Improved Differentiation of Mesenchymal Stem Cells into Hepatocyte-like Cells using FGF4 and IGF-1 in 3D Culture

Human Umbilical Cord Mesenchymal Stem Cells (UCMSCs) are considered as an excellent candidate for cell therapy to treat end-stage liver disease. Fibroblast Growth Factor-4 (FGF4), Hepatocyte Growth Factor, and Insulin-like Growth Factor-1 are some of the critical cytokines involved in liver development and regeneration. To evaluate the differentiation potency of cells into hepatocyte-like cells we used these cytokines. UCMSCs were isolated from Wharton's jelly of fullterm infants. The cells were characterized as MSCs by flow-cytometry and their multilineage differentiation capacity. Then, UCMSCs were cultured in 3D collagen scaffold and hepatogenic media with or without FGF4 for 21 days and the data were compared to control. The expression of liver specific genes was evaluated by real-time quantitative RT-PCR and immunocytochemistry. These cells expressed MSC markers and could differentiate into adipocytes and osteocytes. A non–significant higher level of liver specific genes, such as cytokeratin-18 and 19, alpha-fetoprotein and albumin, and also a significant higher level of CYP2B6 expressed by UCMSCs in hepatogenic medium containing FGF4 compared with control. In some specimens, cytokeratin-19-positive cells surrounded a luminal space within collagen scaffolds. Liver-specific marker expression was increased by pre-exposing the cells to FGF4 before treating with IGF-1 and HGF in 3D collagen scaffold. Abbreviations: UCMSCs: Human Umbilical Cord Mesenchymal Stem Cells; FGF4: Fibroblast Growth Factor 4; HGF: Hepatocyte Growth Factor; IGF-1: Insulin-like Growth Factor-1; MSCs: Mesenchymal Stem Cells; ICG: Indocyanine green; PAS: periodic acid Schiff; CK-18: cytokeratin-18; CK-19: Cytokeratin-19; AFP: alpha-fetoprotein; G6P: glucose 6 phosphatase; PEPCK: phosphoenolpyruvate carboxykinase; TAT: tyrosine amino transferase; FBS: Fetal Bovine Serum; OSM: oncostatin M; RT-PCR: Reverse Transcription Polymerase Chain Reaction; PBS: Phosphate-Buffered Saline; Hep- Par1: Hepatocyte paraffin 1; DAB: Diaminobenzidine; CYP2B6: Cytochrome P450 2B6

The effect of amniotic membrane extract on umbilical cord blood mesenchymal stem cell expansion: is there any need to save the amniotic membrane besides the umbilical cord blood?

Objective(s): Umbilical cord blood is a good source of the mesenchymal stem cells that can be banked, expanded and used in regenerative medicine.  The objective of this study was to test whether amniotic membrane extract, as a rich source of growth factors such as basic-fibroblast growth factor, can promote the proliferation potential of the umbilical cord mesenchymal stem cells. Materials and Methods: The study design was interventional. Umbilical cord mesenchymal stem cells were isolated from voluntary healthy infants from hospitals in Shiraz, Iran, cultured in the presence of basic-fibroblast growth factor and amniotic membrane extracts (from pooled - samples), and compared with control cultures. Proliferation assay was performed and duplication number and time were calculated. The expression of stem cell’s specific markers and the differentiation capacity toward osteogenic and adipogenic lineages were evaluated. Results: Amniotic membrane extract led to a significant increase in the proliferation rate and duplication number and a decrease in the duplication time without any change in the cell morphology. Both amniotic membrane extract and basic-fibroblast growth factor altered the expressing of CD44 and CD105 in cell population. Treating basic-fibroblast growth factor but not the amniotic membrane extract favored the differentiation potential of the stem cells toward osteogenic lineage. Conclusion: The amniotic membrane extract administration accelerated cell proliferation and modified the CD marker characteristics which may be due to the induction of differentiation toward a specific lineage.  Amniotic membrane extract may enhance the proliferation rate and duplication number of the stem cell through changing the duplication time

Growth suppression effect of human mesenchymal stem cells from bone marrow, adipose tissue, and Wharton's jelly of umbilical cord on PBMCs

Objective(s):Immunosuppressive property of mesenchymal stem cells (MSCs) has great attraction in regenerative medicine especially when dealing with tissue damage involving immune reactions. The most attractive tissue sources of human MSCs used in clinical applications are bone marrow (BM), adipose tissue (AT), and Wharton's jelly (WJ) of human umbilical cord. The current study has compared immunomodulatory properties of human BM, AT, and WJ-MSCs. Materials and Methods: Three different types of human MSCs were isolated, cultured, and characterized by flow cytometry and differentiation potentials. The MSCs were co-cultured with allogeneic phytohemagglutinin (PHA) activated peripheral blood mononuclear cells (PBMCs). The proliferation of PBMCs was assessed by flow cytometry of carboxyfluorescein succinimidyl ester (CFSE) stained cells and compared to each other and to the growth of PBMCs in the absence of MSCs, 3 days post co-culture. Additionally, the growth suppression was indirectly assessed by using the transwell culture system. Results: the proliferation of PBMCs reduced to 6.2, 7 and 15.4- fold in cultures with AT-MSCs, WJ-MSCs, and BM-MSCs, respectively, compared to the PHA-activated cells. When the growth suppression was indirectly assessed by using the transwell culture system, it was revealed that AT-MSCs, WJ-MSCs, and BM-MSCs caused growth reduction in PBMCs to 3, 8, and 8 -fold, respectively, compared to the PHA-activated cells. Conclusion:These data collectively conclude that the immunomodulatory effects of MSCs, which may mostly carry out through direct cell to cell contact, are different between various sources. Accordingly results of this study may contribute to the application of these cells in cell therapy and regenerative medicine

