Comparative Analysis of Fluorescent Labeling Techniques for Tracking Canine Amniotic Stem Cells.

The utmost aim of regenerative medicine is to promote the regeneration of injured tissues using stem cells. Amniotic mesenchymal stem cells (AmMSCs) have been used in several studies mainly because of their easy isolation from amniotic tissue postpartum and immunomodulatory and angiogenic properties and the low level of rejection. These cells share characteristics of both embryonic/fetal and adult stem cells and are particularly advantageous because they do not trigger tumorigenic activity when injected into immunocompromised animals. The large-scale use of AmMSCs for cellular therapies would greatly benefit from fluorescence labeling studies to validate their tracking in future therapies. This study evaluated the fluorophore positivity, fluorescence intensity, and longevity of canine AmMSCs. For this purpose, canine AmMSCs from the GDTI/USP biobank were submitted to three labeling conditions, two commercial fluorophores [CellTrace CFSE Cell Proliferation kit - CTrace, and CellTracker Green CMFDA - CTracker (CellTracker Green CMFDA, CT, #C2925, Molecular Probes®; Life Technologies)] and green fluorescent protein (GFP) expression after lentiviral transduction, to select the most suitable tracer in terms of adequate persistence and easy handling and analysis that could be used in studies of domestic animals. Fluorescence was detected in all groups; however, the patterns were different. Specifically, CTrace and CTracker fluorescence was detected 6 h after labeling, while GFP was visualized no earlier than 48 h after transduction. Flow cytometry analysis revealed more than 70% of positive cells on day 7 in the CTrace and CTracker groups, while fluorescence decreased significantly to 10% or less on day 20. Variations between repetitions were observed in the GFP group under the present conditions. Our results showed earlier fluorescence detection and more uniform results across repetitions for the commercial fluorophores. In contrast, fluorescence persisted for more extended periods in the GFP group. These results indicate a promising direction for assessing the roles of canine AmMSCs in regenerative medicine without genomic integration.

Placental scaffolds have the ability to support adipose-derived cells differentiation into osteogenic and chondrogenic lineages.

Prudent choices of cell sources and biomaterials, as well as meticulous cultivation of the tissue microenvironment, are essential to improving outcomes of tissue engineering treatments. With the goal of providing a high-quality alternative for bone and cartilage tissue engineering, we investigated the capability of bovine placental scaffolds to support adipose-derived cell differentiation into osteogenic and chondrogenic lineages. Decellularized bovine placenta, a high-quality scaffold with practical scalability, was chosen as the biomaterial due to its rich extracellular matrix, well-developed vasculature, high availability, low cost, and simplicity of collection. Adipose-derived cells were chosen as the cell source as they are easy to isolate, nontumorigenic, and flexibly differentiable. The bovine model was chosen for its advantages in translational medicine over the mouse model. When seeded onto the scaffolds, the isolated cells adhered to the scaffolds with cell projections, established cell-scaffold communication and proliferated while maintaining cell-cell communication. Throughout a 21-day culture period, osteogenically differentiated cells secreted mineralized matrix, and calcium deposits were observed throughout the scaffold. Under chondrogenic specific differentiation conditions, the cells modified their morphology from fibroblast-like to round cells and cartilage lacunas were observed as well as the deposit of cartilaginous matrix on the placental scaffolds. This experiment provides evidence, for the first time, that bovine placental scaffolds have the potential to support bovine mesenchymal stem cell adherence and differentiation into osteogenic and chondrogenic lineages. Therefore, the constructed material could be used for bone and cartilage tissue engineering.

Characterization of post-edited cells modified in the TFAM gene by CRISPR/Cas9 technology in the bovine model.

Gene editing in large animal models for future applications in translational medicine and food production must be deeply investigated for an increase of knowledge. The mitochondrial transcription factor A (TFAM) is a member of the HMGB subfamily that binds to mtDNA promoters. This gene maintains mtDNA, and it is essential for the initiation of mtDNA transcription. Lately, we generated a new cell line through the disruption of the TFAM gene in bovine fibroblast cells by CRISPR/Cas 9 technology. We showed that the CRISPR/Cas9 design was efficient through the generation of heterozygous mutant clones. In this context, once this gene regulates the mtDNA replication specificity, the study aimed to determine if the post-edited cells are capable of in vitro maintenance and assess if they present changes in mtDNA copies and mitochondrial membrane potential after successive passages in culture. The post-edited cells were expanded in culture, and we performed a growth curve, doubling time, cell viability, mitochondrial DNA copy number, and mitochondrial membrane potential assays. The editing process did not make cell culture unfeasible, even though cell growth rate and viability were decreased compared to control since we observed the cells grow well when cultured in a medium supplemented with uridine and pyruvate. They also exhibited a classical fibroblastoid appearance. The RT-qPCR to determine the mtDNA copy number showed a decrease in the edited clones compared to the non-edited ones (control) in different cell passages. Cell staining with Mitotracker Green and red suggests a reduction in red fluorescence in the edited cells compared to the non-edited cells. Thus, through characterization, we demonstrated that the TFAM gene is critical to mitochondrial maintenance due to its interference in the stability of the mitochondrial DNA copy number in different cell passages and membrane potential confirming the decrease in mitochondrial activity in cells edited in heterozygosis.