We report combination of explants and enzymatic protocol as mixed enzymatic-explant procedure to faster extraction of MSCs from WJ. Umbilical cords (UC) were collected from Imam Khomini Hospital. For explant outgrowth, 6 -9 pieces of WJ were transferred onto tissue culture flask and waited for attachment. For mixed enzymatic-explant, 1 cm 3 pieces WJ were placed in enzymatic cocktail comprising 4 mg/ml Collagenase Type I and 1 mg/ml Hyaluronidase and 0.1% trypsin-EDTA. Then isolated cells were analyzed for surface cell markers such as CD73, CD31. Isolated 1.0 × 10 6 MSCs/ml were encapsulated in alginate hydrogel. Cells with MSCs phenotype were isolated from mixed enzymatic-explant and explant procedures within 24 -48 hrs and 7 -10 days, respectively. Both of procedures were shown to form clumps and colonies with dense centers. Phenotypic changes gradually appeared as round cell in UC pieces into homogeneous spindle-shaped and typical fibroblast-like shape cells. By using flow cytometery MSCs showed positive for CD73, and negative for CD31. the morphology of viable MSCs in the beads did not significantly show a different morphology pattern before and after the bead formation process. These findings are indicated that when mixed enzymatic-explant procedure is performed MSCs can be isolated faster and much higher from WJ. These finding is important in comparing with time consuming explants culture for isolation of MSCs.

MTT assay showed significant difference in the mean of viability rates between groups in day 14 (p < 0.05). Alizarin red S staining was higher in the group 1.8%. In addition, there was statistically significant higher ALP activity in the group 1.8% compared to the group 1.2%.

Background: Mesenchymal stem cells (MSCs) are the most promising tools for cell treatment and human tissue regeneration, e.g., in liver fibrosis. Mesenchymal stem cells repair tissue damage through paracrine mediators such as exosomes. Types and concentrations of inflammatory mediators, including transforming growth factor-beta (TGFβ1), in MSCs microenvironment can affect MSCs’ function and therapeutic potency. Objectives: This experimental study aimed to explore the effects of Wharton jelly MSCs (WJ-MSCs) exosomes on fibrotic gene expression and Smad2/3 phosphorylation (phospho-Smad2/3 (p-Smad2/3)). Moreover, we further investigated whether WJ-MSCs pretreatment with different concentrations of TGFβ1 changes the anti-fibrotic properties of their exosomes. Methods: After isolation from the umbilical cord, WJ-MSCs were characterized by observing differentiation and measuring surface biomarkers using flowcytometry. The WJ-MSC-derived exosomes were extracted and identified using transmission electron microscopy (TEM), dynamic light scattering (DLS), and western blotting. Real-time PCR and western blot for extracellular matrix (ECM) and p-Smad2/3 expression detection were used to investigate the effect of exosomes from untreated and TGFβ1-pretreated WJ-MSCs on activated hepatic stellate cells (HSCs). Results: Phospho-Smad2/3, α-smooth muscle actin (α-SMA), and collagen1α1 levels were enhanced following treatment with TGFβ1, whereas E-cadherin was decreased. However, the outcomes were reversed after treatment with WJ-MSC-derived exosomes. Exosomes from TGFβ1-pretreated WJ-MSCs induced a significant decrease in p-Smad2/3 levels in activated HSCs, accompanied by the upregulation of E-cadherin gene expression and downregulation of α-SMA and collagen1α1 when compared to untreated WJ-MSC-derived exosomes. The p-Smad2/3 proteins were significantly decreased (fold change: 0.23, P-value < 0.0001) after exposure to low-dose TGFβ1-pretreated WJ-MSC-derived exosomes (0.1 ng/mL), showing the best effect on activated HSCs. Conclusions: Exosomes derived from untreated WJ-MSCs could regress TGFβ-Smad2/3 signaling and the expression of fibrotic markers in activated LX-2 cells. However, these effects were significantly profound with applying exosomes derived from 0.1 ng/mL TGFβ-pretreated WJ-MSCs. We also observed the dose-response effects of TGFβ on WJ-MSCs-derived exosomes. Therefore, exosomes derived from TGFβ-pretreated WJ-MSCs may be critical in improving fibrosis and benefit liver fibrosis patients.

Cell encapsulation utilizing biodegradable material has promising outcomes for tissue engineering. From a long time ago, alginate has been generally utilized for drug delivery, cell transplantation and as a scaffold in biomedical applications. The aim of this study was the comparison of cell viability in the presence of two polymerizing ions: Ba2+ and Ca2+ to improvement the quality of alginate scaffold. For this purpose, WJMSCs after three passage were encapsulated in alginate scaffold in the presence of Ba2+ and ca2+. Cell viability was evaluated by WST-8 assay kit after 24, 48 and 72 hours. The results showed that encapsulated cells in the presence of Ca2+ had more viability than Ba2+. It was also found that using the WST-8 assay kit is a convenient and fast method for evaluation the viability of cells. It can be claimed that Calcl2 polymerizing solution provides more favorable conditions for cell viability compared to Bacl2 solution. Running title: Assessing the viability of stem cells by WST-8 assay kit

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