Decellularized Wharton's jelly extracellular matrix as a promising scaffold for promoting hepatic differentiation of human induced pluripotent stem cells.

Liver tissue engineering as a therapeutic option for restoring of damaged liver function has a special focus on using native decellularized liver matrix, but there are limitations such as the shortage of liver donor. Therefore, an appropriate alternative scaffold is needed to circumvent the donor shortage. This study was designed to evaluate hepatic differentiation of human induced pluripotent stem cells (hiPSCs) in decellularized Wharton's jelly (WJ) matrix as an alternative for native liver matrix. WJ matrices were treated with a series of detergents for decellularization. Then hiPSCs were seeded into decellularized WJ scaffold (DWJS) for hepatic differentiation by a defined induction protocol. The DNA quantitative assay and histological evaluation showed that cellular and nuclear materials were efficiently removed and the composition of extracellular matrix was maintained. In DWJS, hiPSCs-derived hepatocyte-like cells (hiPSCs-Heps) efficiently entered into the differentiation phase (G1) and gradually took a polygonal shape, a typical shape of hepatocytes. The expression of hepatic-associated genes (albumin, TAT, Cytokeratin19, and Cyp7A1), albumin and urea secretion in hiPSCs-Heps cultured into DWJS was significantly higher than those cultured in the culture plates (2D). Altogether, our results suggest that DWJS could provide a proper microenvironment that efficiently promotes hepatic differentiation of hiPSCs.

A non-rotational, computer-controlled suspension bioreactor for expansion of umbilical cord blood mononuclear cells.

The proliferation and differentiation characteristics of umbilical cord blood mononuclear cells were examined in a non-rotational suspension bioreactor with a fishtail mixer. The system consisted of a glass vessel, a mixer that moved vertically, a data acquisition and control system to continuously monitor pH, temperature and dissolved O(2). The bioreactor provided superior expansion of total HSCs and not total cell number, as well as expression of stemness-related genes which followed with increasing in number of colony-forming cells during 14 days of culture compared to T -lask culture. Vertical agitation thus reduces the total cell number, which may be related to increased shear stress, but has no effect on HSC function.

Wharton's jelly derived mesenchymal stem cells differentiate into oocyte like cells in vitro by follicular fluid and cumulus cells conditioned medium.

Wharton's jelly derived-mesenchymal stem cells (WJ-MSCs) have a same developmental origin with primordial germ cells. WJ-MSCs perhaps differentiate into oocyte and germ like-cells (OLCs/GLCs) in the presence of appropriate inducers. Human follicular fluid (FF) and cumulus cells conditioned medium (CCM) are naturally rich sources for oocyte development. The aim of this study was to evaluate WJ-MSCs potential for differentiating into OLCs and GLCs exposed to FF and CCM. WJ-MSCs were cultured in two different induction media (10% FF, 10% CCM) for 21 days. Morphological changes and expression of developmental genes were evaluated on days 0, 7, 14 and 21 of culture. Also, on 21st day of culture, the expression of oocyte and germ cell proteins investigated using immunofluorescence staining. Appearance of round shaped cells from 7th day onwards indicated that WJ-MSCs can differentiate into OLCs when exposed to FF and CCM. The size of produced OLCs and expression of oocyte specific genes and proteins were increased more positively in FF group rather than CCM group. Although, WJ-MSCs could differentiate into OLCs by FF and CCM, however, the induction potential of FF for producing OLCs was better than CCM.

Amniotic Membrane Seeded Fetal Fibroblasts as Skin Substitute for Wound Regeneration.

Various natural and synthetic biomaterials have been applied as skin substitutes for regenerating damaged skin. Here, we describe a straightforward method for fabrication of a tissue-engineered skin substitute by seeding human fetal fibroblasts on acellular human amniotic membrane (HAM). Fetal fibroblasts are achieved from the skin of normal and non-macerated fetus of 11-14 weeks old after spontaneous pregnancy termination. Acellular HAM is obtained by separation of the outer membrane of the chorion and removing its epithelial cells.

Cultivation of Adipose-Derived Stromal Cells on Intact Amniotic Membrane-Based Scaffold for Skin Tissue Engineering.

Application of cell-based skin substitutes has recently evolved as a novel treatment for hard-to-heal wounds. Here, we focus on the development of a novel skin substitute by seeding human adipose-derived stromal cells (ASCs) on acellular human amniotic membrane (HAM). This construction is probably associated with higher rates of host cell infiltration and implanted cell engraftment. ASCs are achieved by separation of stromal cells from lipoaspirates using collagenase digestion and acellular HAM was obtained by separation of outer membrane of the chorion and removing its epithelial cells.

Osteogenic differentiation of Wharton's jelly-derived mesenchymal stem cells cultured on WJ-scaffold through conventional signalling mechanism.

