RESUMEN
Cross-linked polymer blends from natural compounds, namely gelatin (Gel), chitosan (CS), and synthetic poly (vinyl alcohol) (PVA), have received increasing scrutiny because of their versatility, biocompatibility, and ease of use for tissue engineering. Previously, Gel/CS/PVA [1:1:1] hydrogel produced via the freeze-drying process presented enhanced mechanical properties. This study aimed to investigate the biocompatibility and chondrogenic potential of a steam-sterilized Gel/CS/PVA hydrogel using differentiation of human adipose-derived mesenchymal stromal cells (AD-hMSC) and cartilage marker expression. AD-hMSC displayed fibroblast-like morphology, 90% viability, and 69% proliferative potential. Mesenchymal profiles CD73 (98.3%), CD90 (98.6%), CD105 (97.0%), CD34 (1.11%), CD45 (0.27%), HLA-DR (0.24%); as well as multilineage potential, were confirmed. Chondrogenic differentiation of AD-hMSC in monolayer revealed the formation of cartilaginous nodules composed of glycosaminoglycans after 21 days. Compared to nonstimulated cells, hMSC-derived chondrocytes shifted the expression of CD49a from 2.82% to 40.6%, CD49e from 51.4% to 92.2%, CD54 from 9.66 to 37.2%, and CD151 from 45.1% to 75.8%. When cultured onto Gel/CS/PVA hydrogel during chondrogenic stimulation, AD-hMSC changed to polygonal morphology, and chondrogenic nodules increased by day 15, six days earlier than monolayer-differentiated cells. SEM analysis showed that hMSC-derived chondrocytes adhered to the surface with extended filopodia and abundant ECM formation. Chondrogenic nodules were positive for aggrecan and type II collagen, two of the most abundant components in cartilage. This study supports the biocompatibility of AD-hMSC onto steam-sterilized GE/CS/PVA hydrogels and its improved potential for chondrocyte differentiation. Hydrogel properties were not altered after steam sterilization, which is relevant for biosafety and biomedical purposes.
RESUMEN
Mesenchymal stem cells are a tool in cell therapies but demand a large cell number per treatment, for that, suitable culture media is required which contains fetal bovine serum (FBS). However, for cell-based therapy applications, the use of FBS is problematic. Several alternatives to FBS have been explored, including human derivatives from platelet-rich plasma (hD-PRP). Although various studies have evaluated the impact of hD-PRP on MSC proliferation and differentiation, few of them have assessed their influence on processes, such as metabolism and gene expression. Here, we cultured human adipose-derived MSCs (hAD-MSCs) in media supplemented with either 10% hD-PRP (hD-PRP-SM) or 10% FBS (FBS-SM) in order to characterize them and evaluate the effect of hD-PRP on cell metabolism, gene expression of associated regenerative factors, as well as chromosome stability during cell expansion. We found that hAD-MSCs cultured in hD-PRP-SM have a greater cell elongation but express similar surface markers; in addition, hD-PRP-SM promoted a significant osteogenic differentiation in the absence of differentiation medium and increased the growth rate, maintaining chromosomal stability. In terms of cell metabolic profile, hAD-MSC behavior did not reveal any differences between both culture conditions. Conversely, significant differences in collagen I and angiopoietin 2 expression were observed between both conditions. The present results suggest that hD-PRP may influence hAD-MSC behavior.
Asunto(s)
Células Madre Mesenquimatosas , Plasma Rico en Plaquetas , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Humanos , Células Madre Mesenquimatosas/metabolismo , Osteogénesis , Plasma Rico en Plaquetas/metabolismoRESUMEN
The study of adipogenesis is essential for understanding and treating obesity, a multifactorial problem related to body fat accumulation that leads to several life-threatening diseases, becoming one of the most critical public health problems worldwide. In this review, we propose to provide the highlights of the adipogenesis study based on in vitro differentiation of human mesenchymal stem cells (hMSCs). We list in silico methods, such as molecular docking for identification of molecular targets, and in vitro approaches, from 2D, more straightforward and applied for screening large libraries of substances, to more representative physiological models, such as 3D and bioprinting models. We also describe the development of physiological models based on microfluidic systems applied to investigate adipogenesis in vitro. We intend to identify the main alternative models for adipogenesis evaluation, contributing to the direction of preclinical research in obesity. Future directions indicate the association of in silico and in vitro techniques to bring a clear picture of alternative methods based on adipogenesis as a tool for obesity research.
