Title : Extensive characterization of umbilical cord-lining stem Ccells and their potential use in tissue engineering and regenerative medicine applications
Abstract:
Background: Mesenchymal Stromal Cells (MSCs) are representing a heterogenous non hematopoietic stem cell population of mesodermal origin, with significant differentiation and immunoregulatory properties. The umbilical cord (UC) represents a perinatal tissue for the isolation of fetal derived MSCs. Moreover, significant differences have been observed between the adult and fetal derived MSCs, in terms of proliferation, differentiation and immunoregulatory potential, with the latter to exhibit better properties. Beyond the Wharton’s Jelly (WJ) tissue of the UC, stem cells can be isolated also from the cord-lining membrane. Cord lining (CL) membrane represents the extension of the placenta, originated from the primitive endoderm formed during the embryogenesis. Typically, the differentiation potential of MSCs has been limited to cellular populations of mesodermal origin including the “osteocytes”, “chondrocytes” and “adipocytes”. However, CL-MSCs may exhibit also endodermal differentiation potential, showing advanced properties regarding the tissue regeneration.
Aim: The aim of this study primarily was to comprehensively explore the biology of CL-MSCs. Moreover, to assess their stemness and plasticity profile to cellular populations beyond mesodermal origin, differentiation to insulin producing cells (IPCs) using defined medium was performed. For this purpose, UC (n=10) were used for the MSCs isolation, derived from end term pregnancies (gestational weeks > 38), after signed informed consent obtained from the parents. The MSCs isolation was performed immediately or in less than 24 hrs after the reception of the UC to the HCBB laboratory. Briefly, the UC were washed to remove any blood clots, and using sterile instruments, the outer layer was isolated, trimmed in small pieces, placed in 6-well plates and incubated at 37o C and 5% CO2. After 18 days of culture, the cells were trypsinized, detached and placed in 75 cm2 flask, until confluency observed. The same procedure was repeated until reached passage 4 (P4). Population Doubling (PD) and cell doubling time (CDT) were estimated. Moreover, CL-MSCs submitted to differentiation to “osteocytes”, “chondrocytes” and “adipocytes” using defined culture media. To validate their successful differentiation histological stains including alizarin Red S, Toluidine Blue and Oil Red O, to detect the presence of calcium deposits, GAGs formation and oil droplets were performed, respectively. Moreover, immunophenotypically analysis using the CD73, CD90, CD105, CD34, CD45, HLA-DR, CD11b and CD326 (EpCam) was performed. Molecular analysis involved the performance of real time PCR for the genes OCT4, KLF4, NANOG, PAX1, FOXA2, REX1, SOX17, ALP and GAPDH. Additionally, CL-MSCs submitted to RNA sec analysis to comprehensively explore their gene expression. Finally, the differentiation of CL-MSCs (n=6) to IPCs using defined medium, was evaluated. The obtained IPCs submitted to low and high glucose assay, and the supernatants were used for detection of insulin with the ELISA method. As control group, WJ-MSCs (n=10) were used for all assays.
Results: The results of this study showed the efficient isolation and expansion of CL-MSCs. The PD and CD values of CL-MSCs were statistically significant difference compared to the WJ-MSCs. Immunophenotyping analysis of CL-MSCs showed high expression of CD73, CD90, CD105 ( >95%) and low expression of CD34, CD45, HLA-DR (< 3%), and were comparable with the WJ-MSCs. On the other hand CD326 appeared to expressed only in the CL-MSCs. CL-MSCs successfully differentiated to osteocytes”, “chondrocytes” and “adipocytes” as it was confirmed by the histological stains. Molecular analysis showed the upregulation of a set of 50 genes including the PAX1, FOXA2 and ALP. Finally, the CL-MSCs presented greater differentiation potential towards IPCs compared to the WJ-MSCs as it was confirmed by the higher insulin production in the ELISA method.
Conclusion: The results presented herein showed the efficient isolation of CL-MSCs from the UCs. CL-MSCs may represent a unique subpopulation of MSCs derived from the UC, which is not limited to mesodermal origin, as it was confirmed by their successful differentiation to IPCs. The understanding of biology of CL-MSCs may represent a fundamental step towards the utilization of this population in tissue engineering and regenerative medicine approaches to combat life-threating human pathological conditions.
