RESUMO
Mammalian testes consist of seminiferous tubules within which Sertoli cells line up at the periphery and nurse germ cells, and of interstitia that harbor various cells such as peritubular myoid cells (PMCs), Leydig cells (LCs), vascular endothelial cells, immune cells such as macrophages, and mesenchymal (stromal) cells. Morphological studies have recently reported the presence of telocytes with telopodes in the interstitium of adult mouse, rat, and human testes. CD34+PDGFRα+ telocytes with long and moniliform telopodes form reticular networks with various cell types such as LCs, PMCs, and vessels, indicating their potential functions in cell-cell communications and tissue homeostasis. Functional studies have recently been performed on testicular interstitial cells and CD34+ cells, using 3D re-aggregate cultures of dissociated testicular cells, and cell cultures. Direct observation of CD34+ cells and adult LCs (ALCs) revealed that CD34+ cells extend thin cytoplasmic processes (telopodes), move toward the LC-CD34+ cell-re-aggregates, and finally enter into the re-aggregates, indicating the chemotactic behavior of CD34+ telocytes toward ALCs. In mammalian testes, important roles of mesenchymal interstitial cells as stem/progenitors in the differentiation and regeneration of LCs have been reported. Here, reports on testicular telocytes so far obtained are reviewed, and future perspectives on the studies of testicular telocytes are noted.
Assuntos
Telócitos , Testículo , Adulto , Animais , Antígenos CD34/metabolismo , Moléculas de Adesão Celular/metabolismo , Células Endoteliais/metabolismo , Humanos , Masculino , Mamíferos/metabolismo , Camundongos , Ratos , Telócitos/metabolismo , Telopódios/metabolismo , Testículo/metabolismoRESUMO
Three-dimensional (3-D) aggregate culturing is useful for investigating the functional properties of mesenchymal stem/stromal cells (MSCs). For 3-D MSC analysis, however, pre-expansion of MSCs with two-dimensional (2-D) monolayer culturing must first be performed, which might abolish their endogenous properties. To avoid the need for 2-D expansion, we used prospectively isolated mouse bone marrow (BM)-MSCs and examined the differences in the biological properties of 2-D and 3-D MSC cultures. The BM-MSCs self-assembled into aggregates on nanoculture plates (NCP) that have nanoimprinted patterns with a low-cellular binding texture. The 3-D MSCs proliferated at the same rate as 2-D-cultured cells by only diffusion culture and secreted higher levels of pro-angiogenic factors such as vascular endothelial growth factor and hepatocyte growth factor (HGF). Conditioned medium from 3-D MSC cultures promoted more capillary formation than that of 2-D MSCs in an in vitro tube formation assay. Matrigel-implanted 3-D MSC aggregates tended to induce angiogenesis in host mice. The 3-D culturing on NCP induced alpha-fetoprotein (AFP) expression in MSCs without the application of AFP- or endodermal-inducible factors, possibly via an HGF-autocrine mechanism, and maintained their differentiation ability for adipocytes, osteocytes, and chondrocytes. Prospectively isolated mouse BM-MSCs expressed low/negative stemness-related genes including Oct3/4, Nanog, and Sox2, which were not enhanced by NCP-based 3-D culturing, suggesting that some of these cells differentiate into meso-endodermal layer cells. Culturing of prospectively isolated MSCs on NCP in 3-D allows the analysis of the biological properties of more closely endogenous BM-MSCs and might contribute to tissue engineering and repair.
Assuntos
Células da Medula Óssea/citologia , Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Imageamento Tridimensional , Células-Tronco Mesenquimais/citologia , Nanotecnologia/métodos , Animais , Biomarcadores/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Agregação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Rastreamento de Células , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/efeitos dos fármacosRESUMO
A mino acids are the essential building blocks for collagen and proteoglycan, which are the main constituents for cartilage extracellular matrix (ECM). Synthesis of ECM proteins requires the uptake of various essential/nonessential amino acids. Analyzing amino acid metabolism during chondrogenesis can help to relate tissue quality to amino acid metabolism under different conditions. In our study, we studied amino acid uptake/secretion using human mesenchymal stem cell (hMSC)-based aggregate chondrogenesis in a serum-free induction medium with a defined chemical formulation. The initial glucose level and medium-change frequency were varied. Our results showed that essential amino acid uptake increased with time during hMSCs chondrogenesis for all initial glucose levels and medium-change frequencies. Essential amino acid uptake rates were initial glucose-level independent. The DNA-normalized glycosaminoglycans and hydroxyproline content of chondrogenic aggregates correlated with cumulative uptake of leucine, valine, and tryptophan regardless of initial glucose levels and medium-change frequencies. Collectively, our results show that amino acid uptake rates during in vitro chondrogenesis were insufficient to produce a tissue with an ECM content similar to that of human neonatal cartilage or adult cartilage. Furthermore, this deficiency was likely related to the downregulation of some key amino acid transporters in the cells. Such deficiency could be partially improved by increasing the amino acid availability in the chondrogenic medium by changing culture conditions.
