A hybrid construct of decellularized matrix and fibrin for differentiating adipose stem cells into insulin-producing cells, an optimized in vitro assessment.

The generation of insulin-producing cells (IPCs) is an attractive approach for replacing damaged ß cells in diabetic patients. In the present work, we introduced a hybrid platform of decellularized amniotic membrane (dAM) and fibrin encapsulation for differentiating adipose tissue-derived stem cells (ASCs) into IPCs. ASCs were isolated from healthy donors and characterized. Human AM was decellularized, and its morphology, DNA, collagen, glycosaminoglycan (GAG) contents, and biocompatibility were evaluated. ASCs were subjected to four IPC differentiation methods, and the most efficient method was selected for the experiment. ASCs were seeded onto dAM, alone or encapsulated in fibrin gel with various thrombin concentrations, and differentiated into IPCs according to a method applying serum-free media containing 2-mercaptoethanol, nicotinamide, and exendin-4. PDX-1, GLUT-2 and insulin expression were evaluated in differentiated cells using real-time PCR. Structural integrity and collagen and GAG contents of AM were preserved after decellularization, while DNA content was minimized. Cultivating ASCs on dAM augmented their attachment, proliferation, and viability and enhanced the expression of PDX-1, GLUT-2, and insulin in differentiated cells. Encapsulating ASCs in fibrin gel containing 2 mg/ml fibrinogen and 10 units/ml thrombin increased their differentiation into IPCs. dAM and fibrin gel synergistically enhanced the differentiation of ASCs into IPCs, which could be considered an appropriate strategy for replacing damaged ß cells.

Assessment of the proteome profile of decellularized human amniotic membrane and its biocompatibility with umbilical cord-derived mesenchymal stem cells.

Extracellular matrix-based bio-scaffolds are useful for tissue engineering as they retain the unique structural, mechanical, and physiological microenvironment of the tissue thus facilitating cellular attachment and matrix activities. However, considering its potential, a comprehensive understanding of the protein profile remains elusive. Herein, we evaluate the impact of decellularization on the human amniotic membrane (hAM) based on its proteome profile, physicochemical features, as well as the attachment, viability, and proliferation of umbilical cord-derived mesenchymal stem cells (hUC-MSC). Proteome profiles of decellularized hAM (D-hAM) were compared with hAM, and gene ontology (GO) enrichment analysis was performed. Proteomic data revealed that D-hAM retained a total of 249 proteins, predominantly comprised of extracellular matrix proteins including collagens (collagen I, collagen IV, collagen VI, collagen VII, and collagen XII), proteoglycans (biglycan, decorin, lumican, mimecan, and versican), glycoproteins (dermatopontin, fibrinogen, fibrillin, laminin, and vitronectin), and growth factors including transforming growth factor beta (TGF-ß) and fibroblast growth factor (FGF) while eliminated most of the intracellular proteins. Scanning electron microscopy was used to analyze the epithelial and basal surfaces of D-hAM. The D-hAM displayed variability in fibril morphology and porosity as compared with hAM, showing loosely packed collagen fibers and prominent large pore areas on the basal side of D-hAM. Both sides of D-hAM supported the growth and proliferation of hUC-MSC. Comparative investigations, however, demonstrated that the basal side of D-hAM displayed higher hUC-MSC proliferation than the epithelial side. These findings highlight the importance of understanding the micro-environmental differences between the two sides of D-hAM while optimizing cell-based therapeutic applications.

Antimicrobial Efficacy Assessment of Human Derived Composite Amnion-Chorion Membrane.

Human derived composite amnion-chorion membrane (ACM) has been used to facilitate wound healing due to reported anti-inflammatory properties and promotion of cell proliferation. This study aimed to assess the antimicrobial properties of the ACM using novel methods to visualize the antimicrobial efficacy of membranes in situ at different time points. Porcine Pericardium Collagen Membranes (PPCM) served as membrane controls. Circular pieces of the membranes were used in three different assays: insert, agar contact and glass-bottom well assays. Streptococcus gordonii were spotted onto the membranes and the plates were subsequently centrifuged to ensure direct bacterial contact with the membranes in the insert and agar contact assays, thus better mimicking bacterial adherence in the oral cavity. After incubation at 37 °C for 8, 24, and 48 hours, the membranes were dyed with the Live/Dead BacLight Bacterial Viability fluorescence stain and analyzed via confocal microscopy. The results demonstrated that the ACM completely inhibited bacterial growth at all time points, whereas the PPCM did not demonstrate any antimicrobial properties. Within the limits of this study, the ACM showed extremely high antimicrobial efficacy against oral streptococci. In addition, our methods may be useful in assessing antimicrobial properties for biomaterials with minimum diffusion ability, when traditional assessment methods are not applicable.

Assessment of the cellular localisation of the annexin A2/S100A10 complex in human placenta.

