A Simple High Yield Technique for Isolation of Wharton's Jelly-derived Mesenchymal Stem Cell.

Background: The isolation of Mesenchymal Stem Cells (MSCs) from various tissues is possible, with the umbilical cord emerging as a competitive alternative to bone marrow. In order to fulfill the demands of cell therapy, it is essential to generate stem cells on a clinical scale while minimizing time, cost, and contamination. Here is a simple and effective protocol for isolating MSC from Wharton's Jelly (WJ-MSC) using the explant method with various supplements. Methods: Utilizing the explant method, small fragments of Wharton's jelly from the human umbilical cord were cultured in a flask. The multipotency of the isolated cells, were confirmed by their differentiation ability to osteocyte and adipocyte. Additionally, the immunophenotyping of WJ-MSCs showed positive expression of CD73, CD90, and CD105, while remaining negative for hematopoietic markers CD34 and CD45, meeting the criteria for WJ-MSC identification. Following that, to evaluate cells' proliferative capacity, various supplements, including basic Fibroblast Growth Factor (bFGF), Non-Essential amino acids (NEA), and L-Glutamine (L-Gln) were added to either alpha-Minimal Essential Medium (α-MEM) or Dulbecco's Modified Eagle's Medium-F12 (DMEM-F12), as the basic culture media. Results: WJ-MSCs isolated by the explant method were removed from the tissue after seven days and transferred to the culture medium. These cells differentiated into adipocyte and osteocyte lineages, expressing CD73, CD90, and CD105 positively and CD34 and CD45 negatively. The results revealed that addition of bFGF to α-MEM or DMEMF12 media significantly increased the proliferation of MSCs when compared to the control group. However, there were no significant differences observed when NEA or LGln were added. Conclusion: Although bFGF considerably enhances cell proliferation, our study demonstrates that MSCs can grow and expand when properly prepared Wharton's jelly tissues of the human umbilical cord.

Harnessing HEK293 cell-derived exosomes for hsa-miR-365a-3p delivery: Potential application in hepatocellular carcinoma therapy.

Hepatocellular carcinoma (HCC) is the most frequent form of liver malignancy, and curing it is very challenging. Restoring tumor suppressor microRNAs could trigger the initiation of cellular anticancer mechanisms. Exosomes are nanosized biocarriers capable of fusing with cell membranes and delivering their cargo. The main goal of the current study was to explore the potential of human embryonic kidney cells (HEK293) cell-derived exosomes to provide an anticancer therapy based on the restoration of tumor suppressor miR-365a downregulated in HepG2 cells. To accomplish this aim, exosomes were isolated from the HEK293 cell line culture and characterized, enriched by Homo sapiens (hsa) miR-365a-3p mimics. Exosomes enabled an efficient loading and intracellular delivery of hsa-miR-365a mimics, which translated into G0/G1 cell cycle arrest, induction of oxidative stress, reduction of migration capacity, and high apoptosis rate. The findings indicate that the delivery of miR-365a-3p by HEK293-derived exosomes may act as an innovative and effective therapeutic strategy against HCC.

Obeticholic acid-loaded exosomes attenuate liver fibrosis through dual targeting of the FXR signaling pathway and ECM remodeling.

