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

Comparison of Cell Viability and Embryoid Body Size of Two Embryonic Stem Cell Lines After Different Exposure Times to Bone Morphogenetic Protein 4

Background: Activation of bone morphogenetic protein 4 (BMP4) signaling pathway in embryonic stem (ES) cells plays an important role in controlling cell proliferation, differentiation, and apoptosis. Adverse effects of BMP4 occur in a time dependent manner; however, little is known about the effect of different time exposure of this growth factor on cell number in culture media. In this study, we investigated the role of two different exposure times to BMP4 in cell viability, embryoid body (EB), size, and cavitation of ES cells. Methods: Embryonic stem cells (R1 and B1 lines) were released from the feeder cell layers and were cultured using EBs protocol by using the hanging drop method and monolayer culture system. The cells were cultured for 5 days with 100 ng/mL BMP4 from the beginning (++BMP4) or after 48 h (+BMP4) of culture and their cell number were counted by trypan blue staining. The data were analyzed using non-parametric two-tailed Mann-Whitney test. P<0.05 was considered as significant. Results: In EB culture protocol, cell number significantly decreased in +BMP4 culture condition with greater cavity size compared to the ++BMP4 condition at day 5 (P=0.009). In contrast, in monolayer culture system, there was no significant difference in the cell number between all groups (P=0.91). Conclusion: The results suggest that short-term exposure of BMP4 is required to promote cavitation in EBs according to lower cell number in +BMP4 condition. Different cell lines showed different behavior in cavitation formation

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

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

The effects of activated-omental extract on nuclear and cytoplasmic in vitro maturation of rat oocytes

Objective: The role of growth factors, including vascular endothelial growth factor of activated omentum on mitosis is clearly known, though not on all the aspects of in vitro oocyte maturation. This study was designed to assess the effect of activated-omental extract (AOE) on in vitro maturation (IVM) of rat cumulus-oocyte complexes (COCs). Materials and Methods: In this experimental study, the COCs were incubated in Ham’s F-10 supplemented with either 20% AOE, 20% fetal bovine serum (FBS) or serum-free media. Post-culture COCs were studied according to the cumulus cells (CCs) expansion, nuclear maturation and cytoplasmic maturation. Cumuli expansion was evaluated by inverted microscope without staining; nuclear maturation was assessed by aceto-orcein staining (light microscope) and cytoplasmic maturation was also observed by TEM. Results: Expansion of CCs and nuclear maturation of the oocytes in in vitro for 24 hr was significantly higher in AOE- and FBS-supplemented groups (P=0.000 and 0.013) and (P=0.004 and 0.014), respectively, compared to serum-free group. At ultra-structural level, after 24 hr, both FBS and AOE-supplemented media showed uniformly wide perivitelline space (PVS). After 12 hr, the cortical granules were found in the oocytes cultured in FBS and AOE-supplemented media. Within 24 hr, both granules and mitochondria were large without any detectable topographic tendency across the ooplasm. In AOE and FBS- supplemented oocytes, the number and size of microvilli were more than those in serum-free one. Conclusion: Although AOE supplementation induced a higher rate of the CCs expansion, and resuming meiosis, it was not as potent as FBS to provide cytoplasmic maturation of rat oocytes

Wharton’s Jelly-derived Mesenchymal Stem Cells can Differentiate into Hepatocyte-like Cells by HepG2 Cell Line Extract

Background: Wharton’s jelly is an unlimited source of stem cells that can be used in cell therapy and tissue engineering without any ethical concern. It has been revealed the cell-free extract could be effective to induce cell differentiation. The objective of this study was to induce Wharton’s jelly-derived mesenchymal stem cells (MSCs) into hepatocyte-like cells by premeabilization of the cells in the presence of HepG2 cell line extract. Methods: MSCs were isolated from the umbilical cord, CD marker profile and their differentiation potential into adipogenic and osteogenic lineages were determined. The cells were then, permeabilized by streptolysin O in the presence of HepG cell extract. The treated cells were cultured for 17 days. The cell phenotype was evaluated and the hepatocyte specific markers were detected by immunofluorescence and immunocytochemistry. The Periodic Acid Schiff (PAS) reaction and the cellular uptake of indocyanine green were performed to evaluate the functional behavior of the differentiated cells. Results: The phenotype of extract-treated MSCs changed into a round or polygonal cells with few short processes and they could express high level of albumin, cytokeratin 18 and 19. The MSCs also could store glycogen and uptake and release indocyanine green. Conclusion: We demonstrated for the first time that Wharton’s jelly-derived MSCs could differentiate into hepatocyte-like cells by premeabilization of them in the presence of HepG2 cell extract. This study suggests a feasible method to differentiate MSCs into functional hepatocyte-like cells

