Introduction: Human umbilical cord (UC) may be a good source of mesenchymal stem cells (MSCs) for musculoskeletal tissue engineering, however their potential to form bone is incompletely understood. The aim of the present study was to evaluate the growth characteristics, the phenotype and the multipotentiality of UC MSCs in comparison to bone marrow (8M) MSCs, with a particular focus on the molecules involved in bone formation and vascular support. The phenotype of UC endothelial cells (ECs) was additionally investigated in order to isolate uncultured fractions of UC MSCs and ECs, and to compare their telomere status. Methodology: UC fragments were enzymatically digested and UC MSC and EC cultures were grown in specialised media. Quantitative in vitro assays were used to study osteogenic, chondrogenic, adipogenic and vessel formation capacities of UC MSCs in comparison to 8M MSCs. Phenotypic characterisation was performed using multiparameter flow cytometry with MSC-, EC-specific and haematopoietic lineage markers. Immunohistochemistry on UC tissue sections was undertaken to study cell topography and cell sorting was performed to purify putative native UC MSCs from whole UC digests. Relative telomere lengths were measured by qPCR in both cultured and purified UC MSCs. Gene expression was analysed using Taqman low density array for 48 transcripts chosen to reflect MSC osteogenenic, angiogenic and other lineage potentials. Results: Compared to BM MSCs, UC MSCs grew slightly faster and had greater telomere stability. Both BM and UC MSCs had a classic MSC phenotype (CD90•, CD73•, CD105•, CD146•, CD31-, CD34-, CD45-, CD235a'), UC MSCs could generate all three mesenchymal lineages but their differentiation levels were inferior to 8M MSCs. MSCs differentiated towards vasculogenesis on 3-D Matrigel scaffold but they did not differentiate towards mature C031+ ECs. Cells expressing MSC markers C090 and C0146 were mainly located in UC perivascular and Wharton's jelly areas. Putative native UC MSCs were electrostatically cell sorted based on the CD146+, CD45-, CD31' phenotype. Their telomere lengths were compared to electrostatically cell sorted CD146-, CD45-, CD31- cells and ECs (CD45-, CD31•) but contrary to the initial hypothesis, no differences in telomere lengths were found. In agreement with functional osteogenesis assays, UC MSCs expressed considerably lower levels (<5-200 fold) of osteogenesis-related transcripts compared to 8M MSCs. Although they were able to respond to osteogenic v stimulation by up-regulation of many osteogenesis-related molecules (up to 100- fold), the transcript expression levels of differentiated 8M MSCs were commonly not achieved. Conclusions: UC MSCs direct differentiation in standard osteogenic assays appeared inferior to BM MSCs, most likely due to their more immature status. However, UC digests could represent a potential source of regenerative cens (MSCs and ECs) for complex tissue engineering where functional and long-lasting vasculature is required. It is possible that bone-forming capacity of UC MSCs can be improved by the development of optimised expansion protocols andlor the use of purified uncultured MSCs.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:590161 |
| Date | January 2012 |
| Creators | Kouroupis, Dimitrios. |
| Publisher | University of Leeds |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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