RESUMEN
Ischemic stroke is a major cause of death and disability, intensely demanding innovative and accessible therapeutic strategies. Approaches presenting a prolonged period for therapeutic intervention and new treatment administration routes are promising tools for stroke treatment. Here, we evaluated the potential neuroprotective properties of nasally administered human adipose tissue mesenchymal stem cell (hAT-MSC)-derived extracellular vesicles (EVs) obtained from healthy individuals who underwent liposuction. After a single intranasal EV (200 µg/kg) administered 24 h after a focal permanent ischemic stroke in rats, a higher number of EVs, improvement of the blood-brain barrier, and re-stabilization of vascularization were observed in the recoverable peri-infarct zone, as well as a significant decrease in infarct volume. In addition, EV treatment recovered long-term motor (front paws symmetry) and behavioral impairment (short- and long-term memory and anxiety-like behavior) induced by ischemic stroke. In line with these findings, our work highlights hAT-MSC-derived EVs as a promising therapeutic strategy for stroke.
Asunto(s)
Vesículas Extracelulares/trasplante , Células Madre Mesenquimatosas , Trasplante de Células Madre/métodos , Accidente Cerebrovascular/terapia , Administración Intranasal , Adulto , Animales , Barrera Hematoencefálica , Encéfalo/irrigación sanguínea , Encéfalo/patología , Prueba de Laberinto Elevado , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neovascularización Fisiológica , Ratas Wistar , Recuperación de la Función , Accidente Cerebrovascular/patologíaRESUMEN
Dermal fibroblasts (DFs) share several qualities with mesenchymal stem cell/multipotent stromal cells (MSCs) derived from various tissues, including adipose-derived stromal/stem cells (ASCs). ASCs and DFs are morphologically comparable and both cell types can be culture expanded through the utilization of their plastic-adherence properties. Despite these similar characteristics, numerous studies indicate that ASC and DF display distinct therapeutic benefits in clinical applications. To more accurately distinguish between these cell types, human DFs and ASCs isolated from three individual donors were analyzed for multipotency and cell surface marker expressions. The detection of cell surface markers, CD29, CD34, CD44, CD73, CD90, and CD105, were used for phenotypic characterization of the DFs and ASCs. Furthermore, both cell types underwent lineage differentiation based on histochemical staining and the expression of adipogenic related genes, CCAAT/Enhancer-Binding Protein alpha (CEBPα), Peroxisome proliferator-activated receptor gamma (PPARγ), UCP1, Leptin (LEP), and Adiponectin (ADIPOQ); and osteogenic related genes, Runt related transcription factor 2 (Runx2), Alkaline phosphatase (ALPL), Osteocalcin (OCN), and Osteopontin (OPN). Evidence provided by this study demonstrates similarities between donor-matched ASC and DF with respect to morphology, surface marker expression, differentiation potential, and gene expression, although appearance of enhanced adipogenesis in the ASC based solely on spectrophotometric analyses with no significant difference in real-time polymerase chain reaction detection of adipogenic biomarkers. Thus, there is substantial overlap between the ASC and DF phenotypes based on biochemical and differentiation metrics.
