RESUMO
Ischemic acute kidney injury (AKI) is associated with high morbidity and frequent complications. Repeated episodes of AKI may lead to end-stage renal failure. The pathobiology of regeneration in AKI is not well understood and there is no effective clinical therapy that improves regeneration. The Notch signaling pathway plays an essential role in kidney development and has been implicated in tissue repair in the adult kidney. Here, we found that kidneys after experimental AKI in mice showed increased expression of Notch receptors, specifically Notch1-3, of the Notch ligands Jagged-1 (Jag1), Jag2 and Delta-like-4 (Dll4) and of the Notch target genes Hes1, Hey2, HeyL, Sox9 and platelet-derived growth factor receptor ß (Pdgfrb). Treatment of ischemic mice with the x03B3;-secretase inhibitor DBZ blocked Notch signaling and specifically downregulated the expression of Notch3 and the Notch target genes Hes1, Hey2, HeyL and Pdgfrb. After DBZ treatment, the mice developed less interstitial edema and displayed altered interstitial inflammation patterns. Furthermore, serum urea and creatinine levels were significantly decreased from 6 h onwards when compared to control mice treated with DMSO only. Our data are consistent with an amelioration of the severity of kidney injury by blocking Notch activation following AKI, and suggest an involvement of Notch-regulated Pdgfrb in AKI pathogenesis.
Assuntos
Injúria Renal Aguda/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Inibidores Enzimáticos/uso terapêutico , Rim/efeitos dos fármacos , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Injúria Renal Aguda/genética , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Animais , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Masculino , CamundongosRESUMO
Beneficial effects of erythropoietin (EPO) have been reported in acute kidney injury (AKI) when administered prior to induction of AKI. We studied the effects of EPO administration on renal function shortly after ischemic AKI. For this purpose, rats were subjected to renal ischemia for 30 min and EPO was administered at a concentration of 500 U/kg either i.v. as a single shot directly after ischemia or with an additional i.p. dose until 3 days after surgery. The results were compared with AKI rats without EPO application and a sham-operated group. Renal function was assessed by measurement of serum biochemical markers, histological grading, and using an isolated perfused kidney (IPK) model. Furthermore, we performed flow cytometry to analyze the concentration of endothelial progenitor cells (EPCs) in the peripheral blood and renal vessels. Following EPO application, there was only a statistically non-significant tendency of serum creatinine and urea to improve, particularly after daily EPO application. Renal vascular resistance and the renal perfusion rate were not significantly altered. In the histological analysis, acute tubular necrosis was only marginally ameliorated following EPO administration. In summary, we could not demonstrate a significant improvement in renal function when EPO was applied after AKI. Interestingly, however, EPO treatment resulted in a highly significant increase in CD133- and CD34-positive EPC both in the peripheral blood and renal vessels.
Assuntos
Injúria Renal Aguda/patologia , Vasos Sanguíneos/citologia , Células Progenitoras Endoteliais/efeitos dos fármacos , Eritropoetina/farmacologia , Antígeno AC133/metabolismo , Injúria Renal Aguda/metabolismo , Animais , Creatinina/sangue , Modelos Animais de Doenças , Células Progenitoras Endoteliais/citologia , Células Progenitoras Endoteliais/metabolismo , Citometria de Fluxo , Taxa de Filtração Glomerular , Hematócrito , Isquemia/patologia , Rim/efeitos dos fármacos , Rim/fisiologia , Masculino , Ratos , Ratos Sprague-Dawley , Ureia/sangueRESUMO
Human adipose-derived stroma cells (ADSCs) have successfully been employed in explorative therapeutic studies. Current evidence suggests that ADSCs are unevenly distributed in subcutaneous adipose tissue; therefore, the anatomical origin of ADSCs may influence clinical outcomes. This study was designed to investigate proliferation and differentiation capacities of ADSCs from the gluteal and abdominal depot of 8 females. All had normal BMI (22.01 ± 0.39 kg/m(2) ) and waist circumference (81.13 ± 2.33 cm). Examination by physicians and analysis of 31 laboratory parameters did not reveal possibly confounding medical disorders. Gluteal and abdominal adipose tissue was sampled by en bloc resection on day 7 (±1) after the last menses. Histological examination did not reveal significant depot-specific differences. As assessed by BrdU assay, proliferation of cells from both depots was similar after 24 h and analysis of 15 cell surface markers by flow cytometry identified the isolated cells as ADSCs, again without depot-specific differences. ADSCs from both depots differentiated poorly to chondroblasts. Gluteal ADSCs displayed significantly higher adipogenic differentiation potential than abdominal cells. Osteogenic differentiation was most pronounced in gluteal cells, whereas differentiation of abdominal ADSCs was severely impaired. Our data demonstrate a depot-specific difference in ADSC differentiation potential with abdominal cells failing to meet the criteria of multipotent ADSCs. This finding should be taken into account in future explorations of ADSC-derived therapeutic strategies.
