Search

In regenerative medicine using transplantation of mesenchymal stem cells (MSCs), the importance of regulating the quality of MSCs has been well recognized; however, there is little information concerning the relationship between the population doubling level (PDL) and the stemness of MSCs in equine medicine. In this study, we showed that the amount of glycosaminoglycan (GAG) secreted by bone marrow-derived MSCs (BMSCs) decreases with increase of PDL. Enzymatic digestion and two-dimensional electrophoresis revealed that a main component of GAG produced by BMSCs was hyaluronan with a small amount of chondroitin sulfate. Increase of PDL downregulated the expression of MSC CD markers, including CD44, CD73, CD90, CD105, and CD146, along with loss of differentiation capacity. Thus, the effect of hyaluronan supplement to the growth medium on both expression of CD markers and the tri-lineage potential of BMSCs was evaluated. Expression of CD73 and CD90 was preserved by continuous addition of hyaluronan to the growth medium, whereas mRNA levels corresponding to CD44, CD105 and CD146 were not preserved by supplementation of hyaluronan. BMSCs subcultured with hyaluronan-supplemented growth medium to PDL-12 showed osteogenic capacity, however adipogenic and chondrogenic activities at PDL-12 were not preserved by exogenous hyaluronan. These results suggest that downregulation of CD44, CD105 and CD146 might not affect the osteogenic capacity. Taken together, the results suggested that supplementation of hyaluronan to the growth medium might be effective at maintaining the osteogenic capacity of equine BMSCs.

Tenomodulin has been recognized as a biomarker for tendon differentiation, and its gene expression is regulated by several transcription factors including Scleraxis and Mohawk. In this study, we found a novel regulatory mechanism of tenomodulin expression. Equine bone marrow-derived mesenchymal stem cells (BMSCs) in monolayer culture showed a low mRNA level of tenomodulin in comparison with the level in the tendon. When cultured in collagen gel containing a glycogen synthase kinase-3 (GSK-3) inhibitor (BIO), expression of tenomodulin in BMSCs increased up to the level in the tendon. Participation of GSK-3 in its gene expression was further demonstrated by a gene silencing experiment with small interference RNA corresponding to GSK-3, suggesting that Wnt/β-catenin signaling mediated expression of tenomodulin. These results were confirmed by nuclear translocation of β-catenin in BIO-treated BMSCs cultured in collagen gel. Under this culture condition, expression of tenomodulin-related transcription factors including Scleraxis and Mohawk was not affected, suggesting that Wnt/β-catenin signaling was independent from these transcription factors. Additionally, BIO strongly enhanced expression of type XIV collagen in collagen-embedded BMSCs up to the level in the tendon, and other tendon-related extracellular matrix components such as decorin and fibromodulin were also upregulated. Taken together, these results indicated that activation of Wnt/β-catenin signaling could induce differentiation of BMSCs into tenomodulin-expressing tendon cells in collagen gel.

We report an autopsy case of treatment by absorbable gelatin sponge and microcoil embolization after perforation of the distal coronary artery by guidewire. Histological examination revealed a foreign body granuloma in the coronary artery with embolization. Foreign body reaction to absorbable gelatin sponge seemed to be stronger than that to the microcoil.

