CD49f+CD34+ cells, a skin epithelial stem cell (EpSC)-rich population, were prepared from adult mouse skin and cultured in the presence of Wnt-3a without feeder cells. CD34 expression was retained in ...about 10% of the cells, which had proliferated about 1,000-fold by day 10, although completely lost by day 14. CD49f+CD34+ cells sorted on day 10 retained canonical Wnt-responsiveness, proliferated markedly in the presence of Wnt-3a, maintained undifferentiated epithelial cell marker expression, and promoted hair follicle development in vivo. Those were subjected to a second 10-day culture with Wnt-3a and sorted, and then the same procedures were repeated a total of 15 times. CD49f+CD34+ cells obtained from each of those cultures retained the same EpSC characteristics as the original cells. CD34+ and CD34− cells were found to produce Wnt-3a and Wnt/β-catenin inhibitors, respectively. CD34+ cells resided as small cellular clusters surrounded by a large amount of CD34− cells. Furthermore, we found that exogenous Wnt-3a delayed the conversion of CD34+ cells to CD34− cells and induced CD34− cells to suppress the production of Wnt/β-catenin inhibitors, likely leading to generation of a microenvironment favorable for maintaining EpSCs. Our results suggest the possibility of partial long-term maintenance of EpSCs in vitro by Wnt-3a.
Purpose: Although the isolation of rat and mouse mesothelial cells has previously been reported, most mesothelial cells used for experimental studies are obtained from peritoneal cells. Here, we ...describe an optimized method for the isolation and in vitro propagation of rodent pleural mesothelial cells without the requirement for specialized surgical techniques. Materials and Methods: To harvest pleural mesothelial cells, the pleural space of 8-9-week-old rats or older mice was filled with 0.25% trypsin in ethylenediaminetetraacetic acid (EDTA) buffer for 20 min at 37 °C. Cells were then harvested, and incubated at 37 °C in a humidified atmosphere with 5% CO
2
. Immunofluorescence analysis of plated pleural mesothelial cells was performed using Alexa 546 (calretinin). To investigate optimal proliferation conditions, medium enriched with various concentrations of fetal calf serum (FCS) was used for pleural mesothelial cell proliferation. Results: By day 10, confluent cell cultures were established, and the cells displayed an obvious cobblestone morphology. Immunofluorescence analysis of the cells demonstrated that all stained positive for Alexa 546 (calretinin) expression. Mesothelial cells grew better in medium containing 20% FCS than with 10% FCS. Conclusions: This is a simple procedure for the efficient collection of primary pleural mesothelial cells, which were obtained in defined culture conditions from the euthanized rodent thoracic cavity using trypsin-EDTA treatment. The ability to easily culture and maintain identifiable pleural mesothelial cells from rodents will be helpful for future experiments using these cells.
Dermal papilla (DP) cells are associated with the development of hair follicles (HFs) and regulation of the hair cycle. However, primary DP cells prepared from cultured HFs are known to lose their ...ability to induce HF after culturing in standard media, for example, fibroblast growth conditions. We explored a new culture condition by which DP cells maintained their HF induction ability. The addition of Wnt-10b to the first culture of primary DP cells promoted their proliferation and maintained their Wnt responsiveness and HF induction ability. Furthermore, DP cells in Wnt-10b-containing medium sustained those characteristics after 10 passages (100 days), which encompassed the entire experimental period. These results suggest that Wnt-10b plays a pivotal role in proliferation and maintenance of DP cells in vitro.
Abstract Ancylostoma (A.) ceylanicum , one of the most common species of hookworms infecting dogs and cats, also causes patent infections in humans and is now considered to be the second most common ...hookworm species infecting populations in southeast Asia. A Japanese patient who returned from a visit to Thailand and Lao People's Democratic Republic (PDR) was presented with intermittent watery diarrhea with eosinophilia. Hookworm eggs were found in feces samples, and adult worms were confirmed to be present in the jejunum with capsule endoscopy and double balloon enteroscopy. A diagnosis of A. ceylanicum infection was made based on the morphology of the adult worms along with findings of a PCR-based molecular study using larvae obtained from a fecal sample culture. The infection was considered likely to have been obtained during a 1-month stay in a Laotian village, where the patient had eaten local food, worn sandals on bare feet, and lived as a local native villager, though he had stayed in modern hotels during the visit to Thailand.
Wnts are deeply involved in the proliferation and differentiation of skin epithelial cells. We previously reported the differentiation of cultured primary skin epithelial cells toward hair shaft and ...inner root sheath (IRS) of the hair follicle via β-catenin stabilization caused by Wnt-10b, however, the effects of Wnt-10b on cultured hair follicles have not been reported. In the present study, we examined the effects of Wnt-10b on shaft growth using organ cultures of whisker hair follicles in serum-free conditions. No hair shaft growth was observed in the absence of Wnt-10b, whereas its addition to the culture promoted elongation of the hair shaft, intensive incorporation of BrdU in matrix cells flanking the dermal papilla (DP), and β-catenin stabilization in DP and IRS cells. These results suggest a promoting effect of Wnt-10b on hair shaft growth that is involved with stimulation of the DP via Wnt-10b/β-catenin signalling, proliferation of matrix cells next to the DP, and differentiation of IRS cells by Wnt-10b.
