RESUMO
Joubert syndrome (JS) is a recessively inherited congenital ataxia characterized by hypotonia, psychomotor delay, abnormal ocular movements, intellectual disability, and a peculiar cerebellar and brainstem malformation, the "molar tooth sign." Over 40 causative genes have been reported, all encoding for proteins implicated in the structure or functioning of the primary cilium, a subcellular organelle widely present in embryonic and adult tissues. In this paper, we developed an in vitro neuronal differentiation model using patient-derived induced pluripotent stem cells (iPSCs), to evaluate possible neurodevelopmental defects in JS. To this end, iPSCs from four JS patients harboring mutations in distinct JS genes (AHI1, CPLANE1, TMEM67, and CC2D2A) were differentiated alongside healthy control cells to obtain mid-hindbrain precursors and cerebellar granule cells. Differentiation was monitored over 31 days through the detection of lineage-specific marker expression by qRT-PCR, immunofluorescence, and transcriptomics analysis. All JS patient-derived iPSCs, regardless of the mutant gene, showed a similar impairment to differentiate into mid-hindbrain and cerebellar granule cells when compared to healthy controls. In addition, analysis of primary cilium count and morphology showed notable ciliary defects in all differentiating JS patient-derived iPSCs compared to controls. These results confirm that patient-derived iPSCs are an accessible and relevant in vitro model to analyze cellular phenotypes connected to the presence of JS gene mutations in a neuronal context.
Assuntos
Anormalidades Múltiplas , Diferenciação Celular , Cerebelo , Cerebelo/anormalidades , Anormalidades do Olho , Células-Tronco Pluripotentes Induzidas , Doenças Renais Císticas , Neurônios , Retina , Retina/anormalidades , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Humanos , Anormalidades do Olho/genética , Anormalidades do Olho/patologia , Cerebelo/patologia , Cerebelo/metabolismo , Neurônios/metabolismo , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Retina/metabolismo , Doenças Renais Císticas/genética , Doenças Renais Císticas/patologia , Doenças Renais Císticas/metabolismo , Masculino , Feminino , Mutação/genética , Cílios/metabolismoRESUMO
Primary ciliopathies are rare inherited disorders caused by structural or functional defects in the primary cilium, a subcellular organelle present on the surface of most cells. Primary ciliopathies show considerable clinical and genetic heterogeneity, with disruption of over 100 genes causing the variable involvement of several organs, including the central nervous system, kidneys, retina, skeleton and liver. Pathogenic variants in one and the same gene may associate with a wide range of ciliopathy phenotypes, supporting the hypothesis that the individual genetic background, with potential additional variants in other ciliary genes, may contribute to a mutational load eventually determining the phenotypic manifestations of each patient. Functional studies in animal models have uncovered some of the pathophysiological mechanisms linking ciliary gene mutations to the observed phenotypes; yet, the lack of reliable human cell models has previously limited preclinical research and the development of new therapeutic strategies for primary ciliopathies. Recent technical advances in the generation of patient-derived two-dimensional (2D) and three-dimensional (3D) cellular models give a new spur to this research, allowing the study of pathomechanisms while maintaining the complexity of the genetic background of each patient, and enabling the development of innovative treatments to target specific pathways. This review provides an overview of available models for primary ciliopathies, from existing in vivo models to more recent patient-derived 2D and 3D in vitro models. We highlight the advantages of each model in understanding the functional basis of primary ciliopathies and facilitating novel regenerative medicine, gene therapy and drug testing strategies for these disorders.
Assuntos
Ciliopatias , Animais , Cílios/patologia , Ciliopatias/metabolismo , Humanos , Mutação , Fenótipo , Retina/patologiaRESUMO
BACKGROUND/AIMS: The skeleton is a metabolically active organ undergoing continuous remodelling initiated by mesenchymal progenitors present in bone and bone marrow. Under certain pathological conditions this remodelling balance shifts towards increased resorption resulting in weaker bone microarchitecture, and there is consequently a therapeutic need to identify pathways that could inversely enhance bone formation from stem cells. Metabolomics approaches recently applied to stem cell characterisation could help identify new biochemical markers involved in osteogenic differentiation. METHODS: Combined intra- and extracellular metabolite profiling was performed by liquid chromatography-mass spectrometry (LC-MS) on human mesenchymal stem cells (MSCs) undergoing osteogenic differentiation in vitro. Using a combination of univariate and multivariate analyses, changes in metabolite and nutrient concentration were monitored in cultures under osteogenic treatment over 10 days. RESULTS: A subset of differentially detected compounds was identified in differentiating cells, suggesting a direct link to metabolic processes involved in osteogenic response. CONCLUSION: These results highlight new metabolite candidates as potential biomarkers to monitor stem cell differentiation towards the bone lineage.
Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais/metabolismo , Metaboloma , Metabolômica , Osteogênese , Linhagem Celular Transformada , HumanosRESUMO
Cancer has been traditionally viewed as a disease characterised by excessive and uncontrolled proliferation, leading to the development of cytotoxic therapies against highly proliferating malignant cells. However, tumours frequently relapse due to the presence of slow-cycling cancer stem cells eluding chemo and radiotherapy. Since these malignant stem cells are largely undifferentiated, inducing their lineage commitment has been proposed as a potential intervention strategy to deplete tumours from their most resistant components. Pro-differentiation approaches have thus far yielded clinical success in the reversion of acute promyelocytic leukaemia (APL), and new developments are fast widening their therapeutic applicability to solid carcinomas. Recent advances in cancer differentiation discussed here highlight the potential and outstanding challenges of differentiation-based approaches.
Assuntos
Diferenciação Celular , Modelos Biológicos , Neoplasias/patologia , HumanosRESUMO
Phosphate-based glasses (PBGs) offer significant therapeutic potential due to their bioactivity, controllable compositions, and degradation rates. Several PBGs have already demonstrated their ability to support direct cell growth and in vivo cytocompatibility for bone repair applications. This study investigated development of PBG formulations with pyro- and orthophosphate species within the glass system (40 - x)P2O5·(16 + x)CaO·20Na2O·24MgO (x = 0, 5, 10 mol%) and their effect on stem cell adhesion properties. Substitution of phosphate for calcium revealed a gradual transition within the glass structure from Q2 to Q0 phosphate species. Human mesenchymal stem cells were cultured directly onto discs made from three PBG compositions. Analysis of cells seeded onto the discs revealed that PBG with higher concentration of pyro- and orthophosphate content (61% Q1 and 39% Q0) supported a 4.3-fold increase in adhered cells compared to glasses with metaphosphate connectivity (49% Q2 and 51% Q1). This study highlights that tuning the composition of PBGs to possess pyro- and orthophosphate species only, enables the possibility to control cell adhesion performance. PBGs with superior cell adhesion profiles represent ideal candidates for biomedical applications, where cell recruitment and support for tissue ingrowth are of critical importance for orthopaedic interventions.
Assuntos
Adesão Celular , Técnicas de Cultura de Células , Vidro/química , Células-Tronco Mesenquimais/efeitos dos fármacos , Fosfatos/química , Materiais Biocompatíveis/química , Cálcio/química , Varredura Diferencial de Calorimetria , Núcleo Celular/metabolismo , Difosfatos/química , Consolidação da Fratura , Proteínas de Fluorescência Verde/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Teste de Materiais , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Difração de Raios XRESUMO
Beige adipocytes possess the morphological and biochemical characteristics of brown adipocytes, including the mitochondrial uncoupling protein (UCP)1. Mesenchymal stem cells (MSCs) are somatic multipotent progenitors which differentiate into lipid-laden adipocytes. Induction of MSC adipogenesis under hypothermic culture conditions (ie 32°C) promotes the appearance of a beige adipogenic phenotype, but the stability of this phenotypic switch after cells are returned to normothermic conditions of 37°C has not been fully examined. Here, cells transferred from 32°C to 37°C retained their multilocular beige-like morphology and exhibited an intermediate gene expression profile, with both beige-like and white adipocyte characteristics while maintaining UCP1 protein expression. Metabolic profile analysis indicated that the bioenergetic status of cells initially differentiated at 32°C adapted post-transfer to 37°C, showing an increase in mitochondrial respiration and glycolysis. The ability of the transferred cells to respond under stress conditions (eg carbonyl cyanide-4-phenylhydrazone (FCCP) treatment) demonstrated higher functional capacity of enzymes involved in the electron transport chain and capability to supply substrate to the mitochondria. Overall, MSC-derived adipocytes incubated at 32°C were able to remain metabolically active and retain brown-like features after 3 weeks of acclimatization at 37°C, indicating these phenotypic characteristics acquired in response to environmental conditions are not fully reversible.