A comparison of the multiple oocyte maturation gene expression patterns between the newborn and adult mouse ovary

Background: The interaction between follicular cells and oocyte leads to a change in gene expression involved in oocyte maturation processes. Objective: The purpose of this study was to quantify the expression of more common genes involved in follicular growth and oocyte developmental competence. Materials and Methods: In this experimental study, the expression of genes was evaluated with qRT-PCR assay in female BALB/c mice pups at 3-day of pre-pubertal and 8 week old virgin adult ovaries. The tissue was prepared by H&E staining for normal morphological appearance. The data were calculated with the 2-ΔCt formula and assessed using non-parametric two-tailed Mann-Whitney test. The p<0.05 was considered as significant. Results: The data showed a significant increase in the level of Stra8 and GDF9 in adult compared with newborn mice ovaries (p=0.049). In contrast, a significant decrease in the level of Mvh, REC8, SCP1, SCP3, and ZP2 was observed in adult mice ovaries compared to those in the newborn mice ovaries (all p=0.049 except SCP1: p=0.046). There was no significant difference in the level of OCT4 and Cx37 expression between adult and newborn mice ovaries. Conclusion: The modifications in gene expression patterns coordinate the follicular developmental processes. Furthermore, the findings showed higher expression level of premeiotic gene (Stra8) and lower level of meiotic entry markers (SCP1, SCP3, and REC8) in juvenile than newborn mouse ovaries

Cardiomyocyte Marker Expression in Mouse Embryonic Fibroblasts by Cell-Free Cardiomyocyte Extract and Epigenetic Manipulation

Background: The regenerative capacity of the mammalian heart is quite limited. Recent reports have focused on reprogramming mesenchymal stem cells into cardiomyocytes. We investigated whether fibroblasts could transdifferentiate into myocardium. Methods: Mouse embryonic fibroblasts were treated with Trichostatin A (TSA) and 5-Aza-2-Deoxycytidine (5-aza-dC). The treated cells were permeabilized with streptolysin O and exposed to the mouse cardiomyocyte extract and cultured for 1, 10, and 21 days. Cardiomyocyte markers were detected by immunohistochemistry. Alkaline phosphatase activity and OCT4 were also detected in cells treated by chromatin-modifying agents. Results: The cells exposed to a combination of 5-aza-dC and TSA and permeabilized in the presence of the cardiomyocyte extract showed morphological changes. The cells were unable to express cardiomyocyte markers after 24 h. Immunocytochemical assays showed a notable degree of myosin heavy chain and α-actinin expressions after 10 days. The expression of the natriuretic factor and troponin T occurred after 21 days in these cells. The cells exposed to chromatin-modifying agents also expressed cardiomyocyte markers; however, the proportion of reprogrammed cells was clearly smaller than that in the cultures exposed to 5-aza-dC , TSA, and extract. Conclusion: It seems that the fibroblasts were able to eliminate the previous epigenetic markers and form new ones according to the factors existing in the extract. Since no beating was observed, at least up to 21 days, the cells may need an appropriate extracellular matrix for their function

Stereological study of the effect of noise stress on the fetus mouse kidney

Background: Sound pollution causes kidney damage in adult but this effect of sound exposure on the fetus is unknown. Therefore, the aim of this study was to find the effect of noise on the structure and volume of fetal kidney. Materials & methods: A total of 32 pregnant mice were divided into four groups. Pregnant mouse in experimental groups were exposed to load 100 dB continuous noise stress, 2.5 hour per day. After birth, fetus kidneys were removed at days 7 and 14. Tissues processing were performed, and sections with 0.5 micron thickness were prepared with orientator method. Total volume and volume of cortex and medulla were calculated according to Cavalier principle. Amount of matrix, inflammatory reaction and necrosis were evaluated by Knodell Scoring system. Results: The results show that there was no significant difference between the volume of different parts of kidney in experimental and control groups (P<0.05). Microscopic evaluations also revealed that sound wave has no effect on the histological structure of kidney. Conclusion: It seems that sound wave has no effect on structure and volume of kidney