FOXA1/2 depletion drives global reprogramming of differentiation state and metabolism in a human liver cell line and inhibits differentiation of human stem cell-derived hepatic progenitor cells.

FOXA factors are critical members of the developmental gene regulatory network (GRN) composed of master transcription factors (TF) which regulate murine cell fate and metabolism in the gut and liver. How FOXA factors dictate human liver cell fate, differentiation, and simultaneously regulate metabolic pathways is poorly understood. Here, we aimed to determine the role of FOXA2 (and FOXA1 which is believed to compensate for FOXA2) in controlling hepatic differentiation and cell metabolism in a human hepatic cell line (HepG2). siRNA mediated knockdown of FOXA1/2 in HepG2 cells significantly downregulated albumin (p < .05) and GRN TF gene expression (HNF4α, HEX, HNF1ß, TBX3) (p < .05) and significantly upregulated endoderm/gut/hepatic endoderm markers (goosecoid [GSC], FOXA3, and GATA4), gut TF (CDX2), pluripotent TF (NANOG), and neuroectodermal TF (PAX6) (p < .05), all consistent with partial/transient reprograming. shFOXA1/2 targeting resulted in similar findings and demonstrated evidence of reversibility of phenotype. RNA-seq followed by bioinformatic analysis of shFOXA1/2 knockdown HepG2 cells demonstrated 235 significant downregulated genes and 448 upregulated genes, including upregulation of markers for alternate germ layers lineages (cardiac, endothelial, muscle) and neurectoderm (eye, neural). We found widespread downregulation of glycolysis, citric acid cycle, mitochondrial genes, and alterations in lipid metabolism, pentose phosphate pathway, and ketogenesis. Functional metabolic analysis agreed with these findings, demonstrating significantly diminished glycolysis and mitochondrial respiration, with concomitant accumulation of lipid droplets. We hypothesized that FOXA1/2 inhibit the initiation of human liver differentiation in vitro. During human pluripotent stem cells (hPSC)-hepatic differentiation, siRNA knockdown demonstrated de-differentiation and unexpectedly, activation of pluripotency factors and neuroectoderm. shRNA knockdown demonstrated similar results and activation of SOX9 (hepatobiliary). These results demonstrate that FOXA1/2 controls hepatic and developmental GRN, and their knockdown leads to reprogramming of both differentiation and metabolism, with applications in studies of cancer, differentiation, and organogenesis.

The science and engineering of stem cell-derived organoids-examples from hepatic, biliary, and pancreatic tissues.

The field of organoid engineering promises to revolutionize medicine with wide-ranging applications of scientific, engineering, and clinical interest, including precision and personalized medicine, gene editing, drug development, disease modelling, cellular therapy, and human development. Organoids are a three-dimensional (3D) miniature representation of a target organ, are initiated with stem/progenitor cells, and are extremely promising tools with which to model organ function. The biological basis for organoids is that they foster stem cell self-renewal, differentiation, and self-organization, recapitulating 3D tissue structure or function better than two-dimensional (2D) systems. In this review, we first discuss the importance of epithelial organs and the general properties of epithelial cells to provide a context and rationale for organoids of the liver, pancreas, and gall bladder. Next, we develop a general framework to understand self-organization, tissue hierarchy, and organoid cultivation. For each of these areas, we provide a historical context, and review a wide range of both biological and mathematical perspectives that enhance understanding of organoids. Next, we review existing techniques and progress in hepatobiliary and pancreatic organoid engineering. To do this, we review organoids from primary tissues, cell lines, and stem cells, and introduce engineering studies when applicable. We discuss non-invasive assessment of organoids, which can reveal the underlying biological mechanisms and enable improved assays for growth, metabolism, and function. Applications of organoids in cell therapy are also discussed. Taken together, we establish a broad scientific foundation for organoids and provide an in-depth review of hepatic, biliary and pancreatic organoids.

In Vivo Differentiation of Stem Cell-derived Human Pancreatic Progenitors to Treat Type 1 Diabetes.