Assuntos
Abdome , Adipogenia/fisiologia , Nádegas , Osteogênese/fisiologia , Células Estromais/citologia , Células Estromais/fisiologia , Gordura Subcutânea/citologia , Adipócitos/citologia , Adipócitos/fisiologia , Adulto , Biópsia , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Feminino , Humanos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/fisiologia , Osteoblastos/citologia , Osteoblastos/fisiologiaRESUMO
Differentiation of embryonic stem (ES) cells via embryoid bodies has been established as an appropriate model to study the development of various cell types in vitro. Here, we show that murine induced pluripotent stem (iPS) cells, reprogrammed by exogenous expression of the two transcription factors Oct4 and Klf4 (2F OK iPS), differentiate into chondrocytes in vitro characterized by the appearance of Alcian blue-stained nodules and the expression of cartilage-associated genes and proteins. Quantitatively, the chondrogenic differentiation potential of 2F OK iPS and ES cells was found to be similar. Further, we demonstrate the induction of chondrogenic iPS cell differentiation by certain members of the transforming growth factor-ß family (BMP-2, TGF-ß(1)). The number of Alcian blue-positive nodules and the expression of the cartilage marker molecule collagen type II increased after application of BMP-2, whereas simultaneous treatment with both BMP-2 and TGF-ß(1) showed no significant effect on gene expression.
Assuntos
Condrócitos/citologia , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Diferenciação Celular/fisiologia , Linhagem Celular , Condrócitos/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Células-Tronco Embrionárias/metabolismo , Expressão Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Camundongos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Mesenchymal stromal cells (MSC) have been introduced into the field of tissue-engineered airway transplantation. Since patients with extensive tracheal defects often require an open tracheotomy, this study investigated if MSC could be obtained from the adipose tissue of the neck during this procedure. Cells were isolated by plastic adherence from the adipose tissue of 8 patients. Cell isolates were analyzed for (i) proliferation, (ii) the expression of CD marker molecules and (iii) multilineage differentiation. The isolated spindle-shaped cells showed a high proliferation capacity and the flow cytometric analysis revealed a distinct population meeting the criteria for MSC. Using classical MSC cultivation protocols the characterized cells showed adipogenic, chondrogenic and osteogenic differentiation for all analyzed cell isolates. This study was able to demonstrate that sufficient amounts of stem/progenitor cells can be easily isolated from adipose tissue of the neck obtained during open tracheotomy. These cells may be a source for future tracheal replacement therapies.
Assuntos
Tecido Adiposo/citologia , Antígenos CD/metabolismo , Células-Tronco Mesenquimais/citologia , Tecido Adiposo/metabolismo , Idoso , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Separação Celular/métodos , Citometria de Fluxo , Humanos , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Pescoço , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Engenharia Tecidual , TraqueotomiaRESUMO
Wound healing and sclerosis are characterized by an increase of extracellular matrix proteins, which are characteristically expressed in the embryo-fetal period. We analyzed the expression of fibrillin-2, which is typically found in embryonic tissues, but only scarcely in adult skin. In wound healing and sclerotic skin diseases such as lipodermatosclerosis and scleroderma, a marked increase of fibrillin-2 expression was found by immunohistology. Double labelling of fibrillin-2 and tenascin-C, which is also expressed in wound healing and sclerosis, showed co-localization of both proteins. Solid-phase and slot blot-overlay assays showed a dose-dependent binding of the recombinant N-terminal half of fibrillin-2 (rFBN2-N) to tenascin-C. Real-time PCR showed an increase of the fibrillin-2 gene expression in cell culture triggered by typical mediators for fibroblast activation such as serum, IL-4, and TGF-beta. By contrast, prolonged hypoxia is not associated with changes in fibrillin-2 expression. Tenascin-C is an anti-adhesive substrate for fibroblasts, whereas fibrillin-2 stimulates cell attachment. Attachment assays using mixed substrates showed decreased cell attachment when tenascin-C and rFBN2-N were coated together, compared with the attachment to rFBN2-N alone. Fibrillins are involved in storage and activation of TGF-beta. Immunohistology with an antibody against the latency-associated peptide (LAP (TGF-beta1)) showed a marked increase of inactive LAP-bound TGF-beta1 in wound healing and sclerotic skin whereas normal skin showed only a weak expression. Double immunofluorescence confirmed a partial colocalization of both proteins. In conclusion, we show that a stimulation of the fibrillin-2 expression is a characteristic feature of fibroblasts present in wound healing and sclerosis, which may be involved in the alteration of cell attachment and storage of inactive TGF-beta in the matrix.