End-stage of liver fibrosis as a precancerous state could lead to cirrhosis and hepatocellular carcinoma which liver transplantation is the only effective treatment. Previous studies have indicated that farnesoid X receptor (FXR) agonists, such as obeticholic acid (OCA) protect against hepatic injuries. However, free OCA administration results in side effects in clinical trials that could be alleviated by applying bio carriers such as MSC-derived exosomes (Exo) with the potential to mimic the biological regenerative effect of their parent cells, as proposed in this study. Loading OCA into the Exo was conducted via water bath sonication. Ex vivo bio distribution studies validated the Exo-loaded OCA more permanently accumulated in the liver. Using CCL4-induced liver fibrosis, we proposed whether Exo isolated from human Warton's Jelly mesenchymal stem cells loaded with a minimal dosage of OCA can facilitate liver recovery. Notably, Exo-loaded OCA exerted additive anti-fibrotic efficacy on histopathological features in CCL4-induced fibrotic mice. Compared to baseline, Exo-mediated delivery OCA results in marked improvements in the fibrotic-related indicators as well as serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) concentrations. Accordingly, the synergistic impact of Exo-loaded OCA as a promising approach is associated with the inactivation of hepatic stellate cells (HSCs), extracellular matrix (ECM) remodeling, and Fxr-Cyp7a1 cascade on CCL4-induced liver fibrosis mice. In conclusion, our data confirmed the additive protective effects of Exo-loaded OCA in fibrotic mice, which suggests a valuable therapeutic strategy to combat liver fibrosis. Furthermore, the use of Exo for accurate drug delivery to the liver tissue can be inspiring.

In vitro evaluation of ferutinin on proliferation and osteogenesis differentiation in human unrestricted Somatic stem cells.

Osteoporosis is a common disease in post-menopausal women. The increased risk of breast cancer and malignancy with hormone replacement, hampers its wide-usage. Phytoestrogens are known to have selective estrogen receptor modulator activity. The present study aims to determine how ferutinin affects unrestricted human Somatic Stem Cells (USSCs) osteogenic differentiation. The effect of ferutinin on USSCs proliferation was assessed by MTT assay while osteogenesis was evaluated using Alkaline Phosphatase Activity (ALP), calcium deposition and Alizarin Red Staining. Quantitative real-time PCR was applied to examine the expression of bone specific genes such as osteocalcin, Runx2, and BMP-2. Ferutinin (5-15 µg/mL) could positively impact on the proliferation of cells in a dose-dependent manner. Also, ALP enzyme activity and calcium deposition were enhanced in the presence of ferutinin. Based on real-time PCR results, ferutinin could increase the expression of bone marker genes. The pattern of ferutinin effect on gene expression is similar to standard synthetic estrogen, 17-ß-estradiol. In the presence of the estrogen activity inhibitor (ICI), the effect of ferutinin on ALP and gene level was diminished. In conclusion, ferutinin may be considered as a potential candidate for the stem cell therapy in osteoporosis.

Ethyl Acetate Extract of Licorice Root (Glycyrrhiza glabra) Enhances Proliferation and Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells.

Glycyrrhiza glabra (G. glabra) has been used as a flavoring and sweetener agent, in addition to its therapeutic properties. It is rich in phytoestrogen and may prevent osteoporosis caused by estrogen deficiency; however, there is no evidence for its effects on proliferation and osteogenesis in mesenchymal stem cells. So, we were encouraged to investigate whether the ethyl acetate extract of licorice root as a source of phytoestrogen can act similar to estrogen in cell culture. Furthermore, the analysis of the licorice extract (LE) based on HPLC-DAD-ESI-MS indicated that LE comprises phytoestrogen compounds, such as glabridin and glabrene. In this study, the effects of LE on proliferation of human bone-marrow mesenchymal stem cells (hBM-MSCs) were investigated using MTT assay. In addition, its effects on the osteogenesis were evaluated using alkaline phosphatase activity (ALP), calcium deposition, and bone specific gene expression such as ALP, osteocalcin, Runx2, and BMP-2. The quantitative gene expression was studied by real-time RT-PCR. Our results showed a significant increase in proliferation in presence of LE in concentration 10-50 µg/mL. The differentiation of hBM-MSCs increased in doses of LE (10-25 µg/mL) compared to the control group. The effects of LE were similar to those of 17ß-estradiol (E2) (10-8 M) and were abolished by ICI 182,780 an antagonist of estrogen receptor (ER) (10-7), indicating that the stimulatory effects of LE occur through estrogen receptor-mediated mechanism . Taking these into account, LE may be a potential candidate for prevention of osteoporosis in menopausal women.