Comparison of Human Mesenchymal Stem Cells Derived from Various Compartments of Human Adipose Tissue and Tunica Adventitia Layer of the Arteries Subsequent to Organ Donation.

BACKGROUND: Mesenchymal stem cells are one of the most interesting cell sources used in regenerative medicine. OBJECTIVE: In the present study, we isolated and characterized the mesenchymal stem cells from various compartments of human adipose tissue and tunica adventitia layer of the arteries. METHODS: Tissue explant culture was done from various compartments of the human adipose tissue and tunica adventitia layer of the arteries, including adipose tissue far from the vessels, perivascular tissues that are completely attached to the vessels, and tunica adventitia layer of the arteries. After the cell culture, characterization of the cells was determined at 3rd-5th passages. Flow cytometry was performed for antigen expression analysis of CD34, CD45, CD44, CD90, CD29, CD73, and CD105. For the evaluation of cell differentiation potential, adipogenic and osteogenic differentiation was conducted under appropriate protocols. RESULTS: The cells were positive for CD44, CD90, CD29, and CD73 and negative for CD34, CD45, and CD105. Adipogenic and osteogenic differentiation potentials were different among the cells from various compartments. The cells derived from perivascular tissue demonstrated better adipogenic and osteogenic differentiation. CONCLUSION: It is essential to characterize the cells from different tissues and compartments for different purposes in regenerative medicine.

Isolation and Characterization of Human Mesenchymal Stem Cells Derived from Human Umbilical Cord Wharton's Jelly and Amniotic Membrane.

BACKGROUND: Mesenchymal stem cells (MSCs) have a capacity for self-renewal and multi-potential differentiations. These cells are considered powerful sources for cell therapy in regenerative medicine and tissue engineering. The cells can be isolated from various tissues; however, harvesting from human umbilical cord and amniotic membrane is easy and accessible source. OBJECTIVE: To isolate and characterize the MSCs derived from human umbilical cord Wharton's jelly (WJ-MSC) and amniotic membrane (AM-MSC) with regard to their morphology, immunophenotype and mesodermal differentiation potential in order to obtain an alternative source of MSC for therapeutic clinical applications. METHODS: Fetal membranes and umbilical cords (n=3) were retrieved from healthy full-term women by elective cesarean delivery. Amniotic membrane and umbilical cord were separately minced and cultured in DMEM supplemented with 10% FBS. After reaching 80% of confluency, the umbilical cord WJ-MSC and AM-MSC were characterized by expression of cell surface markers with flowcytometry, stem cell gene expression with adipogenic/osteogenic potential. RESULTS: Both WJ-MSC and AM-MSC were spindle-shaped cells, expressed MSC surface markers in flowcytometry and stem cell transcriptional factors (OCT4 and NANOG). After induction, the cells differentiated into adipogenic and osteogenic lineages. CONCLUSION: MSC were successfully generated from umbilical cord WJ-MSC and AM-MSC with similar mesenchymal markers and properties.

Angiotensinogen, angiotensine converting enzyme and plasminogen activator inhibitor-1 gene polymorphism in chronic allograft dysfunction.

Despite dramatic improvements in first-year patient and graft survival rates, chronic allograft dysfunction (CAD) remains the leading cause of late renal allograft loss, while current immunologic strategies have little effect on this condition. The renin-angiotensin system (RAS) plays an important role in progression of chronic renal disease. It was shown that plasminogen activator inhibitor-1 (PAI-1) functions in the RAS. This study investigates the possible links between angiotensinogen (AGT M235T), angiotensin-converting enzyme (ACE) and PAI-1 genotypes with CAD. Assessments of polymorphism were performed in 127 renal allograft recipients (77 with CAD and 50 with normal renal function). Fifty healthy subjects were also considered for comparison. Genotypes were determined using polymerase chain reaction (PCR) sequence-specific primers and PCR followed by restriction fragment length polymorphism analysis. Kidney recipients with CAD had significantly higher frequencies of the TT than the recipients without CAD (P < 0.05). The transplant recipients with CAD also had significantly higher frequencies of the DD genotype than those without CAD (P < 0.05). No significant differences were observed between the allelic and genotypic distributions of PAI-1 polymorphisms. Therefore, determination of AGT M235T and ACE genotypes prior to transplantation may be useful to identify patients who are at risk for chronic renal transplant dysfunction.