Assuntos
Proteínas dos Microfilamentos/metabolismo , Pele/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Cicatrização , Adesão Celular , Hipóxia Celular , Células Cultivadas , Meios de Cultura Livres de Soro/farmacologia , Matriz Extracelular/metabolismo , Fibrilina-2 , Fibrilinas , Fibroblastos/metabolismo , Fibroblastos/patologia , Imunofluorescência , Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Interleucina-4/farmacologia , Proteínas dos Microfilamentos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esclerodermia Localizada/genética , Esclerodermia Localizada/metabolismo , Esclerodermia Localizada/patologia , Esclerose , Pele/patologia , Pele/fisiopatologia , Tenascina/genética , Tenascina/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/farmacologiaRESUMO
BACKGROUND AIMS: In vitro cultured mesenchymal stromal cells (MSC) are characterized by a short proliferative lifespan, an increasing loss of proliferation capacity and progressive reduction of differentiation potential. Laminin-1, laminin-5, collagen IV and fibronectin are important constituents of the basement membrane extracellular matrix (ECM) that are involved in a variety of cellular activities, including cell attachment and motility. METHODS AND RESULTS: The in vitro proliferation capacity of MSC was significantly improved when the cells were incubated in the presence of basement membrane ECM proteins. For example, a mixture of proteins improved proliferation capacity 250-fold in comparison with standard conditions after five passages. Furthermore, in colony-forming unit-fibroblast (CFU-F) assays colony numbers and size were significantly extended. Blocking specific integrin cell-surface receptors, positive effects on the proliferation capacity of MSC were inhibited. Additionally, when MSC were co-cultivated with ECM proteins, cells maintained their multipotential differentiation capacity throughout many culture passages in comparison with cells cultivated on plastic. However, expansion of MSC on laminin-5 suppressed any subsequent chondrogenic differentiation. CONCLUSIONS: Our results suggest that expansion of bone marrow-derived MSC in the presence of ECM proteins is a powerful approach for generating large numbers of MSC, showing a prolonged capacity to differentiate into mesodermal cell lineages, with the exception of the lack of chondrogenesis by using laminin-5 coating.
Assuntos
Células da Medula Óssea/metabolismo , Moléculas de Adesão Celular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células Estromais/metabolismo , Anticorpos Bloqueadores/farmacologia , Membrana Basal/metabolismo , Células da Medula Óssea/citologia , Moléculas de Adesão Celular/imunologia , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Condrogênese , Ensaio de Unidades Formadoras de Colônias , Proteínas da Matriz Extracelular/imunologia , Humanos , Células-Tronco Mesenquimais/citologia , Células Estromais/citologia , CalininaRESUMO
The adult bone marrow has been generally considered to be composed of hematopoietic tissue and the associated supporting stroma. Within the latter compartment, a subset of cells with multipotent differentiation capacity exists, usually referred to as mesenchymal stem cells. Mesenchymal stem cells can easily be expanded ex vivo and induced to differentiate into several cell types, including osteoblasts, adipocytes and chondrocytes. Up to now, mesenchymal stem cells have gained wide popularity. Despite the rapid growth in this field, irritations remain with respect to the defining characteristics of these cells, including their differentiation potency, self-renewal and in vivo properties. As a consequence, there is a growing tendency to challenge the term mesenchymal stem cell, especially with respect to the stem cell characteristics. Here, we revisit the experimental origins of mesenchymal stem cells, their classical differentiation capacity into mesodermal lineages and their immunophenotype in order to assess their stemness and function. Based on these essentials, it has to be revisited if the designation as a stem cell remains an appropriate term.