Objective: Evaluate the results from the first 5 years of experience with laparoscopy for diagnosis and treatment of nonpalpable testes., Materials and Methods: Medical records of 51 patients submitted to laparoscopic testicular exploration, during a 5-year period, were retrospectively analyzed. Patients' mean age was 65.7 months (median = 48) on the first procedure. The youngest patient was 10 months and the oldest was 14 years old on the first surgery. Twenty-four (47%) patients presented nonpalpable testes bilaterally, 7 (14%) only at the right side and 20 (39%) at the left, totaling 75 testicular units assessed. Patients who had their testes palpated after anesthetic induction were excluded from the study, and in all other cases, surgical management was based on the testicular position and viability. During the post-operative follow-up, surgical success was classified as palpable testis in scrotal sac, with adequate consistency and volume., Results: Nine (12%) testes were not localized, but their vessels and deferent duct were atrophic. Two (3%) testes were intra-abdominal and atrophic, and 2 (3%) gonads, in the same patient, had a dysmorphic aspect. Nineteen (25%) testicular units were located close to the internal inguinal ring (peeping testes) and, in 22 (29%) units, the spermatic vessels and deferent duct penetrated the internal inguinal ring. Eight (10%) testes were located at a distance of less than 2 cm from the internal inguinal ring and 13 (17%) at a distance greater than 2 cm. The 2 intra-abdominal atrophic testes were removed. Inguinotomy was performed in a total of 41 (54%) cases, reaching a surgical success of 89%. Laparoscopic orchiopexy in one stage, without vascular ligation, was performed in 9 (12%) testes, which presented a distance of less than 2 cm from the internal inguinal ring, also with a surgical success index of 89%. Orchiopexy in 2 stages, with ligation of the spermatic vessels, was performed in 13 (17%) testicular units located at a distance greater than 2 cm from the internal inguinal ring, reaching 77% of good results., Conclusion: Videolaparoscopy is a safe and effective method for diagnosis and treatment of nonpalpable testis.

Trisomy 21 (Ts21) is the most common live-born human aneuploidy; it results in a constellation of features known as Down's syndrome (DS). Ts21 is the most frequent cause of congenital heart defects and the leading genetic cause of mental retardation. To investigate the gene dosage effects of an extra copy of human chromosome 21 (Chr 21) on various phenotypes, we used microcell-mediated chromosome transfer to create embryonic stem (ES) cells containing Chr 21. ES cell lines retaining Chr 21 as an independent chromosome were used to produce chimeric mice with a substantial contribution from Chr 21-containing cells. Fluorescence in situ hybridization and PCR-based DNA analysis revealed that Chr 21 was substationally intact but had sustained a small deletion. The freely segregating Chr 21 was lost during development in some tissues, resulting in a panel of chimeric mice with various mosaicism as regards retention of the Chr 21. These chimeric mice showed a high correlation between retention of Chr 21 in the brain and impairment in learning or emotional behavior by open-field, contextual fear conditioning and forced swim tests. Hypoplastic thymus and cardiac defects, i.e. double outlet right ventricle and riding aorta, were observed in a considerable number of chimeric mouse fetuses with a high contribution of Chr 21. These chimeric mice mimic a wide variety of phenotypic traits of DS, revealing the utility of mice containing Chr 21 as unique models for DS and for the identification of genes responsible for DS.

Epignathus is an extremely rare form of teratoma that arises from the palate or pharynx in the region of the basisphenoid (Rathke's pouch). This condition is associated with a high mortality rate caused by severe airway obstruction in the neonatal period, thus requiring prenatal planning and prompt surgical treatment after birth. The authors describe a case of a giant epignathus that was successfully resected followed by an uneventful recovery.

There have been few pathological investigations of 45,X embryos and fetuses from a developmental point of view. Since most 45,X embryos and fetuses are lost prenatally, it is important to investigate them morphologically in order to elucidate the pathogenesis of the abnormalities. In this study, 13 45,X fetuses with cervical cystic hygroma were examined between 12 and 23 weeks of pregnancy. Every case had a hypoplastic thymus. The aortic valve was bicuspid in 11 cases and unicuspid in 2 cases. The aortic arch showed tubular hypoplasia between the left carotid artery and the left subclavian artery in 12 cases and type B interruption in one case. Smooth muscle cells and elastic fibers were reduced in number in the hypoplastic aortic arch. These results suggest hypoplastic development of the fourth branchial arch. Combined abnormalities between the aortic arch and aortic valve are not infrequently observed in DiGeorge anomaly. A similar developmental mechanism apparently underlies the pathogenesis of 45,X embryos. Possible genes causing the abnormalities are discussed.

a rare case of primary choriocarcinoma of the lung in a male is described with immunohistochemistry for human chorionic gonadotropin (hCG), epidermal growth factor (EGF) and EGF-receptor. The extragonadal trophoblastic origin of this pulmonary carcinoma was definitely confirmed by an autopsy examination, and hCG-production and hCG-positive staining of the tumor cells. Furthermore, the tumor cells clearly expressed EGF and its receptor which play an important role in the proliferation and differentiation of normal and neoplastic trophoblasts of the uterus. Our present case suggests that EGF may act in an autocrine manner in the tumor cells of primary pulmonary choriocarcinoma.

