Figen Kaymaz

Characterization of mesenchymal stem cells from rat bone marrow: ultrastructural properties, differentiation potential and immunophenotypic markers

Bone marrow-derived mesenchymal stem cells (BM-MSCs) can differentiate into many lineages. Although the growing interest in BM-MSCs has led to a number of characterization studies, some important biochemical and immunohistochemical properties are still lacking. In this study, morphological and immunophenotypic properties of BM-MSCs were examined in detail. Differentiation potential and growth kinetics of adult rat BM-MSCs were also determined. Immunohistochemistry and RT-PCR results indicated that BM-MSCs expressed myogenic (desmin, myogenin, myosin IIa, and α-SMA), neurogenic (γ-enolase, MAP2a,b, c-fos, nestin, GFAP and beta III tubulin), and osteogenic (osteonectin, osteocalcin, osteopontin, Runx-2, BMP-2, BMP-4 and type I collagen) markers without stimulation towards differentiation. These expression patterns indicated why these cells can easily differentiate into multiple lineages both in vitro and in vivo. Ultrastructural characteristics of rBM-MSCs showed more developed and metabolically active cells.

Human dental pulp stem cells demonstrate better neural and epithelial stem cell properties than bone marrow-derived mesenchymal stem cells.

Dental pulp stem cells (hDP-SCs) were primarily derived from pulp tissues of primary incisors, exfoliated deciduous and permanent third molar teeth. To understand the characteristics of hDP-SCs from impacted third molar, proliferation capacities, gene expression profiles, phenotypic, ultrastructural, and differentiation characteristics were analyzed in comparison with human bone marrow-derived mesenchymal stem cells (hBM-MSCs), extensively. hDP-SCs showed more developed and metabolically active cells. Contrary to hBM-MSCs, hDP-SCs strongly expressed both cytokeratin (CK)-18 and -19, which could involve in odontoblast differentiation and dentine repair. The intrinsic neuro-glia characteristics of hDP-MSCs were demonstrated by the expression of several specific transcripts and proteins of neural stem cell and neurons. These cells not only differentiate into adipogenic, osteogenic, and chondrogenic lineage, but also share some special characteristics of expressing some neural stem cell and epithelial markers. Under defined conditions, hDP-SCs are able to differentiate into both neural and vascular endothelial cells in vitro. Dental pulp might provide an alternative source for human MSCs. hDP-SCs with a promising differentiation capacity could be easily isolated, and possible clinical use could be developed for neurodegenerative and oral diseases in the future.

Disruption of PTPRO Causes Childhood-Onset Nephrotic Syndrome

Idiopathic nephrotic syndrome (INS) is a genetically heterogeneous group of disorders characterized by proteinuria, hypoalbuminemia, and edema. Because it typically results in end-stage kidney disease, the steroid-resistant subtype (SRNS) of INS is especially important when it occurs in children. The present study included 29 affected and 22 normal individuals from 17 SRNS families; genome-wide analysis was performed with Affymetrix 250K SNP arrays followed by homozygosity mapping. A large homozygous stretch on chromosomal region 12p12 was identified in one consanguineous family with two affected siblings. Direct sequencing of protein tyrosine phosphatase receptor type O (PTPRO; also known as glomerular epithelial protein-1 [GLEPP1]) showed homozygous c.2627+1G>T donor splice-site mutation. This mutation causes skipping of the evolutionarily conserved exon 16 (p.Glu854_Trp876del) at the RNA level. Immunohistochemistry with GLEPP1 antibody showed a similar staining pattern in the podocytes of the diseased and control kidney tissues. We used a highly polymorphic intragenic DNA marker-D12S1303-to search for homozygosity in 120 Turkish and 13 non-Turkish individuals in the PodoNet registry. This analysis yielded 17 candidate families, and a distinct homozygous c.2745+1G>A donor splice-site mutation in PTPRO was further identified via DNA sequencing in a second Turkish family. This mutation causes skipping of exon 19, and this introduces a premature stop codon at the very beginning of exon 20 (p.Asn888Lysfs*3) and causes degradation of mRNA via nonsense-mediated decay. Immunohistochemical analysis showed complete absence of immunoreactive PTPRO. Ultrastructural alterations, such as diffuse foot process fusion and extensive microvillus transformation of podocytes, were observed via electron microscopy in both families. The present study introduces mutations in PTPRO as another cause of autosomal-recessive nephrotic syndrome.