The AnxA2/S100A10 complex has been implicated in various placental functions but although the localisation of these proteins individually has been studied, there is no information about the localisation of their complex in situ at the cellular level. Using the proximity ligation technique, we have investigated the in situ localisation of AnxA2/S100A10 complex in the placenta and have compared this with the location patterns of the individual proteins. High levels of expression of AnxA2/S100A10 complexes were observed in the amniotic membrane and in blood vessel endothelial cells. Lower levels were detected in the brush border area of the syncytium and in the trophoblasts. Immunohistochemical analysis of AnxA2 and S100A10 individually revealed broadly similar patterns of localisation. The brush border staining pattern suggests that in this location at least some of the AnxA2 is not in complex with S100A10. The formal location of the AnxA2/S100A10 complex is compatible with a role in cell-cell interaction, intracellular transport and secretory processes and regulation of cell surface proteases, implying contributions to membrane integrity, nutrient exchange, placentation and vascular remodelling in different parts of the placenta. Future applications will allow specific assessment of the association of the complex with pathophysiological disorders.

In vitro assessment of a novel, hypothermically stored amniotic membrane for use in a chronic wound environment.

Chronic wounds require extensive healing time and place patients at risk of infection and amputation. Recently, a fresh hypothermically stored amniotic membrane (HSAM) was developed and has subsequently shown promise in its ability to effectively heal chronic wounds. The purpose of this study is to investigate the mechanisms of action that contribute to wound-healing responses observed with HSAM. A proteomic analysis was conducted on HSAM, measuring 25 growth factors specific to wound healing within the grafts. The rate of release of these cytokines from HSAMs was also measured. To model the effect of these cytokines and their role in wound healing, proliferation and migration assays with human fibroblasts and keratinocytes were conducted, along with tube formation assays measuring angiogenesis using media conditioned from HSAM. Additionally, the cell-matrix interactions between fibroblasts and HSAM were investigated. Conditioned media from HSAM significantly increased both fibroblast and keratinocyte proliferation and migration and induced more robust tube formation in angiogenesis assays. Fibroblasts cultured on HSAMs were found to migrate into and deposit matrix molecules within the HSAM graft. These collective results suggest that HSAM positively affects various critical pathways in chronic wound healing, lending further support to promising qualitative results seen clinically and providing further validation for ongoing clinical trials.

Assessment of heat shock protein (HSP60, HSP72, HSP90, and HSC70) expression in cultured limbal stem cells following air lifting.

OBJECTIVES: The aim of this study is to create an ex vivo model to examine the expression of major heat-shock protein (HSP) families; HSP60, HSP72, and HSP90, and heat-shock cognate 70 (HCS70) at the mRNA and protein level in differentiating corneal cells from limbal stem cells (LSC) following air exposure. METHODS: Limbal biopsies taken from cadaveric normal human limbus were cultivated as explants on human amniotic membrane (HAM) and plastic dish (PD). Corneal differentiation was induced by air lifting for 16 days. The expression of putative LSC markers (P63 and ATP-binding cassette G2 [ABCG2]), corneal markers (keratin 3 [K3/12] and connexin 43 [CX43]), and HSP60, HSP72, HSP90, and HSC70 were tested by RT-PCR, immunofluorescence, and flow cytometry pre- and post-air exposure. Fresh limbal and corneal tissues were used as control groups. RESULTS: Air lifting induced corneal differentiation with a decrease in the number of P63(+) cells and an increase in the number of K3(+)/CX43(+) cells, which characterized transient amplifying cells (TACs). Moreover, denuded HAM provided a superior niche for LSC proliferation and phenotype maintenance in vitro. Additionally, we have evidence that expressions of HSC70 as well as HSP72 were enhanced through corneal differentiation and HSP90 post-air lifting in vitro and in vivo. HSP60, however, was not detected in either LSC or corneal cells, in vivo and in vitro. CONCLUSIONS: These results suggest that corneal differentiation following air exposure may regulate HSP72 and HSC70 expression. In addition, HSP72 and HSP90 may protect LSC and corneal cells against oxidative stress.

[Assessment of a microarray of solid-phase antibodies to the study of chemokines secreted by the chorioamniotic membrane]. / Evaluación de un microarreglo de anticuerpos en fase sólida para el estudio de quimiocinas secretadas por la membrana corioamniótica.

OBJECTIVE: At the end of pregnancy, a complex signaling network of cytokines, chemokines and proteic effector molecules is started. Due to such complexity, this network is very difficult to analyze. The aim of the present study was to evaluate the effectiveness of an antibody-based proteomic analysis method to distinguish the simultaneous presence of different molecules in biological samples. MATERIAL AND METHOD: Amniochorion explants were obtained from women at term with spontaneous labor (n = 4) and subjected to cesarean section without labor (n = 4). Explants were cultured for 24 h and then homogenated in their own culture media to obtain cell free extracts. Chemokines were identified in these extracts using a commercial array for chemokines that included: eotaxin, eotaxin 2, 1-309, IL-8, IP-10, MCP-1, MCP-2, MIG, MIP-1alpha, MIP-1beta, MIP-1delta and RANTES. RESULTS: All the included chemokines were found in amniochorion extracts, being IL-8 the most abundant. However, once labor is present, all chemokines tend to appear in greater concentrations than those from non-in-labor tissues, except for RANTES, which disappeared when labor began. CONCLUSION: This methodological approach shows that amniochorion from term pregnancies secrete chemokines with a characteristic qualitative profile during labor. This approach would allow the fast evaluation of potential markers of this phenomenon in physiologic or pathologic conditions.