Synergistic impact of platelet rich plasma-heparin sulfate with hydroxyapatite/zirconia on the osteoblast differentiation potential of adipose-derived mesenchymal stem cells.

3D porous hydroxyapatite (HA) has been reinforced by zirconia (ZrO2) coating and impregnation with a combination of platelet rich plasma (PRP) as a source of growth factors (GFs) and Heparin sulfate (HS) to sustain the release of GFs. Adipose mesenchymal stem cells (ADMSCs) were characterized by flow cytometry for CD (cluster of differentiation) 44, CD105, CD106, CD34 and CD144, along with checking the multipotency by differentiation into the adipocytes and osteoblasts. Then, they were cultured on the scaffold treated with and without osteogenic media on days 7, 14 and 21. Electron micrograph and PKH staining show that the ADMSCs have a fusiform phenotype in the absence of osteogenic induction. Cell viability assay shows a higher number of the viable cells on the PRP-containing scaffolds than PRP-free scaffolds on day 7. Colorimetric evaluation, quantitative RT-PCR and immunocytochemistry demonstrate that PRP and HS significantly elevate the alkaline phosphatase enzyme activity and also accelerate the production of both early and mid-osteogenic markers, including collagen I and osteopontin expression with and without osteogenic conditions. The PRP-HS also accelerates the expression of the late osteogenic marker, osteocalcin, in both mRNA and protein level expression with a peak on day 21. In conclusion, supplementation of HA/ZrO2 with PRP/HS has a synergistic impact on the ADMSCs, even in the absence of chemical induction. It seems that HA/ZrO2/PRP/HS scaffold provides a higher osteoconductive microenvironment for stem cell differentiation to osteoblasts.

Promoting hepatogenic differentiation of human mesenchymal stem cells using a novel laminin-containing gelatin cryogel scaffold.

Liver transplantation is the only definitive treatment currently available for acute and chronic liver failure. However, this approach has been restricted by complications including rejection and infection. Tissue engineering approaches using stem cell-derived functional hepatic cells offer a potential alternative. Using biologically compatible scaffolds is an important complementary key to achieve optimal construct for hepatic replacement. In the present study, to optimize the differentiation of human adipose-derived mesenchymal stem cells (ADMSCs) toward hepatocyte-like cells, a previously described gelatin cryogel was optimized and improved by laminin, the major component of basal lamina. The ADMSCs seeded on the scaffold displayed increased attachment in the presence of laminin and the MTT assay showed good compatibility for cell proliferation. The differentiation of stem cells were evaluated using glycogen staining, urea secretion measurement, hepatocyte specific cell surface analysis and gene expression analysis. The results of tests indicated that laminin protein and gelatin cryogel 3D scaffold, each on its own, enhanced hepatogenic differentiation of ADMSCs. However, when laminin immobilized on the gelatin cryogel surface, the differentiation was promoted significantly and the resulting cells showed striking similarity to HepG2 in terms of expressing studied hepatocyte markers.

Subcellular localization of L-selectin ligand in the endometrium implies a novel function for pinopodes in endometrial receptivity.