Association between cyclosporine concentration and genetic polymorphisms of CYP3A5 and MDR1 during the early stage after renal transplantation.

OBJECTIVES: Cyclosporine (CsA) has a narrow therapeutic range, and its pharmacokinetic characteristics vary among individuals. It also is a substrate for cytochrome P450 (CYP) 3A and P-glycoprotein, the product of the multidrug resistance 1 (MDR1) and CYP3A5 genes. Some of the single nucleotide polymorphisms (SNPs) in these genes are associated with deficient protein expression and reduced in vivo activity. We postulated that in renal transplant recipients, these SNPs should be associated with interindividual variations in CsA pharmacokinetics. MATERIALS AND METHODS: In 88 Iranian renal transplant patients receiving CsA, CYP3A5 and MDR1 genotypes were determined by polymerase chain reaction, followed by restriction fragment length polymorphism analysis. Whole blood trough CsA concentrations were measured by radioactive immunosorbent assay. The dose-adjusted concentration (ng/mL per mg/kg/d) was calculated at 1 day (+/-2 days), 7 days, and 1 month after transplantation. RESULTS: The MDR-1 wild-type genotype (3435CC) was observed in 17 patients (19%), whereas 45 patients (51%) were heterozygous (3435CT), and 26 patients (30%) were homozygous (3435 TT) for the mutation. In the days immediately after transplantation, we found a correlation between the concentration/dose ratio and the exon 26 MDR single nucleotide polymorphisms (33.3+/-15.24 microg mg/L/kg in the CT group vs 44.1+/-28.4 microg mg/L/kg in the TT group, P=.019). This ratio was significantly higher in subjects homozygous for the mutation (3435TT). This significant difference was not seen 1 week or 1 month after transplantation. All patients had the CYP3A5*3/*3 genotype, so no differences among the CYP3A5*1/*3 genotypes were found. CONCLUSIONS: MDR-1 (3435CC) polymorphisms are associated with CsA pharmacokinetics and dose requirements in the first few days after renal transplantation. Pharmacogenetic methods could be used to help select the initial dosage and individualize immunosuppressive therapy. According to our results, the major genotype of our recipients is CYP3A5*3/*3. According to the literature, the recommended starting dosage of CsA is 9-14 mg/kg/day; however, the Iranian population has a good response with lower dosages (3-5 mg/kg/day), which may be explained by genetic differences.

Cytokine gene polymorphisms in renal transplant recipients.

OBJECTIVE: Acute rejection remains an important cause of graft loss after renal transplantation, and cytokines are key mediators in the induction and effector phases of all immune and inflammatory responses. However, the influence of gene polymorphisms on the functional immune response of transplant recipient outcomes remains controversial. MATERIALS AND METHODS: The amplification refractory mutation system polymerase chain reaction was used to detect the interleukin-10 (IL-10) (-1082 G/A), tumor necrosis factor-alpha (TNF-alpha) (-308 G/A), and interferon-gamma (IFN-gamma) (+874 T/A) single nucleotide polymorphisms in 100 of the first adult kidney recipients at our institution who were receiving cyclosporine-based immunosuppressive therapy. The diagnosis of acute rejection was based on clinical and histologic findings according to the Banff criteria. RESULTS: The results of multivariate analyses showed no significant association between episodes of acute rejection and single nucleotide polymorphisms in IL- 10, TNF-alpha genes, or dinucleotide repeat polymorphisms in the IFN-gamma gene. CONCLUSIONS: Our results demonstrate that cytokine gene polymorphisms did not influence the early outcome of kidney transplantation.