Effect of Homocysteine Thiolactone on Structure and Aggregation Propensity of Bovine Pancreatic Insulin

Homocysteine thiolactone (HCTL) is a cyclic thioester of homocysteine, showing high reactivity toward lysine residues of proteins. In the present study the structural properties and aggregation propensity of bovine pancreatic insulin were studied in the presences of increasing concentration of HCTL (0–500 μM), using different spectroscopic techniques. As shown in this study, HCTL induces gross structural alterations and subsequently aggregation of insulin in a dose dependent manner. Also induction of insulin aggregation by HCTL occurs in a sequential process, where native protein with alpha-helical abundant structure gradually transforms into partially folded conformations with the significant amount of beta-sheet. Since C-terminal B-chain of insulin plays a critical role in stability of this protein, the structural alteration/aggregation induced by HCTL can be consequence of homocysteinylation of the only Lysine residue (Lys29) on its B-chain. This study may have important implications regarding the effect of HCTL on structure of insulin particularly in the pathological states linked to hyperhomocysteinemia.     

Stepwise morphological changes and cytoskeletal reorganization of human mesenchymal stem cells treated by short-time cyclic uniaxial stretch

This study aimed to investigate stepwise remodeling of human mesenchymal stem cells (hMSCs) in response to cyclic stretch through rearrangement and alignment of cells and cytoskeleton regulation toward smooth muscle cell (SMC) fate in different time spans. Image analysis techniques were utilized to calculate morphological parameters. Cytoskeletal reorganization was observed by investigating F-actin filaments using immunofluorescence staining, and expression level of contractile SMC markers was followed by a quantitative polymerase chain reaction method. Applying cyclic uniaxial stretch on cultured hMSCs, utilizing a costume-made device, led to alteration in fractal dimension (FD) and cytoskeleton structure toward continuous alignment and elongation of cells by elevation of strain duration. Actin filaments became more aligned perpendicular to the axis of mechanical stretch by increasing uniaxial loading duration. At first, FD met a significant decrease in 4 h loading duration then increased significantly by further loading up to 16 h, followed by another decrease up to 1 d of uniaxial stretching. HMSCs subjected to 24 h cyclic uniaxial stretching significantly expressed early and intermediate contractile SM markers. It was hypothesized that the increase in FD after 4 h while cells continuously became more aligned and elongated was due to initiation of change in phenotype that influenced arrangement of cells. At this point, change in cell phenotype started leading to change in morphology while mechanical loading still caused cell alignment and rearrangement. Results can be helpful when optimized engineered cells are needed based on mechanical condition for functional engineered tissue and cell therapy.     

Comparative Evaluation of Cardiac Markers in Differentiated Cells from Menstrual Blood and Bone Marrow-Derived Stem Cells In Vitro

In recent years, menstrual blood-derived stem cells (MenSCs) have been introduced as easily accessible and refreshing stem cell source without ethical considerations in the field of regenerative medicine. The aim of this study was to investigate in vitro cardiac differentiation capacity of MenSCs compared to bone marrow-derived stem cells (BMSCs) under two protocols using 5-aza-2′-deoxycytidine (5-aza) and basic fibroblast growth factor (bFGF). Our data revealed that differentiated MenSCs and BMSCs acquired some features of cardiomyocytes; however, degree of differentiation was dependent on the protocol. In a similar manner with BMSCs, differentiated MenSCs showed upper levels of mRNA/protein of late-stage cardiac markers under 5-aza stimulation and continuous treatment with bFGF (protocol 2) compared to those induced by 5-aza alone (protocol 1) evidencing the key role of bFGF in cardiac development of stem cells. Compared to corresponding undifferentiated cells differentiated MenSCs under protocol 2 showed remarkable expression of connexin-43 and TNNT2 at both gene and protein levels, whereas developed BMSCs under the same condition only expressed connextin-43 at the higher level. Superiority of protocol 2 over protocol 1 was confirmed by assessment of LDH and cTnI production by differentiated cells. Based on the accumulative data, our study provided convincing evidence that MenSCs have relatively higher capability to be differentiated toward cardiomyocyte compared with BMSCs. Furthermore, usage of bFGF and 5-aza to induce in vitro cardiac differentiation of MenSCs is highly recommended.     

