Number Of C-kit Positive Cells Research Articles

Stem Cell Factor Receptor Immunoexpression in Adolescent Varicocele.

Stem cell factor receptor (c-kit) plays a crucial role in regulating proliferation and survival of germ cells. The aim of this study was to find the correlation between the number of c-kit positive germ cells, testicular asymmetry and histological grade in varicocele affected testis samples of adolescents. Twenty testicular biopsy samples of adolescents affected by varicocele and eight normal control testes were included. The relationship between percentage of testicular asymmetry, number of tubular c-kit positive germ cells and severity of spermatogenic failure was assessed. The mean (SD; median) histological grade for spermatogenic failure in controls was 1.37(0.52; 1), while in the varicocele group, it was 2.70(1.08; 3) (P = .0052). Mean(SD; median) number c-kit positive germ cells in the control group were 20.1(2.52; 20), while in the varicocele group it was 12.35(7.16; 12.5) (P = .0059). Spearman test documented a significant positive correlation between percentage of hypotrophy and histological grade of spermatogenic failure (r = 0.5544 , 95% CI: 0.1345 to 0.8055, P = .0112) but a negative correlation with the number of c-kit positive cells (r = - 0.5871, 95% CI: - 0.8219 to -0.1817, P = .0065). Moreover, a significant negative correlation was found between grade of histological changes and number of c-kit positive germ cells (P < .0001). A significant correlation between hypotrophy, histological lesions and c-kit positive germ cells exists in varicocele testes. This finding suggests a possible role for c-kit in the pathogenesis of germ cell impairment in varicocele. Histological changes and lack of c-kit germ cells were also noted in testes not displaying hypotrophy. We believe that reliable markers should be found as better predictors of testicular function in adolescent with varicocele.

The function of hyperpolarization-activated cyclic nucleotide-gated channel in diabetic cystopathy.

We aimed at investigating changes in the expression and physiological function of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in interstitial cells of Cajal (ICC) in diabetic state. Twenty adult female Sprague-Dawley (SD) rats were randomly assigned to control and Zucker diabetic fatty (ZDF) group. The protein and mRNA expression of HCN isoforms and C-kit in the rat bladders were detected using Western blotting and reverse transcription-polymerase chain reaction (RT-PCR). The bladder contraction was evaluated using a bladder smooth muscle strip test. Whole cell patch-clamp techniques were used to detect the activity of HCN channels. Immunofluorescent staining was used to the positive expression of HCN and C-kit in ICC. cAMP, as HCN channel-specific stimulant, could increase the frequency and amplitude of spontaneous contractions in both group, while cAMP inducing contraction of ZDF rats, was still significantly lower compared with the control group. Acute bladder ICCs were isolated by collagenase digestion. Classic Ih current pattern was recorded on ICCs while Ih current amplitude of ICCs from ZDF diabetic rats was significantly lower than the control group. The expression and mRNA of HCN1-4 isoforms in ZDF diabetic rats were both significantly lower compared with the control group. Meanwhile, the number of c-kit positive cells in ZDF diabetic rats showed no significant differences compared with controls. The morphological structure of ICC in the bladder of ZDF rats was relatively loose and the number of their cell process was apparently decreased. The structure of ICCs in ZDF rats was relatively loose, their connection to each other was also diminished. The expression of HCN was down-regulated and its response to cAMP was also decreased. HCN channels in bladder ICCs might regulate detrusor contraction. Changes in HCN expression and activity in bladder ICCs might be one of the most important mechanisms of diabetic cystopathy.

Feasibility of intraperitoneal placental-derived mesenchymal stem cell injection in stomachs of diabetic mice.