Assuntos
Adipócitos Bege/citologia , Temperatura Baixa , Células-Tronco/citologia , Adipócitos Bege/metabolismo , Adipócitos Marrons/citologia , Adipócitos Marrons/metabolismo , Adipogenia/genética , Animais , Biomarcadores/metabolismo , Forma Celular/genética , Regulação da Expressão Gênica , Células-Tronco Mesenquimais/metabolismo , Camundongos , Mitocôndrias/metabolismo , Células-Tronco/metabolismo , Canais de Cátion TRPV/metabolismo , Proteína Desacopladora 1/metabolismoRESUMO
BACKGROUND/AIMS: Glutamine is the most abundant amino acid in the body and has a metabolic role as a precursor for protein, amino sugar and nucleotide synthesis. After glucose, glutamine is the main source of energy in cells and has recently been shown to be an important carbon source for de novo lipogenesis. Glutamine is synthesized by the enzyme glutamine synthetase, a mitochondrial enzyme that is active during adipocyte differentiation suggesting a regulatory role in this process. The aim of our study was therefore to investigate whether glutamine status impacts on the differentiation of adipocytes and lipid droplet accumulation. METHODS: Mouse mesenchymal stem cells (MSCs) were submitted to glutamine deprivation (i.e. glutamine-free adipogenic medium in conjunction with irreversible glutamine synthetase inhibitor, methionine sulfoximine - MSO) during differentiation and their response was compared with MSCs differentiated in glutamine-supplemented medium (5, 10 and 20 mM). Differentiated MSCs were assessed for lipid content using Oil Red O (ORO) staining and gene expression was analysed by qPCR. Intracellular glutamine levels were determined using a colorimetric assay, while extracellular glutamine was measured using liquid chromatography-mass spectrometry (LC-MS). RESULTS: Glutamine deprivation largely abolished adipogenic differentiation and lipid droplet formation. This was accompanied with a reduction in intracellular glutamine concentration, and downregulation of gene expression for classical adipogenic markers including PPARγ. Furthermore, glutamine restriction suppressed isocitrate dehydrogenase 1 (IDH1) gene expression, an enzyme which produces citrate for lipid synthesis. In contrast, glutamine supplementation promoted adipogenic differentiation in a dose-dependent manner. CONCLUSION: These results suggest that the glutamine pathway may have a previously over-looked role in adipogenesis. The underlying mechanism involved the glutamine-IDH1 pathway and could represent a potential therapeutic strategy to treat excessive lipid accumulation and thus obesity.
Assuntos
Adipogenia/genética , Glutamato-Amônia Ligase/metabolismo , Glutamina/biossíntese , Adipócitos/metabolismo , Adipócitos Bege/metabolismo , Adipogenia/fisiologia , Animais , Diferenciação Celular/genética , Células Cultivadas , Meios de Cultura , Glutamato-Amônia Ligase/fisiologia , Glutamina/metabolismo , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/fisiologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , PPAR gama/metabolismo , Células-Tronco/metabolismoRESUMO
Recent osteochondral repair strategies highlight the promise of mesenchymal progenitors, an accessible stem cell source with osteogenic and chondrogenic potential, used in conjunction with biomaterials for tissue engineering. For this, regenerative medicine approaches require robust models to ensure selected cell populations can generate the desired cell type in a reproducible and measurable manner. Techniques for in vitro chondrogenic differentiation are well-established but largely qualitative, relying on sample staining and imaging. To facilitate the in vitro screening of pro-chondrogenic treatments, a 3D micropellet culture combined with three quantitative GAG assays has been developed, with a fourth parallel assay measuring sample content to enable normalisation. The effect of transforming growth factor beta (TGF-ß) used to validate this culture format produced a measurable increase in proteoglycan production in the parallel assays, in both 2D and 3D culture configurations. When compared to traditional micropellets, the monolayer format appeared less able to detect changes in cell differentiation, however in-well 3D cultures displayed a significant differential response. Effects on collagen 2 expression confirmed these observations. Based on these results, a microplate format was optimised for 3D culture, in a high-throughput in-well configuration. This model showed improved sensitivity and confirmed the 3D micropellet in-well quantitative assays as an effective differentiation format compatible with streamlined, high-throughput chondrogenic screens.