BACKGROUND: Apical surfaces of human endometrial epithelium and endothelium are key elements for the initiation of molecular interactions to capture the blastocyst or leukocyte, respectively. The L-selectin adhesion system has been strongly proposed to play an important role in the initial steps of trophoblast adhesion and promotion of integrin-dependent processes, ultimately culminating in the establishment of the embryo-maternal interface. On the basis of these facts, we hypothesized a novel role for pinopodes as the first embryo-fetal contact sites to contain the highest subcellular expression of L-selectin ligand suggesting its role in early adhesion as predicted. Thus, the objective of this study was therefore to determine the subcellular pattern of distribution of the L-selectin ligand (MECA-79) in human endometrial apical membrane region during the window of implantation. METHODS: Endometrial biopsies of secretory phases from fertile females ranging in age between 25 and 42 years were studied using several approaches, including scanning electron microscopy (SEM), immunostaining for light microscopy and transmission electron microscopy (TEM), and immunoblotting as well as statistical analysis of the area-related numerical densities of immunoreactive MECA-79-bound nanogolds to detect the expression pattern and the subcellular distribution pattern of L-selectin ligand (MECA-79) in human endometrium during the window of implantation. RESULTS: The endometrial biopsies were scored according the dating criteria of Noyes et al. by an experienced histologist. The SEM images of the midluteal phase specimens revealed that fully developed pinopodes were abundant in our samples. HRP-immunostaining and immunofluorescent staining as well as immunoblotting revealed that MECA-79 was expressed in the midluteal phase specimens. The results of immunogold TEM illustrated the expression of MECA-79 in human pinopodes in the midluteal phase and a higher area-relate numerical density in pinopodes compared to that of the uterodome-free areas. CONCLUSIONS: This is the first demonstration of the subcellular localization of MECA-79 in the human pinopodes which may indicate a novel role for pinopodes to be capable of shear-stress-dependent tethering-type adhesion in the initial phases of human embryo implantation.

Ectopic expression of OCT4B1 Decreases Fertility Rate and Changes Sperm Parameters in Transgenic Mice.

Background: The octamer-binding transcription factor-4 (OCT4) is known as an established important regulator of pluripotency, as well as a genetic "master switch" in the self-renewal of embryonic stem and germ cells. OCT4B1, one of the three spliced variants of human OCT4, plays crucial roles in the regulation of pluripotency and stemness. Objectives: The present study developed a transgenic mouse model containing an OCT4B1-expressing construct under the transcriptional direction of mouse mammary tumor virus promoter (pMMTV) to evaluate the role of OCT4B1 in the function of male germ cells in terms of fertility potential. Additionally, the effect of ectopic OCT4B1 overexpression on endogenous OCT4 expression was examined in mouse embryonic stem cells (mESCs). Material and Methods: The pMMTV-OCT4B1cDNA construct was injected into the pronuclei of 0.5-day NMRI embryos. Transgenic mice were identified based on the PCR analysis of tail DNA. Further, Diff-Quik staining was applied to assess sperm morphology, while the other sperm parameters were analyzed through a conventional light microscopic evaluation according to World Health Organization (WHO) criteria. The fertility rate was scored by using in vitro frtilization (IVF) method. Furthermore, mESCs was electroporated with the OCT4B1cDNA-containing constructs, followed by analyzing through employing semi-quantitative RT-PCR and western blotting. Results: The results demonstrated the changes in sperm morphology, as well as a statistically significant decrease in the other sperm parameters (count, viability, and motility) and fertility rate (p<0.05) in the transgenic mice compared with the control group. The assessment of the cause of the embryonic stem cell (ESC) death following transfection revealed a significant reduction in the endogenous OCT4 expression at both mRNA and protein levels in the transfected mESCs compared to the control ones. Conclusion: In general, the in vivo results suggested a potential role of OCT4B1 in the spermatogenesis process. These results represented that the overexpression of OCT4B1 may induce its role in spermatogenesis and fertility rate by interfering endogenous OCT4 expression. However, further studies are required to clarify the mechanisms underlying OCT4B1 function.

Polymerization of insulin-like growth factor-binding protein-1 (IGFBP-1) potentiates IGF-I actions in placenta.

Insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1), the main secretory protein of decidua that binds to IGFs and has been shown to inhibit or stimulate IGFs' bioactivities. Polymerization, one of the posttranslational modifications of IGFBP-1, has been shown to lead to loss of inhibiting effect of IGFBP-1 on IGF-I actions. The current studies were undertaken to elucidate the effects of steroid hormones on IGFBP-1 polymerization in trophoblast cell cultures. Placental tissues were obtained during legal, elective procedures of termination of pregnancy performed between 7 and 10 weeks of gestation, and primary trophoblast cells were separated. IGFBP-1 polymerization was analyzed by SDS-PAGE and immunoblotting. IGFBP-1 was polymerized when IGFBP-1 was added to trophoblast cell cultures. Polymerization of IGFBP-1 was inhibited by the addition of anti-tissue transglutaminase antibody into the culture media. There was an increase in the intensity of polymerized IGFBP-1 bands with the addition of medroxyprogesterone acetate (MPA), while no such difference was observed upon treatment with estradiol. MPA also increased the expression of tissue transglutaminase on trophoblast cell membranes. IGF-I stimulated trophoblast cell migration, while IGFBP-1 inhibited this IGF-I-induced trophoblast response. Addition of MPA attenuated the inhibitory effects of IGFBP-1 on IGF-I-induced trophoblast cell migration. IGFBP-1 was polymerized by tissue transglutaminase on the cell surface of trophoblasts, and MPA increased tissue transglutaminase expression on the cell surface and facilitated IGFBP-1 polymerization. These results suggest that progesterone might facilitate polymerization of decidua-secreted IGFBP-1 and increase IGF-I actions at feto-maternal interface, thereby stimulating trophoblast invasion of maternal uterus.

A VHH-Based Anti-MUC1 Chimeric Antigen Receptor for Specific Retargeting of Human Primary T Cells to MUC1-Positive Cancer Cells.

OBJECTIVE: Immunotherapy with redirected T cells that express a chimeric antigen receptor (CAR) is a promising prospect in cancer treatment. Most CARs use murine-derived single-chain variable fragments (scFvs) as an antigen targeting moiety, which may lead to host immunogenic responses and engineered T cell disappearance. It seems that development of less immunogenic CARs, such as CARs composed of the camelid variable domain of heavy chain antibodies (VHHs) may likely overcome this obstacle. Here, we improved the expression of the VHH-based anti-MUC1 CAR gene construct using a third generation lentiviral vector in primary human T cells and assessed its effect on antigen specific targeting, activation and cytotoxicity of redirected human T cells. MATERIALS AND METHODS: In this experimental study, we established a second generation novel CAR (VHH-based anti- MUC1 CAR) that contained a camelid-derived anti-MUC1 VHH followed by an IgG3 hinge, a CD28 transmembrane domain and signalling endodomains of CD28 and CD3ζ. Next, we constructed lentiviral vectors that contained this CAR gene construct using an optimized transiently virus production method and transduced it into human T cells. Cell surface expression of CAR, cytokine secretion and cytotoxic activity were assessed in the transduced CD3+ T cells. RESULTS: The transduced T cells had high levels of surface expression of CAR. T cells that expressed anti-MUC1 CAR showed significantly increased secretion of Th1 cytokines, including IL-2, TNF alpha and IFN-γ, as well as cytotoxic activity upon recognition of MUC1 on tumour cells after co-incubation with T47D or MCF-7 (MUC1-positive) compared with A431 (MUC1-negative) or untransduced T cells. CONCLUSION: Our results suggested that, given the unique properties of VHHs to prevent immunogenic responses and tonic signalling, our novel VHH-based anti-MUC1 CAR might be effective for clinical purposes in cancer immunotherapy.

Evaluation of Fibroblast Viability Seeded on Acellular Human Amniotic Membrane.

BACKGROUND: Investigating the viability and proliferative rates of fibroblast cells on human amniotic membrane (HAM) as a scaffold will be an important subject for further research. The aim of this study was to assess the fibroblast viability seeded on acellular HAM, since foreskin neonatal allogenic fibroblasts seeded on HAM accelerate the wound healing process. METHODS: Fibroblasts were retrieved from the foreskin of a genetically healthy male infant, and we exploited AM of healthy term neonates to prepare the amniotic scaffold for fibroblast transfer. After cell culture, preparation of acellular HAM, and seeding of cells on HAM based on the protocol, different methods including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI), and propidium iodide (PI) staining were employed for assessment of fibroblast viability on HAM. RESULTS: Based on the results obtained from the DAPI and PI staining, the percentage of viable cells in the former staining was clearly higher than that of the dead cells in the latter one. The results of DAPI and PI staining were in accordance with the findings of MTT assay, confirming that fibroblasts were viable and even proliferate on HAM. CONCLUSION: Our findings showed the viability of fibroblasts seeded on the acellular HAM using MTT assay, DAPI, and PI staining; however, this study had some limitations. It would be an interesting subject for future research to compare the viability and proliferation rate of fibroblasts seeded on both cellular and acellular HAM.