Diabetic gastropathy is associated with loss of interstitial cells of Cajal and autonomic neuropathy. Effective management for diabetic gastropathy is still unavailable. This study was aimed to confirm the pathogenetic changes in diabetic gastropathy and to examine the effect of treatment with placental-derived mesenchymal stem cells (PDMSCs) in stomachs of animal models. Fourteen non-obese diabetic/ShiLtJ mice of 8weeks were bled until week 30. Diabetes mellitus developed in 10 out of 14 mice, which all survived with insulin. The mice were grouped into three groups: nondiabetic group (n=4), diabetic sham group (n=5), and diabetic PDMSC group (n=5) all of which were treated with intraperitoneal PDMSCs injection at week 30. All mice were killed at week 34, and the stomachs were examined by immunohistochemical stain with c-kit and neuronal nitric oxide synthase antibodies. The number of c-kit positive cells in stomach decreased significantly in the diabetic sham group compared with that in the nondiabetic group (21.2±6.7 vs 88.0±29.3, P=0.006) but increased with PDMSC treatment (21.2±6.7 vs 64.0±15.1, P=0.02). The positive rate of neuronal nitric oxide synthase in neural plexus was also significantly lower in the diabetic sham group than in the nondiabetic group (22.3%±18.5% vs 48.0%±22.7%, P=0.003) but increased with PDMSC treatment (22.3%±18.5% vs 43.3%±20.5%, P=0.03). Interstitial cells of Cajal and neural plexus decreased in stomachs of mice with diabetes mellitus but were significantly repaired with intraperitoneal injection of PDMSC.

Exploring the dynamics of progenitor cells in the urethra after simulated birth trauma in mice

ObjectiveTo examine the alteration in the cellular dynamics of the urethral tissue after a simulated birth trauma in a mouse model. Materials and MethodsA total of 36 B6 mice received vaginal distention treatment, and four untreated mice were used as controls. Specimens were collected every 24 hours after the injury for 9 consecutive days and examined using immunofluorescent staining for cell markers including c-kit, smooth muscle actin (SMA), and vimentin. Confocal microscopy was used to localize the stained cells and determine the cell number. ResultsThe number of c-kit positive cells increased after the 1st day and peaked on the 3rd day. The amount of SMA positive cells rapidly reduced to its lowest count on the 1st day and maintained a statistically significant low cell number than that at the basal level for 4 days after vaginal distension. The cell number finally returned to basal level on the 9th day. The amount of vimentin positive cells increased dramatically after the 1st day and plateaued from the 3rd day to the 9th day. The number of vimentin positive cells in the plateau phase was significantly higher than that of the control group. ConclusionOur study confirmed that the dynamic change in different cell types after the urethral injury was dependent on the nature and physiology of the wound repairing cells during the tissue healing process. It might be a simple animal model to study birth trauma repair; however, the varied progenitor cell activity in different species should also be considered.

Reduction of c-kit positive cardiac stem cells in patients with atrial fibrillation

BackgroundWe aimed to determine expression patterns of cardiac stem cells in the left atrium (LA) tissue from patients with atrial fibrillation. MethodsLA appendages were obtained during open-heart surgery and processed for explant cell culture and tissue analysis (n=319). The total number of grown cells and c-kit positive cells were analyzed by flow cytometry after 4 weeks of culture. The remaining tissue was used for Masson's trichrome staining to determine the area of the fibrosis. ResultsThe diameter of the LA, as measured by echocardiography, was significantly larger in the AF group than in the sinus rhythm group. Reverse transcription polymerase chain reaction analysis revealed higher expression of collagen in the AF group and an increase in the expression of basic fibrosis growth factor and transforming growth factor-2 and -3. Masson's trichrome staining showed progression of fibrosis in the AF tissue. In addition, the expression of apoptosis-related genes were significantly higher in AF group. There was no difference in the expression of connexin-40 between groups, while the expression of connexin-43 was decreased and that of connexin-45 was increased in the AF group. The total numbers of grown cells as well as c-kit positive cells after 4 weeks of cardiac tissue culture were significantly lower in the AF group. ConclusionProgression of remodeling in LA tissue was observed in AF patients. The number of c-kit positive cells cultured from LA appendages was reduced in AF patients, suggesting impairments in self-renewal.