Assuntos
Bioensaio/métodos , Diferenciação Celular , Condrogênese , Modelos Biológicos , Células-Tronco/citologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Condrogênese/efeitos dos fármacos , Colágeno Tipo II/metabolismo , Genes Reporter , Glucose/farmacologia , Humanos , Células-Tronco/efeitos dos fármacosRESUMO
Human mesenchymal stem cells (MSCs) show promise for musculoskeletal repair applications. Animal-derived serum is extensively used for MSC culture as a source of nutrients, extracellular matrix proteins and growth factors. However, the routine use of fetal calf serum (FCS) is not innocuous due to its animal antigens and ill-defined composition, driving the development of alternatives protocols. The present study sought to reduce exposure to FCS via the transient use of human serum. Transient exposure to animal serum had previously proved successful for the osteogenic differentiation of MSCs but had not yet been tested with alternative serum sources. Here, human serum was used to support the proliferation of MSCs, which retained surface marker expression and presented higher alkaline phosphatase activity than those in FCS-based medium. Addition of osteogenic supplements supported strong mineralisation over a 3-week treatment. When limiting serum exposure to the first five days of treatment, MSCs achieved higher differentiation with human serum than with FCS. Finally, human serum analysis revealed significantly higher levels of osteogenic components such as alkaline phosphatase and 25-Hydroxyvitamin D, consistent with the enhanced osteogenic effect. These results indicate that human serum used at the start of the culture offers an efficient replacement for continuous FCS treatment and could enable short-term exposure to patient-derived serum in the future.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Mesenquimais/citologia , Osteogênese , Soro/química , Fosfatase Alcalina/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/metabolismo , Vitamina D/análogos & derivados , Vitamina D/metabolismoRESUMO
The success of cell therapy approaches is greatly dependent on the ability to precisely deliver and monitor transplanted stem cell grafts at treated sites. Iron oxide particles, traditionally used in vivo for magnetic resonance imaging (MRI), have been shown to also represent a safe and efficient in vitro labelling agent for mesenchymal stem cells (MSCs). Here, stem cells were labelled with magnetic particles, and their resulting response to magnetic forces was studied using 2D and 3D models. Labelled cells exhibited magnetic responsiveness, which promoted localised retention and patterned cell seeding when exposed to magnet arrangements in vitro. Directed migration was observed in 2D culture when adherent cells were exposed to a magnetic field, and also when cells were seeded into a 3D gel. Finally, a model of cell injection into the rodent leg was used to test the enhanced localised retention of labelled stem cells when applying magnetic forces, using whole body imaging to confirm the potential use of magnetic particles in strategies seeking to better control cell distribution for in vivo cell delivery.
Assuntos
Movimento Celular/fisiologia , Nanopartículas de Magnetita/química , Células-Tronco Mesenquimais/citologia , Células-Tronco/citologia , Linhagem Celular , Humanos , Imageamento por Ressonância MagnéticaRESUMO
SOX1 is a member of the SOXB1 subgroup of transcription factors involved in early embryogenesis, CNS development and maintenance of neural stem cells. The structure and regulation of the human SOX1 locus has been less studied than that of SOX2, another member of the SOXB1 subgroup for which an overlapping transcript has been reported. Here we report that the SOX1 locus harbours a SOX1 overlapping transcript (SOX1-OT), and describe expression, splicing variants and detection of SOX1-OT in different stem and cancer cells. RT-PCR and RACE experiments were performed to detect and characterise the structure of SOX1-OT in neuroprogenitor cultures and across different cancer cell lines. SOX1-OT was found to present a complex structure including several unannotated exons, different transcript variants and at least two potential transcription start sites. SOX1-OT was found to be highly expressed in differentiated neural stem cells across different time points of differentiation, and its expression correlated with SOX1 gene expression. Concomitant expression of SOX1 and SOX1-OT was further observed in several cancer cell models. While the function of this transcript is unknown, the regulatory role reported for other lncRNAs strongly suggests a possible role for SOX1-OT in regulating SOX1 expression, as previously observed for SOX2. The elucidation of the genetic and regulatory context governing SOX1 expression will contribute to clarifying its role in stem cell differentiation and tumorigenesis.
