Alternative Methods as Tools for Obesity Research: In Vitro and In Silico Approaches.

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.

Using inhibition of the adipogenesis of adipose-derived stem cells for toxicity prediction.

In vitro stem cell models are used as alternatives to animal models and are important tools for cytotoxicity studies. Researchers can determine the effects of test substances on human cells by evaluating cell viability and differentiation. Here, we describe an in vitro model to quantify adipogenesis based on the Nile red staining of specific lipid droplets and the emission of basic lipids from human adipose tissue-derived mesenchymal stromal cells (AD-MSCs) in the presence of test substances.

Dichloroacetate and Pyruvate Metabolism: Pyruvate Dehydrogenase Kinases as Targets Worth Investigating for Effective Therapy of Toxoplasmosis.

Toxoplasmosis, a protozoan infection caused by , is estimated to affect around 2.5 billion people worldwide. Nevertheless, the side effects of drugs combined with the long period of therapy usually result in discontinuation of the treatment.

The inhibition of adipogenesis via an in vitro assay can reduce animal use by more precisely estimating the starting dose for the acute toxic class method.

In the present work, we established an adipogenesis inhibition assay as an adequate and sensitive in vitro model for reducing animal use by estimating the starting dose for the acute toxic class (ATC) method. First, human adipose-derived stem cells (ADSCs) underwent adipogenic differentiation induction for 14 days. Then, by high-content imaging analysis, we determined the percentage and area of cell differentiation that we considered suitable for negative and positive internal control according to the quality control criteria strictly standardized mean difference (SSMD) and robust SSMD.

Human adipose-derived stem cells (ADSC) and human periodontal ligament stem cells (PDLSC) as cellular substrates of a toxicity prediction assay.

With the increasing need to develop in vitro assays to replace animal use, human stem cell-derived methods are emerging and showing outstanding contributions to the toxicological screening of substances. Adult human stem cells such as adipose-derived stem cells (ADSC) and periodontal ligament stem cells (PDLSC) were used as cell substrates for a cytotoxicity assay and toxicity prediction using the neutral red uptake (NRU) assay. First, primary cell cultures from three independent donors, from each tissue source, were characterized as mesenchymal stem cells (MSC) by plastic adherence and appropriate immunophenotype for MSC markers (positive for CD90, CD73, and CD105 and negative for CD11b, CD34, CD45, HLADR, and CD19).

Tissue-Derived Signals for Mesenchymal Stem Cell Stimulation: Role of Cardiac and Umbilical Cord Microenvironments.

The tissue microenvironment regulates such stem cell behaviors as self-renewal and differentiation. Attempts to mimic components of these microenvironments could provide new strategies for culturing and directing the behavior of stem cells. The aim of the present study was to mimic cardiac and umbilical cord tissue microenvironments in vitro and compare the resulting bone marrow-derived mesenchymal stem cell (BM-MSC) behaviors.

Metabolic switches during the first steps of adipogenic stem cells differentiation.

The understanding of metabolism during cell proliferation and commitment provides a greater insight into the basic biology of cells, allowing future applications. Here we evaluated the energy and oxidative changes during the early adipogenic differentiation of human adipose tissue-derived stromal cells (hASCs). hASCs were maintained under differentiation conditions during 3 and 7days.

The use of human adipose-derived stem cells based cytotoxicity assay for acute toxicity test.

Human adipose-derived stem cells (ADSC) were evaluated as cell culture model for cytotoxicity assay and toxicity prediction by using the neutral red uptake assay (NRU). In this study, we compared ADSC and the murine cell line BALB/c 3T3 clone A31 to predict the toxicity of 12 reference substances as recommended by the Interagency Coordinating Committee on the Validation of Alternative Methods. We predicted the LD50 for RC-rat-only weight and RC-rat-only millimole regressions for both cell culture models.

Polysome profiling shows the identity of human adipose-derived stromal/stem cells in detail and clearly distinguishes them from dermal fibroblasts.