Wharton's jelly-derived extracellular matrix (WJ-ECM) has attracted researcher's attention for its biomedical applications. Previously, we fabricated a biomimetic spongy scaffold from decellularized WJ-ECM and, in this study, we sought to examine the osteogenic inductive potential of this scaffold and its underlying mechanism. To address this question, mesenchymal stem cells (MSCs) were isolated from WJ using a mechanical method and cultured on the scaffold, under dynamic condition, for over 21 days in the presence or absence of osteogenic medium. The status of signalling pathways involved in the osteogenic differentiation and the expression profile of integrins in the WJ-derived MSCs (WJ-MSCs) were examined. WJ-MSCs displayed differentiation capacities and expressed surface antigens, characteristics of MSCs. Histologically, WJ-MSCs seeded on the scaffold showed a proper cellular attachment, penetration and migration. They also exhibited a higher degree of alkaline phosphatase activity, calcium deposition and osteogenic gene expression, than those cultured in 2D condition. The expression of Wnt, BMP and TGF-ß signalling target genes together with that of α2, αv and ß1 integrins was increased in WJ-MSCs in both presence and absence of osteogenic induction medium. Taken together, our results demonstrate that WJ-derived scaffold induces osteogenic differentiation of WJ-MSCs, possibly through activating integrins and subsequently conventional intracellular signalling pathways.

Fabrication of a three dimensional spongy scaffold using human Wharton's jelly derived extra cellular matrix for wound healing.

The Wharton's jelly (WJ) contains significant amounts of extracellular matrix (ECM) components and rich source of endogenous growth factors. In this study, we designed a new biomimetic spongy scaffold from decellularized WJ-derived ECM and used it as a skin substitute. Histological analysis and biochemical assays showed that bio-active molecules preserved in the fabricated scaffolds and that the scaffolds have highly interconnected porous structure. Cytotoxicity and mechanical evaluation of the scaffold indicated that it is non-toxic and has appropriate mechanical properties. MTT assay, SEM and histological analysis of human fibroblast, seeded on the scaffolds, confirmed cellular viability, attachment, penetration and proliferation. The effectiveness of WJ-derived scaffolds in the regeneration of full-thickness wound was assessed through an in vivo experiment. Our results demonstrated that the scaffolds were well integrated into the mouse tissue and absorbed the exudates after one week. Unlike the controls, in WJ group there were not only complete wound closing and disappearance of the scab, but also complete reepithelialization, newly generated epidermal layers and appendages after 12days of implantation. Taken together, our results indicate that WJ-derived scaffolds are able to improve attachment, penetration and growth of the fibroblast cells and speed up the healing processes, which would offer a proper skin graft for wound healing.

Static Magnetic Field Effect on Cell Alignment, Growth, and Differentiation in Human Cord-Derived Mesenchymal Stem Cells.

This investigation is performed to evaluate the impact of static magnetic field on the Cell growth alignment, and differentiation potential in Human Mesenchymal Stem cells derived from human newborn cords. In vitro-cultured mesenchymal stem cells derived from human newborn cords were exposed to SMF up to 24 mT and compared with the control (unexposed) cultures. Viability was assessed via Trypan Blue staining and MTT assay. Cell cycle progression was studied after flow cytometry data analysis. Sox-2, Nanong, and Oct-4 Primers used for RT-PCR experiment. Morphological studies showed that the exposed cells were significantly aligned in parallel bundles in a correlation with the magnetic field lines. Viability measurements showed a significant reduction in cell viability which was noted after exposure to static magnetic field and initiated 36 h after the end of exposure time. Flow cytometric data analysis confirmed a decrease in G1 phase cell population within the treated and cultured groups compared with the corresponding control samples. However, the induced changes were recovered in the cell cultures after the post-exposure culture recovery time which may be attributed to the cellular repair mechanisms. Furthermore, the proliferation rate and Oct-4 gene expression were reduced due to the 18 mT static magnetic field exposure. The significant proliferation rate decrease accompanied by the Sox-2, Nanong, and Oct-4 gene expression decline, suggested the differentiation inducing effects of SMF exposure. Exposure to Static Magnetic fields up to 24 mT affects mesenchymal stem cell alignment and proliferation rate as well as mRNA expression of Sox-2, Nanong, and Oct-4 genes, therefore can be considered as a new differentiation inducer in addition to the other stimulators.

Osteogenic differentiation of mesenchymal stem cells cultured on PLLA scaffold coated with Wharton's Jelly.

Poly-L-lactic acid (PLLA) electrospun nanofiber scaffold is one of the most commonly used synthetic polymer scaffolds for bone tissue engineering application. However, PLLA is hydrophobic in nature, hence does not maintain proper cell adhesion and tissue formation, moreover, it cannot provide the osteo-inductive environment due to inappropriate surface characteristic and the lack of surface motives participating in the first cellular events. To modify these shortcomings different approaches have been used, among those the most commonly used one is coating of the surface of the electrospun nanofiber with natural materials. In this work Wharton's jelly (WJ), a tissue which surrounds the umbilical cord vessels, reaches in high amounts of extracellular matrix (ECM) components mainly; collagen, hyaluronic acid and several sulphated glycosaminoglycans (GAGs) were used to cover the surface of electrospun PLLA nanofiber scaffolds. The surface morphology of the nanofiber scaffold was evaluated via scanning electron microscope, and the in vitro osteogenic differentiation potential was determined by MTT assay and common osteogenic marker tests such as alkaline phosphatase (ALP) activity and calcium deposition tests. Coating of WJ could not change the surface morphology and diameter of the nanofibers. However, WJ-PLLA scaffolds showed higher proliferation of human mesenchymal stem cells (MSC) than tissue culture plate (TCP) and pristine PLLA scaffolds, moreover, WJ-PPLA scaffold demonstrated significant alkaline phosphatase activity and calcium mineralization than either TCP or PLLA nanofiber scaffolds.