Reevaluation of Factors Leading to the Successful Isolation of Mesenchymal Stem Cells From Human Umbilical Cord Blood and Their Differentiation to Mesenchymal Lineages.
Authors: Zhang Xiaohong , Hirai Masako , Cantero Susana , Ciubotariu Rodica , Hirsh Allen , Satoh Hitoshi , Yokomi Izuru , Yoshimura Kotaro , Nishimura Toshihide , Yamaguchi Satoru , Igura Koichi , Rubinstein Pablo , Takahashi, DSc Tsuneo A. 
Published: October 2019

Abstract
Abstract 3232
Poster Board III-169

Introduction
Cord blood derived mesenchymal stem cells (CB-MSC) have been identified as an alternative cell source to bone marrow derived mesenchymal stem cells (BM-MSC) and adipose tissue derived mesenchymal stem cells (AT-MSC) for use in regenerative medicine. However, the low frequency of these cells in cord blood (CB) has led to conflicting reports of its efficacy and this, in turn, has been the main reason limiting their clinical use thus far. We searched for critical factors determining successful isolation of CB-MSC from more than 300 units of CB donated to two public CB banks using a range of different collection methods for CB. We applied several processing and culture methods to identify an optimal method for isolating CB-MSC. Proliferative, in vitro differentiation ability and immunosuppressive ability of CB-MSC were compared with BM- and AT-MSC. CB-MSC cultured with scaffolds were transplanted to nude mice. Additionally, chromosomal stability of CB-MSC after long-term culture was analyzed.

Materials and Methods
CB was collected after obtaining informed consent at two collection facilities: either while the placenta was in utero, or after the delivery of the placenta (ex utero). The mononuclear cells (MNC) were isolated by Ficoll-Paque (FP) density gradient centrifugation or other methods and subjected to a colony forming unit-fibroblast (CFU-F) assay. Their ability to differentiate into osteoblasts, chondorocytes, and adipocytes was tested in vitro and in vivo. Specific genes for differentiation to the mesoderm lineage were identified by RT-PCR. Immunosuppression by CB-MSC was tested by addition of cells to phytohemagglutinin (PHA) activated human T cells and to mixed lymphocyte reactions. Karyotypes of expanded CB-MSC were analyzed. Osteogenesis and chondrogenesis of CB-MSC in vivo were examined by transplantation of CB-MSC with scaffolds (β-TCP block, collagen sponge) subcutaneously to nude mice.

Results
CB-MSCs capable of proliferating were isolated from 121 units of 307 units of CB (63.1 ± 20.7 ml w/o anticoagulant). Two critical factors contributing to the success rate of isolating CB-MSC were: interval between collection of CB and processing of cells, and CB volume. When the interval was less than 2 hours there was a marked increase in success, S, according to the equation S=0.55*t-0.4316, (R2>0.99, n=81). There was also a more modest increase in S from increasing volume: S=0.0034*V (ml) + 0.2244, (R2>0.85, n=249). When both volume was higher than 90 ml and time was shorter than 5 hours, the success rate increased to 84.6%. The mean number of clonies from the units was calculated to be 1.59 ± 1.48 CFU /108 MNC (n=40) and 2.7 ± 2.3 CFU/CB unit. Variation in isolation and culture methods of did not improve the success rate. Most CB-MSC isolated grew rapidly and proliferated at more than 40 PDL (>15 passages), whereas BM-MSC and AT-MSC stopped proliferating at about 10 PDL. The CB-MSC showed higher differentiation ability to chondrocytes more than BM-MSC and AT-MSC. In vivo osteogenesis and chondrogenesis were observed when CB-MSC cultured with scaffolds were transplanted subcutaneously to nude mice. CB-MSC suppressed proliferation of lymphocytes stimilated allogeneically (mixed lymphocyte reaction) and by PHA as the dose of cells increased similar to finding with BM-MSC and AT-MSC. Gene expression related to the differentiation to the mesenchymal lineage indicated that CB-MSC can differentiate towards osteoblasts and chondrocytes. CB-MSC derived cell lines maintained normal karyotypes when the cells were cultured up to 40 PDL.

Conclusions
Among several factors possibly responsible for success in isolating CB-MSC, time between delivery and processing was decisive and volume was also critical. Even though the frequency of CB-MSC was much lower initially than BM-MSC, the high proliferation rate of these cells should allow expansion to cell numbers adequate for clinical use. High proliferation rate combined with high differentiation capability and the karyotype stability after long culture, indicate that CB-MSC should be a potential practical source of MSC for regenerative medicine.

Disclosures
No relevant conflicts of interest to declare.