Although fibroblasts and multipotent stromal/stem cells, including adipose-derived stromal cells (ADSCs), have been extensively studied, they cannot be clearly distinguished from each other. We, therefore, investigated the cellular and molecular characteristics of ADSCs and fibroblasts. ADSCs and fibroblasts share several morphological similarities and surface markers, but were clearly found to be different types of cells.

Polysome profiling shows extensive posttranscriptional regulation during human adipocyte stem cell differentiation into adipocytes.

Adipocyte stem cells (hASCs) can proliferate and self-renew and, due to their multipotent nature, they can differentiate into several tissue-specific lineages, making them ideal candidates for use in cell therapy. Most attempts to determine the mRNA profile of self-renewing or differentiating stem cells have made use of total RNA for gene expression analysis. Several lines of evidence suggest that self-renewal and differentiation are also dependent on the control of protein synthesis by posttranscriptional mechanisms.

Developments on drug discovery and on new therapeutics: highly diluted tinctures act as biological response modifiers.

Background: In the search for new therapies novel drugs and medications are being discovered, developed and tested in laboratories. Highly diluted substances are intended to enhance immune system responses resulting in reduced frequency of various diseases, and often present no risk of serious side-effects due to its low toxicity. Over the past years our research group has been investigating the action of highly diluted substances and tinctures on cells from the immune system.

Mercurius solubilis: actions on macrophages.

Background: Macrophages play central roles in homeostasis as well as host defence in innate and acquired immunity, auto-immunity and immunopathology. Our research group has demonstrated the effects of highly diluted toxic substances in macrophages.

Aim: To investigate if highly diluted Mercurius solubilis (Merc sol), can activate or modulate macrophage functions.

In vitro and in vivo anticancer properties of a Calcarea carbonica derivative complex (M8) treatment in a murine melanoma model.

Background: Melanoma is the most aggressive form of skin cancer and the most rapidly expanding cancer in terms of worldwide incidence. Chemotherapeutic approaches to treat melanoma have had only marginal success. Previous studies in mice demonstrated that a high diluted complex derived from Calcarea carbonica (M8) stimulated the tumoricidal response of activated lymphocytes against B16F10 melanoma cells in vitro.

Treatment with at homeopathic complex medication modulates mononuclear bone marrow cell differentiation.

A homeopathic complex medication (HCM), with immunomodulatory properties, is recommended for patients with depressed immune systems. Previous studies demonstrated that the medication induces an increase in leukocyte number. The bone marrow microenvironment is composed of growth factors, stromal cells, an extracellular matrix and progenitor cells that differentiate into mature blood cells.

Stimulation of lymphocyte anti-melanoma activity by co-cultured macrophages activated by complex homeopathic medication.

Background: Melanoma is the most aggressive form of skin cancer, and the most rapidly expanding cancer in terms of worldwide incidence. Chemotherapeutic approaches to treat melanoma have been uniformly disappointing. A Brazilian complex homeopathic medication (CHM), used as an immune modulator, has been recommended for patients with depressed immune systems.

Activation of mononuclear bone marrow cells treated in vitro with a complex homeopathic medication.

Canova is a Brazilian homeopathic medication with immunomodulatory properties, recommended for patients where the immune system is depressed. Previous studies demonstrated that Canova induces up-regulation in numbers of leukocytes. The bone marrow microenvironment is composed of growth factors, stromal cells, extracellular matrix and progenitor cells that differentiate into mature blood cells.

Activation of bone marrow cells treated with Canova in vitro.

Canova is a Brazilian complex homeopathic medication produced from Aconitum, Thuya, Bryonia, Lachesis and Arsenicum. Previous studies demonstrated that Canova induces up-regulation in numbers of leukocytes. The bone marrow microenvironment is composed of growth factors, stromal cells, extracellular matrix, and progenitor cells that differentiate into mature blood cells.