Use of pimecrolimus to prevent epidural fibrosis in a postlaminectomy rat model

OBJECT Epidural fibrosis is the scar tissue formed over the dura mater after a laminectomy. Extensive epidural fibrosis may be an important underlying cause of failed back syndrome. Pimecrolimus, an ascomycin derivative, is one of the new classes of immunomodulating macrolactams and was specifically developed for the treatment of inflammatory diseases. This study examined the preventive effects of the local application of pimecrolimus in minimizing spinal epidural fibrosis in a rat laminectomy model. METHODS Thirty Wistar rats were divided into 3 equal groups: control, mitomycin C (MMC), and pimecrolimus groups. Each rat underwent a laminectomy at the L-3 lumbar level. In the experimental groups, a cotton pad soaked with MMC (0.5 mg/ml) or 5 mg pimecrolimus was placed on the exposed dura mater. No treatment was performed in the control group rats. Thirty days after surgery, the rats were killed and the dura mater thickness, epidural fibrosis, and arachnoidal involvement were quantified. RESULTS The mean dura thickness was measured at 9.28 +/- 3.39 microm in the MMC group and at 8.69 +/- 2.32 microm in the pimecrolimus group, compared with 14.70 +/- 4.14 microm in the control group. In addition, the epidural fibrosis and arachnoidal involvement were reduced significantly in the treatment groups compared with the control group. CONCLUSIONS In this animal model, it was shown that locally applied pimecrolimus effectively reduces epidural fibrosis and dural adherence in rats that underwent lumbar laminectomy. Mitomycin C was equally effective as pimecrolimus in reducing epidural fibrosis and dural adherence in this study.

Myofibroblasts defined by electron microscopy suggest the dedifferentiation of smooth muscle within the sac walls associated with congenital inguinal hernia

Objective To ascertain the presence of myofibroblasts in sacs associated with inguinal hernia in children, through an ultrastructural evaluation using electron microscopy

Synthesis of microbial elastomers based on soybean oily acids. Biocompatibility studies

Biocompatibility studies of the autoxidized and unoxidized unsaturated medium-long chain length (m-lcl) co-poly-3-hydroxyalkanoates (m-lclPHAs) derived from soya oily acids have been reported. Pseudomonas oleovorans was grown on a series of mixtures of octanoic acid (OA) and soya oily acids (Sy) with weight ratios of 20:80, 28:72 and 50:50 in order to obtain unsaturated m-lcl copolyesters coded PHO-Sy-2080, PHO-Sy-2872 and PHO-Sy-5050, respectively. The PHA films were obtained by solvent cast from CHCl(3). They were all originally sticky and waxy except PHO-Sy-5050. Autoxidation of the unsaturated copolyester films was carried out on exposure to air at room temperature in order to obtain crosslinked polymers. They became a highly flexible elastomer after being autoxidized (about 40 days of autoxidation). The in vivo tissue reactions of the autoxidized PHAs were evaluated by subcutaneous implantation in rats. The rats appeared to be healthy throughout the implantation period. No symptom such as necrosis, abscess or tumorigenesis was observed in the vicinity of the implants. Retrieved materials varied in their physical appearance after 6 weeks of implantation. In vivo biocompatibility studies of the medical applications indicated that the microbial copolyesters obtained were all biocompatible and especially the PHOSy series of copolyesters had the highest biocompatibility among them.

ADCK4-Associated Glomerulopathy Causes Adolescence-Onset FSGS

Hereditary defects of coenzyme Q10 biosynthesis cause steroid-resistant nephrotic syndrome (SRNS) as part of multiorgan involvement but may also contribute to isolated SRNS. Here, we report 26 patients from 12 families with recessive mutations in ADCK4. Mutation detection rate was 1.9% among 534 consecutively screened cases. Patients with ADCK4 mutations showed a largely renal-limited phenotype, with three subjects exhibiting occasional seizures, one subject exhibiting mild mental retardation, and one subject exhibiting retinitis pigmentosa. ADCK4 nephropathy presented during adolescence (median age, 14.1 years) with nephrotic-range proteinuria in 44% of patients and advanced CKD in 46% of patients at time of diagnosis. Renal biopsy specimens uniformly showed FSGS. Whereas 47% and 36% of patients with mutations in WT1 and NPHS2, respectively, progressed to ESRD before 10 years of age, ESRD occurred almost exclusively in the second decade of life in ADCK4 nephropathy. However, CKD progressed much faster during adolescence in ADCK4 than in WT1 and NPHS2 nephropathy, resulting in similar cumulative ESRD rates (>85% for each disorder) in the third decade of life. In conclusion, ADCK4-related glomerulopathy is an important novel differential diagnosis in adolescents with SRNS/FSGS and/or CKD of unknown origin.

