Stem cell-derived polarized hepatocytes

Abstract

Human stem cell-derived hepatocyte-like cells (HLCs) offer an attractive platform to study liver biology. Despite their numerous advantages, HLCs lack critical in vivo characteristics, including cell polarity. Here, we report a stem cell differentiation protocol that uses transwell filters to generate columnar polarized HLCs with clearly defined basolateral and apical membranes separated by tight junctions. We show that polarized HLCs secrete cargo directionally: Albumin, urea, and lipoproteins are secreted basolaterally, whereas bile acids are secreted apically. Further, we show that enterically transmitted hepatitis E virus (HEV) progeny particles are secreted basolaterally as quasi-enveloped particles and apically as naked virions, recapitulating essential steps of the natural infectious cycle in vivo. We also provide proof-of-concept that polarized HLCs can be used for pharmacokinetic and drug-drug interaction studies. This novel system provides a powerful tool to study hepatocyte biology, disease mechanisms, genetic variation, and drug metabolism in a more physiologically relevant setting.

Controversies Surrounding the Origin of Hepatocytes in Adult Livers and the in Vitro Generation or Propagation of Hepatocytes

“…A parallel approach to generate hepatocytes in vitro is to differentiate human pluripotent stem cells (PSCs) into hepatocyte-like cells (HepLCs) using growth factors and small molecules. [131][132][133][134][135][136][137][138][139][140][141][142][143] One major advantage of using human PSCs as a starting source of hepatocytes is that they are highly abundant 144,145 and can be propagated to generate limitless numbers of hepatocytes. A key goal in this field is to generate highly pure and fully functional hepatocytes in vitro representing as counterparts to the hepatocytes in vivo.…”

Virus–Host Cell Interplay during Hepatitis E Virus Infection

“…Within hepatocytes, polarized trafficking and secretion from MVBs results in the egress of eHEV particles from basolateral membranes to spread within the host, and from the apical membranes into the bile, where high concentrations of human bile acids and salts convert eHEV to neHEV virions. In this context, novel HEV-permissive polarized cell models will allow further study of the mechanisms and determinants of directional HEV secretion [ 64 , 65 ].…”

Cell Culture Models for Hepatitis E Virus

“…Stem cell-derived HLCs have been used to study hepatitis viruses and other hepatotropic infectious diseases. For example, HLCs support infection with HAV [96], HBV [91,97,98], and hepatitis C virus (HCV) [88,99,100,101], as well as Dengue [102,103] and Zika virus [104]. HLCs were also shown to be permissive for different Plasmodium species, including P. falciparum [105].…”

“…As described in the introduction, the HEV life cycle and transmission highly depend on hepatocyte polarization: HEV enters hepatocytes at their basolateral side, and progeny virions are mainly secreted from their apical side [96]. HEV ORF3 protein was shown to accumulate at the apical side of hepatocytes in HEV-infected humans [112] and in liver-chimeric humanized mice [26].…”