RESUMEN
Adipose tissue plays a crucial role in metabolic syndrome, autoimmune diseases, and many cancers. Because of adipose's role in so many aspects of human health, there is a critical need for in vitro models that replicate adipose architecture and function. Traditional monolayer models, despite their convenience, are limited, showing heterogeneity and functional differences compared to 3D models. While monolayer cultures struggle with detachment and inefficient differentiation, healthy adipocytes in 3D culture accumulate large lipid droplets, secrete adiponectin, and produce low levels of inflammatory cytokines. The shift from monolayer models to more complex 3D models aims to better replicate the physiology of healthy adipose tissue in culture. This study introduces a simple and accessible protocol for generating adipose organoids using a scaffold-free spheroid model. The method, utilizing either 96-well spheroid plates or agarose micromolds, demonstrates increased throughput, uniformity, and ease of handling compared to previous techniques. This protocol allows for diverse applications, including drug testing, toxin screening, tissue engineering, and co-culturing. The choice between the two methods depends on the experimental goals, with the 96-well plate providing individualized control and the micromold offering scale advantages. The outlined protocol covers isolation, expansion, and characterization of stromal vascular fraction cells, followed by detailed steps for spheroid formation and optional downstream analyses.
Asunto(s)
Adipocitos , Tejido Adiposo , Esferoides Celulares , Esferoides Celulares/metabolismo , Esferoides Celulares/citología , Tejido Adiposo/citología , Tejido Adiposo/metabolismo , Humanos , Adipocitos/metabolismo , Adipocitos/citología , Técnicas de Cultivo de Célula/métodos , Animales , Ingeniería de Tejidos/métodos , Células Cultivadas , Diferenciación Celular , RatonesRESUMEN
BACKGROUND: Immunomodulation by mesenchymal stromal cells (MSCs) can occur through trophic factor mechanisms, however, intravenously infused MSCs are rapidly cleared from the body yet a potent immunotherapeutic response is still observed. Recent work suggests that monocytes contribute to the clearance of MSCs via efferocytosis, the body's natural mechanism for clearing dead and dying cells in a non-inflammatory manner. This begs the questions of how variations in MSC quality affect monocyte phenotype and if viable MSCs are even needed to elicit an immunosuppressive response. METHODS: Herein, we sought to dissect MSC's trophic mechanism from their efferocytic mechanisms and determine if the viability of MSCs prior to efferocytosis influences the resultant phenotype of monocytes. We cultured viable or heat-inactivated human umbilical cord MSCs with human peripheral blood mononuclear cells for 24 h and observed changes in monocyte surface marker expression and secretion profile. To isolate the effect of efferocytosis from MSC trophic factors, we used cell separation techniques to remove non-efferocytosed MSCs before challenging monocytes to suppress T-cells or respond to inflammatory stimuli. For all experiments, viable and heat-inactivated efferocytic-licensing of monocytes were compared to non-efferocytic-licensing control. RESULTS: We found that monocytes efferocytose viable and heat-inactivated MSCs equally, but only viable MSC-licensed monocytes suppress activated T-cells and suppression occurred even after depletion of residual MSCs. This provides direct evidence that monocytes that efferocytose viable MSCs are immunosuppressive. Further characterization of monocytes after efferocytosis showed that uptake of viable-but not heat inactivated-MSC resulted in monocytes secreting IL-10 and producing kynurenine. When monocytes were challenged with LPS, IL-2, and IFN-γ to simulate sepsis, monocytes that had efferocytosed viable MSC had higher levels of IDO while monocytes that efferocytosed heat inactivated-MSCs produced the lowest levels of TNF-α. CONCLUSION: Collectively, these studies show that the quality of MSCs efferocytosed by monocytes polarize monocytes toward distinctive immunosuppressive phenotypes and highlights the need to tailor MSC therapies for specific indications.
Asunto(s)
Leucocitos Mononucleares , Monocitos , Humanos , Fagocitosis , Transporte Biológico , Inmunosupresores , FenotipoRESUMEN
Adipocytes regulate tissues through production of adipokines that can act both locally and systemically. Adipocytes also have been found to play a critical role in regulating the healing process. To better understand this role, we developed a three-dimensional human adipocyte spheroid system that has an adipokine profile similar to in vivo adipose tissues. Previously, we found that conditioned medium from these spheroids induces human dermal fibroblast conversion into highly contractile, collagen-producing myofibroblasts through a transforming growth factor beta-1 (TGF-ß1) independent pathway. Here, we sought to identify how mature adipocytes signal to dermal fibroblasts through adipokines to induce myofibroblast conversion. By using molecular weight fractionation, heat inactivation and lipid depletion, we determined mature adipocytes secrete a factor that is 30-100 kDa, heat labile and lipid associated that induces myofibroblast conversion. We also show that the depletion of the adipokine adiponectin, which fits those physico-chemical parameters, eliminates the ability of adipocyte-conditioned media to induce fibroblast to myofibroblast conversion. Interestingly, native adiponectin secreted by cultured adipocytes consistently elicited a stronger level of α-smooth muscle actin expression than exogenously added adiponectin. Thus, adiponectin secreted by mature adipocytes induces fibroblast to myofibroblast conversion and may lead to a phenotype of myofibroblasts distinct from TGF-ß1-induced myofibroblasts.
