Differentiation of human-induced pluripotent stem cell under flow conditions to mature hepatocytes for liver tissue engineering.

Viktoriia Starokozhko,Layla Larsen,Soumyaranjan Mohanty,Martin Dufva,Anders Wolff,Mette Hemmingsen,Geny Groothuis,Rodrigo C Pimentel,Anders Aspegren,Marjolijn Merema,Jenny Emnéus

doi:10.1002/term.2659

Abstract

Hepatic differentiation of human‐induced pluripotent stem cells (hiPSCs) under flow conditions in a 3D scaffold is expected to be a major step forward for construction of bioartificial livers. The aims of this study were to induce hepatic differentiation of hiPSCs under perfusion conditions and to perform functional comparisons with fresh human precision‐cut liver slices (hPCLS), an excellent benchmark for the human liver in vivo. The majority of the mRNA expression of CYP isoenzymes and transporters and the tested CYP activities, Phase II metabolism, and albumin, urea, and bile acid synthesis in the hiPSC‐derived cells reached values that overlap those of hPCLS, which indicates a higher degree of hepatic differentiation than observed until now. Differentiation under flow compared with static conditions had a strong inducing effect on Phase II metabolism and suppressed AFP expression but resulted in slightly lower activity of some of the Phase I metabolism enzymes. Gene expression data indicate that hiPSCs differentiated into both hepatic and biliary directions. In conclusion, the hiPSC differentiated under flow conditions towards hepatocytes express a wide spectrum of liver functions at levels comparable with hPCLS indicating excellent future perspectives for the development of a bioartificial liver system for toxicity testing or as liver support device for patients.

Highlights

Even though fully matured primary human hepatocytes (PHH) exhibit all the specific liver functions, their limited availability and loss of liver‐specific functions during culturing in vitro are still the major limitations for their application in a bioartificial liver (BAL; Mizumoto et al, 2012; Ordovás, Park, & Verfaillie, 2013)
We investigated the hepatic differentiation of human‐induced pluripotent stem cells (hiPSCs)‐derived definitive endoderm (DE) cells in the 3D scaffold at two different flow rates, 1 and 5 μl/min, and in two different scaffold designs (Figure 1e) and compared the results with cells differentiated under static 2D conditions in standard PS well plates or in well plates coated with a PDMS layer. human precision‐cut liver slices (hPCLS) were used as a benchmark for cells differentiated in the scaffold under perfusion. hPCLS were prepared from 10 individual livers of human donors, aged 20–73 years (60% female) as described
We found that DE cells can be successfully differentiated into hepatocyte‐like cells in a 3D scaffold in a bioreactor under flow conditions, to a similar or only slightly better differentiation grade than under static 2D conditions, especially with respect to Phase II sulfation activity and a lower AFP expression, which can make the production of a BAL easier and more effective in the future

Summary

Introduction

Even though fully matured primary human hepatocytes (PHH) exhibit all the specific liver functions, their limited availability and loss of liver‐specific functions during culturing in vitro are still the major limitations for their application in a bioartificial liver (BAL; Mizumoto et al, 2012; Ordovás, Park, & Verfaillie, 2013). HiPSC‐derived hepatocyte‐like cells have been shown to have certain liver‐specific phenotypic characteristics and exhibit many of the liver‐ specific functions (Chen et al, 2012; Gieseck et al, 2014; Si‐Tayeb et al, 2010; Song et al, 2009), most of these functions are expressed at levels several magnitudes lower than in fresh liver tissue or freshly isolated human hepatocytes (Ulvestad et al, 2013), suggesting that improvements in the differentiation protocols are still warranted In most of these studies, the induced pluripotent stem cell‐derived hepatocyte‐like cells were obtained by maturation in 2D cultures, and the cells are loaded in a bioreactor only after maturation. In another study, perfusion inhibited adipogenic differentiation of adipose‐derived stem cells possibly by washing away autocrine or paracrine factors (Hemmingsen et al, 2013)

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