ABSTRACT
In mesenchymal stem cells (MSCs), it has been reported that prostaglandin E2 (PGE2) stimulation of EP2 and EP4 receptors triggers processes such as migration, self-renewal, survival, and proliferation, and their activation is involved in homing. The aim of this work was to establish a genetically modified adipose (aMSC) model in which receptor genes EP2 and EP4 were edited separately using the CRISPR/Cas9 system. After edition, the genes were evaluated as to if the expression of MSC surface markers was affected, as well as the migration capacity in vitro of the generated cells. Adipose MSCs were obtained from Chilean breed horses and cultured in DMEM High Glucose with 10% fetal bovine serum (FBS). sgRNA were cloned into a linearized LentiCRISPRv2GFP vector and transfected into HEK293FT cells for producing viral particles that were used to transduce aMSCs. GFP-expressing cells were separated by sorting to obtain individual clones. Genomic DNA was amplified, and the site-directed mutation frequency was assessed by T7E1, followed by Sanger sequencing. We selected 11 clones of EP2 and 10 clones of EP4, and by Sanger sequencing we confirmed 1 clone knock-out to aMSC/EP2 and one heterozygous mutant clone of aMSC/EP4. Both edited cells had decreased expression of EP2 and EP4 receptors when compared to the wild type, and the edition of EP2 and EP4 did not affect the expression of MSC surface markers, showing the same pattern in filling the scratch. We can conclude that the edition of these receptors in aMSCs does not affect their surface marker phenotype and migration ability when compared to wild-type cells.
ABSTRACT
In this paper, a new method for prediction of future performance and demand on emergency department (ED) in Spain is presented. Increased life expediency and population aging in Spain, along with their corresponding health conditions such as non-communicable diseases (NCDs), have been suggested to contribute to higher demands on ED. These lead to inferior performance of the department and cause longer ED length of stay (LoS). Prediction and quantification of behavior of ED is, however, challenging as ED is one of the most complex parts of hospitals. Using detailed computational approaches integrated with clinical data behavior of Spain's ED in future years was predicted. First, statistical models were developed to predict how the population and age distribution of patients with non-communicable diseases change in Spain in future years. Then, an agent-based modeling approach was used for simulation of the emergency department to predict impacts of the changes in population and age distribution of patients with NCDs on the performance of ED, reflected in ED LoS, between years 2019 and 2039. Results from different projection scenarios indicated that Spain would experience a continuous increase in total ED LoS from 5.7 million hours in 2019 to 6.2 million hours in 2039 if same human and physical resources, as well as same ED configuration, are used. The results from this study can provide health care provider with quantitative information on required staff and physical resources in the future and allow health care policymakers to improve modifiable factors contributing to the demand and performance of ED.
