RESUMEN
STUDY QUESTION: Is it possible to induce in vitro reorganization of primary human testis cells from testicular sperm extraction (TESE) biopsies, maintain their long-term cultivation in a 2D system and identify cellular compositions? SUMMARY ANSWER: In vitro reorganization of primary human testis cells from TESE biopsies and their long-term cultivation on uncoated cell culture dishes is feasible and the cellular compositions can be uncovered through gene expression and microscopic analyses. WHAT IS KNOWN ALREADY: It has been shown in the rodent model that mixtures of testicular cell types are able to reassemble into clusters when cultivated on different kinds of surfaces or three-dimensional matrices. Two recent publications demonstrated the ability of primary human testicular cells to assemble into testicular organoids and their cultivation for a period of 3-4 weeks. STUDY DESIGN SIZE, DURATION: Primary human testis cells from TESE biopsies from 16 patients were reorganized in vitro and the clusters were cultivated long term on uncoated cell culture dishes, providing a solid ground for in vitro spermatogenesis. Gene expression analysis as well as fluorescence/transmission electron microscopy (TEM) were employed to uncover the cellular composition of the clusters. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testis biopsies from adult, normogonadotropic patients displaying full spermatogenesis (n = 11), hypospermatogenesis (n = 2), predominantly full spermatogenesis with some hypospermatogenic tubules (n = 1), meiotic arrest (n = 1) or mixed atrophy (n = 1) were enzymatically digested and dispersed cells were cultivated on 96-well plates or chamber dishes as aggregate-free cell suspensions. Time-lapse imaging of cluster formation was performed over a period of 48 h. For receptor tyrosine kinase inhibition of cluster formation, cells were treated twice with K252a within 2-3 days. Immunofluorescence staining and confocal microscopy was carried out on clusters after 1-3 weeks of cultivation to identify the presence of Sertoli cells (SC) (SOX9), peritubular myoid cells (SMA), Leydig cells (LC) (STAR), undifferentiated spermatogonia (FGFR3), differentiating spermatogonia/spermatocytes (DDX4) and postmeiotic germ cells (PRM1). Single clusters from four patients and a pool of eight larger clusters from another patient were manually picked and subjected to quantitative real-time PCR to evaluate the presence of SC (SOX9, AR), LC (INSL3, STAR, HSD3B1), peritubular myoid cells (ACTA2), fibroblasts (FSP1), endothelial cells (CD34), macrophages (CD68), undifferentiated spermatogonia (FGFR3), differentiating spermatogonia/spermatocytes (DDX4) and postmeiotic germ cells (PRM1). Finally, an ultrastructural investigation was conducted based on TEM of clusters from six different patients, among them 3-month cultivated large clusters from two patients. MAIN RESULTS AND THE ROLE OF CHANCE: Quantitative PCR-based analysis of single-picked testicular cell clusters identified SC, peritubular myoid cells, endothelial cells, fibroblasts, macrophages, spermatids and LC after 1, 2 or 3 weeks or 3 months of cultivation. Immunofluorescence positivity for SC and peritubular myoid cells corroborated the presence of these two kinds of testis niche cells. In addition, round as well as elongated spermatids were frequently encountered in 1 and 2 weeks old clusters. Transmission electron microscopical classification confirmed all these cell types together with a few spermatogonia. Macrophages were found to be of the proinflammatory M1 subtype, as revealed by CD68+/CD163-/IL6+ expression. Time-lapse imaging uncovered the specific dynamics of cluster fusion and enlargement, which could be prevented by addition of protein kinase inhibitor K252a. LARGE SCALE DATA: N/A. LIMITATIONS REASON FOR CAUTION: Cell composition of the clusters varied based on the spermatogenic state of the TESE patient. Although spermatids could be observed with all applied methods, spermatogonia were only detected by TEM in single cases. Hence, a direct maintenance of these germ cell types by our system in its current state cannot be postulated. Moreover, putative dedifferentiation and malignant degeneration of cells in long-term cluster cultivation needs to be investigated in the future. WIDER IMPLICATIONS OF THE FINDINGS: This work demonstrates that the reorganization of testicular cells can be achieved with TESE biopsies obtained from men enroled in a standard clinical assisted reproduction program. The formed clusters can be cultivated for at least 3 months and are composed, to a large extent, of the most important somatic cell types that are essential to support spermatogenesis. These findings may provide the cellular basis for advances in human in vitro spermatogenesis and/or the possibility for propagation of spermatogonia within a natural stem cell niche-like environment. STUDY FUNDING AND COMPETING INTERESTS: The project was funded by a DFG grant to K.v.K. (KO 4769/2-1). The authors declare they have no conflicts of interest.
