Disease modeling using human induced pluripotent stem cells: lessons from the liver

Human pluripotent stem cells (hPSCs) have the capacity to differentiate into any of the hundreds of distinct cell types that comprise the human body. This unique characteristic has resulted in considerable interest in the field of regenerative medicine, given the potential for these cells to be used to protect, repair, or replace diseased, injured, and aged cells within the human body. In addition to their potential in therapeutics, hPSCs can be used to study the earliest stages of human development and to provide a platform for both drug screening and disease modeling using human cells. Recently, the description of human induced pluripotent stem cells (hIPSCs) has allowed the field of disease modeling to become far more accessible and physiologically relevant, as pluripotent cells can be generated from patients of any genetic background. Disease models derived from hIPSCs that manifest cellular disease phenotypes have been established to study several monogenic diseases; furthermore, hIPSCs can be used for phenotype-based drug screens to investigate complex diseases for which the underlying genetic mechanism is unknown. As a result, the use of stem cells as research tools has seen an unprecedented growth within the last decade as researchers look for in vitro disease models which closely mimic in vivo responses in humans. Here, we discuss the beginnings of hPSCs, starting with isolation of human embryonic stem cells, moving into the development and optimization of hIPSC technology, and ending with the application of hIPSCs towards disease modeling and drug screening applications, with specific examples highlighting the modeling of inherited metabolic disorders of the liver. This article is part of a Special Issue entitled Linking transcription to physiology in lipodomics

Maturation of induced pluripotent stem cell derived hepatocytes by 3D-culture

Induced pluripotent stem cell derived hepatocytes (IPSC-Heps) have the potential to reduce the demand for a dwindling number of primary cells used in applications ranging from therapeutic cell infusions to in vitro toxicology studies. However, current differentiation protocols and culture methods produce cells with reduced functionality and fetal-like properties compared to adult hepatocytes. We report a culture method for the maturation of IPSC-Heps using 3-Dimensional (3D) collagen matrices compatible with high throughput screening. This culture method significantly increases functional maturation of IPSC-Heps towards an adult phenotype when compared to conventional 2D systems. Additionally, this approach spontaneously results in the presence of polarized structures necessary for drug metabolism and improves functional longevity to over 75 days. Overall, this research reveals a method to shift the phenotype of existing IPSC-Heps towards primary adult hepatocytes allowing such cells to be a more relevant replacement for the current primary standard

Pharmacological inhibition of IRAK4 kinase activity does not prevent cachexia in mice with pancreatic cancer

Abstract Background Inflammation is a hallmark of cachexia; however, effective anti‐inflammatory treatments have not yet been identified. Interleukin‐1 receptor‐associated kinase 4 (IRAK4) is a key signalling node linking interleukin‐1 receptor (IL‐1R) and toll‐like receptor (TLR) activation to the production of multiple proinflammatory cytokines that are elevated in cancer cachexia. The purpose of this work is to evaluate whether pharmacological inhibition of IRAK4 kinase activity with PF‐06426779 could prevent cachexia using a model of pancreatic cancer. The effect of appetite stimulation via the ghrelin receptor agonist anamorelin was also examined as a benchmark of clinically validated mechanisms. Methods Female C57Bl/6J mice were given an intraperitoneal injection of KrasG12D; p53R172H; Pdx1‐Cre (KPC) pancreatic tumour cells. PF‐06426779 or anamorelin treatment was initiated at the onset of anorexia. Body weight and food intake were measured throughout the study. Body composition, muscle function (force), and physical activity (treadmill running endurance) were assessed at the end of the study. Results Chronic treatment with PF‐06426779, at doses covering in vitro IC50 and IC90 at Cmin, did not increase body weight, food intake, and muscle function in the KPC tumour model. In contrast, anamorelin (vs. vehicle) increased food intake (P < 0.01), hindlimb skeletal muscle mass (P < 0.01), and muscle strength (P < 0.05); however, treadmill running endurance was not increased. Conclusions These data suggest that inhibition of IRAK4 kinase activity is not sufficient to treat cachexia, at least in pancreatic cancer, and exploration of alternative anti‐inflammatory strategies that increase appetite is required

Functional and transcriptional comparison of IPSC-Hep 3D cultures plated as single cells or clumps.

<p>(<b>A</b>) Secreted albumin, alpha-fetoprotein, and alpha-1-antitrypsin levels as evaluated by immunoassays (mean ± s.d.; n = 3 biological replicates). (<b>B</b>) qPCR heatmap of 39 hepatic genes comparing the two 3D culture conditions to adult and fetal hepatocytes (range of expression shown as sample extrema for each gene; quantitative values shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086372#pone.0086372.s002" target="_blank">Figures S2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086372#pone.0086372.s004" target="_blank">S4</a></b>). (<b>C</b>) Confocal micrograph highlighting the loss of detectable albumin in 3D single cell cultures and the spontaneous polarization of IPSC-Heps within 3D clump cultures (scalebar = 100 microns).</p

Functional comparison of IPSC-Hep 3D clump culture versus traditional 2D culture.

<p>(<b>A</b>) Oil red O and periodic acid staining demonstrating lipid storage and glycogen synthesis in both 2D and 3D clump cultures. (<b>B</b>) qPCR analysis of select phase I and phase II enzymes, hepatic transporters, and other hepatic markers demonstrating a shift towards a more mature phenotype in the 3D clump cultures (fold expression to undifferentiated IPSCs; mean ± s.d.; n = 3 biological replicates). (<b>C</b>) Confocal micrographs comparing the presence and localization of hepatic markers within the two culture systems (scale bar = 100 microns). (<b>D</b>) CYP3A4 activity of the two culture conditions measured over a period of 75 days (mean ± s.d.; n = 3 biological replicates).</p