C-Kit immunoexpression patterns differ in melanotic and amelanotic canine oral melanomas

&lt;p&gt;Melanomas são as mais frequentes neoplasias malignas da cavidade bucal de cães. Sabe-se que a proliferação de células e expressão de conexina diferem em melanomas melanóticos e amelanóticos da cavidade bucal de cães. Este estudo analisou a expressão da proteína c-Kit em melanomas melanóticos e amelanóticos da cavidade bucal canina. Um total de 34 melanomas bucais caninos (19 melanóticos e 15 amelanóticos) foram coletados. Os melanomas amelanóticos apresentaram evolução mais rápida e maior incidência de metástase. Foi constatado um número significativamente maior de células positivas para c-Kit em neoplasias amelanóticas. Além disso, a intensidade de imunomarcação de c-Kit foi predominantemente mais forte em melanomas amelanóticos. Estes resultados confirmam um papel potencial para c-Kit em melanomas orais caninos, com diferenças claras em padrões de expressão entre os dois tipos histológicos de tumor, melanóticos e amelanóticos. Este trabalho destaca a importância de um estudo detalhado das mutações c-Kit em melanomas orais caninos para ser possível a melhor compreensão dos mecanismos moleculares envolvidos no desenvolvimento da doença.&lt;/p&gt;

Establishment of Isolation and Expansion Protocols for Human Cardiac C-kit-Positive Progenitor Cells for Stem Cell Therapy

Although cardiac stem cells (CSCs) have emerged in regeneration research, the number of isolated CSCs is low, making a sufficient supply of functional elements an important consideration in cardiovascular research. In this study, we established an efficient method for CSC isolation. We directly compared cultures of single cells to human cardiac-derived c-kit-positive progenitor cells (hCPCsc-kit+). The two protocols employed enzymatically digested hCPCsc-kit+ (ED-hCPCs) with tissue-expanded hCPCc-kit+ (TE-hCPCs). Using fluorescence-activated cell sorting, we showed the concentration of c-kit in TE-hCPCs to be higher than in ED-hCPCs, although the total number of c-kit positive cells resulting from ED-hCPCs was similar to that resulting from TE-hCPCs. The cardiomyocyte-associated proteins, GATA4 and Nkx2-5, which were expressed during hCPCs expansion, did not differ between the isolation methods. Importantly, the expression of the CSC stem cell marker, c-kit, was more efficiently preserved using the ED-hCPCs versus the TE-hCPCs method. In a cell proliferation assay, the ED-hCPCs method produced a significantly greater number of cells. Finally, hCPCs derived using both protocols differentiated into endothelial, smooth muscle, and cardiomyocyte lineages. In conclusion, the single-cell culture protocol using an enzymatic digestion method may be more useful to isolate human cardiac-derived c-kit-positive elements compared with the tissue expansion method.

Characterization of c-Kit receptor function in cardiac regeneration by using transgenic mouse models

Background. Cardiac stem cells expressing the tyrosine kinase receptor c-kit have been recently used in in vivo and in vitro cardiac regenerative studies. However, it remains to be clarified whether the c-kit receptor itself plays a critical role in the process of cardiac regeneration. In order to clarify this point, we will explore whether c-Kit receptor affects cardiac stem cells proliferation, survival, migration and differentiation after heart injury. Methods and Results. We have generated transgenic mice in which an activatory point mutation (c-KitD814Y mice) has been introduced in the kinase domain of the c-kit gene. Initially, we have analyzed c-kit expression in tissues and organs at different stages of embryonal and post-natal development through immunohystochemical and biochemical analyses. We have found that in two transgenic lines the receptor is highly expressed and activated in heart, testis and cerebellum, compared to wild type mice. In order to follow the fate of the c-Kit transgenic stem cells we crossed c-KitD814Y mice with mice expressing GFP under c-Kit regulative sequences control. By cytofluorimetric and fluorescence microscopy analyses, we observed a 2 fold of increase in the number of c-kit positive cells on heart samples from double transgenic mice at different ages. To verify the c-kit role in cardiac regeneration we performed a necrotic heart damage in vivo and monitored cardiac repair in transgenic mice versus wild-type mice. After 9 days the wounded hearts of transgenic mice presented a larger connectival tissue area compared to wild-type mice. On the contrary, after 45 days a consistent reduction of fibrotic area was observed in transgenic mice. These preliminary results suggest a faster repair of damaged heart area that contain stem cells with an activated c-kit receptor. Further in vitro and in vivo experiments will be performed to assess whether transgenic c-kit cells directly transdifferentiate into cardiomyocytes or whether they act in a paracrine manner. In summary, the generation of transgenic mice carrying a constitutively activated c-kit in cardiac stem cells, will allow to investigate the role of the receptor and to highlight the molecular mechanism underlying heart regeneration.