RESUMO
BACKGROUND: Because specific marker molecules for phenotypical identification of mesenchymal stem and progenitor cells are missing, the assessment of the in vitro-differentiation capacity is a prerequisite to characterize these cells. However, classical differentiation protocols are often cell-consuming and time intensive. Therefore, the establishment of novel strategies for differentiation is one topic of current efforts in stem cell biology. The goal of this study was to demonstrate the practicability of a new differentiation test using plastic adherent cell isolates from different tissues. RESULTS: We introduced the mesenchymal microsphere method as a feasible time- and cell saving screening method to analyse multilineage differentiation properties of adult progenitor cells in a three-dimensional system. For this purpose we isolated, characterized and analyzed new sources of adult murine mesenchymal progenitor cells from perirenal adipose tissue and mediastinal stromal tissue in comparison to bone marrow progenitor cells. The proliferation capacity of the cells was demonstrated by determination of the daily doubling index. Although the flow cytometry analysis of undifferentiated cells revealed differences in the expression of CD marker molecules, all isolates have the capacity for multilineage differentiation following the mesenchymal microsphere protocol as well as the classical "micro mass body" protocol for chondrogenic and the monolayer cultivation protocol for osteogenic and adipogenic differentiation. Differentiation was characterized using histochemical and immunhistochemical staining as well as RT-PCR. CONCLUSIONS: We were able to show that the mesenchymal microsphere method is an efficient test system for chondro-, osteo- and adipogenic differentiation of adult progenitor cells. The advantage of this system in comparison to classical protocols is that approximately 7 times lower cell numbers are necessary. Since classical culture procedures are time intensive because high cell numbers have to be obtained, the new differentiation method may also save cells and time in future clinical applications using human mesenchymal stromal cells.
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Mesoderma/citologia , Animais , Células Cultivadas , Feminino , Camundongos , MicroesferasRESUMO
The transcription factor Sox9 plays an important role during chondrogenesis. After early conditional inactivation of Sox9 in mesenchymal limb bud cells of mice, mesenchymal condensations as well as cartilage and bone are completely absent in the developing limbs. We analyzed chondrogenic differentiation of Sox9-/- mouse embryonic stem cells in vitro, using two clones with different targeted mutations. We found that the development of mature and hypertrophic chondrocytes is completely inhibited in the absence of Sox9 confirming that Sox9 is required for the formation of cartilage. In contrast, Sox9+/- mouse embryonic stem cells showed continuous but reduced differentiation into mature chondrocytes. Interestingly, the formation of early chondrogenic condensations expressing characteristic marker genes such as scleraxis, Sox5 and Sox6 was not inhibited in the absence of Sox9 in vitro. Thus, we propose that the earliest step of chondrogenesis could be regulated by a non cell-autonomous function of Sox9.
Assuntos
Condrócitos/química , Condrócitos/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Proteínas de Grupo de Alta Mobilidade/deficiência , Fatores de Transcrição/deficiência , Sequência de Aminoácidos , Animais , Sequência de Bases , Cartilagem/citologia , Cartilagem/crescimento & desenvolvimento , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Condrogênese/genética , Condrogênese/fisiologia , Células Clonais , Colágeno Tipo II/genética , DNA/genética , Éxons , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/fisiologia , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOX9 , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologiaRESUMO
Currently, adult stem cells are attracting significant interest in regenerative medicine and tissue engineering. These cells have been isolated from various tissue sources; however, in most cases, adult stem cells useful for tissue engineering and regeneration are present at a low frequency. High numbers of stem cells with an effective and reliable potential for differentiation are needed for clinical applications. Thus, the identification of new stem cell sources and the establishment of optimized cell culture conditions that allow for the amplification of stem cells are of utmost relevance. In addition, the isolation procedure should ideally be minimally invasive and possibly be performed under local anesthesia. We report here for the first time on the identification of adult stem cells with mesenchymal characteristics in human parotid gland tissue. Cells were isolated from freshly resected specimens of parotid glands using enzymatic digestion and plastic adhesion protocols. Following an initial proliferation period and short-term culture for four passages, immunophenotyping revealed the presence of mesenchymal stem cell markers. In the presence of tissue-specificinduction medium, stem cells could be differentiated into adipogenic, osteogenic, and chondrogenic cell types. Tissue-specific differentiation was confirmed by histochemical and immunocytochemical staining as well as by RT-PCR for defined marker genes. This study is, to the best of our knowledge, the first report on the isolation and differentiation of stem cells from adult human parotid glands. Although isolated from an endodermal tissue source, these stem cells share many characteristics with MSCs. Easy accessibility and a high differentiation potential make salivary gland-derived stem cells a promising source for future applications in regenerative medicine.