Asunto(s)
Espermatogonias/metabolismo , Testículo/metabolismo , Biopsia , Células Cultivadas , ARN Helicasas DEAD-box/metabolismo , Humanos , Masculino , Reacción en Cadena de la Polimerasa , Protaminas/metabolismo , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/metabolismo , Células de Sertoli/citología , Células de Sertoli/metabolismo , Espermatogénesis/fisiología , Espermatogonias/citología , Espermatozoides/citología , Espermatozoides/fisiología , Testículo/citologíaRESUMEN
BACKGROUND: Neuroblastoma is a common pediatric solid tumor with poor outcome for metastatic disease. Thus, novel therapeutic options are of main interest. The anti-neoplastic properties of taurolidine have been demonstrated on a variety of human cancer cells. However, data on neuroblastoma is lacking. Therefore, our aim was to evaluate the effect of taurolidine on growth of neuroblastoma cell lines. MATERIALS AND METHODS: Neuroblastoma SK-N-BE(2)-M17 and SK-N-SH cells and nonmalignant human umbilical vein endothelial cells as controls were incubated with increasing concentrations of taurolidine (100, 250, 500 µM). Cell growth was examined after 12, 24, and 48 hours of exposure. RESULTS: Inhibition of cell growth by taurolidine was seen in both malignant cell lines. When compared with human umbilical vein endothelial cells, the neuroblastoma cell lines were significantly more responsive to taurolidine. CONCLUSIONS: The observed negative impact on cell growth, highly distinctive in SK-N-BE(2)-M17 and SK-N-SH, implies a taurolidine-specific mode of action that appears dependent on differences on cellular and molecular level. Further investigations are warranted to evaluate its mechanism and probable clinical use.
Asunto(s)
Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/metabolismo , Taurina/análogos & derivados , Tiadiazinas/farmacología , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales/métodos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Neuroblastoma/patología , Taurina/farmacologíaRESUMEN
BACKGROUND: The pathogenesis of intestinal dysmotility in gastroschisis is not completely understood. Peel formation and disorganization of interstitial Cajal cells (ICC) have been proposed in humans. The aim of this study was to evaluate the impact of prenatal coverage of gastroschisis on gut inflammation and expression of ICC in a fetal lamb model. METHODS: Twenty-one German blackhead sheep with an abdominal wall defect that was created fetoscopically on day 77 of 145 days gestation were used in this study. Intrauterine surgery with the aim to cover the defect was performed 3 weeks later; two fetuses were covered completely, 5 partially and 11 remained uncovered. Three fetuses without gastroschisis were used as controls. All fetuses were retrieved by cesarean section at day 135. Samples of the small intestine were stained with hematoxylin and eosin for histologic analysis of peel formation and serosal and muscular thickness. For ICC detection, immunohistochemistry using anti-CD117 (c-Kit) antibody was used. RESULTS: In all samples with exposure to amniotic fluid, peel formation and significantly decreased ICC were found. Complete coverage reduced peel formation and disorganization of ICC compared to uncovered animals almost to the level of controls. CONCLUSIONS: Peel formation and ICC derangement were significantly reduced by prenatal coverage of gastroschisis. Moreover, this animal model mimics the histopathological bowel changes as seen in human gastroschisis and may, therefore, be used for further research on the pathophysiology and fetal therapy of this malformation.
Asunto(s)
Fetoscopía , Gastrosquisis/cirugía , Inflamación/patología , Células Intersticiales de Cajal/metabolismo , Animales , Recuento de Células , Modelos Animales de Enfermedad , Femenino , Gastrosquisis/patología , Inmunohistoquímica , Inflamación/metabolismo , Mucosa Intestinal/patología , Embarazo , OvinosRESUMEN
INTRODUCTION: The embryogenesis of gastroschisis is not completely understood. The aim of our study was to evaluate the impact of a simple abdominal wall defect versus a defect including eviscerated intestine or omentum for the development of gastroschisis in a fetal lamb model. MATERIAL AND METHODS: At mid-gestation (day 77) an abdominal wall defect was fetoscopically created with three different approaches in 19 German blackhead sheep. The intestine was eviscerated in 7 fetuses (group 1). The peritoneum was incised and a patch of the omentum pulled through the incision in 5 fetuses (group 2). In 7 fetuses (group 3) the skin and rectus muscle were incised until the peritoneum was visible. In this group, no abdominal contents were exteriorized and the peritoneum was left intact. A second fetoscopic procedure was performed 21 days later, assessing the condition and extension of eviscerated bowel. The fetus was retrieved by Cesarean section on day 132 and evaluated. RESULTS: The second fetoscopy acting as a control for the creation of gastroschisis demonstrated eviscerated and inflamed intestine in all 3 groups. The amount of eviscerated intestine did not appear to depend on the size of the defect nor on its duration. DISCUSSION: It appears that a simple incision of the abdominal wall with intact peritoneum is sufficient for the development of gastroschisis in a fetal sheep model. This finding may improve the understanding of the etiology of gastroschisis.
