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.

3D Bioprinted Osteogenic Tissue Models for In Vitro Drug Screening.

Metabolic bone disease affects hundreds of millions of people worldwide, and as a result, in vitro models of bone tissue have become essential tools to help analyze bone pathogenesis, develop drug screening, and test potential therapeutic strategies. Drugs that either promote or impair bone formation are in high demand for the treatment of metabolic bone diseases. These drugs work by targeting numerous signaling pathways responsible for regulating osteogenesis such as Hedgehog, Wnt/ß-catenin, and PI3K-AKT. In this study, differentiated bone marrow-derived mesenchymal stem cell (BM-MSC) scaffold-free 3D bioprinted constructs and 2D monolayer cultures were utilized to screen four drugs predicted to either promote (Icariin and Purmorphamine) or impair osteogenesis (PD98059 and U0126). Osteogenic differentiation capacity was analyzed over a four week culture period by evaluating mineralization, alkaline phosphatase (ALP) activity, and osteogenesis related gene expression. Responses to drug treatment were observed in both 3D differentiated constructs and 2D monolayer cultures. After four weeks in culture, 3D differentiated constructs and 2D monolayer cultures treated with Icariin or Purmorphamine showed increased mineralization, ALP activity, and the gene expression of bone formation markers (BGLAP, SSP1, and COL1A1), signaling molecules (MAPK1, WNT1, and AKT1), and transcription factors (RUNX2 and GLI1) that regulate osteogenic differentiation relative to untreated. 3D differentiated constructs and 2D monolayer cultures treated with PD98059 or U0126 showed decreased mineralization, ALP activity, and the expression of the aforementioned genes BGLAP, SPP1, COL1A1, MAPK1, AKT1, RUNX2, and GLI1 relative to untreated. Differences in ALP activity and osteogenesis related gene expression relative to untreated cells cultured in a 2D monolayer were greater in 3D constructs compared to 2D monolayer cultures. These findings suggest that our bioprinted bone model system offers a more sensitive, biologically relevant drug screening platform than traditional 2D monolayer in vitro testing platforms.

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.

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.

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.

Human placenta hydrogel reduces scarring in a rat model of cardiac ischemia and enhances cardiomyocyte and stem cell cultures.

INTRODUCTION: Xenogeneic extracellular matrix (ECM) hydrogels have shown promise in remediating cardiac ischemia damage in animal models, yet analogous human ECM hydrogels have not been well development. An original human placenta-derived hydrogel (hpECM) preparation was thus generated for assessment in cardiomyocyte cell culture and therapeutic cardiac injection applications. METHODS AND RESULTS: Hybrid orbitrap-quadrupole mass spectrometry and ELISAs showed hpECM to be rich in collagens, basement membrane proteins, and regenerative growth factors (e.g. VEGF-B, HGF). Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes synchronized and electrically coupled on hpECM faster than on conventional cell culture environments, as validated by intracellular calcium measurements. In vivo, injections using biotin-labeled hpECM confirmed its spatially discrete localization to the myocardium proximal to the injection site. hpECM was injected into rat myocardium following an acute myocardium infarction induced by left anterior descending artery ligation. Compared to sham treated animals, which exhibited aberrant electrical activity and larger myocardial scars, hpECM injected rat hearts showed a significant reduction in scar volume along with normal electrical activity of the surviving tissue, as determined by optical mapping. CONCLUSION: Placental matrix and growth factors can be extracted as a hydrogel that effectively supports cardiomyocytes in vitro, and in vivo reduces scar formation while maintaining electrophysiological activity when injected into ischemic myocardium. STATEMENT OF SIGNIFICANCE: This is the first report of an original extracellular matrix hydrogel preparation isolated from human placentas (hpECM). hpECM is rich in collagens, laminin, fibronectin, glycoproteins, and growth factors, including known pro-regenerative, pro-angiogenic, anti-scarring, anti-inflammatory, and stem cell-recruiting factors. hpECM supports the culture of cardiomyocytes, stem cells and blood vessels assembly from endothelial cells. In a rat model of myocardial infarction, hpECM injections were safely deliverable to the ischemic myocardium. hpECM injections repaired the myocardium, resulting in a significant reduction in infarct size, more viable myocardium, and a normal electrophysiological contraction profile. hpECM thus has potential in therapeutic cardiovascular applications, in cellular therapies (as a delivery vehicle), and is a promising biomaterial for advancing basic cell-based research and regenerative medicine applications.

Demineralized bone matrix fibers formable as general and custom 3D printed mold-based implants for promoting bone regeneration.

INTRODUCTION: Bone repair frequently requires time-consuming implant construction, particularly when using un-formed implants with poor handling properties. We therefore developed osteoinductive, micro-fibrous surface patterned demineralized bone matrix (DBM) fibers for engineering both defect-matched and general three-dimensional implants. METHODS AND RESULTS: Implant molds were filled with demineralized human cortical bone fibers there were compressed and lyophilized, forming mechanically strong shaped DBM scaffolds. Enzyme linked immunosorbent assays and mass spectrometry confirmed that DBM fibers contained abundant osteogenic growth factors (bone morphogenetic proteins, insulin-like growth factor-I) and extracellular matrix proteins. Mercury porosimetry and mechanical testing showed interconnected pores within the mechanically stable, custom DBM fiber scaffolds. Mesenchymal stem cells readily attached to the DBM and showed increasing metabolic activity over time. DBM fibers further increased alkaline phosphatase activity in C2C12 cells. In vivo, DBM implants elicited osteoinductive potential in a mouse muscle pouch, and also promoted spine fusion in a rat arthrodesis model. SIGNIFICANCE: DBM fibers can be engineered into custom-shaped, osteoinductive and osteoconductive implants with potential for repairing osseous defects with precise fitment, potentially reducing operating time. By providing pre-formed and custom implants, this regenerative allograft may improve patient outcomes following surgical bone repair, while further advancing personalized orthopedic and craniomaxillofacial medicine using three-dimensional-printed tissue molds.