Aperito Journal of Cellular and Molecular Biology

Mesenchymal Stem Stem Cells (MSCs) could provide alternative therapeutic solutions for a number of bone and cartilage tissue diseases, playing an important role in regenerative medicine. Several sources of stem cells are currently available. In recent years, researchers attention was focused on the isolation of Adipose Stem Cells (ASCs) because they are easily obtainable in large quantity trough a minimally invasive procedure. Furthermore, Adipose Derived Stem Cells (ADSCs) have multiple properties, including self-renewal, differentiation potential, secretion of active factors that might influence surrounding cells and immune modulatory effects useful in the treatment of several degenerative diseases. The aim of this study was to present a new “in vitro” ADSCs isolation and characterization protocol using a 
mechanical method in comparison to the enzymatic digestion that has represented the gold standard procedure to isolate adult mesenchymal stem cells. 

Methods Lipoaspirate from forty human donors were obtained by minimally invasive liposuction procedures: by definition, lipoaspirate contains a relatively high content of ASCs (1-5% of isolated nucleated cells). A minimum starting volume of 30 mL of lipoaspirate was obtained in all procedures for a sufficient yield of mesenchymal stem cells (MSCs). Lipoaspirate was stored at 2-8º C for a maximum of 24 hours before use. Tissue from each donor was split in two samples and each sample was treated by two different in vitro procedures with the goal to isolate ADSCs: enzymatic digestion and experimental mechanical isolation procedure. Tissue from both methods were first diluted with an equal volume of Phosphate-Buffered Saline (PBS), centrifuged at 500 xg at 22° for 5 minutes, digested adding 1 ml of collagenase digestion solution for every 1 ml volume of tissue, incubated at 37ºC in a pre-warmed orbital shaker for 30 minutes, centrifuged at 22°C for 5 minutes at 500xg and finally suspended for expansion in alpha-MEM medium adding 10% of bovine fetal serum. Subsequently, MSCs were characterized following the guidelines of the Society for Cellular Therapy (SCT). 

Results Characterization of the processed tissue was performed after digestion and “in vitro” culture expansion of plastic adherent cells. Our results showed that cells from both study groups were adherent to the plastic having fibroblast morphology. The cells were immune-characterized by Flow cytometry. Results showed a phenotypic mutation during the process of cellular expansion, from the isolation of the stromal vascular fraction to the selection of a purely population with mesenchymal stem cells properties in the subsequent passages. After the isolation of MSCs, the obtained ADSC showed a subpopulation (immuno phenotype differentiation) expressing specific markers such us CD 34, a marker associated with the state of vascular and endothelial progenitor, and CD 146, a pericytic markers. Finally, the evaluation of the cells differentiation potential showed that ADSCs, under controlled condition, were able to differentiate into adipogenic, chondrogenic and osteogenic cells. The results were similar for both differential methods. 

Conclusion Our experience suggests that ADSCs enzymatically and mechanically isolated from Lipoaspirate respond to the three criteria of the Society for Cellular Therapy (SCT) and, for this reason, can be considered as MSCs. New methods to treat fat tissue and obtain ADSCs, as the mechanical method described in this article, can be safely developed to isolate in an easier way these promising cells. Therefore, improved characterization protocols are still necessary in order to make a final population of ADSCs as an homogeneous cellular product ready for clinical application in the field of osteogenic and chondrogenic tissue engineering. Finally, more studies are necessary to understand the immunomodulatory and anti-inflammatory properties of ADSCs before clinical applications to treat different orthopaedic pathologies

Niemann-Pick C1-Like 1 (NPC1L1) is expressed in human liver, but its mechanism of action and how it is regulated in this tissue is not well-understood.  Herein, we decided to examine the effects of the inhibitor of NPC1L1, ezetimibe, in the presence and absence of a low cholesterol medium (MITO+ medium), on the expression of NPC1L1 in the human hepatocyte like C3A cell line.  It was demonstrated that ezetimibe and MITO+ medium were able to independently decrease NPC1L1 protein expression while increased the mRNA expression of this transporter.  Treating with ezetimibe and MITO+ medium together additively decreased NPC1L1 protein levels, but their effects on NPC1L1 mRNA expression were counteracting. Cycloheximide studies showed that these treatments enhanced the stability of the NPC1L1 protein in an additive manner, supporting the possibility that the translation efficiency of the NPC1L1 mRNA was decreased under these conditions or that the factors required to degrade the NPC1L1 transporter were inhibited by the presence of cycloheximide.  In addition, the data confirmed that growing the cells in flasks coated with poly-D-lysine (should induce polarization) was required for maximum expression levels of NPC1L1 protein but not for localization of this transporter to the apical membrane.  The information derived from this research is critical to our understanding of the role of hepatic NPC1L1 in removing cholesterol from the bile and in cholesterol homeostasis in general.  

High Low Density Lipoprotein (LDL) cholesterol levels are well-known to cause atherosclerotic diseases. The hepatic LDL receptor is the major determinant of plasma LDL-cholesterol levels, and as a result, a greater understanding of the regulatory mechanisms that control the expression and function of this gene is essential. Herein, we decided to examine whether the function of the LDL receptor increased in response to the cholesterol biosynthesis inhibitor, Zaragozic Acid A (ZA), which has been shown to significantly decrease serum cholesterol levels in rats.  The results demonstrated that ZA increased the binding and internalization of Very Low Density Lipoprotein (VLDL), but not of LDL. This ZA-dependent increase in the internalization rate of VLDL was unrelated to an increase in the overall expression levels of the LDL receptor at the cell surface. These results suggest that ZA could reduce plasma cholesterol levels by preventing the synthesis of LDL from VLDL.   

Introduction Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency is a common human enzyme deficiency. Molecular abnormalities data from Egypt are scarce, and have not been extensively investigated. To fill this gap, we investigated the frequency of the presence of certain known G6PD mutations among Egyptian patients with G6PD-deficiency. 

Methods DNA was extracted from 50 G6PD-deficient unrelated male subjects. We have analyzed the G6PD gene mutations in those with a history of favism by using the appropriate PCR- restriction enzyme digestion technique (PCR/RFLP analysis). 

Results The G6PD Mediterranean mutation was found in 16 patients (32%). The African A-variant (202 G→A & 376 A→G) were detected in 5 (10%) G6PD-deficient patients, the Chatham variant was detected in 2 (4%) of the patients and the Aures variant was not detected in any of the patients. Enzymatic activity was shown to be a poor predictive parameter of acute hemolytic crisis and was not correlated with clinical features. 

Conclusion The findings suggest that gene flow from the Indian subcontinent, sub-Saharan African, and other parts of the Mediterranean may have contributed to the observed G6PD mutations seen in the Egyptian population. The PCR-RFLP technique can be used for rapid molecular screening of the to ndividual variability. As a result, cases can be misdiagnosed, but in patients who have other mutations in the G6PD gene, these should be subjected to direct sequencing, in an attempt to fully characterize their genotypes and to search for other novel mutations.