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PURPOSE: There is a long history of research dealing with the embryology of the testicular descent. However, important aspects like the role of the gubernaculum and the development of the processus vaginalis peritonei are not understood. Micro-computed tomography (µCT) is an established tool for anatomical studies in rodents. Our study applied µCT imaging to visualize the testicular descent in rats and focused on the role of the gubernacular bulb and the development of the processus vaginalis peritonei. METHODS: Rats from embryonic day 15 (ED15) to ED21 and newborns (N0) were fixed and dried using the "critical point" technique. We ran a SkyScan® µCT system and scans were analyzed for gender-specific differentiation of the genital ridge and used for 3D visualization of relevant anatomic structures. RESULTS: µCT imaging confirmed the intraperitoneal location of the testicles from ED15 to N0. The components of the inner genital moved closer together while the intestinal volume expanded. The gubernacular bulb seemed to be involved in the formation of the processus vaginalis peritonei. CONCLUSION: Here, we utilized µCT imaging to visualize the testicular descent in the rat. Imaging provides new morphologic aspects on the development of the processus vaginalis peritonei.
Assuntos
Genitália , Testículo , Ratos , Animais , Masculino , Humanos , Feminino , Gravidez , Microtomografia por Raio-X , Testículo/diagnóstico por imagem , Cuidado Pré-NatalRESUMO
Understanding of normal fetal organ development is crucial for the evaluation of the pathogenesis of congenital anomalies. Various techniques have been used to generate imaging of fetal rat organogenesis, such as histological dissection with 3-dimensional reconstruction and scanning electron microscopy. However, these techniques did not imply quantitative measurements of developing organs (volumes, surface areas of organs). Furthermore, a partial or total destruction of the embryos prior to analysis was inevitable. Recently, micro-computed tomography (micro-CT) has been established as a novel tool to investigate embryonic development in non-dissected embryos of rodents. In this study, we used the micro-CT technique to generate 4D datasets of rat embryos aged between embryonic day 15-22 and newborns. Lungs, hearts, diaphragms, and livers were digitally segmented in order to measure organ volumes and analyze organ development as well as generate high-resolution 3D images. These data provide objective values compiling a 4D atlas of pulmonary, cardiac, diaphragmatic, and hepatic development in the fetal rat.
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BACKGROUND: Intestinal obstruction in newborns is associated with intestinal motility disorders after surgery. Alterations in the enteric nervous system (ENS) might cause abnormal peristalsis, which may then result in intestinal motility disorders. We aimed to quantify alterations in the myenteric plexus after a ligation and to test if these alterations were reversible. METHODS: Small intestines of chicken embryos were ligated in ovo at embryonic day (ED) 11 for either 4 d (ED 11-15) or 8 d (ED 11-19). Both treated groups and control group were sacrificed and intestinal segments examined by means of both light and electron microscopy. RESULTS: The number of proximal myenteric ganglia increased (ED 19, 30.7 ± 3.16 versus 23.1 ± 2.03; P < 0.001) in the 8-d ligature group but had values similar to the control group in the 4-d ligature group. The size distribution was skewed toward small ganglia in the 8-d ligature group (ED 19, 83.71 ± 11.60% versus 3.88 ± 4.74% in the control group; P < 0.001) but comparable with the control group in the 4-d ligature group. Subcellular alterations in the 4-d ligature group were reversible. CONCLUSIONS: The pathologic alterations in the ENS were fully reversible in the 4-d ligature group. This reversibility might be linked to the degree of immaturity of the ENS.
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Sistema Nervoso Entérico/embriologia , Regeneração Nervosa , Animais , Embrião de Galinha , Sistema Nervoso Entérico/ultraestruturaRESUMO
Introduction: During embryonic development, the trachea emerges from an area of the foregut, which is often referred to as "anterior" or "common" foregut tube or simply foregut. To explain this process of differentiation, four competing models exist to date. The outgrowth and watershed models propose a foregut that remains constant in length. In the outgrowth model, the trachea buds off and elongates from the foregut, while in the watershed model, a mesenchymal wedge splits the growing foregut into the trachea and esophagus. In contrast, the septation model proposes a cranial splitting and thus a shortening of the "common" foregut tube into the trachea and esophagus by an emerging septum. Finally, the splitting and extension model describes an interaction of cranial splitting of the foregut and simultaneous caudal tracheal and esophageal growth. Methods: Here we examine the development of the undifferentiated foregut by micro computed tomography, which allows precise measurements. Results: Our results show that this area of the foregut transforms into the larynx, a process, which is independent from tracheal and esophageal development. Discussion: These observations are only consistent with the outgrowth model.