Assuntos
Fatores de Transcrição SOXB1/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Mapeamento Cromossômico , Loci Gênicos , Humanos , Modelos Biológicos , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição SOXB1/genéticaRESUMO
Raman micro-spectroscopy (RMS) is a non-invasive technique for imaging live cells in vitro. However, obtaining quantitative molecular information from Raman spectra is difficult because the intensity of a Raman band is proportional to the number of molecules in the sampled volume, which depends on the local molecular concentration and the thickness of the cell. In order to understand these effects, we combined RMS with atomic force microscopy (AFM), a technique that can measure accurately the thickness profile of the cells. Solution-based calibration models for RNA and albumin were developed to create quantitative maps of RNA and proteins in individual fixed cells. The maps were built by applying the solution-based calibration models, based on partial least squares fitting (PLS), on raster-scan Raman maps, after accounting for the local cell height obtained from the AFM. We found that concentrations of RNA in the cytoplasm of mouse neuroprogenitor stem cells (NSCs) were as high as 25 ± 6 mg ml(-1), while proteins were distributed more uniformly and reached concentrations as high as â¼50 ± 12 mg ml(-1). The combined AFM-Raman datasets from fixed cells were also used to investigate potential improvements for normalization of Raman spectral maps. For all Raman maps of fixed cells (n = 10), we found a linear relationship between the scores corresponding to the first component (PC1) and the cell height profile obtained by AFM. We used PC1 scores to reconstruct the relative height profiles of independent cells (n = 10), and obtained correlation coefficients with AFM maps higher than 0.99. Using this normalization method, qualitative maps of RNA and protein were used to obtain concentrations for live NSCs. While this study demonstrates the potential of using AFM and RMS for measuring concentration maps for individual NSCs in vitro, further studies are required to establish the robustness of the normalization method based on principal component analysis when comparing Raman spectra of cells with large morphological differences.
Assuntos
Microscopia de Força Atômica , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Análise Espectral Raman , Animais , Análise dos Mínimos Quadrados , Camundongos , Células-Tronco Neurais/química , Análise de Componente Principal , Proteínas/análise , RNA/análiseRESUMO
Bone regeneration is a complex biological process where major cellular changes take place to support the osteogenic differentiation of mesenchymal bone progenitors. To characterise these biological changes and better understand the pathways regulating the formation of mature bone cells, the metabolic profile of mesenchymal stem cell (MSC) differentiation in vitro has been assessed non-invasively during osteogenic (OS) treatment using a footprinting technique. Liquid chromatography (LC)-mass spectrometry (MS)-based metabolite profiling of the culture medium was carried out in parallel to mineral deposition and alkaline phosphatase activity which are two hallmarks of osteogenesis in vitro. Metabolic profiles of spent culture media with a combination of univariate and multivariate analyses investigated concentration changes of extracellular metabolites and nutrients linked to the presence of MSCs in culture media. This non-invasive LC-MS-based analytical approach revealed significant metabolic changes between the media from control and OS-treated cells showing distinct effects of MSC differentiation on the environmental footprint of the cells in different conditions (control vs. OS treatment). A subset of compounds was directly linked to the osteogenic time-course of differentiation, and represent interesting metabolite candidates as non-invasive biomarkers for characterising the differentiation of MSCs in a culture medium.
Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Osteogênese , Fosfatase Alcalina/análise , Animais , Células Cultivadas , Cromatografia Líquida , Espectrometria de Massas , CamundongosRESUMO
Raman microscopy was used as a label-free method to study the mineralisation of bone nodules formed by mesenchymal stem cells cultured in osteogenic medium in vitro. Monitoring individual bone nodules over 28 days revealed temporal and spatial changes in the crystalline phase of the hydroxyapatite components of the nodules.