We sought to establish a more efficient technique for induction of inner ear hair cell-like cells (HC-like cells) from embryonic stem cells (ES cells) by using a combination of two previously ...reported methods; ST2 stromal cell-conditioned medium, known to be favorable for HC-like cell induction (HIST2 method), and ES cells with transfer of the Math1 gene (Math1-ES cells). Math1-ES cells carrying Tet-inducible Math1 were cultured for 14days with doxycycline in conditioned medium from cultures of ST2 stromal cells following formation of 4-day embryoid bodies (EBs). Although each of the previously introduced methods have been reported to induce approximately 20% HC-like cells and 10% HC-like cells in their respective populations in EB outgrowths at the end of the culture periods, the present combined method was able to generate approximately 30% HC-like cells expressing HC-related markers (myosin6, myosin7a, calretinin, α9AchR, Brn3c), which showed remarkable formation of stereocilia-like structures. Analysis of expressions of marker genes specific for cochlear (Lmod3, Emcn) and vestibular (Dnah5, Ptgds) cells indicated that our HIST2 method may lead to induction of cochlear- and vestibular-type cells. In addition, continuous Math1 induction by doxycycline without use of the HIST2 method preferentially induced cochlear markers with negligible effects on vestibular marker induction.
•Inner ear hair cells from embryonic stem cells in vitro•Efficient induction method by combination of conditioned medium and gene regulation•Math1 expression regulates the production of hair cells from embryonic stem cells.
Neurogenesis in the subventricular zone (SVZ), subgranular zone (SGZ), and cerebral cortex is now a familiar event to confirm by cerebral arterial ischemia in rat models. However, it remains unclear ...whether cerebral venous ischemia (CVI) alone causes neurogenesis, and where that neurogenesis occurs. After creating CVI rat models via a two-vein occlusion (2-VO) method, neurogenesis was immunohistochemically evaluated by double-labeling 5-bromo-2′-deoxyuridine (BrdU)-positive cells with neuronal nuclei (NeuN) or doublecortin (DCX) antibody. Fifty Wistar rats were divided into two major groups (BrdU-NeuN and BrdU-DCX) and then separated into two subgroups (2-VO or sham). The total number of double-positive cells expressed inside a predefined region of interest (ROI) covering the ischemic area was compared between the two subgroups. Then, we divided the ROI into six sections to evaluate and compare the distribution of double-positive cells generated in each section between the two subgroups. The 2-VO subgroup presented more double-positive cells than the sham group in both BrdU-NeuN and BrdU-DCX groups, while the BrdU-DCX+2-VO group showed a characteristic distribution of double-positive cells in ROI 2 and ROI 3, suggesting areas of the ischemic core and penumbra, with a significant difference compared to the BrdU-DCX+sham group. This study demonstrates that CVI has the potential to induce endogenous neurogenesis, with significant numbers of both newly generated neurons and precursors observed in the ischemic area. The distribution of these cells suggests that the cortex could be the main origin of neurogenesis after cortical CVI.
Decellularization of tissues is a recently developed technique mostly used to provide a 3-dimensional matrix structure of the original organ, including decellularized lung tissues for lung ...transplantation. Based on the results of the present study, we propose new utilization of decellularized tissues as inducers of stem cell differentiation. Decellularized lung matrix (L-Mat) samples were prepared from mouse lungs by SDS treatment, then the effects of L-Mat on differentiation of ES cells into lung cells were investigated. ES cell derived-embryoid bodies (EBs) were transplanted into L-Mat samples and cultured for 2 weeks. At the end of the culture, expressions of lung cell-related markers, such as TTF-1 and SP-C (alveolar type II cells), AQP5 (alveolar type I cells), and CC10 (club cells), were detected in EB outgrowths in L-Mat, while those were not found in EB outgrowths attached to the dish. Our results demonstrated that L-Mat has an ability to induce differentiation of ES cells into lung-like cells.
Vestibular hair cells (V-HCs) in the inner ear have important roles and various functions. When V-HCs are damaged, crippling symptoms, such as vertigo, visual field oscillation, and imbalance, are ...often seen. Recently, several studies have reported differentiation of embryonic stem (ES) cells, as pluripotent stem cells, to HCs, though a method for producing V-HCs has yet to be established. In the present study, we used vestibular cell conditioned medium (V-CM) and effectively induced ES cells to differentiate into V-HCs. Expressions of V-HC-related markers (
,
,
,
) were significantly increased in ES cells cultured in V-CM for 2 weeks, while those were not observed in ES cells cultured without V-CM. On the other hand, the cochlear HC-related marker
was either not detected or detected only faintly in those cells when cultured in V-CM. Our results demonstrate that V-CM has an ability to specifically induce differentiation of ES cells into V-HCs.
Abstract We present 3 adult cases of visceral toxocariasis from the same family, who each consumed thin slices of raw bovine liver weekly, and developed eosinophilia and multiple small lesions in ...their livers and lungs. Serological examinations using the larval excretory–secretory product of Toxocara canis strongly indicated infection with Toxocara species larvae. The patients responded well to treatment with albendazole. Ingestion of raw liver from paratenic animals is considered to be a common transmission route of human toxocariasis, especially in adults.
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