Type 1 diabetes mellitus (T1DM) is an autoimmune disease that results from the loss of the pancreatic ß-cells. The autoimmune destruction of the ß-cells causes the loss of insulin production from the islets of the pancreas, resulting in the loss of blood glucose regulation. This loss of regulation, if not treated, can lead to a plethora of long-term complications in patients. Subsequently, T1DM patients rely on the administration of exogenous insulin sources to maintain their blood glucose levels. In this review, we summarize the history of T1DM therapy and current treatment options. Although treatments for T1DM have progressed substantially, none of the available treatment options allow the patient to live autonomously. Therefore, the challenge to develop a therapy that will fully reverse the disease still remains. A promising field of T1DM therapies is cell replacement therapies derived from human pluripotent stem cells. Here, we specifically review studies that employ stem-cell derived pancreatic progenitors transplanted for in vivo differentiation/maturation and discuss, in detail, the complications that arise post transplantation, including heterogeneity, graft immaturity, and host foreign bodyresponse. We also discuss efforts to induce human stem cell-derived mature ß-cells in vitro and compare strategies regarding transplantation of pancreatic progenitors versus mature ß-cells cells. Finally, we review key approaches that address critical limitations of in vivo progenitor differentiation including vascularization, oxygenation, and transplant location. The field of islet replacement therapy has made tremendous progress in the last two decades. If the strengths and limitations of the field continue to be identified and addressed, future studies will lead to an ideal treatment for T1DM. Graphical abstract.

The role of PAK1 in the sensitivity of kidney epithelial cells to ischemia-like conditions.

Kidney ischemia, characterized by insufficient supply of oxygen and nutrients to renal epithelial cells, is the main cause of acute kidney injury and an important contributor to mortality world-wide. Earlier research implicated a G-protein coupled receptor (NK1R) in the death of kidney epithelial cells in ischemia-like conditions. P21-associated kinase 1 (PAK1) is involved in signalling by several G-proteins. We explored the consequences of PAK1 inhibition for cell survival under the conditions of reduced glucose and oxygen. Inhibition of PAK1 by RNA interference, expression of a dominant-negative mutant or treatment with small molecule inhibitors greatly reduced the death of cultured kidney epithelial cells. Similar protection was achieved by treating the cells with inhibitors of MEK1, in agreement with the prior reports on PAK1-MEK1 connection. Concomitant inhibition of NK1R and PAK1 offered no better protection than inhibition of NK1R alone, consistent with the two proteins being members of the same pathway. Furthermore, NK1R, PAK and MEK inhibitors reduced the induction of TRAIL in ischemia-like conditions. Considering the emerging role of TRAIL in ischemia-mediated cell death, this phenomenon may contribute to the protective effects of these small molecules. Our findings support further exploration of PAK and MEK inhibitors as possible agents to avert ischemic kidney injury.

Optimization of norovirus virus-like particle production in Pichia pastoris using a real-time near-infrared bioprocess monitor.

The production of norovirus virus-like particles (NoV VLPs) displaying NY-ESO-1 cancer testis antigen in Pichia pastoris BG11 Mut(+) has been enhanced through feed-strategy optimization using a near-infrared bioprocess monitor (RTBio(®) Bioprocess Monitor, ASL Analytical, Inc.), capable of monitoring and controlling the concentrations of glycerol and methanol in real-time. The production of NoV VLPs displaying NY-ESO-1 in P. pastoris has potential as a novel cancer vaccine platform. Optimization of the growth conditions resulted in an almost two-fold increase in the expression levels in the fermentation supernatant of P. pastoris as compared to the starting conditions. We investigated the effect of methanol concentration, batch phase time, and batch to induction transition on NoV VLP-NY-ESO-1 production. The optimized process included a glycerol transition phase during the first 2 h of induction and a methanol concentration set point of 4 g L(-1) during induction. Utilizing the bioprocess monitor to control the glycerol and methanol concentrations during induction resulted in a maximum NoV VP1-NY-ESO-1 yield of 0.85 g L(-1) . © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:518-526, 2016.