Assuntos
Células-Tronco Adultas/citologia , Glândulas Salivares/citologia , Adipogenia , Biomarcadores/metabolismo , Linhagem da Célula , Proliferação de Células , Separação Celular , Células Cultivadas , Condrogênese , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Imunofenotipagem , Masculino , Células-Tronco Mesenquimais/citologia , Pessoa de Meia-Idade , Especificidade de Órgãos , Osteogênese , Glândula Parótida/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The human iPS cell line VUZUZLi001-A (hVH-1) was generated from human foreskin fibroblasts to be used as a control line. Reprogramming was performed by retroviral transduction of reprogramming factors OCT4, SOX2, KLF4 and c-MYC. Resource table.
Assuntos
Fibroblastos/metabolismo , Prepúcio do Pênis/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Humanos , Fator 4 Semelhante a Kruppel , MasculinoRESUMO
The model system of embryonic stem (ES) cell differentiation in vitro via cellular aggregates (embryoid bodies, EBs) can be used to analyze cell differentiation from a pluripotent stem cell via progenitor cells up to terminally differentiated cell types. ES cells are known to be pluripotent; they have the capacity to differentiate into any cell lineage of the three germ layers. Using various ES cell lines, we characterized chondrogenic and osteogenic differentiation in EBs by histochemical staining, immunostaining, mRNA-in situ hybridization, and reverse transcriptase polymerase chain reaction analysis. Here, we describe in detail our established protocols to analyze chondrogenic differentiation of ES cells. We summarize different ways to modulate ES cell-derived chondrogenic differentiation.
Assuntos
Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Condrócitos/fisiologia , Células-Tronco/fisiologia , Animais , Linhagem da Célula , Células Cultivadas , Condrócitos/citologia , Embrião de Mamíferos/citologia , Hibridização In Situ , Camundongos , Camundongos Knockout , Células-Tronco/citologiaRESUMO
Pluripotent embryonic stem (ES) cells cultivated as cellular aggregates, so called embryoid bodies (EBs), differentiate spontaneously into different cell types of all three germ layers in vitro resembling processes of cellular differentiation during embryonic development. Regarding chondrogenic differentiation, murine ES cells differentiate into progenitor cells, which form pre-cartilaginous condensations in the EB-outgrowths and express marker molecules characteristic for mesenchymal cell types such as Sox5 and Sox6. Later, mature chondrocytes appear which express collagen type II, and the collagen fibers show a typical morphology as demonstrated by electron-microscopical analysis. These mature chondrogenic cells are organized in cartilage nodules and produce large amounts of extracellular proteoglycans as revealed by staining with cupromeronic blue. Finally, cells organized in nodules express collagen type X, indicating the hypertrophic stage. In conclusion, differentiation of murine ES cells into chondrocytes proceeds from the undifferentiated stem cell via progenitor cells up to mature chondrogenic cells, which then undergo hypertrophy. Furthermore, because the ES-cell-derived chondrocytes did not express elastin, a marker for elastic cartilage tissue, we suggest the cartilage nodules to resemble hyaline cartilage tissue.