Asunto(s)
Tejido Adiposo , Células del Estroma , Adipogénesis , Diferenciación Celular , Células Cultivadas , Fibroblastos , Humanos , Osteogénesis , Células MadreRESUMEN
In recent years, stem cell therapy has shown promise in regenerative medicine. The lack of standardized protocols for cell isolation and differentiation generates conflicting results in this field. Mesenchymal stem cells derived from adipose tissue (ASC) and fibroblasts (FIB) share very similar cell membrane markers. In this context, the distinction of mesenchymal stem cells from fibroblasts has been crucial for safe clinical application of these cells. In the present study, we developed aptamers capable of specifically recognize ASC using the Cell-SELEX technique. We tested the affinity of ASC aptamers compared to dermal FIB. Quantitative PCR was advantageous for the in vitro validation of four candidate aptamers. The binding capabilities of Apta 2 and Apta 42 could not distinguish both cell types. At the same time, Apta 21 and Apta 99 showed a better binding capacity to ASC with dissociation constants (Kd) of 50.46 ± 2.28 nM and 72.71 ± 10.3 nM, respectively. However, Apta 21 showed a Kd of 86.78 ± 9.14 nM when incubated with FIB. Therefore, only Apta 99 showed specificity to detect ASC by total internal reflection microscopy (TIRF). This aptamer is a promising tool for the in vitro identification of ASC. These results will help understand the differences between these two cell types for more specific and precise cell therapies.
Asunto(s)
Tejido Adiposo/metabolismo , Aptámeros de Nucleótidos/farmacología , Diferenciación Celular/efectos de los fármacos , Fibroblastos/metabolismo , Células Madre Mesenquimatosas/metabolismo , Tejido Adiposo/citología , Aptámeros de Nucleótidos/química , Células Cultivadas , Fibroblastos/citología , Humanos , Células Madre Mesenquimatosas/citologíaRESUMEN
BACKGROUND: Bariatric surgery, especially Roux-en-Y gastric bypass (RYGB), is the most effective and durable treatment option for severe obesity. The mechanisms involving adipose tissue may be important to explain the effects of surgery. METHODS: We aimed to identify the genetic signatures of adipose tissue in patients undergoing RYGB. We evaluated 13 obese, non-diabetic patients (mean age 37 years, 100% women, Body mass index (BMI) 42.2 kg/m2) one day before surgery, 3 and 6 months (M) after RYGB. RESULTS: Analysis of gene expression in adipose tissue collected at surgery compared with samples collected at 3 M and 6 M Post-RYGB showed that interleukins [Interleukin 6, Tumor necrosis factor-α (TNF-α), and Monocyte chemoattractant protein-1(MCP1)] and endoplasmic reticulum stress (ERS) genes [Eukaryotic translation initiation factor 2 alpha kinase 3 (EIF2AK3) and Calreticulin (CALR)] decreased during the follow-up (P ≤ 0.01 for all). Otherwise, genes involved in energy homeostasis [Adiponectin and AMP-activated protein kinase (AMPK)], cellular response to oxidative stress [Sirtuin 1, Sirtuin 3, and Nuclear factor erythroid 2-related factor 2 (NRF2)], mitochondrial biogenesis [Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α)] and amino acids metabolism [General control nonderepressible 2 (GCN2)] increased from baseline to all other time points evaluated (P ≤ 0.01 for all). Also, expression of Peroxisome proliferator-activated receptor gamma (PPARÏ) (adipogenesis regulation) was significantly decreased after RYGB (P < 0.05). Additionally, we observed that PGC1α, SIRT1 and AMPK strongly correlated to BMI at 3 M (P ≤ 0.01 for all), as well as ADIPOQ and SIRT1 to BMI at 6 M (P ≤ 0.01 for all). CONCLUSIONS: Our findings demonstrate that weight loss is associated with amelioration of inflammation and ERS and increased protection against oxidative stress in adipose tissue. These observations are strongly correlated with a decrease in BMI and essential genes that control cellular energy homeostasis, suggesting an adaptive process on a gene expression level during the caloric restriction and weight loss period after RYGB. Trial registration CAAE: 73,585,317.0.0000.5440.