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INTRODUCTION: Micro-computed tomography (micro-CT) is an established tool to study fetal development in rodents. This study aimed to use micro-CT imaging to visualize the development of the urinary tract in fetal rats. MATERIALS AND METHODS: Fetal rats from embryonic day (ED) 15, ED17, ED19, ED21, and N0 (newborn) (n = 6 per group; 3 males) were fixed and desiccated using the "critical point" technique. We utilized the micro-CT system (SkyScan) and analyzed the resulting scans with CTAn, DataViewer, and ImageJ to visualize the morphology and quantify the volumes of kidney, bladder, adrenal gland, as well as length of the ureter. RESULTS: High-resolution micro-CT showed continuous growth of both kidneys from ED15 to N0, with the highest increase between ED19 and ED21. The length of the ureter increased from ED15 to ED21 and remained stable until birth. The volume of the bladder steadily increased from ED15 to N0.In females, a statistically higher volume of the adrenal gland on ED21 was observed, whereas no sex-specific differences were seen for kidney, ureter, and bladder development. CONCLUSION: Micro-CT depicts an excellent tool to study urinary tract development in the fetal and neonatal rat. It enables the metric quantification of longitudinal anatomic changes in high definition without previous destructive tissue preparation. The present study revealed sex-specific differences of the adrenal gland development and provides comprehensive data for the understanding of fetal urinary tract development, inspiring future research on congenital urological malformations.
Assuntos
Feto , Sistema Urinário , Gravidez , Masculino , Humanos , Feminino , Ratos , Animais , Microtomografia por Raio-X/métodos , Feto/diagnóstico por imagem , Sistema Urinário/diagnóstico por imagem , Cuidado Pré-Natal , RimRESUMO
(1) Background: Accessory liver lobes are a rare finding and only a few case reports of accessory liver lobes in abdominal wall defects have been reported so far. In the case of a congenital wall defect including liver parenchyma, there is still an ongoing debate on the definition of the abdominal wall defect and best care practice. Even though congenital abdominal wall defects are frequently diagnosed in prenatal screenings, controversy on the underlying etiology, embryology and underlying anatomy remains. Prenatal distinction between omphalocele and hernia into the cord cannot always be obtained; however, due to its clinical relevance for postnatal management and counseling of parents, accurate diagnosis is essential. (2) Case Presentation: We describe the uncommon postnatal finding of a pediculated accessory liver lobe with gallbladder in a preterm with umbilical cord hernia, which was prenatally diagnosed as omphalocele. Postnatal examination revealed an amniotic sac with a diameter of six and a small abdominal wall defect of three centimeters in diameter. Postnatal management included resection of the accessory liver lobe and gallbladder and closure of the defect. (3) Results and (4) Conclusions: Throughout the literature, the distinction between umbilical cord hernia and omphalocele has been variable. This has led to confusion and difficulties regarding postnatal treatment options. In order to achieve an accurate prenatal and/or postnatal diagnosis, the morphological differences and clinical manifestation of umbilical cord hernia and omphalocele need to be assessed. Further embryological studies are warranted to understand the underlying embryological pathology of omphalocele and umbilical cord hernia and offer appropriate treatment. In consideration of possibly severe complications in the case of the torsion of a pedunculated accessory liver lobe, we strongly recommend primary removal once pre- or intraoperative identification has been made.
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The development of the mammalian gut was first described more than a century ago. Since then, it has been believed that a series of highly orchestrated developmental processes occur before the intestine achieves its final formation. The key steps include the formation of the umbilicus, the so-called "physiological herniation" of the midgut into the umbilical cord, an intestinal "rotation", and the "return of the gut" into the abdominal cavity. However, this sequence of events is predominantly based on histological sections of dissected embryos, a 2D technique with methodological limitations. For a better understanding of spatial relationships in the embryo, we utilized microcomputed tomography (µCT), a nondestructive 3D imaging method. Here, we show the detailed processes and mechanisms of intestinal development in rat embryos, including the development of the umbilicus, the formation of loops inside the umbilical coelom, and the subsequent shift of these loops into the abdominal cavity. Our 3D datasets of developing intestines will substantially advance the understanding of normal mammalian midgut embryology and offer new possibilities to reveal unknown mechanisms in the pathogenesis of congenital disorders.