Properties of L-type amino acid transporter 1 in epidermal ovarian cancer.

HYPOTHESIS: To investigate the expression and the functional properties of L-type amino acid transporter 1 (LAT1) in human epithelial ovarian cancer to provide a basis for potential new therapies to control the growth and the metastasis of ovarian cancer. METHODS: The material used comprised 63 surgically resected specimens obtained from female patients undergoing gynecologic surgery at Kyorin University School of Medicine (Tokyo, Japan). The expression of LAT1 in 53 cases of ovarian cancers was determined by Western blot and immunohistochemical staining, and results were compared with those of normal ovarian tissues (5 cases) and benign ovarian tumors (5 cases). Furthermore, we examined the effect of 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), the classic inhibitor of system L on the survival, the migration, and the uptake of l-leucine by human epithelial ovarian cancer cell line (OVCAR-3). RESULTS: The LAT1 was significantly up-regulated in various human epithelial ovarian cancers that was localized predominantly on their plasma membrane and in the plasma membrane of the ovarian cancer cell line in conjunction with 4F2hc via disulfide bonds. The BCH inhibited the proliferation and the migration of the OVCAR-3 cells and the uptake of [14C]l-leucine by these cells in a dose-dependent manner. The OVCAR-3 cells did not express LAT2, and the uptake of [14C]l-leucine by these cells was Na-independent and almost completely inhibited by BCH. Thus, our findings indicated that most l-leucine uptake in OVCAR-3 cells was mediated by LAT1. CONCLUSIONS: The LAT1 plays significant roles in nutrition, proliferation, and migration of ovarian cancer. Then, LAT1 inhibition would be useful for anticancer therapy in suppressing tumor growth without affecting normal tissues.

Effects of clomiphene citrate plus estradiol or progesterone on endometrial ultrastructure: An RCT.

BACKGROUND: Pinopods concentrations in endometrial surface is a marker of implantation. Estradiol valerate (EV) was used to change the adverse effects of Clomiphene Citrate (CC) on the endometrium. OBJECTIVE: The goal was to assess whether there is a significant difference in the endometrial pinopods concentrations and other parameters after adding EV and progesterone to higher doses of CC. MATERIALS AND METHODS: In this prospective randomized clinical trial, a total of 30 women who did not respond to 100 mg of CC from February 2016 to June 2016 were evaluated. They were divided into three groups: group I) received 150 mg of CC alone, group II) CC with EV, and group III) CC plus progesterone. On day 21 of the menstrual cycle, endometrial biopsy, a blood sampling, and a scanning by electron microscopy were performed. RESULTS: On day 21 of the menstrual cycle, there was no significant difference in the pinopods concentrations (p = 0.641) and serum estrogen levels (p = 0.276) between groups. However, the Serum progesterone levels in group I was higher than the other two groups (p = 0.007) in the same day. CONCLUSION: Since the addition of EV and progesterone to higher dosages of CC did not change the pinopods concentration and serum estrogen levels on day 21 of the menstrual cycle, and the serum progesterone levels was higher in CC alone group (i.e. group I) compared to other groups, it can be concluded that the anti-estrogenic effects of CC just appear on the endometrium and not on the plasma levels.

The membrane-spanning domain of CD98 heavy chain promotes alpha(v)beta3 integrin signals in human extravillous trophoblasts.