RESUMO
The contact poison VX (O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothioate) is a chemical warfare agent that is one of the most toxic organophosphorus compounds known. Its primary mechanism of toxic action is through the inhibition of acetylcholinesterase and resultant respiratory paralysis. The majority of work on VX has thus concentrated on its potent anticholinesterase activity and acute toxicity, with few studies investigating potential long-term effects. In this report we describe the effects of VX in aggregating rat brain cell cultures out to 28 days post-exposure. Cholinesterase activity was rapidly inhibited (60 min IC50 = 0.73 +/- 0.27 nM), but recovered towards baseline values over the next four weeks. Apoptotic cell death, as measured using caspase-3 activity was evident only at 100 µM concentrations. Cell type specific enzymatic markers (glutamine synthase, choline acetyltransferase and 2',3'-cyclic nucleotide 3'-phosphodiesterase) showed no significant changes. Total Akt levels were unchanged, while an increased phosphorylation of this protein was noted only at the highest VX concentration on the first day post-exposure. In contrast, significant and delayed (28 days post-exposure) decreases were noted in vascular endothelial growth factor (VEGF) levels, a protein whose reduced levels are known to contribute to neurodegenerative disorders. These observations may indicate that the long-term effects noted in some survivors of nerve agent intoxication may be due to VX-induced declines in brain VEGF levels.
Assuntos
Encéfalo/efeitos dos fármacos , Substâncias para a Guerra Química/toxicidade , Compostos Organotiofosforados/toxicidade , Acetilcolinesterase/sangue , Acetilcolinesterase/efeitos dos fármacos , Animais , Apoptose , Encéfalo/enzimologia , Agregação Celular , Células Cultivadas , Inibidores da Colinesterase/toxicidade , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Testes de Toxicidade Aguda , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Understanding in vitro chondrogenesis of human mesenchymal stem cells (hMSCs) is important as it holds great promise for cartilage tissue engineering and other applications. The current technology produces the end tissue quality that is highly variable and dependent on culture conditions. We investigated the effect of nutrient availability on hMSC chondrogenesis in a static aggregate culture system by varying the medium-change frequency together with starting glucose levels. Glucose uptake and lactate secretion profiles were obtained to monitor the metabolism change during hMSC chondrogenesis with different culture conditions. Higher medium-change frequency led to increases in cumulative glucose uptake for all starting glucose levels. Furthermore, increase in glucose uptake by aggregates led to increased end tissue glycosaminoglycan (GAG) and hydroxyproline (HYP) content. The results suggest that increased glucose availability either through increased medium-change frequency or higher initial glucose levels lead to improved chondrogenesis. Also, cumulative glucose uptake and lactate secretion were found to correlate well with GAG and HYP content, indicating both molecules are promising biomarkers for noninvasive assessment of hMSC chondrogenesis. Collectively, our results can be used to design optimal culture conditions and develop dynamic assessment strategies for cartilage tissue engineering applications. Impact statement In this study, we investigated how culture conditions, medium-change frequency and glucose levels, affect chondrogenesis of human mesenchymal stem cells in an aggregate culture model. Doubling the medium-change frequency significantly increased the biochemical quality of the resultant tissue aggregates, as measured by their glycosaminoglycan and hydroxyproline content. We attribute this to increased glucose uptake through the glycolysis pathway, as secretion of lactate, a key endpoint product of the glycolysis pathway, increased concurrently. These findings can be used to design optimal culture conditions for tissue engineering and regenerative medicine applications.
Assuntos
Condrogênese , Glucose , Matriz Extracelular , HumanosRESUMO
Sulphur mustard (H; bis(2-chloroethyl) sulphide) is a vesicant chemical warfare (CW) agent that has been well documented as causing acute injury to the skin, eyes and respiratory system. Although a great deal of research effort has been expended to understand how H exerts these effects, its mechanism of action is still poorly understood. At high exposures, H also causes systemic toxicity with chronic and long-term effects to the immune, cardiovascular and central nervous systems, and these aspects of H poisoning are much less studied and comprehended. Rat aggregate cultures comprised of multiple brain cell types were exposed to H and followed for four weeks post-exposure to assess neurotoxicity. Toxicity (LDH, caspase-3 and aggregate diameter) was progressive with time post-exposure. In addition, statistically significant changes in neurofilament heavy chain (NFH), glial fibrillary acidic protein (GFAP), Akt phosphorylation, IL-6, GRO-KC and TNF-α were noted that were time- and concentration-dependent. Myelin basic protein, CNPase and vascular endothelial growth factor (VEGF) were found to be especially sensitive to H exposure in a time- and concentration-dependent fashion, with levels falling to â¼50 % of control values at â¼10 µM H by 8 days post-exposure. Demyelination and VEGF inhibition may be causal in the long-term neuropsychological illnesses that have been documented in casualties exposed to high concentrations of H, and may also play a role in the peripheral neuropathy that has been observed in some of these individuals.