Assuntos
Embrião de Mamíferos/diagnóstico por imagem , Intestinos/diagnóstico por imagem , Microtomografia por Raio-X , Animais , Feminino , Idade Gestacional , Imageamento Tridimensional , Intestinos/embriologia , Morfogênese , Valor Preditivo dos Testes , Gravidez , Interpretação de Imagem Radiográfica Assistida por Computador , Ratos Sprague-DawleyRESUMO
PURPOSE: Numerous researchers studied the morphology of testicular descent including the possible function of gubernaculum. However, a clear illustration of this process is still missing. The aim of this study was to illustrate testicular descent using scanning electron microscopy (SEM) in a rat model. METHODS: The abdomen of rat fetuses between gestational day (E) 15 and E 22 and newborns at postnatal day (D) 0 and D 1.5 was opened by microsurgery. Standard preparation for SEM was carried out. The position of the testis and gubernaculum testis was documented. RESULTS: The gubernaculum was obvious in male rat embryos at E 17.5. In a first phase (E 16-E 21) the testis moved from cranio-lateral and dorsal to caudo-medial and ventral, while clear signs of an active role of the gubernaculum were missing. In a second phase (E 22-D 1.5) the processus vaginalis peritonei (PVP) developed, while the conus of the gubernaculum disappeared, after which, the testis moved out of the abdominal cavity and entered the PVP. CONCLUSION: In our study, we could not specify the role of gubernaculum for testicular descent. However, our data showed that the testis lay intraperitoneal throughout the descensus testis.
Assuntos
Microscopia Eletrônica de Varredura , Testículo/fisiologia , Testículo/ultraestrutura , Animais , Animais Recém-Nascidos , Embrião de Mamíferos , Feto , Masculino , Modelos Animais , Ratos , Ratos Sprague-DawleyRESUMO
Previous studies in developing Xenopus and zebrafish reported that the phosphate transporter slc20a1a is expressed in pronephric kidneys. The recent identification of SLC20A1 as a monoallelic candidate gene for cloacal exstrophy further suggests its involvement in the urinary tract and urorectal development. However, little is known of the functional role of SLC20A1 in urinary tract development. Here, we investigated this using morpholino oligonucleotide knockdown of the zebrafish ortholog slc20a1a. This caused kidney cysts and malformations of the cloaca. Moreover, in morphants we demonstrated dysfunctional voiding and hindgut opening defects mimicking imperforate anus in human cloacal exstrophy. Furthermore, we performed immunohistochemistry of an unaffected 6-week-old human embryo and detected SLC20A1 in the urinary tract and the abdominal midline, structures implicated in the pathogenesis of cloacal exstrophy. Additionally, we resequenced SLC20A1 in 690 individuals with bladder exstrophy-epispadias complex (BEEC) including 84 individuals with cloacal exstrophy. We identified two additional monoallelic de novo variants. One was identified in a case-parent trio with classic bladder exstrophy, and one additional novel de novo variant was detected in an affected mother who transmitted this variant to her affected son. To study the potential cellular impact of SLC20A1 variants, we expressed them in HEK293 cells. Here, phosphate transport was not compromised, suggesting that it is not a disease mechanism. However, there was a tendency for lower levels of cleaved caspase-3, perhaps implicating apoptosis pathways in the disease. Our results suggest SLC20A1 is involved in urinary tract and urorectal development and implicate SLC20A1 as a disease-gene for BEEC.