Assuntos
Densidade Óssea/fisiologia , Células-Tronco Mesenquimais/química , Células-Tronco Mesenquimais/fisiologia , Análise Espectral Raman/métodos , Tamanho Celular , Células Cultivadas , Humanos , Fatores de TempoRESUMO
Bioresorbable chitosan scaffolds have shown potential for osteochondral repair applications. Thein vivodegradation of chitosan, mediated by lysozyme and releasing glucosamine, enables progressive replacement by ingrowing tissue. Here the degradation process of a chitosan-nHA based bioresorbable scaffold was investigated for mass loss, mechanical properties and degradation products released from the scaffold when subjected to clinically relevant enzyme concentrations. The scaffold showed accelerated mass loss during the early stages of degradation but without substantial reduction in mechanical strength or structure deterioration. Although not cytotoxic, the medium in which the scaffold was degraded for over 2 weeks showed a transient decrease in mesenchymal stem cell viability, and the main degradation product (glucosamine) demonstrated a possible adverse effect on viability when added at its peak concentration. This study has implications for the design and biomedical application of chitosan scaffolds, underlining the importance of modelling degradation products to determine suitability for clinical translation.
Assuntos
Sobrevivência Celular , Quitosana , Teste de Materiais , Células-Tronco Mesenquimais , Engenharia Tecidual , Alicerces Teciduais , Quitosana/química , Sobrevivência Celular/efeitos dos fármacos , Alicerces Teciduais/química , Células-Tronco Mesenquimais/citologia , Animais , Engenharia Tecidual/métodos , Materiais Biocompatíveis/química , Células Cultivadas , Glucosamina/química , Humanos , Muramidase/química , Implantes AbsorvíveisRESUMO
Osteochondral grafts are used for repair of focal osteochondral lesions. Autologous grafts are the gold standard treatment; however, limited graft availability and donor site morbidity restrict use. Therefore, there is a clinical need for different graft sources/materials which replicate natural cartilage function. Chitosan has been proposed for this application. The aim of this study was to assess the biomechanics and biotribology of a bioresorbable chitosan/chitosan-nano-hydroxyapatite osteochondral construct (OCC), implanted in an in vitro porcine knee experimental simulation model. The OCC implanted in different surgical positions (flush, proud and inverted) was compared to predicate grafts in current clinical use and a positive control consisting of a stainless steel graft implanted proud of the cartilage surface. After 3 h (10 800 cycles) wear simulation under a walking gait, subsidence occurred in all OCC samples irrespective of surgical positioning, but with no apparent loss of material and low meniscus wear. Half the predicate grafts exhibited delamination and scratching of the cartilage surfaces. No graft subsidence occurred in the positive controls but wear and deformation of the meniscus were apparent. Implanting a new chitosan-based OCC either optimally (flush), inverted or proud of the cartilage surface resulted in minimal wear, damage and deformation of the meniscus.
RESUMO
Cells in the pluripotent ground state can give rise to somatic cells and germ cells, and the acquisition of pluripotency is dependent on the expression of Nanog. Pluripotency is conserved in the primitive ectoderm of embryos from mammals and urodele amphibians, and here we report the isolation of a Nanog ortholog from axolotls (axNanog). axNanog does not contain a tryptophan repeat domain and is expressed as a monomer in the axolotl animal cap. The monomeric form is sufficient to regulate pluripotency in mouse embryonic stem cells, but axNanog dimers are required to rescue LIF-independent self-renewal. Our results show that protein interactions mediated by Nanog dimerization promote proliferation. More importantly, they demonstrate that the mechanisms governing pluripotency are conserved from urodele amphibians to mammals.
