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.

In vitro proliferation and differentiation of mouse spermatogonial stem cells in decellularized human placenta matrix.

Utilizing natural scaffold production derived from extracellular matrix components presents a promising strategy for advancing in vitro spermatogenesis. In this study, we employed decellularized human placental tissue as a scaffold, upon which neonatal mouse spermatogonial cells (SCs) were cultured three-dimensional (3D) configuration. To assess cellular proliferation, we examined the expression of key markers (Id4 and Gfrα1) at both 1 and 14 days into the culture. Our quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis revealed a notable increase in Gfrα1 gene expression, with the 3D culture group exhibiting the highest levels. Furthermore, the relative frequency of Gfrα1-positive cells significantly rose from 38.1% in isolated SCs to 46.13% and 76.93% in the two-dimensional (2D) and 3D culture systems, respectively. Moving forward to days 14 and 35 of the culture period, we evaluated the expression of differentiating markers (Sycp3, acrosin, and Protamine 1). Sycp3 and Prm1 gene expression levels were upregulated in both 2D and 3D cultures, with the 3D group displaying the highest expression. Additionally, acrosin gene expression increased notably within the 3D culture. Notably, at the 35-day mark, the percentage of Prm1-positive cells in the 3D group (36.4%) significantly surpassed that in the 2D group (10.96%). This study suggests that the utilization of placental scaffolds holds significant promise as a bio-scaffold for enhancing mouse in vitro spermatogenesis.

In vitro development of a muscle-tendon junction construct using decellularised extracellular matrix: Effect of cyclic tensile loading.

The muscle tendon junction (MTJ) plays a crucial role in transmitting the force generated by muscles to the tendon and then to the bone. Injuries such as tears and strains frequently happen at the MTJ, where the regenerative process is limited due to poor vascularization and the complex structure of the tissue. Current solutions for a complete tear at the MTJ have not been successful and therefore, the development of a tissue-engineered MTJ may provide a more effective treatment. In this study, decellularised extracellular matrix (DECM) derived from sheep MTJ was used to provide a scaffold for the MTJ with the relevant mechanical properties and differentiation cues such as the relase of growth factors. Human mesenchymal stem cells (MSCs) were seeded on DECM and 10 % cyclic strain was applied using a bioreactor. MSCs cultured on DECM showed significantly higher gene and protein expression of MTJ markers such as collagen 22, paxillin and talin, than MSCs in 2D culture. Although collagen 22 protein expression was higher in the cells with strain than without strain, reduced gene expression of other MTJ markers was observed when the strain was applied. DECM combined with 10 % strain enhanced myogenic differentiation, while tenogenic differentiation was reduced when compared to static cultures of MSCs on DECM. For the first time, these results showed that DECM derived from the MTJ can induce MTJ marker gene and protein expression by MSCs, however, the effect of strain on the MTJ development in DECM culture needs further investigation.