Effect of high cyclic hydrostatic pressure on osteogenesis of mesenchymal stem cells cultured in liquefied micro-compartments.

Bone resident cells are constantly subjected to a range of distinct mechanical loadings, which generates a complex microenvironment. In particular, hydrostatic pressure (HP) has a key impact on modulation of cell function and fate determination. Although HP is a constant mechanical stimulus, its role in regulating the osteogenesis process within a defined 3D microenvironment has not been comprehensively elucidated. Perceiving how environmental factors regulate the differentiation of stem cells is essential for expanding their regenerative potential. Inspired by the mechanical environment of bone, this study attempted to investigate the influence of different ranges of cyclic HP on human adipose-derived mesenchymal stem cells (MSCs) encapsulated within a compartmentalized liquefied microenvironment. Taking advantage of the liquefied environment of microcapsules, MSCs were exposed to cyclic HP of 5 or 50 MPa, 3 times/week at 37 °C. Biological tests using fluorescence staining of F-actin filaments showed a noticeable improvement in cell-cell interactions and cellular network formation of MSCs. These observations were more pronounced in osteogenic (OST) condition, as confirmed by fluorescent staining of vinculin. More interestingly, there was a significant increase in alkaline phosphatase activity of MSCs exposed to 50 MPa magnitude of HP, even in the absence of osteoinductive factors. In addition, a greater staining area of both osteopontin and hydroxyapatite was detected in the 50 MPa/OST group. These findings highlight the benefit of hydrostatic pressure to regulate osteogenesis of MSCs as well as the importance of employing simultaneous biochemical and mechanical stimulation to accelerate the osteogenic potential of MSCs for biomedical purposes.

Viscous Microcapsules as Microbioreactors to Study Mesenchymal Stem/Stromal Cells Osteolineage Commitment.

It is essential to design a multifunctional well-controlled platform to transfer mechanical cues to the cells in different magnitudes. This study introduces a platform, a miniaturized bioreactor, which enables to study the effect of shear stress in microsized compartmentalized structures. In this system, the well-established cell encapsulation system of liquefied capsules (LCs) is used as microbioreactors in which the encapsulated cells are exposed to variable core viscosities to experience different mechanical forces under a 3D dynamic culture. The LC technology is joined with electrospraying to produce such microbioreactors at high rates, thus allowing the application of microcapsules for high-throughput screening. Using this platform for osteogenic differentiation as an example, shows that microbioreactors with higher core viscosity which produce higher shear stress lead to significantly higher osteogenic characteristics. Moreover, in this system the forces experienced by cells in each LC are simulated by computational modeling. The maximum wall shear stress applied to the cells inside the bioreactor with low, and high core viscosity environment is estimated to be 297 and 1367 mPa, respectively, for the experimental setup employed. This work outlines the potential of LC microbioreactors as a reliable in vitro customizable platform with a wide range of applications.

Cell encapsulation in liquified compartments: Protocol optimization and challenges.

Cell encapsulation is a widely used technique in the field of Tissue Engineering and Regenerative Medicine (TERM). However, for the particular case of liquefied compartmentalised systems, only a limited number of studies have been reported in the literature. We have been exploring a unique cell encapsulation system composed by liquefied and multilayered capsules. This system transfigured the concept of 3D scaffolds for TERM, and was already successfully applied for bone and cartilage regeneration. Due to a number of appealing features, we envisage that it can be applied in many other fields, including in advanced therapies or as disease models for drug discovery. In this review, we intend to highlight the advantages of this new system, while discussing the methodology, and sharing the protocol optimization and results. The different liquefied systems for cell encapsulation reported in the literature will be also discussed, considering the different encapsulation matrixes as core templates, the types of membranes, and the core liquefaction treatments.

Functional Germ Cells From Non-Testicular Adult Stem Cells: A Dream or Reality?

BACKGROUND: Some research studies provided evidence for the differentiation capacity of adult stem cells (ASCs) into germ cells (GCs). Since the generation of GCs from stem cells (SCs) has been proposed as a potential way for treatment of infertility, many research groups have begun their creative studies on generation of new GCs both in vitro and in vivo, and utilized different ASC types such as bone marrow mesenchymal stem cells (BM-MSCs), skin stem cells, pancreatic stem cells, and adipose tissue MSCs. Despite many interesting reports with promising results, an obvious problem in the research projects was the functionality of the produced GCs. OBJECTIVE: In this paper, we have reviewed the results of almost all previously published reports on derivation of male and female GCs from ASCs to provide a better insight into this field of research. RESULTS: The most evaluated papers have shown that ASCs from various tissues can differentiate into GCs but rarely were the produced GCs functional and could form fertile gametes neither in vitro, nor in vivo (after transplantation into the gonads). CONCLUSION: There are still so many unknown issues about gametogenesis. Perhaps making alterations in treatment methods and utilizing creative techniques like tissue engineering and gene targeting help to achieve a standard method of in vitro GC production from ASCs.

Male and female rat bone marrow-derived mesenchymal stem cells are different in terms of the expression of germ cell specific genes.

Recent studies have shown that mesenchymal stem cells (MSCs), under appropriate conditions, can differentiate into cell types including germ cells (GCs). These studies also show that MSCs without any induction express some GC-specific genes innately. Moreover, one report suggests that female MSCs have a greater tendency to differentiate into female instead of male GCs. Therefore, for the first time, this study attempts to assay and determine the differences between the expression levels of some important GC-specific genes (Stra8, Vasa, Dazl, Stella, Piwil2, Oct4, Fragilis, Rnf17 and c-Kit) in male and female bone marrow (BM)-MSCs of rats. BM sampling of the rate was performed by a newly established method. We cultured rat BM samples, then characterized male and female MSCs according to their adhesion onto the culture dish, their differentiation potential into bone, cartilage and fat cells, and phenotype analysis by flow cytometry. The expression of GC-specific genes and their expression levels were evaluated with reverse transcription polymerase chain reaction (RT-PCR) and real-time RT-PCR. Our results showed that Dazl and Rnf17 did not express in the cells. The majority of examined genes, except Piwil2, expressed at almost the same levels in male and female MSCs. Piwil2 had higher expression in male MSCs which was probably related to the more prominent role of Piwil2 in the male GC development process. Male BM-MSCs appeared more prone to differentiate into male rather than female GCs. Additional research should be performed to determine the exact role of different genes in the male and female GC development process.