Assuntos
Células-Tronco Embrionárias/metabolismo , Proteínas de Homeodomínio/metabolismo , Células-Tronco Pluripotentes/metabolismo , Ambystoma mexicanum , Anfíbios , Animais , Linhagem Celular , Proliferação de Células , Proteínas de Homeodomínio/genética , Humanos , Mamíferos , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Ligação ProteicaRESUMO
Adipocytes and osteoblasts derive from a common mesenchymal progenitor present in a range of connective tissues. Differentiation of the progenitors toward the two cell lineages can be induced in vitro through well-established protocols, and leads to the appearance of lipid-laden adipocytes and osteoblasts embedded in a mineralized matrix. The formation of these two lineages in cell cultures can be monitored using lipophilic dyes such as Oil Red O and substances binding to mineral deposits such as Alizarin Red S, respectively. However, these common staining techniques require cell fixation and are thus incompatible with live analyses. Recently, alternative approaches using vital stains have allowed the dual visualization and fluorescence imaging of adipogenic and osteogenic lineages in live cultures. Here we present the concomitant analysis of cultures containing adipogenic and osteogenic cell types using live staining, combining LipidTox Red and tetracycline with NucRed nuclear counterstain for confocal imaging. This approach can be applied to visualize the kinetics and 3D structure of differentiating mesenchymal cultures over time and highlights the interaction of adipose and mineralized compartments associated with bone marrow stroma.
Assuntos
Células-Tronco Mesenquimais , Adipogenia , Diferenciação Celular , Células Cultivadas , Corantes/metabolismo , Lipídeos , Minerais , Osteogênese , Células-Tronco , Tetraciclinas/metabolismoRESUMO
Although phenotypically different, brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT) are able to produce heat through non-shivering thermogenesis due to the presence of mitochondrial uncoupling protein 1 (UCP1). The appearance of thermogenically active beige adipocytes in iWAT is known as browning. Both brown and beige cells originate from mesenchymal stem cells (MSCs), and in culture conditions a browning response can be induced with hypothermia (i.e. 32 °C) during which nuclear leptin immunodetection was observed. The central role of leptin in regulating food intake and energy consumption is well recognised, but its importance in the browning process at the cellular level is unclear. Here, immunocytochemical analysis of MSC-derived adipocytes established nuclear localization of both leptin and leptin receptor suggesting an involvement of the leptin pathway in the browning response. In order to elucidate whether leptin modulates the expression of brown and beige adipocyte markers, BAT and iWAT samples from leptin-deficient (ob/ob) mice were analysed and exhibited reduced brown/beige marker expression compared to wild-type controls. When MSCs were isolated and differentiated into adipocytes, leptin deficiency was observed to induce a white phenotype, especially when incubated at 32 °C. These adaptations were accompanied with morphological signs of impaired adipogenic differentiation. Overall, our results indicate that leptin supports adipocyte browning and suggest a potential role for leptin in adipogenesis and browning.
Assuntos
Adipogenia , Leptina , Animais , Camundongos , Adipócitos/metabolismo , Adipócitos Marrons/metabolismo , Adipogenia/genética , Diferenciação Celular , Leptina/metabolismo , Transdução de SinaisRESUMO
Raman microspectroscopy (rms) was used to identify, image, and quantify potential molecular markers for label-free monitoring the differentiation status of live neural stem cells (NSCs) in vitro. Label-free noninvasive techniques for characterization of NCSs in vitro are needed as they can be developed for real-time monitoring of live cells. Principal component analysis (PCA) and linear discriminant analysis (LDA) models based on Raman spectra of undifferentiated NSCs and NSC-derived glial cells enabled discrimination of NSCs with 89.4% sensitivity and 96.4% specificity. The differences between Raman spectra of NSCs and glial cells indicated that the discrimination of the NSCs was based on higher concentration of nucleic acids in NSCs. Spectral images corresponding to Raman bands assigned to nucleic acids for individual NSCs and glial cells were compared with fluorescence staining of cell nuclei and cytoplasm to show that the origin of the spectral differences were related to cytoplasmic RNA. On the basis of calibration models, the concentration of the RNA was quantified and mapped in individual cells at a resolution of ~700 nm. The spectral maps revealed cytoplasmic regions with concentrations of RNA as high as 4 mg/mL for NSCs while the RNA concentration in the cytoplasm of the glial cells was below the detection limit of our instrument (~1 mg/mL). In the light of recent reports describing the importance of the RNAs in stem cell populations, we propose that the observed high concentration of cytoplasmic RNAs in NSCs compared to glial cells is related to the repressed translation of mRNAs, higher concentrations of large noncoding RNAs in the cytoplasm as well as their lower cytoplasm volume. While this study demonstrates the potential of using rms for label-free assessment of live NSCs in vitro, further studies are required to establish the exact origin of the increased contribution of the cytoplasmic RNA.