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BACKGROUND: Microcirculatory disorders play a major part in the pathogenesis of acute pancreatitis. Improvement of microcirculation is hypothesized to open a therapeutic window. The aim of this study was to evaluate the effects of small-volume resuscitation in acute pancreatitis. METHODS: In rats, acute pancreatitis of graded severity was induced and pancreatic microcirculation was observed in vivo with an epiluminescent microscope. Primary outcome measures were microcirculation, leukocyte adherence, concentration of trypsinogen-activating peptide, amylase activity and histopathologic tissue damage. RESULTS: In necrotizing pancreatitis patients receiving prophylactic intervention with 7.5% hypertonic saline the functional capillary density was 76%. Postcapillary venular leukocyte adherence was 45% of vein cross-section. The median histopathologic damage scored 8 points. In controls, a complete microcirculatory breakdown was observed, and in the group with therapeutic intervention no significant difference was detected. In intermediate pancreatitis, the number of perfused capillaries remained 55.0 versus 23.3% in controls. Leukocyte adherence was 40.0 versus 51.7%. The histopathologic damage scored 6.0 versus 9.0 points. Trypsinogen-activating peptide concentration was reduced to 164 versus 402 nM in controls. In cerulein pancreatitis, the number of perfused capillaries was equally preserved in both groups. CONCLUSION: Small-volume resuscitation preserves capillary microcirculation and prevents pancreatic injury in intermediate necrotizing pancreatitis.
Assuntos
Pâncreas/irrigação sanguínea , Pancreatite Necrosante Aguda/tratamento farmacológico , Pancreatite/tratamento farmacológico , Solução Salina Hipertônica/uso terapêutico , Animais , Capilares/patologia , Capilares/ultraestrutura , Feminino , Microcirculação , Microscopia de Fluorescência , Pancreatite/patologia , Pancreatite Necrosante Aguda/patologia , Ratos , Ratos WistarRESUMO
Liver transplantation is still the only treatment for end-staged liver diseases in children. However, donor organ shortage and immunosuppression are major limitations. Thus, approaches of hepatocyte transplantation are under investigation. Using cells might permit mass expansion, cryopreservation, and the ex vivo genetic modification of cells. For the development of cell-transplantation techniques, the use of three-dimensional scaffolds as carrier was shown to be advantageous. Polymeric matrices permit the formation of a neo-tissue and stimulation by the modification of the matrix surface. Another important issue is to define the right cell type for transplantation. Adult hepatocytes have a limited growth and differentiation potential. In contrast, fetal liver cells (FLC) possess an enormous growth and a bipotential differentiation potential. Thus, these cells may be very attractive as a cell resource for developing cell-based liver replacement. A third major issue in this approach is the neo-vascularization. Therefore, the transplantation in a recently developed model using a microsurgically created arterioveno-venous (AV) loop as a central vessel for the neo-tissue was used for transplantation of FLC in a fibrin-matrix. Initial results indicated that the transplantation of FLC using the AV-loop transplantation model may be promising for the development of highly vascularized in vivo tissue-engineered liver support systems.
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Insuficiência Hepática/terapia , Transplante de Fígado , Fígado/fisiologia , Engenharia Tecidual , Feto , Hepatócitos/transplante , Humanos , Fígado/irrigação sanguínea , Alicerces TeciduaisRESUMO
BACKGROUND: Neuroblastoma is the most common solid tumor in childhood with unconventional clinical behavior. L1, a neuronal cell adhesion molecule, is associated with poor survival in malignant adult tumors. The aim of the current study was to determine expression of L1 in pediatric neuroblastoma. METHODS: L1 expression was assessed on a tissue microarray with 66 surgically resected neuroblastoma samples by immunohistochemistry with a monoclonal antibody and peroxidase method. Additionally, mRNA expression was analyzed by reverse transcriptase-polymerase chain reaction with L1-specific primers. Data were correlated survival data by log rank test and Cox regression multivariate analysis. RESULTS: L1 was detected in 57 (86%) of 66 neuroblastomas, whereas 9 (14%) were L1 negative. Median survival of all children was 72 months. Analysis with Kaplan-Meier method revealed a surprising and contrary finding to adult tumor entities: an association of L1 positivity with better event-free and overall survival (P < .001 and P < .01 by log rank test). Multivariate Cox regression analysis showed an independent prognostic impact of L1 negativity for event-free and overall survival of the children (P < .05). CONCLUSIONS: In contrast to adult tumor entities, where L1 is associated with aggressive clinical behavior, our data show that L1 predicts good outcome in children with neuroblastoma. This novel finding suggests an inverse role of L1 in neuroblastoma. Future studies might focus on the molecular basis of the varying effect of L1 in different tumors.