RESUMEN
Human adipose-derived stromal/stem cells (ASC) have immunomodulatory properties and the potential to differentiate into several cell lines, important for application in regenerative medicine. However, the contamination with dermal fibroblasts (FIB) can impair the beneficial effects of ASC in cell therapy. It is then essential to develop new strategies that contribute to the distinction between these two cell types. In this study, we performed functional assays, high-throughput RNA sequencing (RNA-Seq) and quantitative PCR (qPCR) to find new markers that can distinguish ASC and FIB. We showed that ASC have adipogenic and osteogenic differentiation capacity and alkaline phosphatase activity, not observed in FIB. Gene expression variation analysis identified more than 2000 differentially expressed genes (DEG) between these two cell types. We validated 16 genes present in the list of DEG, including the alkaline phosphatase gene (ALPL). In conclusion, we showed that ASC and FIB have distinct biological properties as demonstrated by alkaline phosphatase activity and differentiation capacity, besides having different gene expression profiles. SIGNIFICANCE OF THE STUDY: Although many differences between stromal stem cells derived from human adipose tissue (ASC) and human dermal fibroblasts (FIB) are described, it is still difficult to find specific markers to differentiate them. This problem can interfere with the therapeutic use of ASC. This work aimed to find new markers to differentiate these two cell populations. Our findings suggest that these cells can be distinguished by biological and molecular characteristics, such as adipogenic and osteogenic differentiation, alkaline phosphatase activity and differential gene expression profiles. The DEG were related to the regulation of the cell cycle, development process, structural organization of the cell and synthesis of the extracellular matrix. This study helps to find new cellular markers to distinguish the two populations and to better understand the properties of these cells, which can improve cell therapy.
Asunto(s)
Tejido Adiposo/metabolismo , Dermis/metabolismo , Fibroblastos/metabolismo , RNA-Seq , Células Madre/metabolismo , Tejido Adiposo/citología , Dermis/citología , Fibroblastos/citología , Humanos , Especificidad de Órganos , Células Madre/citología , Células del Estroma/citología , Células del Estroma/metabolismoRESUMEN
Obesity is a chronic inflammatory disease associated with adipose tissue macrophage (ATM) activation. ATMs from lean mice contribute to tissue homeostasis by their M2-oriented polarization, whereas obesity leads to an increase of M1 inflammatory ATMs that underlies obesity-related metabolic disorders. In humans, studies characterizing ATMs and their functional status are limited. Here we investigated ATM phenotype in visceral (VAT) and subcutaneous (SAT) adipose tissue from healthy lean and obese individuals using two molecules previously identified as markers of M1-like and M2-like/tissue-resident macrophages, the C-type lectin CLEC5A and the scavenger receptor CD163L1, respectively. CD163L1 was expressed by the majority of ATMs, and CD163L1+ ATM density was greater with respect to cells expressing the pan-macrophage markers CD68 or CD11b. ATM counts in SAT, but not in VAT, increased in obese compared to lean individuals, measured with the three markers. Accordingly, CD163L1, CD68 and ITGAM gene expression was significantly enhanced in obese with respect to control individuals only in SAT. CLEC5A+ ATMs had a proinflammatory profile and were abundant in the lean VAT, but their density diminished in obesity. The only ATM subset that increased its counts in the obese VAT had a mixed M1-like (CD11c+ CD163- CD209- ) and M2-like (CLEC5A- CD206+ ) phenotype. ATM expansion was dominated by a subset of M2-like macrophages (CD11c- CLEC5A- CD163+ CD206+ CD209+ ) in the obese SAT, with a minor contribution of a CD11c+ CLEC5A- ATM subpopulation. Thus, both SAT and VAT seems to limit inflammation during obesity by differentially altering their ATM subset composition.