CD98 heavy chain (CD98hc) is expressed highly in developing human placental trophoblast. CD98hc is an amino acid transporter and is thought to function in cell fusion, adhesion, and invasion by interacting with integrins. In invasive extravillous trophoblast, alpha(v)beta(3) integrin is expressed in a temporally and spatially specific manner, which prompted us to investigate the potential role of CD98hc in signal transduction of alpha(v)beta(3) integrin. Immunocytochemistry of extravillous trophoblast derived from human placenta revealed that CD98hc colocalized with alpha(v)beta(3) integrin and with alpha(v)beta(3)-associated cytoplasmic proteins including paxillin, vinculin, and focal adhesion kinase. Coimmunoprecipitation of CD98hc and its mutants revealed that the transmembrane domain of CD98hc is necessary for the association of CD98hc with alpha(v)beta(3) integrin. When CD98hc negative liver cells (FLC4) were stably transfected with CD98hc and the extracellular domain of CD98hc was cross-linked by anti-CD98 antibody, FLC4 cells binding affinity to fibronectin and cell motility increased. The anti-CD98 antibody cross-linking promoted actin stress fiber formation and activation of signal transduction downstream of RhoA GTPase, and elevated the phosphorylation of focal adhesion kinase, paxillin, and protein kinase B. Pretreatment of transfected FLC4 cells with specific inhibitors for alpha(v)beta(3)integrin, phosphatidylinositol 3-kinase, and RhoA diminished these effects caused by anti-CD98 antibody cross-linking. These results suggest that notoriously invasive activity of extravillous trophoblast is mediated by CD98hc, which promotes alpha(v)beta(3) integrin-dependent signals.

Induction of Chondrogenic Differentiation in Human Mesenchymal Stem Cells Cultured on Human Demineralized Bone Matrix Scaffold under Hydrostatic Pressure.

BACKGROUND: Articular cartilage damage is still a troublesome problem. Hence, several researches have been performed for cartilage repair. The aim of this study was to evaluate the chondrogenicity of demineralized bone matrix (DBM) scaffolds under cyclic hydrostatic pressure (CHP) in vitro. METHODS: In this study, CHP was applied to human bone marrow mesenchymal stem cells (hBMSCs) seeded on DBM scaffolds at a pressure of 5 MPa with a frequency of 0.5 Hz and 4 h per day for 1 week. Changes in chondrogenic and osteogenic gene expressions were analyzed by quantifying mRNA signal level of Sox9, collagen type I, collagen type II, aggrecan (ACAN), Osteocalcin, and Runx2. Histological analysis was carried out by hematoxylin and eosin, and Alcian blue staining. Moreover, DMMB and immunofluorescence staining were used for glycosaminoglycan (GAG) and collagen type II detection, respectively. RESULTS: Real-time PCR demonstrated that applying CHP to hBMSCs in DBM scaffolds increased mRNA levels by 1.3-fold, 1.2-fold, and 1.7-fold (p < 0.005) for Sox9, Col2, and ACAN, respectively by day 21, whereas it decreased mRNA levels by 0.7-fold and 0.8-fold (p < 0.05) for Runx2 and osteocalcin, respectively. Additionally, in the presence of TGF-ß1 growth factor (10 ng/ml), CHP further increased mRNA levels for the mentioned genes (Sox9, Col2, and ACAN) by 1.4-fold, 1.3-fold and 2.5-fold (p < 0.005), respectively. Furthermore, in histological assessment, it was observed that the extracellular matrix contained GAG and type II collagen in scaffolds under CHP and CHP with TGF-ß1, respectively. CONCLUSION: The osteo-inductive DBM scaffolds showed chondrogenic characteristics under hydrostatic pressure. Our study can be a fundamental study for the use of DBM in articular cartilage defects in vivo and lead to production of novel scaffolds with two different characteristics to regenerate both bone and cartilage simultaneously.

Induction of trophinin in human endometrial surface epithelia by CGbeta and IL-1beta.

