Percentage Of C-kit Research Articles

Research Highlights

Research Highlights

Prolonged elevated levels of c-kit+ progenitor cells after a myocardial infarction by beta 2 adrenergic receptor priming.

Endogenous progenitor cells may participate in cardiac repair after a myocardial infarction (MI). The beta 2 adrenergic receptor (ß2-AR) pathway induces proliferation of c-kit+ cardiac progenitor cells (CPC) in vitro. We investigated if ß2-AR pharmacological stimulation could ameliorate endogenous CPC-mediated regeneration after a MI. C-kit+ CPC ß1-AR and ß2-AR expression was evaluated in vivo and in vitro. A significant increase in the percentage of CPCs expressing ß1-AR and ß2-AR was measured 7 days post-MI. Accordingly, 24hrs of low serum and hypoxia in vitro significantly increased CPC ß2-AR expression. Cell viability and differentiation assays validated a functional role of CPC ß2-AR. The effect of pharmacological activation of ß2-AR was studied in C57 mice using fenoterol administered in the drinking water 1 week before MI or sham surgery or at the time of the surgery. MI induced a significant increase in the percentage of c-kit+ progenitor cells at 7 days, whereas pretreatment with fenoterol prolonged this response resulting in a significant elevated number of CPC up to 21 days post-MI. This increased number of CPC correlated with a decrease in infarct size. The immunofluorescence analysis of the heart tissue for proliferation, apoptosis, macrophage infiltration, cardiomyocytes surface area, and vessel density showed significant changes on the basis of surgery but no benefit due to fenoterol treatment. Cardiac function was not ameliorated by fenoterol administration when evaluated by echocardiography. Our results suggest that ß2-AR stimulation may improve the cardiac repair process by supporting an endogenous progenitor cell response but is not sufficient to improve the cardiac function.

Wnt signaling pathway regulates differentiation of chicken embryonic stem cells into spermatogonial stem cells via Wnt5a.

In this study, we investigated the mechanism of signaling pathway-mediated differentiation of embryonic stem cells (ESCs) into spermatogonial stem cells (SSCs) in chicken. The Wnt signaling pathway was identified based on previous RNA Sequencing results and was proven a crucial signaling pathway that participates in the differentiation of ESCs into SSCs. In retinoic acid (RA) induction experiments in vitro, we found that Wnt signaling expression was inhibited by Wnt5a-shRNA, resulting in decreased expression of corresponding marker genes in SSCs, C-kit, Cvh, integrin α6 and integrin β1, but it was significantly promoted by RA treatment. Immunofluorescence assay showed that percentage of C-kit, Cvh, and integrin α6 and integrin β1-positive cells in RA treatment group and Wnt5a overexpression group was significantly higher than that in Wnt5a signaling interference group. Results of fluorescence-activated cell sorting analysis (FACS) also showed that proportion of germ-like cells was reduced by 14.3% (from 18.3% to 4.0%) at day 4 and 15.4% (from 18.6% to 3.2%) at day 12 after transfection, respectively. In experiments in vivo, shRNA-Wnt5a was stably expressed in fertilized chicken embryos and significantly reduced germ cell formation by 11.3% (from 21.7% to 10.4%) and 3.7% (6.4% from 10.1%). Results of quantitative PCR (qRT-PCR) and western blot assays showed that the expression of some specific germ cell marker genes, integrin α6 and integrin β1, was significantly suppressed following Wnt5a signaling interference in vivo. Taken together, our study suggests that Wnt signaling pathway could regulate positively the differentiation of chicken ESCs into SSCs through Wnt5a.