Asunto(s)
Grasa Intraabdominal/citología , Macrófagos/citología , Obesidad , Grasa Subcutánea/citología , Humanos , Inflamación , Lectinas Tipo C , Activación de Macrófagos , Glicoproteínas de Membrana , Obesidad/inmunología , Receptores de Superficie Celular , Receptores DepuradoresRESUMEN
DDX6 helicase is an RNA-binding protein involved in different aspects of gene expression regulation. The roles played by DDX6 depend on the complexes associated with it. Here, for the first time, we characterize the protein complexes associated with DDX6 in human adipose tissue-derived stem cells (hASCs) and analyze the dynamics of this helicase under different conditions of translational activity and differentiation. The results obtained demonstrated that the DDX6 helicase is associated with proteins involved in the control of mRNA localization, translation and metabolism in hASCs. DDX6 complexes may also assemble into more complex structures, such as RNA-dependent granules, the abundance and composition of which change upon inhibited translational activity. This finding supports the supposition that DDX6 is possibly involved in the regulation of the mRNA life cycle in hASCs. Although there was no significant variation in the protein composition of these complexes during early adipogenic or osteogenic induction, there was a change in the distribution pattern of DDX6: the number of DDX6 granules per cell was reduced during adipogenesis and was enhanced during osteogenesis.
Asunto(s)
Adipogénesis , Tejido Adiposo/citología , Proteínas Portadoras/metabolismo , ARN Helicasas DEAD-box/metabolismo , Osteogénesis , Proteínas Proto-Oncogénicas/metabolismo , Células Madre/citología , Células Madre/metabolismo , Adipogénesis/genética , Adolescente , Adulto , Proteínas Portadoras/genética , Biología Computacional/métodos , Gránulos Citoplasmáticos/metabolismo , ARN Helicasas DEAD-box/genética , Femenino , Técnica del Anticuerpo Fluorescente , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Persona de Mediana Edad , Osteogénesis/genética , Unión Proteica , Transporte de Proteínas , Proteómica , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Adulto JovenRESUMEN
An important tool to study the regulation of gene expression is the sequencing and the analysis of different RNA fractions: total, ribosome-free, monosomal and polysomal. By comparing these different populations, it is possible to identity which genes are differentially expressed and to get information on how transcriptional and translational regulation modulates cellular function. Therefore, we used this strategy to analyze the regulation of gene expression of human adipose-derived stem cells during the triggering of the adipogenic and osteogenic differentiation. Here, we have focused on analyzing the differential expression of mRNAs during early adipogenic and osteogenic differentiation, and presented the detailed data concerning the experimental design, the RNA-Seq quality data, the raw data obtained and the RT-qPCR validation data. This information is important to confirm the accuracy of the data considering a future reuse of the data provided. Moreover, this study may be used as groundwork for future characterization of the transcriptome and the translatome regulation of different cell types.
RESUMEN
Abstract Background Mesenchymal stem cells have immense potential in stem cell-based therapies, however there is a pre-requisite to develop a curative cell dose. Adipose tissue-derived mesenchymal stem cells are promising mainly due to their potential abundance, immunomodulatory effect and remarkable differentiation potential. Nevertheless, senescence may develop during their in vitro expansion due to the incidence of genetic instability. Hence, it is important to attain an ideal balance between mesenchymal stem cell growth, quality and genetic integrity before their clinical use. Methods Stromal vascular fraction was obtained from omentum tissue of patients undergoing liposuction procedures for morbid obesity. This study standardized a closed system protocol which can be utilized for clinical grade stem cell derivation. Stages of cell growth and characterization of human adipose tissue-derived mesenchymal stem cells were also assessed along with the chromosomal stability in these in vitro cultures. Results Human adipose tissue-derived mesenchymal stem cells maintained their spindle-shaped morphology and were able to proliferate and renew, confirming their suitability for in vitro cultivation and generate clinical grade mesenchymal stem cells. Immunophenotyping indicates that the cells expressed cluster of differentiation (CD)73/CD90/CD105, mesenchymal stem-cell markers, while lacked CD34/CD45/ Human Leukocyte antigen-antigen D related (HLA-DR) expression (hematopoietic cell markers). A cell cycle study demonstrated growth kinetics under in vitro culture conditions. Human adipose tissue derived mesenchymal stem cells expressed normal cell karyotype by chromosomal G-banding indicating their genetic stability at Passage 5. Mesenchymal stem cells also demonstrated trilineage differentiation. Conclusions Availability of adipose tissue in abundance is a major advantage for clinical applications. Furthermore, detailed characterization of human adipose tissue-derived mesenchymal stem cells, their genomic stability and differentiation potential from stromal vascular fraction of human adipose tissue would help assist in tissue regeneration and repair.