Effects of L-Carnitine and Pentoxifylline on Carbohydrate Distribution of Mouse Testicular Sperm Membrane

Background: The glycoconjugate content of sperms indicates their physiological and fertility properties. Lectin reactivity is indicative of intact, capacitated, and acrosome-reacted sperms. In the epididymis, sperms experience maturation, glycoconjugate modification, and simultaneously, higher L-carnitine (LC) concentrations. The aim of this project was to evaluate the effects of LC and Pentoxifylline (PF) on the integrity, capacitation, and acrosomal reaction of sperms by studying their lectin reactivity.Methods: Mouse testicular sperm samples were divided into three parts. Each sample was added Ham’s F10 (control) or media containing 1.76 mM LC or PF. At 30 and 90 minutes after incubation, sperm motility was assessed. Peanut agglutinin (PNA), wheat germ agglutinin (WGA), and Concanavalin A (Con A) were used to detect non-acrosome-reacted, non-capacitated, and acrosome-reacted sperms, respectively and the frequency was evaluated by flow cytometry. Statistical analysis was performed using the ANOVA. Results: Sperm motility increased after 30 and 90 minutes of incubation in the LC- and PF-treated cultures (P=0.001). LC administration created a significant increase in the percentage of the non-acrosome-reacted sperms compared to the control sperms after 30 and 90 minutes (P=0.02 and P=0.03, respectively). The frequency of the non-capacitated sperms in the LC-treated group increased compared to the control sperms after 30 minutes significantly (P=0.01). Conclusion: Although the administration of LC and PF enhanced sperm motility, LC also impacted glycoconjugates on the sperm surface. Glycoconjugates are involved in the interaction between the sperm and the zona pellucida and subsequently fertilization, thereby probably influencing the male fertility state

Differentiation of Human Breast-Milk Stem Cells to Neural Stem Cells and Neurons

Objectives. Human breast milk contains a heterogeneous population of cells that have the potential to provide a noninvasive source of cells for cell therapy in many neurodegenerative diseases without any ethical concern. The objectives of this study were to differentiate the breast milk-derived stem cells (BMDSC) toward neural stem cells and then into the neurons and neuroglia. Materials and Methods. To do this, the BMDSC were isolated from human breast milk and cultured in Dulbecco’s modified Eagle medium/F12 (DMEM/F12) containing fibroblast growth factor (bFGF). The cells were then characterized by evaluation of the embryonic and stem cell markers. Then, the cells were exposed to culture medium containing 1% B27 and 2% N2 for 7–10 days followed by medium supplemented with B27, N2, bFGF 10 µg/mL, and endothelial growth factor (EGF) 20 µg/mL. Then, the sphere-forming assay was performed. The spheres were then differentiated into three neural lineages by withdrawing growth factor in the presence of 5% FBS (fetal bovine serum). The immunofluorescence was done for β-tubulin III, O4, and GFAP (glial fibrillary acidic protein). Results. The results indicated that the cells expressed both embryonic and mesenchymal stem cell (MSC) markers. They also showed neurospheres formation that was nestin-positive. The cells were also differentiated into all three neural lineages. Conclusion. The BMDSC can behave in the same way with neural stem cells. They were differentiated into oligodendrocytes, and astrocytes as well as neurons

Effect of Pre-Incubation of Cryopreserved Sperm with either Kisspeptin or Glutathione to Mitigate Freeze-Thaw Damage

Background: Sperm cryopreservation reduces sperm quality. Kisspeptin (KP) has beneficial effects on sperm functions. This study compares the effect of KP and Glutathione (GSH) on mitigating the detrimental effects of the freeze-thaw cycle on sperm. Methods: An experimental study was conducted in Birjand (Iran) during 2018-2020. Thirty normal swim-up semen samples were treated with Ham’s F10 medium (negative control), 1 mM GSH (positive control), or KP (10 µM) for 30 min before freezing. The motility, acrosome reaction, capacitation, and DNA quality of the frozen-thawed sperms were assessed according to the WHO guidelines. Statistical analysis was performed using paired t test, one-way analysis of variance, and least significant difference.Results: Pre-incubation with KP significantly increased the percentage of sperm motility (34.00±6.7, P=0.003) compared to the control (20.44±7.4) and GSH-treated (31.25±12.2) aliquots. The frequency of non-capacitated spermatozoa was significantly higher in the KP-treated group (98.73%) than in the control (96.46%) and GSH-treated (96.49%) aliquots (P<0.001). The percentage of acrosome-intact spermatozoa in the KP-treated group (77.44%) was significantly higher than the control (74.3%) and GSH-treated (74.54%) groups (P<0.001). The sperm frequency with normal histone in the KP-treated group (51.86%) and with normal protamine (65.39%) was significantly higher than the controls (P=0.001 and P=0.002, respectively). The percentage of TUNEL-positive sperm was significantly lower in the KP-treated group (9.09±2.71) than both GSH-treated (11.22±2.73) and control (11.31±2.2) groups (both P=0.002).Conclusion: Pre-incubation with KP protects sperm motility and DNA integrity from the detrimental effect of the freeze-thaw cycle. KP is suitable as a pre-treatment to control sperm quality during freezing-thawing