Asunto(s)
Modelos Animales de Enfermedad , Gastrosquisis/embriología , Ovinos/embriología , Pared Abdominal/cirugía , Animales , Desarrollo Embrionario , Femenino , Laparotomía , EmbarazoRESUMEN
BACKGROUND: Gastroschisis is a malformation consisting of an abdominal wall defect with eviscerated bowel. Its standard treatment is postnatal repositioning or temporary prosthetic bag placement. The aim of our study is to evaluate the feasibility of its fetoscopic management in a lamb model. METHODS: At mid-gestation (day 75), gastroschisis was created in eight German blackhead sheep. A second fetoscopic procedure was performed on day 105, assessing the viability, extension, and potential for repositioning of eviscerated bowel. The fetus was retrieved by Cesarean section on day 132 and evaluated. RESULTS: In six fetuses gastroschisis could be successfully created and assessed with fetoscopy. Two fetuses were lost due to technical complications. Primary repair by repositioning the intestine into the abdomen was not possible because it grew into an inflammatory conglomerate too large for the small fetal abdominal cavity. CONCLUSIONS: Although technically demanding, we were able to produce and reassess six cases of gastroschisis by fetoscopy. As primary repositioning appears unfavorable, fetoscopic prosthetic bag placement may become an alternative.
Asunto(s)
Sufrimiento Fetal/cirugía , Fetoscopía/métodos , Gastrosquisis/cirugía , Animales , Complicaciones Posoperatorias/etiología , Oveja Doméstica , Factores de TiempoRESUMEN
Cell transplantation and tissue engineering with liver cells are currently under investigation as experimental therapies for certain liver diseases. In this study we evaluated a fibrin-based gel matrix as carrier for hepatocytes in culture. Furthermore, a novel technique for direct intrahepatic injection of fibrin gel-immobilized hepatocytes was developed and evaluated in a rat model. Hepatocytes were harvested from rats. Fibrin matrix was generated with modified fibrin sealant. Cells, in medium containing epidermal growth factor and insulin, were seeded in a drop of fibrin matrix onto plastic culture dishes. Cell numbers were assessed by DNA content. Hepatocyte differentiation was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistology (IH) for cytokeratin (CK)-18 and albumin. PKH26-labeled fibrin gel-immobilized hepatocytes were transplanted into liver by direct injection underneath the capsule. Fluorescence microscopy of explanted liver was performed to identify PKH26+ donor cells. Neotissue was characterized by IH for the markers CK-18, ED1, and desmin. Culture in a fibrin matrix allowed stable cell numbers and three-dimensional neotissue formation. RT-PCR and IH showed preservation of liver-specific markers CK-18 and albumin in vitro. Transplanted cells were identified by fluorescence microscopy after 2 and 7 days. CK-18 and desmin staining showed integration of hepatocytes and hepatic stellate cells into the host liver. Fibrin matrix is an appropriate environment for hepatocytes in culture. Direct intrahepatic injection of fibrin gel-immobilized hepatocytes is technically feasible. We conclude that fibrin gel immobilization is an attractive tool for the development of tissue engineering-based liver support systems.