Asunto(s)
Humanos , Garantía de la Calidad de Atención de Salud , Estándares de Referencia , Tejido Adiposo , Células Madre Mesenquimatosas , CariotipificaciónRESUMEN
BACKGROUND: Mesenchymal stem cells have immense potential in stem cell-based therapies, however there is a pre-requisite to develop a curative cell dose. Adipose tissue-derived mesenchymal stem cells are promising mainly due to their potential abundance, immunomodulatory effect and remarkable differentiation potential. Nevertheless, senescence may develop during their in vitro expansion due to the incidence of genetic instability. Hence, it is important to attain an ideal balance between mesenchymal stem cell growth, quality and genetic integrity before their clinical use. METHODS: Stromal vascular fraction was obtained from omentum tissue of patients undergoing liposuction procedures for morbid obesity. This study standardized a closed system protocol which can be utilized for clinical grade stem cell derivation. Stages of cell growth and characterization of human adipose tissue-derived mesenchymal stem cells were also assessed along with the chromosomal stability in these in vitro cultures. RESULTS: Human adipose tissue-derived mesenchymal stem cells maintained their spindle-shaped morphology and were able to proliferate and renew, confirming their suitability for in vitro cultivation and generate clinical grade mesenchymal stem cells. Immunophenotyping indicates that the cells expressed cluster of differentiation (CD)73/CD90/CD105, mesenchymal stem-cell markers, while lacked CD34/CD45/ Human Leukocyte antigen-antigen D related (HLA-DR) expression (hematopoietic cell markers). A cell cycle study demonstrated growth kinetics under in vitro culture conditions. Human adipose tissue derived mesenchymal stem cells expressed normal cell karyotype by chromosomal G-banding indicating their genetic stability at Passage 5. Mesenchymal stem cells also demonstrated trilineage differentiation. CONCLUSIONS: Availability of adipose tissue in abundance is a major advantage for clinical applications. Furthermore, detailed characterization of human adipose tissue-derived mesenchymal stem cells, their genomic stability and differentiation potential from stromal vascular fraction of human adipose tissue would help assist in tissue regeneration and repair.
RESUMEN
Activity of the human long interspersed nuclear elements-1 (LINE-1) retrotransposon occurs mainly in early embryonic development and during hippocampal neurogenesis. SOX-11, a transcription factor relevant to neuronal development, has unknown functions in the control of LINE-1 retrotransposon activity during neuronal differentiation. To study the dependence of LINE-1 activity on SOX-11 during neuronal differentiation, we induced differentiation of human SH-SY5Y neuroblastoma cells and adult adipose mesenchymal stem cells (hASCs) to a neuronal fate and found increased LINE-1 activity. We also show that SOX-11 protein binding to the LINE-1 promoter is higher in differentiating neuroblastoma cells, while knock-down of SOX-11 inhibits the induction of LINE-1 transcription in differentiating conditions. These results suggest that activation of LINE-1 retrotransposition during neuronal differentiation is mediated by SOX-11.