During embryo implantation, trophinin mediates cell adhesion by homophilic binding at the apical surfaces of trophectoderm and endometrium. Trophinin is expressed on the human endometrial epithelia in rare occasions. We developed hCG-coated agarose beads that mimic the physical and physiological features of an implantation-stage human blastocyst. When hCG-coated beads were applied to human endometrial epithelial cells in the presence of IL-1beta, endometrial cells acquired strong trophinin expression and the ability for apical cell adhesion with trophinin-expressing human trophoblastic cells. These results provide a mechanism for trophinin-mediated adhesion of human blastocyst to endometrium by a spatially and temporally restricted paracrine effect of hCG derived from the blastocyst.

Expression and localization of galectin-9 in the human uterodome.

Galectin-9 has been recently considered as a novel marker for the mid- and late-secretory phases of human endometrium and decidua. The aim of this study was to investigate the subcellular distribution of galectin-9 in the endometrial epithelium, especially during the frame of the implantation window. Endometrial biopsies in the proliferative, early, and mid-secretory phases from women with regular menstrual cycle were studied using several approaches, including scanning electron microscopy, immunostaining for light and transmission electron microscopies (TEM), immunoblotting, and statistical analysis of the area-related numerical densities of galectin-9-bound nanogold. Images of immunostaining for light microscopy demonstrated a strong expression of galectin-9 at the luminal and glandular endometrial epithelium in the mid-secretory phase compared to the proliferative and early secretory phases. Data of immunoblotting revealed a molecular weight of 36 kDa band with high intensity in the mid-secretory samples. Photomicrographs of immunogold staining for TEM illustrated the localization of galectin-9 in the uterodomes. Statistical and morphometric analysis showed a significantly higher area-related numerical density of galectin-9-bound nano-golds in the uterodomes compared to that of the uterodome-free areas of the luminal epithelium (p<0.001). This is the first demonstration of the molecular localization of galectin-9 in the bulbous ultrastructure of the human endometrial epithelium, called uterodomes. High expression of galectin-9 at uterodomes during the frame of implantation window suggests that galectin-9 can be considered as a marker of endometrial receptivity and should play an important role during the initial events of human embryo implantation.

Improvement of hepatogenic differentiation of iPS cells on an aligned polyethersulfone compared to random nanofibers.

The application of stem cells holds great promises in cell and tissue transplants. This study was designed to compare the hepatogenic differentiation of iPSCs on aligned PES/COL versus random. Aligned and random PES/COL nanofibrus scaffolds were fabricated by electrospining and their surface modified through plasma treatment and collagen coating. The scaffolds were characterized using scanning electron microscopy (SEM) and ATR-FTIR. Morphology and biochemical activities of the differentiated hepatocyte-like cells (HLCs) were examined after 5 and 20 days of differentiation. Real-Time RT-PCR and ICC showed no significant difference in the mRNA and protein levels of two important definitive endoderm specific markers, including Sox17 and Foxa2 between two scaffolds. However, Real-Time RT-PCR analysis indicated an increase in the expression of Cyp7A1 gene over the period of the differentiation procedure on the aligned nanofibers but there was no difference in other genes such as Albumin and CK19. Moreover, comparison of hepatogenic differentiation evaluated by Albumin production in conditioned media of HLCs differentiated on aligned PES/COL, showed increase expression of these markers after 20 days compared to that of the random nanofibers. Taken together, the results of this study may indicate that aligned PES/COL nanofibrous scaffolds can improve terminal differentiation of HLCs from iPSCs.

Hepatogenic Differentiation of Human Induced Pluripotent Stem cells on Collagen-Coated Polyethersulfone Nanofibers.