Asunto(s)
Miofibroblastos , Factor de Crecimiento Transformador beta1 , Humanos , Miofibroblastos/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Factor de Crecimiento Transformador beta1/metabolismo , Adiponectina/metabolismo , Transducción de Señal/fisiología , Fibroblastos/metabolismo , Adipocitos/metabolismo , Lípidos , Actinas/metabolismo , Diferenciación Celular , Células CultivadasRESUMEN
Polychlorinated biphenyl (PCB) accumulates in adipose where it may impact the growth and function of cells within the tissue. This is particularly concerning during adolescence when adipocytes expand rapidly. Herein, we sought to understand how exposure to PCB mixtures found in U.S. schools affects human adipose mesenchymal stem/stromal cell (MSC) health and function. We investigated how exposure to Aroclor 1016 and Aroclor 1254, as well as a newly characterized non-Aroclor mixture that resembles the PCB profile found in cabinets, Cabinet Mixture, affects adipose MSC growth, viability, and function in vitro. We found that exposure to all three mixtures resulted in two distinct types of toxicity. At PCB concentrations >20 µM, the majority of MSCs die, while at 1-10 µM, MSCs remained viable but display numerous alterations to their phenotype. At these sublethal concentrations, the MSC rate of expansion slowed and morphology changed. Further assessment revealed that PCB-exposed MSCs had impaired adipogenesis and a modest decrease in immunosuppressive capabilities. Thus, exposure to PCB mixtures found in schools negatively impacts the health and function of adipose MSCs. This work has implications for human health due to MSCs' role in supporting the growth and maintenance of adipose tissue.
Asunto(s)
Bifenilos Policlorados , Humanos , Bifenilos Policlorados/toxicidad , Bifenilos Policlorados/metabolismo , Arocloros/metabolismo , Arocloros/toxicidad , Tejido Adiposo , Células del Estroma/metabolismoRESUMEN
As MSC products move from early development to clinical translation, culture conditions shift from xeno- to xeno-free systems. However, the impact of isolation and culture-expansion methods on the long-term resiliency of MSCs within challenging transplant environments is not fully understood. Recent work in our lab has shown that palmitate, a saturated fatty acid elevated in the serum of patients with obesity, causes MSCs to convert from an immunosuppressive to an immunostimulatory state at moderate to high physiological levels. This demonstrated that metabolically-diseased environments, like obesity, alter the immunomodulatory efficacy of healthy donor MSCs. In addition, it highlighted the need to test MSC efficacy not only in ideal conditions, but within challenging metabolic environments. To determine how the choice of xeno- vs. xeno-free media during isolation and expansion would affect future immunosuppressive function, umbilical cord explants from seven donors were subdivided and cultured within xeno- (fetal bovine serum, FBS) or xeno-free (human platelet lysate, PLT) medias, creating 14 distinct MSC preparations. After isolation and primary expansion, umbilical cord MSCs (ucMSC) were evaluated according to the ISCT minimal criteria for MSCs. Following baseline characterization, ucMSC were exposed to physiological doses of palmitate and analyzed for metabolic health, apoptotic induction, and immunomodulatory potency in co-cultures with stimulated human peripheral blood mononuclear cells. The paired experimental design (each ucMSC donor grown in two distinct culture environments) allowed us to delineate the contribution of inherent (nature) vs. environmentally-driven (nurture) donor characteristics to the phenotypic response of ucMSC during palmitate exposure. Culturing MSCs in PLT-media led to more consistent growth characteristics during the isolation and expansion for all donors, resulting in faster doubling times and higher cell yields compared to FBS. Upon palmitate challenge, PLT-ucMSCs showed a higher susceptibility to palmitate-induced metabolic disturbance, but less susceptibility to palmitate-induced apoptosis. Most striking however, was that the PLT-ucMSCs resisted the conversion to an immunostimulatory phenotype better than their FBS counterparts. Interestingly, examining MSC suppression of PBMC proliferation at physiologic doses of palmitate magnified the differences between donors, highlighting the utility of evaluating MSC products in stress-based assays that reflect the challenges MSCs may encounter post-transplantation.