Absent semilunar valve was found in three fetal cases with cystic hygroma. Two cases which simultaneously showed absent aortic and pulmonary valves (AAV and APV, respectively) had double-outlet right ventricle. The third case, which lacked only the aortic valve, had atrioventricular septal defect and anomalous origin of the right subclavian artery. Two of the three cases had a markedly hypoplastic thymus. Fifteen AAV and 179 APV cases, including the above-mentioned cases and others reported elsewhere, were discussed with special reference to the pathogenesis of absent semilunar valve. Of the 15 AAV cases, hypoplasia of the left heart was observed in 11 cases (73.3%), double-outlet right ventricle in 5 (33.3%), and aortic arch malformations in 6 (40.0%). In the 179 APV cases, there were 111 tetralogy of Fallot (62.0%) and 44 right-sided aortic arch (24.6%). DiGeorge anomaly was found in one AAV and eight APV cases. These results indicate a pathogenesis that is possibly related to hemodynamic abnormalities or abnormal neural crest cells. Further investigation will be needed to elucidate a more definite pathogenesis of absent semilunar valve.

The purpose of this study is to reconstruct an alveolus-like structure from alveolar type II epithelial cells in a culture condition. Isolated alveolar type II epithelial cells of the rat were cultured in a three-dimensional collagen gel matrix. Single type II cells formed cellular aggregates that had a lumen after cell division in this culture condition. Through proliferation of the component cells, these aggregates grew to assume a globular or branching structure, part of which in turn developed into a large, cystic alveolus-like structure. This structure consisted of flattened epithelial cells intermingled with cuboidal epithelial cells. In these structures, the surfactant production was confirmed by immunohistochemistry and electron microscopy. To our knowledge, this is the first report of a reconstruction of an alveolus-like structure in a three-dimensional collagen gel matrix culture. This culture system seems to provide an appropriate physiological environment in which to study the differentiation and disorders of pulmonary alveoli.

Subcapsular small cortical cells (SC cells) and subcapsular large cortical cells (LC cells) of bovine adrenal cortex were cultured separately after purification by unit gravity sedimentation, and then compared with inner-half layer cells (IL cells) prepared by the same method. Both SC and LC cells were polygonal in shape and their mitochondria were elongated with lamellar cristae. SC cells became as large as LC cells on day 6 of culture with increased cytoplasmic lipid droplets, whereas IL cells showed no change in size. IL cells were spindle-shaped and had mitochondria with tubulovesicular cristae. Both SC and LC cells produced 11 beta-deoxycorticosterone, corticosterone, aldosterone and small amounts of 17 alpha-hydroxyprogesterone (17 alpha-OH-Prog) and cortisol (F). IL cells produced much more 17 alpha-OH-Prog and F than SC or LC cells. When stimulated with ACTH, cortical cells in each group showed cellular retraction and their mitochondria became spherical. The amounts of 17 alpha-OH-Prog and F increased in all groups, especially in IL cells. These results show that LC cells have similar characteristics to SC cells in both morphology and function, and that they differ from IL cells, which correspond to classical fasciculata-reticularis cells.

Setleis bitemporal "forceps marks" syndrome is characterized by "forceps marks" and a peculiar facies. The syndrome has previously been reported mainly in the Puerto Rican population. We describe here a Japanese boy with the syndrome. In addition, the hypothesis is presented that the Setleis syndrome may result from an insufficient migration of neural crest cells into the frontonasal process and the first branchial arch.

The skin comprises three layers: epidermis, dermis, and hypodermis. We report here on a skin, reconstructed in vitro, that is composed of all three layers. The top-most layer, epidermis, was exposed to air by a new method. The exposure induced an extensive proliferation, and differentiation, i.e. keratinization was eventually observed in the cultured epidermal cells. Skin thus cultured will be a useful graft of transplantation and provide an ideal model system in which to study diseases of the skin.