Many scientists have been fascinated with induced pluripotent stem cells (iPSCs) for cell replacement therapies. Nanofibrous biocompatible scaffolds have been shown to foster better cell adhesion and improve stem cell differentiation. In the current study, after fabrication using electrospinning technique and surface modifications, the characteristics of polyethersulfone (PES) nanofibers were determined by scanning electron microscopy, attenuated total reflection Fourier transform infrared spectroscopy, and 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyltetrazolium bromide (MTT) assay. Then, the hepatogenic potential of iPSCs was evaluated using real-time reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry (ICC) after culture on collagen-coated polyethersulfone (PES/COL) scaffolds. After scaffolds characterization, analysis of two important definitive endoderm specific markers (Sox17 and Foxa2) using real-time RT-PCR and ICC indicated increase in their mRNA and protein levels after 5 days of hepatogenic induction. In addition, to determine hepatic differentiation of iPSCs cultured on PES/COL, the expression of albumin and α-fetoprotein was evaluated by ICC after 20 days. Real-time RT-PCR analysis showed increased expression of albumin, TAT, cytokeratin 19, and Cyp7A1 genes during the course of differentiation program. Finally, enzyme-linked immunosorbent assay analysis demonstrated an increased expression of albumin in the protein level after 28 days of differentiation. In conclusion, our results demonstrated that PES/COL nanofibrous scaffolds could be a proper substrate to significantly increase the hepatogenic differentiation potential of iPSCs and could also be introduced as a promising candidate for liver tissue engineering applications.

Laminin matrix promotes hepatogenic terminal differentiation of human bone marrow mesenchymal stem cells.

OBJECTIVES: The application of stem cells holds great promises in cell transplants. Considering the lack of optimal in vitro model for hepatogenic differentiation, this study was designed to examine the effects of laminin matrix on the improvement of in vitro differentiation of human bone marrow mesenchymal stem cells (hBM-MSC) into the more functional hepatocyte-like cells. MATERIALS AND METHODS: Characterization of the hBM-MSCs was performed by immunophenotyping and their differentiation into the mesenchymal-derived lineage. Then, cells were seeded on the laminin-coated or tissue culture polystyrene (TCPS). The differentiation was carried out during two steps. Afterward, the expression of hepatocyte markers such as AFP, ALB, CK-18, and CK-19 as well as the expression of C-MET, the secretion of urea, and the activity of CYP3A4 enzyme were determined. Moreover, the cytoplasmic glycogen storage was examined by periodic acid-Schiff (PAS) staining. RESULTS: The results demonstrated that the culture of hBM-MSC on laminin considerably improved hepatogenic differentiation compared to TCP group. A significant elevated level of urea biosynthesis and CYP3A4 enzyme activity was observed in the media of the laminin-coated differentiated cells (P<0.05). Furthermore higher expressions of both AFP and ALB were determined in cells differentiated on laminin matrix. Glycogen accumulation was not detected in the undifferentiated hBM-MSCs, however, both differentiated cells in laminin and TCPS groups demonstrated the intracellular glycogen accumulation on day 21 of hepatogenic differentiation. CONCLUSION: Taken together, these findings may indicate that laminin matrix can improve terminal differentiation of hepatocyte-like cells from hBM-MSCs. Thus, laminin might be considered as a suitable coating in hepatic tissue engineering designs.

Improving the Growth Rate of Human Adipose-Derived Mesenchymal Stem Cells in Alginate/Gelatin Versus Alginate Hydrogels.

BACKGROUND: Expansion and differentiation of stem cells relies on the soluble materials as well as the physical conditions of their microenvironment. Several methods have been studied in attempt to enhance the growth and differentiation rates of different adult stem cells extracted from different sources. OBJECTIVES: The purpose was to improve the three-dimensional (3D) culture condition of the semi-permeable polymeric beads for encapsulation of the human adipose-derived mesenchymal stem cells (hADSCs) by modifying the ratio of the alginate-gelatin composition. MATERIALS AND METHODS: Following isolation and characterization of hADSCs by flow cytometry and their functional differentiation, encapsulation in the alginate and alginate/gelatin compositions were performed. Moreover, the stability, swelling, size frequency, growth kinetics, and cytotoxicity of the beads were measured to meet proper condition in the designed experimental and control culture conditions. Finally, the growth rates of the cells in different experimental groups and control were measured and analyzed statistically. RESULTS: Viability decreased in 2 and 3 percent alginate once compared to 1% alginate in beads (p≤0.05). Moreover swelling of the beads in the alginate/gelatin compositions (50:50 and 70:30) were higher than the pure alginate beads (p≤0.05). Finally, the cell growth rate in alginate/gelatin (50:50) beads was significantly higher than alginate and alginate/gelatin (70:30) beads (p≤0.05). CONCLUSIONS: These findings suggested for the first time that the composite of alginate/gelatin beads with the ratio of 50:50 might provide a suitable culture condition for the encapsulation and in vitro expansion of the hADSCs.