Effect of insulin on small intestinal smooth muscle in diabetic mice

Objective To investigate the effects of regular insulin (RI)on duodenal smooth muscle in diabetic mice. Methods Diabetes mellitus (DM) model was established by intraperitoneal injection of 150 mg/kg streptozotocin (STZ) in male BALB/c mice. The model mice were divided into DM group and DM treated with RI group with 6 each. Meanwhile, 6 normal mice were served as controls. The mice in treatment group were intraperitoneally injected with 40 U/kg of RI daily.Whereas the mice in DM and control groups were intraperitoneally injected with phosphate buffer solution (pH = 7. 40). After 6 weeks, the small intestinal transit rate of mice was determined by lavage of Indian ink. Interstitial cells of cajal (ICC) in duodenal myenteric plexus were counted using immunohistochemical staining. Slow waves of duodenal smooth muscle cells were recorded with intracellular recordings. Data were analysed by SPSS 17.0 software, and comparisons among three groups were done using LSD test. Results After intervention for 6 months, the clinical presentations,such as more water and food intake and polyuria, were improved in treatment group. The body weight was increased in treatment group [(23.33±3.13) g] compared with DM group [(15.42±1.40) g,P＜0.01] ,but dereased compared with control group [(26.78 ± 2.09) g, P＜0.05]. The level of blood glucose in DM group was significantly higher than that in control and treatment groups(P＜0.01). Small intestine transmission rate was significantly reduced in DM group than that in control and treatment groups (P＜0.01), but it was slower in treatment group than that in control group (P＜ 0. 01 ). Immunohistochemical study showed that the number of c-kit positive cells reduced obviously in DM group than that in control group and treatment group (P＜0.05), whereas it was lower in treatment group than that in control group (P ＜ 0.05). The slow wave frequency and amplitude of duodenal smooth muscle cells in DM group were reduced when compared with control and treatment groups (P＜0.01) and both were lower in treatment group than that in control group (P＜0. 01 ). Conclusion The findings indicate that DM mice have gastrointestinal dysmotility and exogenous insulin may improve small intestinal dysmotility in DM mice. Key words: Diabetes mellitus; Insulin; Duodenum; Myocytes, smooth muscle

Spatial and temporal patterns of c-kit positive cells in embryonic lungs

It has been reported that the smooth muscles in fetal airways exhibit spontaneous phasic contractions throughout gestation. However, the mechanism of these spontaneous contractions is unknown. In the bowel wall, interstitial cells of Cajal (ICCs), which are derived from c-kit positive precursor cells, play an important role as pacemaker cells responsible for the spontaneous, rhythmic activity in the smooth muscle cells. In this study, we investigated the spatial and temporal expression patterns of c-kit positive cells in the embryonic lung and its relationship to the smooth muscle cells surrounding the trachea and the bronchus. Rat fetuses were removed from timed pregnant dams on embryonic days (E) 11.5, 13.5, 15.5, and 17.5. Immunohistochemical studies with anti c-kit antibody and anti α-SMA antibody were carried out using frozen sections. A small number of c-kit positive cells were observed in the mesenchyme of the lung bud on day E 11.5. They were markedly increased in number on day E 13.5. On day E 15.5 and on day E 17.5, strong c-kit expressions were observed on the vascular wall and moderate expressions in the mesenchyme. C-kit positive cells co-localized with α-SMA positive smooth muscle cells surrounding the airway epithelium. Co-localization of c-kit positive cells and airway smooth muscles in the fetal lung suggests that c-kit positive cells may play an important role in the spontaneous contractions of fetal airways. C-kit expressions in the fetal pulmonary vascular wall suggest that these cells may play an important role in vasculogenesis and angiogenesis of the embryonic lung.

