Human umbilical cord blood (CB) is a potential source for mesenchymal stem cells (MSC) capable of forming specific tissues, for example, bone, cartilage, or muscle. However, difficulty isolating MSC from CB (CB-MSC) has impeded their clinical application. Using more than 450 CB units donated to two public CB banks, we found that successful cell recovery fits a hyper-exponential function of time since birth with very high fidelity. Additionally, significant improvement in the isolation of CB-MSC was achieved by selecting cord blood units having a volume ≥90 ml and time ≤2 h after donor's birth. This resulted in 90% success in isolation of CB-MSC by density gradient purification and without a requirement for immunoaffinity methods as previously reported. Using MSC isolated from bone marrow (BM-MSC) and adipose tissue (AT-MSC) as reference controls, we observed that CB-MSC exhibited a higher proliferation rate and expanded to the order of the 1 × 10(9) cells required for cell therapies. CB-MSC showed karyotype stability after prolonged expansion. Functionally, CB-MSC could be more readily induced to differentiate into chondrocytes than could BM-MSC and AT-MSC. CB-MSC showed immunosuppressive activity equal to that of BM-MSC and AT-MSC. Collectively, our data indicate that viable CB-MSC could be obtained consistently and that CB should be reconsidered as a practical source of MSC for cell therapy and regenerative medicine using the well established CB banking system.
Rats with dwarfism accompanied by skeletal abnormalities, such as shortness of the limbs, tail, and body (dwarf rats), emerged in a Jcl-derived Sprague-Dawley rat colony maintained at the Institute for Animal Experimentation, St. Marianna University Graduate School of Medicine. Since the dwarfism was assumed to be due to a genetic mutation based on its frequency, we bred the dwarf rats and investigated their characteristics in order to identify the causative factors of their phenotypes and whether they could be used as a human disease model. One male and female that produced dwarf progeny were selected, and reproduction was initiated by mating the pair. The incidence of dwarfism was 25.8% among the resultant litter, and dwarfism occurred in both genders, suggesting that it was inherited in an autosomal recessive manner. At 12 weeks of age, the body weights of the male and female dwarf rats were 40% and 57% of those of the normal rats, respectively. In soft X-ray radiographic and histological examinations, shortening and hypoplasia of the long bones, such as the tibia and femur, were observed, which were suggestive of endochondral ossification abnormalities. An immunohistochemical examination detected an aggrecan synthesis disorder, which might have led to delayed calcification and increased growth plate thickening in the dwarf rats. We hypothesized that the principal characteristics of the dwarf rats were systemically induced by insufficient cartilage calcification in their long bones; thus, we named them cartilage calcification insufficient (CCI) rats.
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.
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