Microbial-derived lithocholic acid and vitamin K2 drive the metabolic maturation of pluripotent stem cells-derived and fetal hepatocytes.

UNLABELLED: The liver is the main organ responsible for the modification, clearance, and transformational toxicity of most xenobiotics owing to its abundance in cytochrome P450 (CYP450) enzymes. However, the scarcity and variability of primary hepatocytes currently limits their utility. Human pluripotent stem cells (hPSCs) represent an excellent source of differentiated hepatocytes; however, current protocols still produce fetal-like hepatocytes with limited mature function. Interestingly, fetal hepatocytes acquire mature CYP450 expression only postpartum, suggesting that nutritional cues may drive hepatic maturation. We show that vitamin K2 and lithocholic acid, a by-product of intestinal flora, activate pregnane X receptor (PXR) and subsequent CYP3A4 and CYP2C9 expression in hPSC-derived and isolated fetal hepatocytes. Differentiated cells produce albumin and apolipoprotein B100 at levels equivalent to primary human hepatocytes, while demonstrating an 8-fold induction of CYP450 activity in response to aryl hydrocarbon receptor (AhR) agonist omeprazole and a 10-fold induction in response to PXR agonist rifampicin. Flow cytometry showed that over 83% of cells were albumin and hepatocyte nuclear factor 4 alpha (HNF4α) positive, permitting high-content screening in a 96-well plate format. Analysis of 12 compounds showed an R(2) correlation of 0.94 between TC50 values obtained in stem cell-derived hepatocytes and primary cells, compared to 0.62 for HepG2 cells. Finally, stem cell-derived hepatocytes demonstrate all toxicological endpoints examined, including steatosis, apoptosis, and cholestasis, when exposed to nine known hepatotoxins. CONCLUSION: Our work provides fresh insights into liver development, suggesting that microbial-derived cues may drive the maturation of CYP450 enzymes postpartum. Addition of these cues results in the first functional, inducible, hPSC-derived hepatocyte for predictive toxicology.

Selection for CD26- and CD49A+ Cells From Pluripotent Stem Cells-Derived Islet-Like Clusters Improves Therapeutic Activity in Diabetic Mice.

Background: Cell therapy of diabetes aims at restoring the physiological control of blood glucose by transplantation of functional pancreatic islet cells. A potentially unlimited source of cells for such transplantations would be islet cells derived from an in vitro differentiation of human pluripotent stem cells (hESC/hiPSC). The islet-like clusters (ILC) produced by the known differentiation protocols contain various cell populations. Among these, the ß-cells that express both insulin and the transcription factor Nkx6.1 seem to be the most efficient to restore normoglycemia in diabetes animal models. Our aim was to find markers allowing selection of these efficient cells. Methods: Functional Cell-Capture Screening (FCCS) was used to identify markers that preferentially capture the cells expressing both insulin and Nkx6.1, from hESC-derived ILC cells. In order to test whether selection for such markers could improve cell therapy in diabetic mouse models, we used ILC produced from a clinical-grade line of hESC by a refined differentiation protocol adapted to up-scalable bioreactors. Re-aggregated MACS sorted cells were encapsulated in microspheres made of alginate modified to reduce foreign body reaction. Implantation was done intraperitoneally in STZ-treated C57BL/6 immuno-competent mice. Results: CD49A (integrin alpha1) was identified by FCCS as a marker for cells that express insulin (or C-peptide) as well as Nkx6.1 in ILC derived by hESC differentiation. The ILC fraction enriched in CD49A + cells rapidly reduced glycemia when implanted in diabetic mice, whereas mice receiving the CD49A depleted population remained highly diabetic. CD49A-enriched ILC cells also produced higher levels of human C-peptide in the blood of transplanted mice. However, the difference between CD49A-enriched and total ILC cells remained small. Another marker, CD26 (DPP4), was identified by FCCS as binding insulin-expressing cells which are Nkx6.1 negative. Depletion of CD26 + cells followed by enrichment for CD49A + cells increased insulin+/Nkx6.1+ cells fraction to ~70%. The CD26 - /CD49A + enriched ILC exhibited improved function over non-sorted ILC or CD49A + cells in diabetic mice and maintain prolonged blood C-peptide levels. Conclusions: Refining the composition of ILC differentiated from hPSC by negative selection to remove cells expressing CD26 and positive selection for CD49A expressing cells could enable more effective cell therapy of diabetes.

Scalable Expansion of Pluripotent Stem Cells.

Large-scale expansion of pluripotent stem cells (PSC) in a robust, well-defined, and monitored process is essential for production of cell-based therapeutic products. The transition from laboratory-scale protocols to industrial-scale production is one of the first milestones to be achieved in order to use both human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) as the starting material for cellular products. The methods to be developed require adjustment of the culture platforms, optimization of culture parameters, and adaptation of downstream procedures. Optimization of expansion protocols and their scalability has become much easier with the design of bioreactor systems that enable continuous monitoring of culture parameters, continuous media change, and support software for automated control. This chapter highlights the common properties that are required for production of scalable, reproducible, homogeneous, and clinically suitable cell therapy products. We describe the available platforms for large-scale expansion of PSCs and parameters that should be considered when optimizing the expansion protocols in a scalable bioreactor. All the above are detailed in the light of the requirements and challenges of bringing a cell-based therapeutic product to the clinic and ultimately to the market. We discuss some considerations that should be taken into account, such as cost-effectiveness, good manufacturing practice, and regulatory guidelines. Graphical Abstract.

Microfluidic Concentric Gradient Generator Design for High-Throughput Cell-Based Studies.

Gradients of diffusible signaling molecules play important role in various processes, ranging from cell differentiation to toxicological evaluation. Microfluidic technology provides an accurate control of tempospatial conditions. However, current microfluidic platforms are not designed to handle multiple gradients and cell populations simultaneously. Here, we demonstrate a rapidly adaptable microfluidic design able to expose multiple cell populations to an array of chemical gradients. Our design is based on pressure-equilibrated concentric channels and a pressure-dissipating control layer, facilitating the seeding of multiple cell populations in a single device. The design was numerically evaluated and experimentally validated. The device consists of 8 radiating stimuli channels and 12 circular cell culture channels, creating an array of 96 different continuous gradients that can be simultaneously monitored over time.