Assuntos
Cartilagem/embriologia , Condrócitos/citologia , Embrião de Mamíferos/citologia , Camundongos/embriologia , Células-Tronco Pluripotentes/citologia , Animais , Biomarcadores/metabolismo , Cartilagem/citologia , Diferenciação Celular , Linhagem Celular , Condrócitos/metabolismo , Colágeno Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Matriz Extracelular/ultraestrutura , Proteínas de Grupo de Alta Mobilidade/metabolismo , Hibridização in Situ Fluorescente , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Proteínas Nucleares/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição SOXD , Fatores de Transcrição/metabolismoRESUMO
The aim of this study was to examine the influence of matrix elasticity on the maintenance of the chondrogenic phenotype of chondrocytes cultured in monolayer. We used a two-dimensional culturing system in which polyacrylamide gels with different concentrations of bis-acrylamide were coated with collagen type I. Matrices with a Young's modulus of 4, 10, 40, and 100 kPa were produced, as determined by atomic force microscopy. Porcine chondrocytes were cultivated on these matrices at a low density for 7 days. The proliferation of cells was analyzed by 5-Bromo-2'-deoxy-uridine incorporation. Maintenance of the chondrogenic phenotype was analyzed by measuring collagen type I, type II, and aggrecan gene expression, immunofluorescence staining for collagen type II, and phalloidin staining for actin filaments. Cellular proliferation and actin organization were decreased on matrices of 4 kPa compared with stiffer substrates. The differentiated phenotype of the chondrocytes grown on matrices of 4 kPa was stabilized, indicated by higher collagen type II and aggrecan, and lower collagen type I expression. These findings indicate that chondrocytes sense the elasticity of the matrix and might be used for the design of scaffolds with mechanical properties specifically tailored to support the chondrogenic phenotype in tissue engineering applications.
Assuntos
Condrócitos/citologia , Condrócitos/fisiologia , Matriz Extracelular/fisiologia , Engenharia Tecidual/métodos , Resinas Acrílicas , Actinas/metabolismo , Animais , Sequência de Bases , Proliferação de Células , Células Cultivadas , Condrogênese/genética , Condrogênese/fisiologia , Colágeno , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Primers do DNA/genética , Módulo de Elasticidade , Elasticidade , Géis , Fenótipo , Sus scrofa , Alicerces TeciduaisAssuntos
Osso e Ossos/citologia , Cartilagem/citologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Embrião de Mamíferos/citologia , Células-Tronco/citologia , Animais , Condrócitos/citologia , Meios de Cultura/farmacologia , Fibroblastos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Hibridização in Situ Fluorescente , Camundongos , Hibridização de Ácido Nucleico , Osteoblastos/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
This unit describes the protocols used for cultivation of murine embryonic stem (ES) cells and their differentiation into chondrogenic cell types in vitro. ES cells cultivated as cellular aggregates, so-called embryoid bodies (EBs), differentiate spontaneously into chondrogenic cell types recapitulating cellular events of chondro- and osteogenesis. The undifferentiated ES cells differentiate into mesenchymal prechondrogenic cells in the EB outgrowths. These progenitor cells aggregate and form mesenchymal condensations. During further cultivation, these cells form cartilage nodules, show a phenotype typical for chondroblasts, and start to express marker molecules of cartilage tissue. Later, the chondrocytes become hypertrophic, and finally, marker molecules indicating bone formation can be detected in the nodules. This unit also contains protocols for characterization of the differentiated cells by immunostaining, mRNA-in situ hybridization, electron microscopy, and RT-PCR analysis.
Assuntos
Técnicas de Cultura de Células/métodos , Condrócitos/citologia , Células-Tronco Embrionárias/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Pluripotentes/citologia , Animais , Biomarcadores/análise , Técnicas de Cultura de Células/instrumentação , Diferenciação Celular , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Corantes , Meios de Cultura/farmacologia , Células-Tronco Embrionárias/efeitos dos fármacos , Fibroblastos/citologia , Perfilação da Expressão Gênica/métodos , Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente/métodos , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Microscopia Eletrônica/métodos , Osteócitos/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Coloração e Rotulagem/métodosRESUMO
Differentiation of mouse embryonic stem (ES) cells via embryoid bodies (EB) is established as a suitable model to study cellular processes of development in vitro. ES cells are known to be pluripotent because of their capability to differentiate into cell types of all three germ layers including germ cells. Here, we show that ES cells differentiate into renal cell types in vitro. We found that genes were expressed during EB cultivation, which have been previously described to be involved in renal development. Marker molecules characteristic for terminally differentiated renal cell types were found to be expressed predominantly during late stages of EB cultivation, while marker molecules involved in the initiation of nephrogenesis were already expressed during early steps of EB development. On the cellular level--using immunostaining--we detected cells expressing podocin, nephrin and wt-1, characteristic for differentiated podocytes and other cells, which expressed Tamm-Horsfall protein, a marker for distal tubule epithelial cells of kidney tissue. Furthermore, the proximal tubule marker molecules renal-specific oxido reductase, kidney androgen-related protein and 25-hydroxyvitamin D3alpha-hydroxylase were found to be expressed in EBs. In particular, we could demonstrate that cells expressing podocyte marker molecules assemble to distinct ring-like structures within the EBs. Because the differentiation efficiency into these cell types is still relatively low, application of fibroblast growth factor (FGF)-2 in combination with leukaemia inhibitory factor was tested for induction, but did not enhance ES cell-derived renal differentiation in vitro.