Assuntos
Encéfalo/efeitos dos fármacos , Agregação Celular/efeitos dos fármacos , Substâncias para a Guerra Química/toxicidade , Doenças Desmielinizantes/induzido quimicamente , Gás de Mostarda/toxicidade , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Agregação Celular/fisiologia , Células Cultivadas , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Relação Dose-Resposta a Droga , Feminino , Gravidez , Ratos , Ratos Sprague-Dawley , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The acquisition of a specific cell fate is one of the core aims of tissue engineering and regenerative medicine. Significant evidence shows that aggregate cultures have a positive influence on cell fate decisions, presumably through cell-cell interactions, but little is known about the specific mechanisms. To investigate the difference between cells cultured as a monolayer and as aggregates, we started by looking at cadherin expression, an important protein involved in cell adhesion, during the differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs) in aggregate and monolayer cultures. We observed that proliferating hMSCs in monolayer culture expressed lower levels of cadherin-2 and increased cadherin-11 expression at cell-cell contact sites over time, which was not evident in the aggregate cultures. By knocking down cadherin-2 and cadherin-11, we found that both cadherins were required for adipogenic differentiation in a monolayer as well as aggregate culture. However, during osteogenic differentiation, low levels of cadherin-2 were found to be favorable for cells cultured as a monolayer and as aggregates, whereas cadherin- 11 was dispensable for cells cultured as aggregates. Together, these results provide compelling evidence for the important role that cadherins play in regulating the differentiation of hMSCs and how this is affected by the dimensionality of cell culture.
Assuntos
Células-Tronco Mesenquimais , Caderinas/genética , Técnicas de Cultura de Células , Diferenciação Celular , Células Cultivadas , Humanos , OsteogêneseRESUMO
Gut CD4 T cells are major targets of HIV-1 and are massively depleted early during infection. To better understand the mechanisms governing HIV-1-mediated CD4 T cell death, we developed the physiologically-relevant Lamina Propria Aggregate Culture (LPAC) model. The LPAC model is ideal for studying CD4 T cell death induced by clinically-relevant Transmitted/Founder (TF) HIV-1 strains and is also suitable for studying how enteric microbes and soluble factors (e.g., Type I Interferons) impact LP CD4 T cell death and function. Here, we detail the protocol to establish LP CD4 T cell infection using a process of spinoculation, the subsequent evaluation of infection levels using multicolor flow cytometry and the determination of overall LP CD4 T cell death using absolute LP CD4 T cell counts. We also describe the preparation of virus stocks of Transmitted/Founder (TF) HIV-1 infectious molecular clones that were successfully used in the LPAC model.
RESUMO
BACKGROUND: Current experimental models using either human or mouse cell lines, are not representative of the complex features of GBM. In particular, there is no model to study patient-derived iPSCs to generate a GBM model. Overexpression of c-met gene is one of the molecular features of GBM leading to increased signaling via STAT3 phosphorylation. We generated an iPSC line from a patient with c-met mutation and we asked whether we could use it to generate neuronal-like organoids mimicking features of GBM. METHODS: We have generated iPSC-aggregates differentiating towards organoids. We analyzed them by gene expression profiling, immunostaining and transmission electronic microscopy analyses (TEM). RESULTS: Herein we describe that c-met-mutated iPSC aggregates spontaneously differentiate into dopaminergic neurons more rapidly than control iPSC aggregates in culture. Gene expression profiling of c-met-mutated iPSC aggregates at day +90 showed neuronal- and GBM-related genes, reproducing a genomic network described in primary human GBM. Comparative TEM analyses confirmed the enrichment of these structures in intermediate filaments and abnormal cilia, a feature described in human GBM. The c-met-mutated iPSC-derived organoids, as compared to controls expressed high levels of glial fibrillary acidic protein (GFAP), which is a typical marker of human GBM, as well as high levels of phospho-MET and phospho-STAT3. The use of temozolomide (TMZ) showed a preferential cytotoxicity of this drug in c-met-mutated neuronal-like organoids. GENERAL SIGNIFICANCE: This study shows the feasibility of generating "off-the shelf" neuronal-like organoid model mimicking GBM using c-met-mutated iPSC aggregates and its potential future use in research.