0509 : Beta 2 adrenegic receptor expression and activation of endogenous progenitor cells

Endogenous progenitor cells may participate in cardiac repair after a myocardial infarction. The beta adrenergic pathway has been proposed to induce proliferation and migration of progenitor cells. However the mechanisms have not yet been clarified. The mechanism underlying beta adrenergic signaling on endogenous c-kit+/CD45 – cardiac cells was investigated by inducing myocardial infarction in adult mice. Hearts were dissociated and flow cytometry analysis demonstrated that one week after ligation, the percentage of c-kit+/ CD45 – cells expressing beta 1 or beta 2 adrenergic receptor was significantly increased (88.1±3% and 106.8±36.5% increase compared to sham respectively). Flow cytometry studies on cultured cardiac c-kit+/CD45 – cells confirmed increased beta 1 and 2 adrenergic receptor expression in response to stress conditions, specifically hypoxia (5%) or serum starvation. Interestingly, stress conditions altered localization of the beta 2 adrenergic receptor by increasing membrane expression. The beta 2 adrenergic receptor signaling pathway was stimulated in adult sham mice with the agonist fenoterol (0.25 mg/kg/day) administered in drinking water. Seven days after treatment the mice and non-treated controls were sacrificed and progenitor cells were measured by flow cytometry in the heart and blood. Fenoterol increased the proliferation and percentage of c-kit+/CD45 – cells in the heart (123.3±86.2% and 70.9±44.6% increase compared to control respectively). Fenoterol treatment also elevated levels of circulating endothelial progenitor cells (158.5±87.9% compared to control) and c-kit/CD45 – cells (70.6±33.5% increase) in the peripheral blood. Beta adrenergic receptor expression is increased after coronary ligation in vivo and in stress conditions in vitro . A beta 2 adrenergic receptor agonist may be used to improve endogenous cardiac repair through the activation of progenitor cells. The author hereby declares no conflict of interest

The influence of disease and age on human cardiac stem cells

Recent studies have found that cardiac stem cells (CSCs) are present in the adult heart. CSCs play an important role in maintaining the balance of the number of myocardial cells. The purpose of this study was to examine characteristics of human CSCs and their correlation with clinical characteristics of patients. We collected heart auricles of 105 patients (age range, 1-78 years; mean, 55.6 ± 17.0 years) undergoing cardiac surgery to obtain CSCs. We assayed the percentage of c-kit positive (c-kit(+)) CSCs with flow cytometry. Plasma N(ɛ)-(carboxymethyl)lysine (CML) concentrations were measured by enzyme-linked immunosorbent assay. The percentage of c-kit(+) CSCs was 4.96 ± 3.12% (0.98-17.17%), and this was significantly negatively correlated with age, the presence of diabetes mellitus (DM) and coronary heart disease (CHD) (r values were -0.797 [P < 0.01], -0.500 [P < 0.01] and -0.250 [P = 0.011], respectively). The percentage of c-kit(+) CSCs was significantly negatively correlated with CML concentrations (r = -0.859, P < 0.01). The percentage of c-kit(+) CSCs decreases with ageing and is further decreased in patients with DM and/or CHD. Furthermore, plasma CML concentrations may have potential as an indicator of the number of c-kit(+) CSCs.

Abstract 41: Explant-Derived Cells from Chronic Heart Failure Activated Endothelial-Mesenchymal Transition and Attenuated Pluripotency Genes Expression