The effects of short-term uniaxial strain on the mechanical properties of mesenchymal stem cells upon TGF-β<Subscript>1</Subscript> stimulation

Cellular mechanical characteristics represent cell ability to produce tissue-specific metabolites. Therefore, to achieve effective cell therapy, a better understanding of the effects of chemo-mechanical stimuli on the mechanical properties of in vitro-treated cells is essential. Herein, we investigated the effects of uniaxial strain on the mechanical properties of mesenchymal stem cells (MSCs) upon transforming growth factor beta 1 (TGF-β₁) stimulation. The MSCs were categorized into control and test groups. In one test group, the MSCs were treated by TGF-β₁ for 6 d, and in the other, they were additionally subjected to 1-d uniaxial strain on day 2. The cell mechanical properties and smooth muscle (SM) gene expression were assessed on days 2, 4, and 6. During the entire experiment, the MSCs treated by TGF-β₁ ± uniaxial strain were induced to differentiate into SM-like cells by significantly upregulation of α-actin, SM22α, and h1-calponin in respect to the control samples. When the MSCs were treated with TGF-β₁ alone, their stiffness and viscosity decreased significantly on day 2 and then increased by increase in culture time. When the cells were subjected to 1-d uniaxial strain upon TGF-β₁ stimulation, their stiffness and viscosity significantly increased on days 2 and 4 and then decreased on day 6 to a level comparable to that of TGF-β₁ group. Different paths were noticeable among the treated samples to reach nearly similar states on day 6. It seems that uniaxial strain activates mechanobiological cascades by which cellular mechanical behavior can be regulated after its removal. However, these effects are transient and would diminish over time. The findings may be helpful in the chemo-mechanical regulation of MSCs.     

Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) improved osteogenic differentiation of the human induced pluripotent stem cells while considered as an artificial extracellular matrix

Cocell polymers can be the best implants for replacing bone defects in patients. The pluripotent stem cells produced from the patient and the nanofibrous polymeric scaffold that can be completely degraded in the body and its produced monomers could be also usable are the best options for this implant. In this study, electrospun poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanofibers were fabricated and characterized and then osteogenic differentiation of the human-induced pluripotent stem cells (iPSCs) was investigated while cultured on PHBV scaffold. MTT results showed that cultured iPSCs on PHBV proliferation were increased compared to those cultured on tissue culture polystyrene (TCPS) as the control. Alkaline phosphatase (ALP) activity and calcium content were also significantly increased in iPSCs cultured on PHBV compared to the cultured on TCPS under osteogenic medium. Gene expression evaluation demonstrated that Runx2, collagen type I, ALP, osteonectin, and osteocalcin were upregulated in iPSCs cultured on PHBV scaffold in comparison with those cultured on TCPS for 2 weeks. Western blot analysis have shown that osteocalcin and osteopontin expression as two major osteogenic markers were increased in iPSCs cultured on PHBV scaffold. According to the results, nanofiber-based PHBV has a promising potential to increase osteogenic differentiation of the stem cells and iPSCs-PHBV as a cell-co-polymer construct demonstrated that has a great efficiency for use as a bone tissue engineered bioimplant.     

Adipose-derived stem cells-conditioned medium improved osteogenic differentiation of induced pluripotent stem cells when grown on polycaprolactone nanofibers

Considering that the common osteogenic growth factors cannot be transplanted with stem cells to the patients, many studies are underway to find a replacement for these factors. Recently, it has been determined that mesenchymal stem cell (MSC)-derived conditioned medium (CM) contains effective factors in the bone formation process. In the current study, the synergistic effect of adipose-derived MSC’s CM, and polycaprolactone (PCL) scaffold was investigated on the osteogenic differentiation potential of human induced pluripotent stem cells (iPSCs). After scaffold fabrication by electrospinning and characterization by scanning electron microscopy, iPSCs proliferation in the presence of CM, PCL, and both was evaluated using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide. Then, iPSCs osteogenic differentiation was investigated while cultured on tissue culture plate and PCL under CM compared with the osteogenic medium using alizarin red staining, calcium content, alkaline phosphatase activity and gene and protein expression analysis. Proliferation rate of the iPSCs was increased while cultured under CM and its effect was synergistically enhanced by culture on PCL. Evaluation of the osteogenic markers was showed CM alone could induce osteogenic differentiation into the iPSCs and this potential was significantly increased while combined with PCL nanofibrous scaffold. According to the results, it was demonstrated that CM has an osteogenic induction property almost the same of the common osteogenic medium and it can also be used potentially with stem cells when transplant to the patients. CM can also help by prolonging cell survival at the site of the defect as well as accelerating healing process.     

Aggregation and Fibrillation of Eye Lens Crystallins by Homocysteinylation; Implication in the Eye Pathological Disorders

There are several evidences, suggesting a relationship between hyperhomocysteinemia and various diseases of the visual system. Therefore in this study the effects of homocysteinylation on aggregation and fibrillation of lens crystallins were studied using spectroscopic techniques, SDS-PAGE and western blot analysis. The results of UV?CVis absorption studies suggest an induction of lens protein aggregation after homocysteinylation. Furthermore, the existence of fibril in the aggregate of lens proteins confirmed by Congo red absorption measurement and Thioflavin-T fluorescence assay. Taken together the results of SDS-PAGE and Western blotting, it is suggested that almost all detectable eye lens crystallins are prone to aggregation by homocysteinylation, while ??-Crystallin comprises the main portion of lens protein aggregate. Overall this study may suggest lens protein homocysteinylation as a possible mechanism to explain the relationship between hyperhomocysteinimia and some impairments of the visual system.