By means of a combination of immunosurgery and a modified method of microsurgery, blastocysts were reconstructed to produce viable chimeric fetal-placental units. Reciprocal reconstituted blastocysts were produced using euploid and trisomy 16 blastocysts. Reconstructed blastocysts yielded significantly smaller fetuses at day 17 of pregnancy than simultaneously transferred control blastocysts (mean body weight 0.49 g vs 0.64 g, P less than 0.01). However, apart from reduced size, no abnormalities were observed for any euploid fetus-euploid placenta construct. The three reconstructed blastocysts that yielded a trisomic fetus-trisomic placenta were viable when examined on day 17 and displayed the abnormalities typical of mouse trisomy 16. No reconstructed blastocyst that yielded a trisomic fetus-euploid placenta or a euploid fetus-trisomic placenta was viable beyond day 13 of development. One case in which a trisomic fetus had a placenta that was chimeric (euploid/trisomic) examined on day 17 displayed the abnormalities typical of a trisomic fetus but the placenta appeared histologically normal. The findings suggest that there is a coordination of the development of the fetus and the placenta that is essential for the development of the fetus.

The effect of alpha-melanocyte-stimulating hormone (alpha-MSH) on the fetal adrenal cortex in rat was studied in vivo and in vitro. In vivo, an injection of alpha-MSH stimulated the adrenal cortex slightly. Treatment of the adrenal cortex with alpha-MSH in organ culture caused hyperfunctional change in the innermost layer of the cortex, the detection of which was done by electron microscope. This treatment caused at the same time an increase in corticosterone in the culture medium. Furthermore, comparison was made between alpha-MSH and ACTH with respect to their effect on the fetal adrenal cortex.

Murine trisomy (Ts) 16 occurs in the fetal and neonatal progeny of males doubly heterozygous for the Robertsonian metacentric chromosomes Rb(16.17)7Bnr/Rb(9.16)9Rma and "all acrocentric" females. The developmental aspects of this trisomy were studied between day 12 of gestation and birth. So far, postnatal survival longer than a few hours after birth has not been observed. The frequency of Ts 16 among all implants decreased from more than 20% on day 14 to values between 4% and 7% shortly before term. Main features of Ts 16 are moderate general hypoplasia, slight developmental retardation, and cardiovascular anomalies. These latter were found in 96% of the trisomies, the great majority belonging to the transposition type, i.e., riding aorta, double outlet right ventricle (DORV) and transposition of the great arteries (TGA). Association with common atrio ventricular (AV)-canal was frequent. Other anomalies as "open eyelid", hydronephrosis, and hydroureter seem to be attributable to the effects of retardation. Generalized transient edema was frequent in the later gestational stages of Ts 16. Severe cardiovascular malformation is possible one of the factors responsible for late fetal or neonatal death in some cases. Another factor probably contributing to Ts 16 fetal mortality is insufficiency of placental function due to hypoplasia of the fetal vasculature of this organ. The teratological study of Ts 16 demands interest since evidence has been forwarded to consider this trisomy as an animal model of human trisomy 21.

Under lowered Ca2+ content and in the post-treatment state of low Ca2+, we studied the cellular structure and functioning in mouse adrenocortical tumor cells, Y-1. These cells had been maintained in Ham F12 medium containing 10% fetal calf serum. The Ca2+ present in this complete medium was 0.39 mM. Under a slightly lowered Ca2+ (0.29 mM) produced by EGTA, the cells had many blebs on their surfaces and specific functional activity decreased in steroidogenesis as did ACTH reactivity. In the post-treatment state of a low Ca2+, the cellular surface was covered with many short microvilli and there was greater cellular activity than in the control cells. When the Ca2+ concentration was below 0.17 mM, the cellular structure and functioning were disturbed, and there was no recovery even at the physiological Ca2+ after the removal of EGTA.

Malignant testicular tumors occurring in non-twin brothers are reported. Both of the brothers suffered from amentia and epilepsy and were the product of a consanguineous marriage. One brother presented with teratocarcinoma and the other seminoma. With a review of the literature, genetic roles in the etiology of testicular neoplasia are discussed.