Asunto(s)
Fibrina , Hepatocitos/trasplante , Hígado Artificial , Ingeniería de Tejidos , Animales , Trasplante de Células/métodos , Células Inmovilizadas/citología , Células Inmovilizadas/fisiología , Células Inmovilizadas/trasplante , Hepatocitos/citología , Hepatocitos/fisiología , Humanos , Ratas , Ratas Sprague-Dawley , Ingeniería de Tejidos/métodosRESUMEN
UNLABELLED: BACKGROUND/PURPOSE; The embryology of ventral body wall malformations is only partially understood, although their incidence is relatively common. As only few experimental data exist on the development of those defects, the aim of our study was to compare the teratogenic effect of trypan blue (TB) and suramin (SA) in their capability to induce umbilical and supraumbilical abdominal wall malformations in a chicken egg model. MATERIALS AND METHODS: A total of 255 fertilized chicken eggs were incubated at 38 °C and 75% relative humidity. Embryos were treated in ovo on incubation day 2.5 (Hamburger/Hamilton (HH) stage 13). The eggshell was windowed, and solutions of TB or SA were injected into the coelomic cavity at the region of the umbilicus. The window was closed and the embryos reincubated until examination on day 8 (HH 34). RESULTS: A total of 60 embryos survived in each group. The largest number of embryos presented with defects in the umbilical and supraumbilical region (25% in the SA group and 40% in the TB group). A combination of both defects (thoracoabdominoschisis) was seen in 20% of the TB and 8.3% of the SA groups, respectively. Associated anomalies found in both groups were head and eye defects, abnormal pelvic configurations, leg deformities, and mild forms of cloacal exstrophies. CONCLUSIONS: TB and SA have both a high potential to induce umbilical and supraumbilical ventral body wall malformations in chicken embryos. This novel animal model might help to establish a more profound understanding of the developmental steps in ventral body wall formation and the embryology for its malformations.
Asunto(s)
Pared Abdominal/anomalías , Embrión de Pollo , Modelos Animales , Suramina/administración & dosificación , Teratógenos , Azul de Tripano/administración & dosificación , Pared Abdominal/embriología , Animales , Cloaca , Hernia Umbilical/embriologíaRESUMEN
Hepatoblastoma is a pediatric liver tumor with epithelial components resembling embryonal and fetal liver cells. The existence of teratoid hepatoblastoma suggests the presence of stem cells in hepatoblastoma. The aim of this study was to analyze the expression of stem cell markers in hepatoblastomas. We studied specimens from 10 hepatoblastomas. Five of the hepatoblastomas were of epithelial and five of mixed type. Immunohistochemistry (IHC) for the stem cell markers CD34, Thy1, c-kit, and the hepatic or biliary lineage markers CK-18, OCH, CK-7, and CD56 was performed. Double IHC for stem cell and lineage markers was used to identify putative liver stem cells. The different markers showed distinct distributions on the tumor cells. Cells in atypical ducts were found to express simultaneously stem cell markers and hepatocytic or biliary lineage markers. Other cells in connective tissue showed c-kit expression, but not hepatic or biliary marker expression. The data show the presence of different cell populations bearing stem cell markers in human hepatoblastoma. Ductal cells co-expressing stem cell markers and hepatic lineage markers phenotypically resemble hepatic stem-like cells. These findings support the thesis that stem cells play a role in the histogenesis of hepatoblastoma.
Asunto(s)
Hepatoblastoma/patología , Neoplasias Hepáticas/patología , Células Madre/metabolismo , Antígenos CD34/biosíntesis , Biomarcadores/metabolismo , Antígeno CD56/biosíntesis , Niño , Preescolar , Humanos , Inmunohistoquímica , Lactante , Recién Nacido , Queratina-7 , Queratinas/biosíntesis , Proteínas Proto-Oncogénicas c-kit/biosíntesis , Antígenos Thy-1/biosíntesisRESUMEN
INTRODUCTION: The aim of our study is to establish a reliable neonatal rat model by formula feeding only for evaluation of early surgical intervention on the course of experimental necrotizing enterocolitis (NEC). MATERIAL AND METHODS: Newborn Sprague-Dawley rats were divided into 50 breast-fed (group 1) and 38 formula fed (Similac/Esbilac, group 2) animals. The pups were sacrificed on the 4th, 5th, and 6th day of life and the terminal intestine examined for macroscopic and histologic changes as well as cytokine expression. RESULTS: The histological mucosal damage was significantly higher of group 2 compared to group 1. The area of the vital mucosa of group 2 was significantly (58.57%, p<0.001) lower compared to group 1 (75.12%). The mRNA expression of the inflammatory cytokines IL-6, IL-8 and COX-2 was significantly 2-, 5- and 10-fold increased in group 2 compared to group 1. DISCUSSION: Formula fed newborn rats displayed an inflammatory enterocolitis similar to human NEC. Our study demonstrates a significant loss of mucosa in animals with NEC having increased expression levels of IL-6, IL-8 and COX-2. Mucosal loss appears to be a distinct feature of experimental NEC and has to be correlated with the human disease.