Tissue transglutaminase at embryo-maternal interface.

CONTEXT: Tissue transglutaminase (tTG) has a high affinity for fibronectin (FN) and is a coreceptor of both beta1 and beta3 integrin subunits. Considering the notion that FN and integrins have critical roles during the implantation process, this study was undertaken to elucidate the expression pattern and the potential physiological function of tTG at the embryo-maternal interface. METHODS: The primary cultures of human placentas from 15 legal elective abortions at the first trimester of normal pregnancies and endometrial biopsies of 12 female patients in the midluteal phase as well as normal trophoblastic cell lines (CRL) were employed to address these issues using several approaches, such as scanning and transmission electron microscopies, immunostaining for light and electron microscopies, western blotting, and function assays using GRGDSP hexapeptide and an antibody against tTG. RESULTS: The results demonstrated tTG expression on uterine pinopodes and lamellipodia of extravillous trophoblasts. The colocalization of tTG with beta1 and beta3 integrins and its interaction with alpha(v)beta3 integrin and integrin-associated proteins at focal adhesions of the extravillous trophoblasts were illustrated in the results of immunofluorescence, immunoblot, and coimmunoprecipitation studies. Furthermore, function assays revealed that tTG mediated the adhesion and spread of the placental cells on intact FN-coated and 42- and 110-kDa FN fragment-coated wells. CONCLUSION: In conclusion, our findings demonstrated for the first time that tTG actively participates in adhesion events at the embryo-maternal interface through its interaction with FN, at least in part, by activating integrin-signaling pathways.

Alphavbeta3 integrin signaling pathway is involved in insulin-like growth factor I-stimulated human extravillous trophoblast cell migration.

IGF-I and -II provide paracrine and autocrine stimuli, respectively, for extravillous trophoblast (EVT) cell migration. This study examined the role of alpha(v)beta(3) integrin and its signaling pathway in IGF-I-stimulated migration. Migration assays were conducted using cultured EVT cells treated with or without IGF-I in the presence or absence of alphaIR3, Arg-Gly-Asp (RGD) hexapeptide, and antibody against alpha(v)beta(3) integrin. Morphological changes were studied using scanning electron microscopy. Colocalization of alpha(5)beta(1) alpha(v)beta(3) integrins, vinculin, focal adhesion kinase, and paxillin were determined by immuno-cytochemistry and immunoblotting. The results showed that IGF-I could stimulate EVT cell migration in a time- and dose-dependent manner and addition of alphaIR3, Arg-Gly-Asp hexapeptide, and antibody against alpha(v)beta(3) integrin attenuated the IGF-I migratory effect. Scanning electron microscopy images revealed that IGF-I promoted lamellipodia formation. Immunostaining and immunoblotting exhibited the colocalization of alpha(v)beta(3) integrin with phosphorylated focal adhesion kinase, paxillin, and vinculin at focal adhesions after IGF-I treatment. Immunoblotting demonstrated an increase in focal adhesion kinase and paxillin tyrosine phosphorylation followed by tyrosine phosphorylation of IGF-I receptor in a time- and dose-dependent manner. These findings indicated alpha(v)beta(3) integrin localization in the core of focal adhesions of EVT cells and that alpha(v)beta(3) integrin signaling pathways are activated in IGF-I-mediated migration of these cells.