Assuntos
Embrião de Mamíferos/citologia , Rim/citologia , Células-Tronco/citologia , Animais , Biomarcadores/análise , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem Celular , Expressão Gênica , Imuno-Histoquímica , Rim/embriologia , Rim/metabolismo , Túbulos Renais/citologia , Túbulos Renais/metabolismo , Túbulos Renais/ultraestrutura , Camundongos , Podócitos/citologia , Podócitos/metabolismo , Podócitos/ultraestrutura , Células-Tronco/metabolismo , Células-Tronco/ultraestruturaRESUMO
Pluripotent mouse embryonic stem (ES) cells differentiate in vitro spontaneously into cell types of all three primary germ layers when cultivated as cell aggregates, so-called 'embryoid bodies'. Many reports have shown that this system recapitulates cellular developmental processes and gene expression patterns of early embryogenesis. During ES cell differentiation, efficient and directed differentiation into a specific cell type is influenced by many parameters, for example, the batch of the serum used or the application of growth factors and signalling molecules. Because all ES cell lines are considered to be pluripotent, one should not expect remarkable differences regarding their spontaneous differentiation efficiencies. However, here we show that different ES cell lines exhibit a variable degree of spontaneous chondrogenic differentiation indicating that lines with a specific differentiation capacity could be selected. This is an important aspect if ES cells are applied for tissue regeneration.
Assuntos
Linhagem Celular , Condrócitos/citologia , Embrião de Mamíferos/citologia , Camundongos/embriologia , Células-Tronco Pluripotentes/citologia , Azul Alciano/química , Animais , Cartilagem/citologia , Cartilagem/metabolismo , Diferenciação Celular , Condrócitos/metabolismo , Corantes , Expressão Gênica , Mesoderma/citologiaRESUMO
Our knowledge of cellular differentiation processes during chondro- and osteogenesis, in particular the complex interaction of differentiation factors, is still limited. We used the model system of embryonic stem (ES) cell differentiation in vitro via cellular aggregates, so called embryoid bodies (EBs), to analyze chondrogenic and osteogenic differentiation. ES cells differentiated into chondrocytes and osteocytes throughout a series of developmental stages resembling cellular differentiation events during skeletal development in vivo. A lineage from pluripotent ES cells via mesenchymal, prechondrogenic cells, chondrocytes and hypertrophicchondrocytes up to osteogenic cells was characterized. Furthermore, we found evidence for another osteogenic lineage, bypassing the chondrogenic stage. Together our results suggest that this in vitro system will be helpful to answer so far unacknowledged questions regarding chondrogenic and osteogenic differentiation. For example, we isolated an as yet unknown cDNA fragment from ES cell-derived chondrocytes, which showed a developmentally regulated expression pattern during EB differentiation. Considering ES cell differentiation as an alternative approach for cellular therapy, we used two different methods to obtain pure chondrocyte cultures from the heterogenous EBs. First, members of the transforming growth factor (TGF)-beta family were applied and found to modulate chondrogenic differentiation but were not effective enough to produce sufficient amounts of chondrocytes. Second, chondrocytes were isolated from EBs by micro-manipulation. These cells initially showed dedifferentiation into fiboblastoid cells in culture, but later redifferentiated into mature chondrocytes. However, a small amount of chondrocytes isolated from EBs transdifferentiated into other mesenchymal cell types, indicating that chondrocytes derived from ES cells posses a distinct differentiation plasticity.