Hair stem cells for bladder regeneration in rats: preliminary results.

BACKGROUND: A variety of tissue engineering techniques are currently under development or investigation for bladder augmentation, but so far no approach is clearly superior. The aim of this study was to compare the suitability for cystoplasty augmentation in rats of in vivo implanted acellular bladder matrices (BAM) previously seeded with hair follicle stem cells and that of matrices implanted without the cells. MATERIALS AND METHODS: The rat hair follicle stem cell line was positive for CD34, p63, and Ki-67. 1 x 10(6) cells from 34 to 40 passages seeded onto nine BAM scaffolds were cultured for one week. Nine other scaffolds were left unseeded. Scaffolds were grafted into a surgically created defect within the anterior bladder wall: nine rats with acellular matrices and nine with cell-seeded BAM. Rats observed for six months were killed in monthly intervals. We performed gross examination, X-ray cystography, and hematoxylin-eosin, cytokine (CK)-7, CK-20, myoglobin, and desmin staining of the excised bladders. RESULTS: Minimal adhesions were observed and urinary leakage was noted in one case. Two animals died in the acellular group. Rats developed stone disease in bladders reconstructed with acellular BAM. Bladder capacity was similar, but the shape was regular and characteristically oval only in bladders grafted with cell-seeded BAM. Muscle layers in the apical parts of the reconstructed bladder walls were extremely thin in the cases of acellular grafts and thicker in bladders reconstructed with cell-seeded grafts. Muscle layer regeneration was better in the cell-seeded group. Urothelium regenerated in all animals. CONCLUSIONS: We have shown that hair follicle stem cells may be used for rat bladder wall regeneration.

Primary cultures from rat vibrissae as a potential cell source for in vitro construction of urinary bladder wall grafts.

BACKGROUND: In vitro-constructed grafts can be used for human bladder augmentation. There are many diseases in which autologous cells cannot be used for this purpose. The aim of the present study was to examine the potential of rat vibrissae hair follicle cells to form cultures, which could serve as a source for in vitro creation of urinary bladder wall grafts. METHODS: Two hundred vibrissae were excised from young Wistar male rats. Two different digestions were performed, in dispase and in collagenase. All follicles were additionally incubated in trypsin and ethylenediamine tetraacetic acid. Two different culture media based on DMEM (Dulbecco's Modified Eagle's Medium) were used: the first was supplemented with keratinocyte growth factor (KGF) and the second with epidermal growth factor. Immunocytochemical detection of cytokeratin, CD34, p63, Ki-67 (proliferation index), and HMB45 (Human Melanoma Black 45) was performed. RESULTS: Forty-eight primary cultures of rat follicle vibrissae cells were established from 200 hair follicles (24% successful rate). Twenty-four primary cultures were obtained after dispase digestion and 24 after collagenase treatment. Each group was cultured in 2 different media. A heterogeneity of primary cultures was observed. Cells formed a monolayer within a period of 2 to 4 weeks. The 24 primary cultures established after dispase treatment exhibited monolayers of small cuboid cells expressing cytokeratin and CD34. In the 40th passage 20%-40% of cells expressed p63; 85% of these cells from late passages were positive for Ki-67, indicating preserved mitotic potential. Epithelial-like phenotype was observed after dispase digestion and cultivation in KGF-supplemented medium. After 3 weeks, the morphology of these cells changed into fibroblast-like. These cultures were negative for CD34. Fibroblast-like cell growth was observed after collagenase treatment in both KGF- and EGF-supplemented media. These cells were positive for the melanocyte cell marker (HMB45). CONCLUSIONS: Culture media and isolation conditions influence hair follicle stem cell differentiation. The stem cell niche within the hair follicles is a reservoir of cells, which can be potentially used for in vitro creation of urinary bladder wall grafts.

Influence of two Pt(IV) complexes on viability, apoptosis and cell cycle of B16 mouse melanoma tumors.

UNLABELLED: Several platinum(IV) complexes are showing considerable promise in initial trials, producing reactive intermediates that then interact with DNA. AIM: To perform in vitro study of two new platinum(IV) complexes cytotoxic effect on B16 mouse melanoma cells. METHODS: PtCl(4)(dbtp)(2) and PtCl(2)(6mp)(2) complexes were prepared. PtCl(4)(dbtp)(2) was created as modification of PtCl(4)(dmtp) test previously. Apoptosis and necrosis were examined using flow cytometry, upon Annexin V/PI staining. RESULTS: LC(10), LC(50) and LC(90) parameters established for PtCl(4)(dbtp)(2) were as following: 2.6, 17.0, 58.0 mumol/L. However LC(10) and LC(50) established for PtCl(2)(6mp)(2) were 1.2 and 14.0 micromol/l respectively. The both complexes induced apoptosis. PtCl(2)(6mp)(2) induced cell cycle arrest in G0/G1, while PtCl(4)(dbtp)(2) - in S-phase. CONCLUSIONS: PtCl(4)(dbtp)(2) appeared to be more cytotoxic against B16 cells than PtCl(2)(6mp)(2). Apoptosis was the main mechanism of cell loss in cultures incubated with both tested complexes.

Bone marrow progenitors from animals with chronic renal failure lack capacity of in vitro proliferation.

BACKGROUND: An interesting way to regenerate a kidney is an autologous bone marrow transplantation. The aim of this study was to examine whether chronic kidney disease influenced bone marrow progenitors. METHODS: Wistar male rats included group I (n = 4, chronic kidney disease 1/2, CKD 1/2) that underwent right nephrectomy. In group II (n = 3, chronic kidney disease 5/6, CKD 5/6) underwent removal of the right kidney and approximately one-third of the cortex of the left kidney. Animals in the control group (n = 4) were intact. Bone marrow cells obtained from femurs were separated using a CD34 Micro-Beads magnetic isolation kit. Isolated cells were counted using a trypan blue exclusion test. Numbers of isolated cells were presented as mean values with standard deviation with P < .05 considered significant. CD34(-) cells were cultivated and observed to the passage 6. RESULTS: The CKD rat model was used for in vitro experiments. There were no differences in cell numbers isolated from control rats versus both CKD rats. No differences were observed in CD34(-) cells after separation when compared to controls. Cell morphology was similar in primary CD34(-) cultures during the first days of primary culture. CD34(-) primary cultures established from chronic renal failure rats collapsed within 2 weeks. No differences were found in CD34(+) cell number after isolation when compared with controls. These cells did not form a monolayer. Cells in cultures established from control animals resembled normal fibroblast-like morphology of mesenchymal stem cells during 3 months. CONCLUSIONS: Bone marrow cells from chronic renal failure rats showed no capacity for in vitro proliferation. We speculated that bone marrow cells obtained from renal chronic failure patients may not be useful for autologous cell transplantation.