Polypoid squamous cell carcinoma of the larynx consists of two components, namely, squamous cell carcinoma and sarcomatoid spindle cells. To further investigate the histogenesis of the spindle cell component, we studied two cases of polypoid squamous cell carcinoma of the larynx by immunohistochemistry and ultrastructural analysis. Positive staining for ras oncogene p21 and cytokeratin was demonstrated in both squamous cell carcinoma and spindle cell component. Only the latter component was positive for vimentin. Electron microscopic examination showed well-developed desmosomes in spindle cells. These results suggest that the spindle cell component is epithelial in origin and malignantly neoplastic; it originates from mesenchymal metaplasia or squamous cell carcinoma.

Fetuses with cystic hygroma or loose skin of the neck were studied chromosomally and phenotypically to clarify the relation between neck abnormality and cardiovascular malformations. Of 12 fetuses, 9 had chromosome abnormalities: 4 with 45,X, 3 with trisomy 21, one each with trisomy 13, dup 6q. One had normal chromosomes. Two cases, in which chromosome analysis was unsuccessful, were morphologically suspected to be trisomy 13. Nine of the 12 fetuses had either bilateral cystic hygroma of the neck (7 cases) or nuchal bleb (2 cases: trisomy 13 and dup 6q). Two of the 3 remaining cases (trisomy 21) had loose skin of the neck, and one had edematous swelling of the skin of the neck. Except for the last case of trisomy 21, 11 fetuses (91.7%) had severe and/or rare cardiovascular malformations. They were divided into 3 major groups: a) spectrum of hypoplastic left heart syndrome (45,X and dup 6q), b) double outlet right ventricle, agenesis of semilunar valve (trisomy 13), and c) abnormality of atrioventricular orifice or valves (trisomy 21). One fetus with normal chromosomes had persistent left superior vena cava instead of absent right one and calcification of myocardium. Histological observation of edematous skin demonstrated the abnormal distribution of lymph vessels, including their absence. Some cases showed hypoplastic thymus. To integrate the findings of the present study and the descriptions in the literature, a pathogenesis is hypothesized in relation to migration of neural crest cells and extracellular matrix.

Mature white fat tissue consists primarily of unilocular fat cells. Clearly, the study of the biology of these cells would be most helpful for the elucidation of the mechanism of obesity. We describe a new method termed 'ceiling culture' for culturing in vitro unilocular fat cells obtained from humans and rats. These cells can be maintained in culture for long periods of time and, under such conditions, continue to exhibit specific functions such as lipogenesis and lipolysis. Under the culture conditions described, unilocular fat cells change into multilocular fat cells or cells with a fibroblast-like appearance. These cells then proliferate, form a cell monolayer attached to the substratum, and after becoming confluent, exhibit accumulations of intracytoplasmic lipid droplets. These attached dedifferentiated cells continue to exhibit lipogenesis and lipolysis.

A 44-year-old male was admitted to hospital with a complaint of abdominal distention. A CT scan revealed a giant multicystic mass in the abdominal cavity, a tumor that measured 34 cm in diameter, which had risen from the gastric body and grew in an exophytic fashion with a stalk. Histologic findings exhibited a malignant leiomyoblastoma with cellular pleomorphism and a intracytoplasmic cystic change, which included disseminated nests of immature small round cells. After excisional surgery, the patient has had no sign of a recurrence for the past two years.

An autopsy case of polysplenia with absence of the hepatic segment of the inferior vena cava in a 53-year-old female is reported. The venous blood from the lower parts of the body was drained through the azygos vein and the superior vena cava into the right atrium. Other than acquired pathological change, the heart was found to be normal. Except for the partial absence of the inferior vena cava, isomerism of asymmetric organs, and heterotaxia of abdominal organs, characteristic of typical polysplenia, were not present. This case of atypical polysplenia suggests the presence of a transitional form in the complex.

Micromys minutus japonicus (Japanese harvest mouse) is one of the smallest known mammals and is easily maintainable in the laboratory. The adrenal cortex of this animal is described here for the first time. The morphological structure with histochemistry was essentially the same as that of the mouse or the rat. However, the histochemical activity of steroid 3 beta-OH dehydrogenase in the zona glomerulosa was characteristically positive in this animal and electron microscopically Golgi's apparatus and endoplasmic reticulum were prominent in the glomerular cells. The concentration of aldosterone was high in the serum. These findings suggest the hyperfunction of the zona glomerulosa in this animal.