Asunto(s)
Diferenciación Celular/genética , Elementos de Nucleótido Esparcido Largo/genética , Neuronas/metabolismo , Factores de Transcripción SOXC/genética , Tejido Adiposo/citología , Línea Celular Tumoral , Células Cultivadas , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Neurogénesis/genética , Neuronas/citología , Interferencia de ARN , Factores de Transcripción SOXC/metabolismoRESUMEN
Processing stable polysaccharide membranes with suitable mechanical properties has been challenging for applications in wound healing and tissue engineering. Here we expand the characterization of pectin/chitosan (PT/CS) membranes (without covalent crosslinking), which we recently reported. Membranes containing pectin (PT) excess were formed, and PT/CS ratio can be tuned to enhance the mechanical strength, and to modulate hydrophilicity and cytocompatibility. The surface wettability and swelling properties of the polyelectrolyte complexes (PECs) played an important role to promote the attachment of stem cells. These PECs membranes have ultimate tensile strength similar to that of human skin, which is on the order of ten times higher than similar previously reported polysaccharide materials. We show for the first time that these new PT/CS membranes may promote anchorage, adhesion and support human stem cell growth, making them candidate materials for tissue engineering purposes.
Asunto(s)
Quitosano/farmacología , Pectinas/farmacología , Células Madre/citología , Adhesión Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Quitosano/química , Humanos , Tamaño de la Partícula , Pectinas/química , Espectroscopía de Fotoelectrones , Estrés Mecánico , Propiedades de Superficie , Ingeniería de Tejidos , HumectabilidadRESUMEN
We evaluated the effects of conditioned media (CMs) of human adipose tissue from renal cell carcinoma located near the tumor (hRATnT) or farther away from the tumor (hRATfT), on proliferation, adhesion and migration of tumor (786-O and ACHN) and non-tumor (HK-2) human renal epithelial cell lines. Human adipose tissues were obtained from patients with renal cell carcinoma (RCC) and CMs from hRATnT and hRATfT incubation. Proliferation, adhesion and migration were quantified in 786-O, ACHN and HK-2 cell lines incubated with hRATnT-, hRATfT- or control-CMs. We evaluated versican, adiponectin and leptin expression in CMs from hRATnT and hRATfT. We evaluated AdipoR1/2, ObR, pERK, pAkt y pPI3K expression on cell lines incubated with CMs. No differences in proliferation of cell lines was found after 24 h of treatment with CMs. All cell lines showed a significant decrease in cell adhesion and increase in cell migration after incubation with hRATnT-CMs vs. hRATfT- or control-CMs. hRATnT-CMs showed increased levels of versican and leptin, compared to hRATfT-CMs. AdipoR2 in 786-O and ACHN cells decreased significantly after incubation with hRATfT- and hRATnT-CMs vs. control-CMs. We observed a decrease in the expression of pAkt in HK-2, 786-O and ACHN incubated with hRATnT-CMs. This result could partially explain the observed changes in migration and cell adhesion. We conclude that hRATnT released factors, such as leptin and versican, could enhance the invasive potential of renal epithelial cell lines and could modulate the progression of the disease.
RESUMEN
AIMS: Several alternative cellular approaches using biomaterials to host insulin-producing cells derived from stem cells have been developed to overcome the limitations of type 1 diabetes treatment (exogenous insulin injection). However, none seem to fulfill all requirements needed to induce pancreatic cells successful colonization of the scaffolds. Here, we report a polymeric platform adherent to the native mice pancreas filled with human adipose stem cells (hASCs) that was able to induce growth of pancreatic parenchyma. MAIN METHODS: Synthetic polyether-polyurethane discs were placed adjacent to pancreas of normoglycemic and streptozotocin-induced diabetic mice. At day 4 post implantation, 1×106 hASCs were injected intra-implant in groups of normoglycemic and diabetic mice. Immunohistochemistry analysis of the implants was performed to identify insulin positive cells in the newly formed tissue. In addition, metabolic, inflammatory and angiogenic parameters were carried out in those mice. KEY FINDINGS: This study provides evidence of the ability of a biohybrid device to induce the growth of differentiated pancreas parenchyma in both normoglycemic and streptozotocin-induced diabetic mice as detected by histological analysis. Glucose metabolism and body weight of hyperglycemic mice bearing hASCs implants improved. SIGNIFICANCE: The synthetic porous scaffold bearing hASC cells placed adjacent to the native animal pancreas exhibits the potential to be exploited in future cell-based type 1 diabetes therapies.
