regulatory requirements in the field of cell therapy strongly recommend avoiding any animal-derived reagents and sustain a search for alternatives. In this context, the use of human platelet lysate has gained much attention in the last few years (21., 22., 23.).

The aim of the present work was to establish a good manufacturing practice (GMP)-compliant protocol for the isolation and expansion of large quantities of UC-MSC; to our knowledge, this is the first report on the feasibility of clinical-scale production of UC-MSC using only clinical-grade reagents and human platelet lysate. Furthermore, only minimal mechanical manipulation was performed, to avoid the possible selection of cellular subpopulations (24., 25., 26.). The projected theoretical number of final UC-MSC yields justifies the hypothesis that this procedure may support the exploitation of small clinical trials with cells derived from one single cord unit.