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Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neuroblastoma/metabolismo , Adulto , Biomarcadores Tumorais/genética , Pré-Escolar , Humanos , Técnicas Imunoenzimáticas , Lactente , Estadiamento de Neoplasias , Molécula L1 de Adesão de Célula Nervosa/genética , Neuroblastoma/genética , Neuroblastoma/cirurgia , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Análise Serial de TecidosRESUMO
PURPOSE: Only few immunohistochemical markers besides c-kit exist for gastrointestinal stromal tumors (GISTs). Thy-1, a cell-surface glycoprotein, is a marker for several types of stem cells and particularly for neuronal precursor cells. The aim of this study was to determine Thy-1 expression in GISTs. MATERIALS AND METHODS: Fifty-seven surgically resected and paraffin-embedded GIST samples were analyzed by immunohistochemistry with peroxidase method for Thy-1 molecule. RESULTS: Thy-1 was detected in the majority of 57 GIST samples (54 out of 57 patients, 95%). All samples were c-kit positive and 90% were CD34 positive. All three Thy-1 negative samples were CD34 positive, had a low proliferative index (Ki-67 Assuntos
Biomarcadores Tumorais/análise
, Neoplasias Gastrointestinais/diagnóstico
, Tumores do Estroma Gastrointestinal/diagnóstico
, Antígenos Thy-1/análise
, Feminino
, Humanos
, Imuno-Histoquímica
, Masculino
, Pessoa de Meia-Idade
, Prognóstico
, Análise de Sobrevida
RESUMO
PURPOSE: Midkine (MK), a heparin-binding growth factor, has an important role in cancer progression. The outcome of patients with gastrointestinal stromal tumors (GISTs) is correlated with tumor size and mitotic count. The aim of this study was to determine MK expression in GISTs. METHODS: Midkine was detected in 31 (55%) of 57 surgically resected GISTs by immunohistochemistry with a rabbit antibody against MK and peroxidase method. RESULTS: A significant worse outcome of MK-positive patients was found (P < 0.05; log rank test). Multivariate Cox regression analysis showed an independent prognostic impact (relative risk for overall survival 3.64; P < 0.05). Interestingly, MK expression was significantly associated with mitotic rate (P < 0.05; Chi-squared test), but not with tumor size (P = 0.97). CONCLUSIONS: Taken together, MK is a prognostic marker for GIST patients. MK might also be a useful peripheral tumor marker since it can be detected in peripheral serum. Future studies should involve higher GIST patient numbers including tumor and serum samples for detection of MK.
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Citocinas/metabolismo , Tumores do Estroma Gastrointestinal/metabolismo , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Midkina , Prognóstico , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
BACKGROUND: Surfactant proteins (SP's) have been described as inherent proteins of the human central nervous system (CNS). Their distribution pattern in brain tissue and altered cerebrospinal fluid (CSF) - concentrations in different CNS pathologies are indicative of their immunological and rheological importance. The aim of this study has been to investigate when - compared to the lungs - SP's are expressed in the developing rat brain and which functional components in the CNS participate in their production. MATERIAL AND METHODS: Brain and lung tissue from embryonal (days 10, 12, 14, 16, 17 and 20), newborn, and adult rats were harvested and investigated for expression of SP-A, SP-B, SP-C and SP-D using immunofluorescence microscopy in order to identify and compare the time points of their occurence in the respective tissue. To better identify the location of SP expression in the rat brain, SP's were colocalized with use of an astrocyte marker (GFAP), a neuronal marker (NeuN), an endothelial marker (CD31) and an axonal marker (NF). RESULTS AND CONCLUSION: SP-A and SP-C are expressed in the CNS of rats during early embryonic age whereas SP-B and SP-D are first present in the adult rat brain. All SP's are expressed in cells adjacent to CSF spaces, probably influencing and maintaining physiological CSF flow. SP's A and C are abundant at the site of the blood brain barrier (BBB).
