Abstract
Bone narrow (BM)- derived mesenchymal stem/stromal cells (MSCs) represent the most extensively studied population of adult MSCs and are considered as the gold-standard for MSC-based clinical applications. Yet, it is now becoming increasingly clear that BM may not represent the most suitable source for MSC collection. Indeed, Umbilical cord (UC) has emerged as a more abundant and easily attainable source of MSCs and several reports have shown that MSCs can be efficiently isolated from the connective tissue that surrounds UC vessels, namely the Wharton's jelly (WJ). According to the existing literature, WJ-MSCs display typical MSC characteristics, however a head-to-head comparison with BM-MSCs is still lacking.
Provided that ex vivo MSC expansion is a prerequisite for clinical MSC-applications, in the present study we seek to comparatively investigate the characteristics of WJ- and BM-MSCs, cultured under identical conditions.
MSCs were isolated and expanded from consenting healthy donors’ BM aspirates (n=5) and from the WJ of full-term neonates (n=10) after written informed consent of the family. MSCs were in vitro expanded and re-seeded for a total of 10 passages (P) and phenotypically characterized by flow cytometry (FC). MSCs were induced to differentiate in vitro to adipocytes and osteoblasts. Differentiation was assessed by cytochemical stains and by the expression of adipocyte- and osteocyte-specific genes. Relative gene expression was calculated by the ΔCt method. MSC growth characteristics were assessed by evaluating the population doubling time (DT) and by a methyl-triazolyl-tetrazolium (MTT)-assay throughout passages. Cell-cycle analysis was performed using propidium iodide (PI) staining. MSC survival was evaluated by FC with 7-Aminoactinomycin D (7-AAD) and senescence was estimated by the percentage of SA-b-gal+ cells in cultures. Moreover, MSC karyotypic stability was assessed with classic G-banding. Finally the expression of genes related to Wnt-mediated signal transduction was also investigated, using a PCR array. Total RNA was thus isolated from 6 representative BM- and 6 WJ-MSC cultures at P2. The fold change (FC) for each gene between the group of WJ- and the group of BM-MSCs was calculated with the ΔΔCt method (FC=2-ΔΔCt).
WJ-MSCs displayed a spindle-shape morphology, similar to BM-MSCs. Furthermore, WJ- and BM-MSCs displayed identical immunophenotype, as evidenced by the expression of CD90,CD105,CD44,CD29,CD73 and the lack of expression of CD45,CD14,CD34,CD31. WJ-MSCs displayed superior proliferative potential compared to BM-MSCs throughout passages (p<0.05). Moreover, the proportion of proliferating (S/G2/M) WJ-MSCs was higher compared to BM-MSCs at P4 (p<0.001), while there was no significant difference between two MSC populations in the proportion of 7-AADbright/dim –cells at P4. Regarding senescence, significantly fewer SA-b-gal+ cells were observed in WJ-MSC cultures, as compared to BM-MSCs at P10 (p<0.05). Compared to their bone marrow counterparts, WJ-MSCs displayed inferior capacity to differentiate into adipocytes and osteoblasts as evidenced by Oil Red O and Alizarin Red staining, respectively, and also by the weaker expression of adipocyte- (PPAR-g, p<0.0002; CEBP-a, p<0.0001) and osteocyte-specific markers (RUNX2, p<0.0006; DLX5, p<0.0001; ALP, p<0.0042). No chromosomal abnormalities were observed in either WJ- or BM-MSCs during in vitro expansion. Regarding the Wnt-pathway signaling molecules, the Wnt antagonist sFRP4, which induces adipogenesis, as well the Wnt/b-catenin target gene Wisp-1, a regulator of osteogenesis were significantly down-regulated in WJ-MSCs (FC=22.3825, p<0,05; FC=20.18, p<0.0001, respectively). On the other hand, the expression of Wnt/b-catenin target gene Cyclin D1, which induces MSC proliferation and represses adipogenesis, was up-regulated in WJ-MSCs (FC=2.8, p<0.05).
Taken together WJ-MSCs display decreased cellular senescence after extended in vitro culture, increased proliferative capacity and reduced potential to differentiate in vitro to adipocytes and osteocytes, as compared to BM-MSCs. The last two observations can be explained, at least partly, by the aberrant expression of Wnt-signaling molecules in WJ-MSCs. The emerging role of Wnt-signaling pathway in WJ-MSC biology is currently under investigation.
Disclosures:
No relevant conflicts of interest to declare.
Single-cell RNA-seq highlights heterogeneity in human primary Wharton’s jelly mesenchymal stem/stromal cells cultured in vitro