Rosuvastatin induces apoptosis in cultured human papillary thyroid cancer cells

Statins show antiproliferative activity in various cancer cells. The aim of this study was to evaluate the effects of rosuvastatin treatment on papillary thyroid carcinoma. The papillary thyroid carcinoma (B-CPAP) and normal (Nthy-ori 3-1) thyroid cell lines were treated with rosuvastatin at 12.5, 18.5, 25, 50, 100, and 200 μM concentrations. After 48 and 72 h of rosuvastatin treatment, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Ki-67 immunolabeling, FACS analysis, electron microscopy, caspase-3, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) analysis were performed. Decreased cell viability and G1 phase arrest were detected in papillary thyroid cell line treated with rosuvastatin. Positive immunoreactivity of Ki-67 and dose-dependent increase in S phase on Nthy-ori 3-1 cells were also detected. B-CPAP cells showed intense vacuolisation and autophagosomes with low concentrations and 48 h incubations, while Nthy-ori 3-1 cells showed these changes at higher concentrations. A decrease in the percentage of cells showing autophagy was determined with increasing concentrations of rosuvastatin in B-CPAP cells. Rosuvastatin treatment also caused a dose- and time-dependent increase in caspase-3 activity and apoptotic index by TUNEL assay in B-CPAP cells compared with the Nthy-ori 3-1 cells. Apoptotic cells with nuclear condensation and fragmentation were observed in B-CPAP cell line. Rosuvastatin induced autophagic changes in B-CPAP papillary thyroid cancer cells in lower doses and caused a shift from autophagy to apoptosis. Rosuvastatin may be an alternative treatment for refractory papillary thyroid cancer. Further in vivo studies are necessary to clarify the effects of rosuvastatin in papillary thyroid carcinoma and the clinical implications of rosuvastatin treatment.

Effects of intra-articular administration of autologous bone marrow aspirate on healing of full-thickness meniscal tear: an experimental study on sheep*

OBJECTIVE The aim of this study was to evaluate the effects of bone marrow-derived mesenchymal stem cell and bone marrow elements on the healing of meniscal tears. METHODS This study was performed on twelve, 2-year-old male Tahirova sheep. In each subject, one knee was used for experiment purposes and the other knee was used as a control. After creating a longitudinal full-thickness tear in the red-white zone of the medial meniscus, aspirated autologous bone marrow material was injected into the tear site in the experiment group. The control group received no intervention for secondary healing. RESULTS In the macroscopic evaluation of meniscus, a bridging reparation tissue and adhesion were observed between the rims of the tear in the experiment group. There was no statistical difference in collagen fibril formation between the groups (p=0.16). There was significantly more neovascularization in the experiment group than the control group (p=0.003). The cell count was also a significantly higher in the experiment group (p=0.004) and formation of cartilage plaques was more frequent in the experiment group (p=0.016). There was no evidence suggesting intrinsic repair in the meniscus of control group by light and electron microscopy. CONCLUSION An injection of bone marrow into the meniscus tear site improves healing in a meniscal tear model as demonstrated by both light and electron microscopic findings.

Isolation and characterization of stem cells from pancreatic islet: pluripotency, differentiation potential and ultrastructural characteristics

BACKGROUND AIMS Stem cells (SC) in different locations have individual characteristics. Important questions to be answered include how these specialties are generated, what the mechanism underlying their generation is, and what their biologic and clinical merits are. A basic approach to answering these questions is to make comparisons between the differences and similarities among the various SC types. They may focus on aspects of biologic marker discovery, capacity of proliferation and differentiation, along with other characteristics. The aim of this study was to characterize in detail the SC isolated from pancreatic islet (PI) and compare their properties with bone marrow (BM)-derived mesenchymal stromal cells (MSC) of the rat. METHODS Immunophenotypic characteristics, proliferation capacities, telomerase activities, pluripotent-related gene expressions, ultrastructure and the potential for multilineage differentiation of PI SC and BM MSC were studied. RESULTS We found that PI SC expressed markers of embryonic SC (Oct-4, Sox-2 and Rex-1) and had a high proliferation capacity, proven also by high telomerase activities. Surprisingly, markers belonging to differentiated cells were expressed by these cells in a constitutive manner. PI SC ultrastructure showed more developed and metabolically active cells. CONCLUSIONS The immunocytochemical identification of both PI SC and BM MSC was demonstrated to be typical MSC. Without stimulation of differentiation markers of adipogenic, chondrogenic, neurogenic, myogenic and osteogenic cells in these SC, the expression of those markers might explain their multilineage differentiation potential. We suggest that, by reason of the respectively high telomerase activity in PI SC, they could be better candidates than BM MSC for cell replacement therapy of type 1 diabetes.