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Química Encefálica/fisiologia , Encéfalo/crescimento & desenvolvimento , Proteínas Associadas a Surfactantes Pulmonares/metabolismo , Animais , Animais Recém-Nascidos , Biomarcadores , Desenvolvimento Embrionário , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
Stem cells are interesting candidates as a new source for cell/organ culture or cell transplantation concepts. So far it is believed that the hepatoblast is the common progenitor cell during fetal liver development. In previous studies two distinct fractions of liver cells were found during development: cells co-expressing Thy1 and CK-18 (cytokeratin-18) and cells expressing CK-18 only. In this study we cultured Thy1-positive and Thy1-negative hepatic progenitors isolated from collagenase digested fetal rat livers after depletion of OX43/OX44-positive hematopoietic cells. The cells were cultured on a collagen-I matrix in a medium containing epidermal growth factor, insulin, and fetal calf serum. Thy1-positive cells isolated from ED16, ED18, or ED20 livers showed significantly enhanced cell growth compared with Thy1-negative cells during the culture period. Both cell types showed expression of the liver-specific genes CK-18, albumin and alpha-feto-protein at the beginning of the culture period, as assessed by reverse-transcription polymerase chain reaction and immunocytochemistry. The growth of Thy1-positive cells was significantly higher when compared with Thy1-negative cells and declined with maturation of the liver. The data suggest a stem cell-like growth potential of Thy1-positive fetal hepatic cells. Thus, these cells might be useful for concepts of cell-based therapies. However, further efforts must be undertaken to define the biological, ethical, and legal aspects of using fetal cells.
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Diferenciação Celular/fisiologia , Proliferação de Células , Fígado/citologia , Células-Tronco/citologia , Albuminas/biossíntese , Albuminas/metabolismo , Animais , Separação Celular , Células Cultivadas , Feto , Imuno-Histoquímica , Queratinas/biossíntese , Queratinas/metabolismo , Fígado/metabolismo , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/metabolismo , Antígenos Thy-1/biossíntese , Antígenos Thy-1/metabolismo , alfa-Fetoproteínas/biossíntese , alfa-Fetoproteínas/metabolismoRESUMO
AIM: To investigate the hepatocytic differentiation of mesenchymal stem cells (MSCs) in co-cultures with fetal liver cells (FLC) and the possibility to expand differentiated hepatocytic cells. METHODS: MSCs were marked with green fluorescent protein (GFP) by retroviral gene transduction. Clonal marked MSCs were either cultured under liver stimulating conditions using fibronectin-coated culture dishes and medium supplemented with stem cell factor (SCF), hepatocyte growth factor (HGF), epidermal growth factor (EGF), and fibroblast growth factor 4 (FGF-4) alone, or in presence of freshly isolated FLC. Cells in co-cultures were harvested, and GFP+ or GFP- cells were separated using fluorescence activated cell sorting. Reverse transcription-polymerase chain reaction (RT-PCR) for the liver specific markers cytokeratin-18 (CK-18), albumin, and alpha-fetoprotein (AFP) was performed in different cell populations. RESULTS: Under the specified culture conditions, rat MSCs co-cultured with FLC expressed albumin, CK-18, and AFP-RNA over two weeks. At wk 3, MSCs lost hepatocytic gene expression, probably due to overgrowth of the cocultured FLC. FLC also showed a stable liver specific gene expression in the co-cultures and a very high growth potential. CONCLUSION: The rat MSCs from bone marrow can differentiate hepatocytic cells in the presence of FLC in vitro and the presence of MSCs in co-cultures also provides a beneficial environment for expansion and differentiation of FLC.
Assuntos
Hepatócitos/citologia , Células-Tronco Mesenquimais/citologia , Albuminas/genética , Animais , Diferenciação Celular , Separação Celular , Técnicas de Cocultura , Feto/citologia , Citometria de Fluxo , Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Hepatócitos/metabolismo , Queratinas/genética , Células-Tronco Mesenquimais/metabolismo , Ratos , Proteínas Recombinantes/metabolismo , alfa-Fetoproteínas/genéticaRESUMO
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.