Cardiac progenitor cells are generated from atria explants; however the cellular origin and the mechanisms of cell outgrowth are unclear. Using transgenic tamoxifen-induced Willms tumor 1 (Wt1)-Cre/ERT and Cre-activated GFP reporter mice, we found approximately 40% of explant-derived cells and 74% of explant-derived c-Kit+ cells originated from the epicardium. In atria from sham hearts, Wt1+ cells were located in a thin epicardial layer, while c-Kit+ cells were primarily found within both the sub-epicardium and the myocardium, albeit at low frequency. No overlap between c-Kit+ and Wt1+ cells was observed, suggesting that epicardial Wt1+ cells do not express c-Kit marker in vivo, but more likely the c-Kit marker was acquired in culture. Compared with 4 days in culture, at day 21 we observed 7 folds increase in Snail gene expression; 32% increase in α-smooth muscle actin (SMA) marker, and 30% decrease in E-cadherin marker, suggesting that the explant-derived cells underwent epithelial to mesenchymal transition (EMT) in vitro. Cell outgrowths released TGF-β (1036.4 ± 1.18 pm/ml) and exhibited active TGF-β signaling, which might triggered the EMT. Compared to shams, CHF cell outgrowths exhibited elevated levels of EMT markers, SMA (49% vs. 34%) and Snail (2 folds), and reduced level of Wt1 (11% vs. 22%). In addition, CHF cell outgrowths had two folds increase in Pai1 gene expression, a direct target of TGF-β signaling. In c-Kit+ cells derived from CHF explants, Nanog gene expression was 4 folds lower and Sox 2 was 2 folds lower compared with cells from shams. Suppression of EMT in cell outgrowth increased the percentage of c-Kit+ and Wt1+ cells by 17%, and 15%, respectively. Also suppression of EMT in c-Kit+ cells resulted in 4 folds increase in Nanog and 3 fold increase in Sox2 gene expressions. Our results showed that CHF may further exuberates EMT while diminishes the re-activation of pluripotency genes. Thus, EMT modulation in CHF is a possible strategy to regulate both the yield and the pluripotency of cardiac-explant-derived progenitor cells.

Changes in Interstitial Cells of Cajal at the Deep Muscular Plexus Are Associated with Loss of Distention-Induced Burst-Type Muscle Activity in Mice Infected by Trichinella spiralis

The physiology and pathophysiology of the network of interstitial cells of Cajal associated with the deep muscular plexus (ICC-DMP) of the small intestine are still poorly understood. The objectives of the present study were to evaluate the effects of inflammation associated with Trichinella spiralis infection on the ICC-DMP and to correlate loss of function with structural changes in these cells and associated structures. We used immunohistochemistry, electron microscopy, and assessment of distention-inducing electrophysiological parameters in vitro. Damage to ICC-DMP was associated with a loss of distention-induced patterns of electrical activity normally associated with distention-induced peristalsis. Consistently, the timing of recovery of ICC-DMP paralleled the timing of recovery of the distention-induced activity. Nerve varicosities associated with ICC-DMP including cholinergic nerves, assessed by immunoelectron microscopy and whole mount double labeling, paralleled injury to ICC-DMP thus contributing to impaired excitatory innervation of smooth muscle cells. Major additional changes included a remodeling of the inner circular muscle layer, which may affect long-term sensitivity to distention after infection. In conclusion, transient injury to ICC-DMP in response to T. spiralis infection is severe and associated with a complete lack of distention-induced burst-type muscle activity.

Changes in markers, receptors and adhesion molecules expressed on murine hemopoietic stem cells after a single injection of 5-fluorouracil.

Cytokines play a crucial role in the differentiation and proliferation of hemopoietic cells, and it has recently been found that adhesion molecules play crucial roles not only in differentiation and proliferation, but also in the homing and other functions of hemopoietic cells. We have very recently established a new method for purifying pluripotent hemopoietic stem cells (P-HSC) in mice by injecting 5-fluorouracil (5-FU). The P-HSC were found to be low-density, lineage marker-negative (Lin-), CD71- and major histocompatibility complex class I(high). In the present study, we analyze changes in the expression of various HSC markers (Sca-1 and CD34), receptors (c-kit and interleukin-6 receptor [IL-6R]) and adhesion molecules (very late activation antigen-4 [VLA-4], lymphocyte function-associated antigen-1 [LFA-1], and CD44) after 5-FU injection. The percentage of Sca-1+ cells increases after 5-FU treatment, reaching a maximum on day 3, whereas the percentage of IL-6R+ cells decreases, reaching a minimum on day 3. The percentage of CD34+ cells does not change after 5-FU treatment. The percentages of both c-kit(low) and c-kit(high) cells decrease, reaching a minimum on day 3 after 5-FU treatment, whereas the percentage of c-kit- cells reciprocally increases, reaching a maximum on day 3. However, there is no change in the expression of adhesion molecules (VLA-4, LFA-1 and CD44) on the P-HSC.