Assuntos
Glioblastoma/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Neurônios/metabolismo , Organoides/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Glioblastoma/tratamento farmacológico , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Microscopia Eletrônica de Transmissão , Neurônios/efeitos dos fármacos , Organoides/efeitos dos fármacos , Temozolomida/efeitos adversosRESUMO
UNLABELLED: Chondrocyte dedifferentiation presents a major barrier in engineering functional cartilage constructs. To mitigate the effects of dedifferentiation, this study employed a post-expansion aggregate culture step to enhance the chondrogenic phenotype of passaged articular chondrocytes (ACs) before their integration into self-assembled neocartilage constructs. The objective was twofold: (1) to explore how passage number (P2, P3, P4, P5, P6, and P7), with or without aggregate culture, affected construct properties; and (2) to determine the highest passage number that could form neocartilage with functional properties. Juvenile leporine ACs were passaged to P2-P7, with or without aggregate culture, and self-assembled into 5mm discs in non-adhesive agarose molds without using any exogenous scaffolds. Construct biochemical and biomechanical properties were assessed. With aggregate culture, neocartilage constructs had significantly higher collagen content, higher tensile properties, and flatter morphologies. These beneficial effects were most obvious at higher passage numbers. Specifically, collagen content, Young's modulus, and instantaneous compressive modulus in the P7, aggregate group were 53%, 116%, and 178% higher than those in the P7, non-aggregate group. Most interestingly, these extensively passaged P7 ACs (expansion factor of 85,000), which are typically highly dedifferentiated, were able to form constructs with properties similar to or higher than those formed by lower passage number cells. This study not only demonstrated that post-expansion aggregate culture could significantly improve the properties of self-assembled neocartilage, but also that chondrocytes of exceedingly high passage numbers, expanded using the methods in this study, could be used in cartilage engineering applications. STATEMENT OF SIGNIFICANCE: This work demonstrated that extensively passaged chondrocytes (up to passage 7 (P7); expansion factor of 85,000) could potentially be used for cartilage tissue engineering applications. Specifically, an aggregate culture step, employed after cell expansion and before cell integration into a neocartilage construct, was shown to enhance the ability of the chondrocytes to form neocartilage with better biochemical and biomechanical properties. The beneficial effects of this aggregate culture step was especially noticeable at the high passage numbers. Most interestingly, P7 chondrocytes, which are typically highly dedifferentiated, were able to form neocartilage with properties similar to or higher than those formed by lower passage number cells. The ability to obtain high chondrocyte yields with an enhanced chondrogenic potential could have a broad, beneficial impact in improving current therapies (e.g., using higher cell seeding densities for repair) or developing new strategies that require high cell numbers, such as a scaffold-free approach in forming engineered cartilage.
Assuntos
Cartilagem Articular/citologia , Técnicas de Cultura de Células/métodos , Engenharia Tecidual/métodos , Animais , Fenômenos Biomecânicos , Cartilagem Articular/anatomia & histologia , Agregação Celular , Proliferação de Células , Tamanho Celular , Células Cultivadas , Condrócitos/citologia , Força Compressiva , Imuno-Histoquímica , Coelhos , Resistência à Tração , Alicerces Teciduais/químicaRESUMO
Adipose-derived stem cells (ADSCs) are an attractive cell source for tissue engineering, and recently a modified aggregate culture of human ADSCs (hADSCs) was established based on preparation of three-dimensional (3D) cell aggregates in growth factor-enriched low serum medium using the hanging droplet method. Growth and differentiation factor 5 (GDF5) plays a critical role in chondrogenesis and cartilage development. In the present study, we examine (1) whether the modified aggregate culture is feasible for chondrogenic induction of hADSCs, (2) whether overexpressed GDF5 can promote chondrogenesis, and (3) the gene expression profile during chondrogenesis in this aggregate culture. hADSCs were infected with an adenovirus carrying the GDF5 gene (Ad-GDF5). Cells were cultured with chondrogenic media either in a modified aggregate culture or in an attached micromass culture that served as a control. The chondrogenic phenotype was assessed by morphology (n=8), biochemistry (n=3), and histology (n=2). Expression of 12 genes was determined by quantitative real-time polymerase chain reaction (n=3). We found that ADSCs cultured in the modified aggregates exhibited denser pellets and higher content of sulfated glycosaminoglycan (sGAG) compared with those cultured in the micromass. Infection of cells with Ad-GDF5 increased the aggregate size and sGAG content. It also up-regulated expression of GDF5, aggrecan, and leptin and down-regulated expression of COL I, while expression of COL II and COL 10 remained unchanged. We concluded that the modified aggregate culture is feasible for chondrogenic induction of human ADSCs. Infection with Ad-GDF5 appears to promote the chondrogenesis. These findings suggest that genetic modification of ADSCs with GDF5 in the modified aggregate culture could be useful for treating diseases with cartilage defects.