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Fibrina , Hepatócitos/transplante , Fígado Artificial , Engenharia Tecidual , Animais , Transplante de Células/métodos , Células Imobilizadas/citologia , Células Imobilizadas/fisiologia , Células Imobilizadas/transplante , Hepatócitos/citologia , Hepatócitos/fisiologia , Humanos , Ratos , Ratos Sprague-Dawley , Engenharia Tecidual/métodosRESUMO
BACKGROUND: The term exstrophy-epispadias complex (EEC) has been coined for a group of congenital malformations that includes epispadias, bladder exstrophy and cloacal exstrophy. It is usually thought that these malformations develop against a similar embryological background. This background, however, is still obscure. This is mainly due to the lack of availability of abnormal human or non-human embryos showing the crucial developmental steps in the morphogenesis of EEC malformations. In this paper, we present chick embryos that show cloacal exstrophy at early developmental stages. To the best of our knowledge, this is the first documentation of this rare malformation in young embryos. MATERIALS AND METHODS: Embryos with cloacal exstrophy (n=4) were found among embryos from two experimental series (n=50) that were primarily performed to document the early morphogenesis of facial and cardiovascular malformations. The malformations were induced by the administration of suramin according to established protocols. Suramin can induce a spectrum of malformations including facial clefts, heart defects, and cloacal exstrophy. RESULTS AND CONCLUSIONS: Besides the presence of an abnormal opening into the cloaca, all embryos were characterised by an abnormal broadening of the caudal trunk at the level of the leg buds, which, in the youngest embryos, was associated with the abnormal presence of large aneurysmatic swellings of the dorsal aortae at this side. We postulate that these aneurysmatic swellings might be the primary defects leading to the development of EEC malformations. These space-occupying anomalies seem to cause abnormal distensions of the developing pelvis and of the infra-umbilical portion of the developing body wall. In consequence, the mid-portion of the developing ventral body wall between the origin of the umbilical cord and the cloacal plate becomes stretched and thinned out. Tension and thinning of the ventral body wall might ultimately lead to its rupture with exposure of the lumen of the embryonic cloaca and allantois. This new concept on the morphogenesis of the EEC is the first not to be inferred from the conditions seen in fetal or postnatal human cases but is based entirely on data from malformed embryos.
Assuntos
Extrofia Vesical/patologia , Cloaca/anormalidades , Epispadia/patologia , Organogênese/fisiologia , Animais , Antineoplásicos/efeitos adversos , Aorta Abdominal/anormalidades , Aorta Abdominal/efeitos dos fármacos , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/complicações , Aneurisma da Aorta Abdominal/fisiopatologia , Extrofia Vesical/induzido quimicamente , Extrofia Vesical/fisiopatologia , Padronização Corporal/efeitos dos fármacos , Padronização Corporal/fisiologia , Embrião de Galinha , Cloaca/efeitos dos fármacos , Cloaca/patologia , Modelos Animais de Doenças , Epispadia/induzido quimicamente , Epispadia/fisiopatologia , Microscopia Eletrônica de Varredura , Organogênese/efeitos dos fármacos , Suramina/efeitos adversos , Teratogênicos/farmacologia , Uretra/anormalidades , Uretra/patologia , Uretra/fisiopatologia , Bexiga Urinária/anormalidades , Bexiga Urinária/patologia , Bexiga Urinária/fisiopatologiaRESUMO
AIM: The origin of putative liver cells from distinct bone marrow stem cells, e.g. hematopoietic stem cells or multipotent adult progenitor cells was found in recent in vitro studies. Cell culture experiments revealed a key role of growth factors for the induction of liver-specific genes in stem cell cultures. We investigated the potential of rat mesenchymal stem cells (MSC) from bone marrow to differentiate into hepatocytic cells in vitro. Furthermore, we assessed the influence of cocultured liver cells on induction of liver-specific gene expression. METHODS: Mesenchymal stem cells were marked with green fluorescent protein (GFP) by retroviral gene transduction. Clonal marked MSC were either cultured under liver stimulating conditions using fibronectin-coated culture dishes and medium supplemented with SCF, HGF, EGF, and FGF-4 alone, or in presence of freshly isolated rat liver cells. Cells in cocultures were harvested and GFP+ or GFP- cells were separated using fluorescence activated cell sorting. RT-PCR analysis for the stem cell marker Thy1 and the hepatocytic markers CK-18, albumin, CK-19, and AFP was performed in the different cell populations. RESULTS: Under the specified culture conditions, rat MSC cocultured with liver cells expressed albumin-, CK-18, CK-19, and AFP-RNA over 3 weeks, whereas MSC cultured alone did not show liver specific gene expression. CONCLUSION: The results indicate that (1) rat MSC from bone marrow can differentiate towards hepatocytic lineage in vitro, and (2) that the microenvironment plays a decisive role for the induction of hepatic differentiation of rMSC.