Emerging Roles of Wisp-1 and SFRP4 in Proliferation and Differentiation Potential of Wharton's Jelly Mesenchymal Stem/Stromal Cells
Authors: Batsali Aristea , Pontikoglou Charalampos , Agrafiotis Emmanuel , Kouvidi Elisavet , Mavroudi Irene , Damianaki Athina , Kastrinaki Maria-Christina , Papadaki Helen 
Published: October 2019

Abstract
We have previously shown (Batsali A et al., Blood 2013:122, 1212) that ex vivo expanded human mesenchymal stem/stromal cells (MSCs) derived from the Wharton's jelly (WJ) of the umbilical cord exhibit increased proliferative capacity and reduced potential to differentiate in vitro to adipocytes and osteocytes, compared to bone marrow (BM) derived-MSCs. Provided that the WNT-pathways are involved in proliferation and differentiation of BM-MSCs, we assumed that the aforementioned findings might be attributed, at least in part, to aberrant WNT-signaling in WJ-MSCs. In support of this hypothesis, we found that gene expression of the Wnt antagonist sFRP4, a promoter of adipogenesis in human adipose tissue-derived MSCs, was significantly down-regulated in WJ-MSCs and that mRNA levels of WNT-induced secreted protein-1, (WISP-1), a regulator of osteogenesis in BM-MSCs, were also significantly reduced in WJ-MSCs. These observations imply a connection between these WNT-associated molecules and the biological properties of WJ-MSCs, which requires, however, further investigation.
The present study was undertaken so as to explore the effects of WISP-1 and sFRP4 in growth and differentiation of ex-vivo expanded WJ-MSCs.
MSCs were isolated from consenting healthy donors’ BM aspirates (n=5) and from the WJ of full-term neonates (n=5) after written informed consent of the family. MSCs were in vitro expanded, re-seeded for a total of 4 passages (P) and phenotypically characterized by flow cytometry at P3. WJ-MSCs were grown in the absence or presence of rhWISP-1 or rhsFRP4 and cell proliferation was assessed by a methyl-triazolyl-tetrazolium (MTT)-assay. In addition, WJ-MSCs were induced to differentiate in vitro to osteoblasts and adipocytes, in the absence or presence of rhWISP-1 or rhsFRP4 respectively. Differentiation was quantified by cytochemical stains and by the expression of adipocyte- and osteocyte-specific genes by real time RT-PCR. Relative gene expression was calculated by the ΔCt method. The expression of WISP-1 and sFRP4 by non-differentiated WJ- and BM-MSCs as well as by WJ-MSCs during osteogenesis and adipogenesis, respectively, was also evaluated by real time RT-PCR.
Culture-expanded cells from both WJ and BM displayed typical morphological and immunophenotypic MSC characteristics and were able to differentiate into osteoblasts and adipocytes. In line with our previous work WISP-1 and sFRP4 mRNA were significantly decreased in WJ-MSCs, compared to BM-MSCs.
To explore the role of WISP-1 in WJ-MSCs' growth we cultured cells in the presence of 50 ng/ml or 100 ng/ml rhWISP1 and assessed cell proliferation at multiple time points, throughout a 14-day culture. WISP-1 treatment did not lead to any significant effect in cell numbers. Next, we investigated the time course of WISP1 gene expression during osteoinduction. In all samples, WISP1 mRNA levels increased during osteogenesis. As compared to day0 (exposure to osteogenic medium), the increase in gene expression reached statistical significance at days 7 and 14. Furthermore, WISP-1 gene expression was significantly higher at day 14, compared to day 7. To investigate the functional effects of WISP1 on the osteoblastic differentiation of WJ-MSCs, cells were cultured for 7 days in osteogenic medium supplemented with 50ng/ml rh-WISP1. A significant increase in the expression of RUNX2 and ALP was detected, compared to non-treated cells.
To investigate the impact of sFRP4 in WJ-MSC's proliferation we exposed cells to 20nM rhsFRP4 for 14 days. Live cell numbers, at various time points, were significantly reduced in treated cells. Regarding the time course of sFRP4 expression during adipogenic differentiation, sFRP4 mRNA levels increased during adipogenesis reaching statistical significance at days7 and 14, as compared to day0. In addition, sFRP4 gene expression was significantly higher at day 14 as compared to day 7. Finally, when cells underwent adipogenic differentiation in the presence of rhSFRP4, a significant increase in PPARG and CEBPA mRNA levels was detected at day 14, as compared to non-treated cells
Collectively, our results suggest that WISP-1 and sFRP4 may be actively implicated in proliferation and differentiation of WJ-MSCs. The functional role of these WNT-related molecules in the biology of WJ-MSCs requires deeper understanding, in view of the growing interest for the use of WJ-MSCs in cell-based therapies.

Disclosures
No relevant conflicts of interest to declare.