Characterization of diseased primary human hepatocytes in an all-human cell-based triculture system.

Liver diseases, including NAFLD, are a growing worldwide health concern. Currently, there is a lack of suitable in vitro models that sustain basic primary human hepatocyte (PHH) morphology and functionality while supporting presentation of disease-associated phenotypic characteristics such as lipid accumulation and inflammasome activation. In TruVivo, an all-human triculture system (hTCS), basic metabolic functions were characterized in PHHs isolated from normal or diseased livers during two-weeks of culture. Decreases in albumin and urea levels and CYP3A4 activity were seen in diseased-origin PHHs compared to normal PHHs along with higher CYP2E1 expression. Positive expression of the macrophage markers CD68 and CD163 were seen in the diseased PHH preparations. Elevated levels of the pro-inflammatory cytokines IL-6 and MCP-1 and the fibrotic markers CK-18 and TGF-ß were also measured. Gene expression of FASN, PCK1, and G6PC in the diseased PHHs was decreased compared to the normal PHHs. Further characterization revealed differences in lipogenesis and accumulation of intracellular lipids in normal and diseased PHHs when cultured with oleic acid and high glucose. TruVivo represents a promising new platform to study lipogenic mechanisms in normal and diseased populations due to the preservation of phenotypic differences over a prolonged culture period.

An increase in inflammation and islet dysfunction is a feature of prediabetes.

AIMS: Type 2 diabetes (T2D) is a global health problem that will be diagnosed in almost 300 million people by 2025 according to the World Health Organization. Before being diagnosed with T2D, individuals may have glucose levels above normal but below the diabetic range. This condition is known as prediabetes. Studies showed that people with prediabetes had an increase in several pro-inflammatory cytokines in their serum and in their fasting glucose levels. The answer remains unclear when inflammation begins in the pancreas and islets, and what is the extent of this inflammation. METHODS: Subjects with haemoglobin A1c levels from 5.7% to 6.4% were classified as pre-diabetic. Sections of pancreas and isolated islets from normal donors and donors with prediabetes were tested for markers of inflammation and glucose-stimulated insulin secretion (GSIS). RESULTS: Gene and protein expression of the inflammatory markers resistin, interleukin-1 beta, tumour necrosis factor-alpha, interleukin-6, and monocyte chemoattractant protein-1 increased in donors with prediabetes compared to normal donors. GSIS response was significantly decreased in pre-diabetic islets compared to normal islets. Donors with prediabetes also had decreased expression of CD163+ cells but not CD68+ cells. CONCLUSIONS: Based on our findings, inflammation and islet dysfunction may be more significant than originally thought in people with prediabetes. Rather than being in a normal state before diabetes occurs, it appears that subjects are already in an early diabetic condition resembling more closely T2D.

Human Placenta-Derived ECM Supports Tri-Lineage Differentiation of Human Induced Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) hold great promise for future applications in drug discovery and cell therapies. hPSC culture protocols require specific substrates and medium supplements to support cell expansion and lineage specific differentiation. The animal origin of these substrates is a severe limitation when considering the translation of hPSC derivatives to the clinic and in vitro disease modeling. The present study evaluates the use of a human placenta-derived extracellular matrix (ECM) hydrogel, HuGentraⓇ, to support tri-lineage differentiation of human induced pluripotent stem cells (hiPSCs). Lineage-specific embryoid bodies (EBs) were plated onto three separate matrices, and differentiation efficiency was evaluated based on morphology, protein, and gene expression. HuGentra was found to support the differentiation of hiPSCs to all three germ layers: ectodermal, mesodermal, and endodermal lineages. hiPSCs differentiated into neurons, cardiomyocytes, and hepatocytes on HuGentra had similar morphology, protein, and gene expression compared to differentiation on Matrigel or other cell preferred matrices. HuGentra can be considered as a suitable human substrate for hiPSC differentiation.

Scaffold-free bioprinted osteogenic and chondrogenic systems to model osteochondral physiology.

Three-dimensional bioprinted culture platforms mimic the native microenvironment of tissues more accurately than two-dimensional cell cultures or animal models. Scaffold-free bioprinting eliminates many complications associated with traditional scaffold-dependent printing as well as provides better cell-to-cell interactions and long-term functionality. In this study, constructs were produced from bone marrow derived mesenchymal stem cells (BM-MSCs) using a scaffold-free bioprinter. These constructs were cultured in either osteogenic, chondrogenic, a 50:50 mixture of osteogenic and chondrogenic ('osteo-chondro'), or BM-MSC growth medium. Osteogenic and chondrogenic differentiation capacity was determined over an 8-week culture period using histological and immunohistochemical staining and RT-qPCR (Phase I). After 6 weeks in culture, individual osteogenic and chondrogenic differentiated constructs were adhered to create a bone-cartilage interaction model. Adhered differentiated constructs were cultured for an additional 8 weeks in either chondrogenic or osteo-chondro medium to evaluate sustainability of lineage specification and transdifferentiation potential (Phase II). Constructs cultured in their respective osteogenic and/or chondrogenic medium differentiated directly into bone (model of intramembranous ossification) or cartilage. Positive histological and immunohistochemical staining for bone or cartilage identification was shown after 4 and 8 weeks in culture. Expression of osteogenesis and chondrogenesis associated genes increased between weeks 2 and 6. Adhered individual osteogenic and chondrogenic differentiated constructs sustained their differentiated phenotype when cultured in chondrogenic medium. However, adhered individual chondrogenic differentiated constructs cultured in osteo-chondro medium were converted to bone (model of metaplastic transformation). These bioprinted models of bone-cartilage interaction, intramembranous ossification, and metaplastic transformation of cartilage into bone offer a useful and promising approach for bone and cartilage tissue engineering research. Specifically, these models can be potentially used as functional tissue systems for studying osteochondral defect repair, drug discovery and response, and many other potential applications.

Multiple roles of soluble sugars in the establishment of Gunnera-Nostoc endosymbiosis.

Gunnera plants have the unique ability to form endosymbioses with N(2)-fixing cyanobacteria, primarily Nostoc. Cyanobacteria enter Gunnera through transiently active mucilage-secreting glands on stems. We took advantage of the nitrogen (N)-limitation-induced gland development in Gunnera manicata to identify factors that may enable plant tissue to attract and maintain cyanobacteria colonies. Cortical cells in stems of N-stressed Gunnera plants were found to accumulate a copious amount of starch, while starch in the neighboring mature glands was nearly undetectable. Instead, mature glands accumulated millimolar concentrations of glucose (Glc) and fructose (Fru). Successful colonization by Nostoc drastically reduced sugar accumulation in the surrounding tissue. Consistent with the abundance of Glc and Fru in the gland prior to Nostoc colonization, genes encoding key enzymes for sucrose and starch hydrolysis (e.g. cell wall invertase, α-amylase, and starch phosphorylase) were expressed at higher levels in stem segments with glands than those without. In contrast, soluble sugars were barely detectable in mucilage freshly secreted from glands. Different sugars affected Nostoc's ability to differentiate motile hormogonia in a manner consistent with their locations. Galactose and arabinose, the predominant constituents of polysaccharides in the mucilage, had little or no inhibitory effect on hormogonia differentiation. On the other hand, soluble sugars that accumulated in gland tissue, namely sucrose, Glc, and Fru, inhibited hormogonia differentiation and enhanced vegetative growth. Results from this study suggest that, in an N-limited environment, mature Gunnera stem glands may employ different soluble sugars to attract Nostoc and, once the cyanobacteria are internalized, to maintain them in the N(2)-fixing vegetative state.