Decreased Interstitial Cells of Cajal-like Cells, Possible Cause of Congenital Refluxing Megaureters: Histopathologic Differences in Refluxing and Obstructive Megaureters

To evaluate the location of interstitial cells of Cajal-like cells and the ureteral aspect using histopathologic studies of obstructive and refluxing megaureters to reveal the different pathogenesis of megaureters in the human urinary tract. The underlying pathophysiology of obstructive megaureter and refluxing megaureter is poorly understood. The data from 14 patients with obstructive megaureter (7 boys and 7 girls), with a mean age of 12 months (range 2-84), and 9 patients with refluxing megaureter (7 boys and 2 girls), with a mean age of 11 months (range 4-24), were compared. We investigated the difference in the histopathologic aspects using Masson's trichrome, terminal uridine deoxynucleotidyl transferase dUTP nick end labeling (for apoptosis), and human c-kit antibody (CD117 for interstitial cells of Cajal-like cells) between the obstructive and refluxing megaureters. The proportion of smooth muscle was significantly lower in segments of refluxing megaureter (32.04% +/- 4.96%) than in the segments of obstructive megaureter (52.48% +/- 3.46%; P < .01). The number of apoptotic cells was significantly increased in the obstructive megaureter (mean 1661 +/- 135.1 cells) compared with the refluxing megaureter (mean 375.2 +/- 65.14 cells; P < .0001). The number of c-kit positive cells was significantly lower in the refluxing megaureter (mean 83.60 +/- 48.84 cells) than in the obstructive megaureter (mean 463.6 +/- 100.4 cells; P < .05). The differences in the histopathologic aspects can provide information on the possible pathophysiology of obstructive and refluxing megaureters. Ureteral peristalsis can be affected by the increased myocyte apoptosis in the obstructive megaureter and by the decreased number of interstitial cells of Cajal-like cells and smooth muscle content in refluxing megaureters. Additional research is warranted to elucidate the exact pathophysiology.

Chronological Change in Intramural Components in Severe Proximally Dilated Jejunal Atresia: An Immunohistochemical Study

Chronological Change in Intramural Components in Severe Proximally Dilated Jejunal Atresia: An Immunohistochemical Study

An Abnormal Distribution of C-Kit Positive Cells in the Normoganglionic Segment Can Predict a Poor Clinical Outcome in Patients with Hirschsprung's Disease

The loss or decrease of interstitial cells of Cajal (ICCs) has been implicated in several disorders of human intestinal motility. We have encountered a few cases suffering from severe constipation or enterocolitis resulting in patient death after a definitive operation for HD, even though the normoganglionic intestine had been successfully pulled through. We investigated the distribution of ICCs using c-kit immunostaining in the normoganglionic segment and compared these findings with the clinical outcome after a definitive operation in each case. The distributions of ICCs were investigated by using c-kit immunostaining in the normoganglionic segment in the resected bowel in 15 cases with HD. The distributions of protein gene product 9.5 (PGP 9.5) as a general neuronal marker and those of NADPH-diaphorase (NADPH-d) as a marker of nitric-oxide neurons were also examined. The numbers of ICCs and neurons were evaluated quantitatively. The histopathological results were compared with the clinical outcome after definitive operation in each case. C-kit immunoreactive cells showed a normal distribution in the normoganglionic segment in 13 cases, while they were markedly (less than 50% compared with the other cases) decreased in 2 cases. The distributions of PGP 9.5 and NADPH-d were almost the same in all cases. The bowel movements of 13 cases showing normal c-kit distribution were satisfactory. In contrast, the bowel movements were impaired in 2 cases with a decreased number of c-kit positive cells. One infant suffered from severe persistent constipation and thus had to undergo a resection of a dilated colon. The other infant died of sepsis due to postoperative enterocolitis and showed a markedly dilated colon. A decreased number of c-kit positive cells in the normoganglionic segment can thus allow us to predict a poor clinical outcome after definitive surgery, probably due to poor intestinal motility. Therefore examining the c-kit distribution in a resected bowel specimen in patients with HD should be mandatory in order to select the optimal postoperative treatment regimen for each case.