Asunto(s)
Tejido Adiposo/metabolismo , Diabetes Mellitus Experimental , Matriz Extracelular/química , Células Secretoras de Insulina/metabolismo , Poliuretanos/química , Regeneración , Trasplante de Células Madre , Células Madre/metabolismo , Animales , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/terapia , Xenoinjertos , Humanos , Células Secretoras de Insulina/patología , Masculino , Ratones , Células Madre/patologíaRESUMEN
Tissue engineering is a multidisciplinary science that combines a structural scaffold and cells to form a construct able to promote regeneration of injured tissue. Bioactive glass foam produced by sol-gel is an osteoinductive material with a network of interconnected macropores necessary for cell colonization. The use of human adipose-derived stem cell (hASC) presents advantages as the potential for a large number of cells, rapid expansion in vitro and the capability of differentiating into osteoblasts. The use of a bioreactor in three-dimensional cell culture enables greater efficiency for cell nutrition and application of mechanical forces, important modulators of bone physiology. The hASC seeded in a bioactive glass scaffold and cultured in osteogenic Leibovitz L-15 medium in a bioreactor with a flow rate of 0.1 mL min(-1) demonstrated a significant increase in cell proliferation and viability and alkaline phosphatase (ALP) activity peak after 14 days. The immunofluorescence assay revealed an expression of osteopontin, osteocalcin and type I collagen from 7 to 21 days after culture. The cells changed from a spindle shape to a cuboidal morphology characteristic of osteoblasts. The polymerase chain reaction assay confirmed that osteopontin, osteocalcin, and ALP genes were expressed. These results indicate that hASCs differentiated into an osteogenic phenotype when cultured in bioactive glass scaffold, osteogenic Leibovitz L-15 medium and a perfusion bioreactor. Therefore, these results highlight the synergism between a bioactive glass scaffold and the effect of perfusion on cells and indicate the differentiation into an osteogenic phenotype.
Asunto(s)
Tejido Adiposo/citología , Materiales Biocompatibles/química , Reactores Biológicos , Vidrio/química , Osteogénesis , Células Madre/citología , Ingeniería de Tejidos/instrumentación , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Femenino , Humanos , Osteoblastos/citologíaRESUMEN
The objective of our study was to investigate chondrogenesis potential of human adipose-derived mesenchymal stromal cells (MSCs), using as a positive control a human source of cartilage-derived progenitor cells (PCs). This source of PCs was recently described by our group and dwells on the surface of nasoseptal cartilage. Histological analysis using Safranin O staining and immunofluorescence for actin filaments and collagen type II was performed on three-dimensional (3D) pellet cultures. Cartilage PCs and adipose MSCs showed similarities in monolayer culture related to cell morphology and proliferation. Our 3D pellet cultures substantially reduced the actin stress and after 21 days under chondrogenic medium, we observed an increase in the pellet diameter for cartilage PCs (7.4%) and adipose MSCs (21.2%). Adipose-derived MSCs responded to chondrogenic stimulus, as seen by positive areas for collagen type II, but they were not able to recreate a mature extracellular matrix. Using semi-quantitative analysis, we observed a majority of Safranin O areas rising from blue (no stain) to orange (moderate staining) and no changes in fibroblastic morphology (P < 0.0001). For cartilage PCs, chondrogenic induction is responsible for morphological changes and a high percentage of matrix area/number of cells (P ≤ 0.0001), evaluated by computerized histomorphometry. Morphological analyses reveal that adipose-derived MSCs were not able to recreate a bioengineered cartilage. The cost of culture was reduced, as the cartilage PCs under growth-factor free medium exhibit a high score for cartilage formation compared with the induced adipose mesenchymal stromal cells (P = 0.0021). Using a pellet 3D culture, our cartilage PCs were able to produce a cartilage tissue in vitro, leading to the future development of bioengineered products.