Stem cell-derived liver cells for drug testing and disease modeling

Abstract

Differences between animals and humans in liver pathways now necessitate the use of in vitro models of the human liver for several applications such as drug screening. However, isolated primary human hepatocytes (PHHs) are a limited resource for building such models given shortages of donor organs. In contrast, human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) can be propagated nearly indefinitely and differentiated into hepatocyte-like cells in vitro using soluble factors inspired from liver development. Additionally, iPSCs can be generated from patients with specific genetic backgrounds to study genotype-phenotype relationships. While current protocols to differentiate hESCs and iPSCs into human hepatocyte-like cells (hESC-HHs and iPSC-HHs) still need improvement to yield cells functionally similar to the adult liver, proof-of-concept studies have already shown utility of these cells in drug development and modeling liver diseases such as α1-antitrypsin deficiency, hepatitis B/C viral infections, and malaria. Here, we present an overview of hESC-HH and iPSC-HH culture platforms that have been utilized for the aforementioned applications. We also discuss the use of semiconductor-driven microfabrication tools to precisely control the microenvironment around these cells to enable higher and longer-term liver functions in vitro. Finally, we discuss areas for improvement in creating next generation stem cell-derived liver models. In the future, stem cell-derived hepatocyte-like cells could provide a sustainable cell source for high-throughput drug screening, enabling better mechanistic understanding of human liver diseases for the development of more efficacious and safer therapeutics, and personalized cell-based therapies in the clinic.

Figure 1. Generation of human hepatocyte-like cells from somatic cells

(A) Flow diagram of various methods for generating human hepatocyte-like cells. (Top) A series of growth factors and small molecules convert somatic cells into endoderm progenitor cells, which then enter hepatic differentiation (Zhu et al., 2014). (Middle) Somatic cells are converted to an induced pluripotent stem cell (iPSC) intermediate via non-integrating plasmid vectors (i.e. OCT3/4, SOX2, C-MYC, KLF4, NANOG, LIN28) and differentiated down the hepatic lineage (Schwartz et al., 2014). (Bottom) Somatic cells are converted directly to hepatocyte like cells via transcription factors (Huang et al., 2014). (B) Typical protocol for the directed differentiation of iPSCs down the hepatic lineage along with representative growth factors used by various groups (Schwartz et al., 2014; Gerbal-Chaloin et al., 2014).

Figure 2. Engineered cultures of induced pluripotent stem cell-derived human hepatocyte-like cells (iPSC-HHs)

(A) Generation of iPSC-HH-based micropatterned co-cultures (iMPCCs) with stromal fibroblasts. Tissue culture polystyrene (TCPS) wells coated with collagen and then subjected to soft lithography-based patterning. iPSC-HHs are seeded to fill the islands, and 3T3-J2 murine embryonic fibroblasts complete the iMPCC model (Berger et al., 2014). Adapted with permission from Wiley. (B) Nanopillar plate used to generate 3D spheroids of iPSC-HHs (Takayama et al., 2013). Adapted with permission from Elsevier.

Figure 3. Utility of induced pluripotent stem cell-derived human hepatocyte-like cells (iPSC-HHs) in preclinical drug toxicity screens

(A) Albumin secretions in micropatterned co-cultures (iMPCCs) containing iPSC-HHs and 3T3-J2 murine embryonic fibroblasts after treatment for 8 days with either diclofenac (hepatotoxic) or aspirin (non-toxic) at various doses. Data was normalized to dimethyl sulfoxide (DMSO)-only controls. C_max represents the maximum concentration of a given drug measured in human plasma. (B) Representative images of iMPCCs before and after treatment with drugs. DMSO-treated cultures did not appear morphologically different than aspirin-treated cultures. Scale bars are 400 µm. (C) Comparison of sensitivity (percent of 37 clinical liver toxins correctly identified in various in vitro models) and specificity (percent of 10 non-liver-toxic drugs correctly identified) in extracellular matrix (ECM) sandwich cultures of primary human hepatocytes (SCHH), micropatterned co-cultures containing primary human hepatocytes and 3T3-J2 fibroblasts (PHH-MPCCs), and iMPCCs (Khetani et al., 2013; Ware et al., 2015).

Figure 4. Drug screening platform development and infection of induced pluripotent stem cell-derived human hepatocyte-like cells (iPSC-HHs) with malaria parasite

(A) Flow diagram of infectious disease-specific drug screening platform development. (B) Characterization of iPSC-HH morphology and host entry factors for malaria sporozoite infection. (C) Representative fluorescent images of iPSC-HHs (large blue nuclei) infected with sporozoites from a human-specific malaria strain (red), Plasmodium falciparum, at day 3 (left image) and day 6 post infection (middle image). Right image shows immunofluorescence of the Plasmodium maturation marker, MSP1 (malaria merozoite surface protein 1), in iPSC-HHs at day 6 post infection. The right panel shows the size distributions of P. falciparum exoerythrocytic forms (EEFs), or colonies, in iPSC-HHs at day 4 and day 6 post infection. Scale bars are 5 µm. (D) iPSC-HHs were treated with small molecule maturation factor, FPH1 (functional proliferation hit 1), or solvent vehicle, DMSO (dimethyl sulfoxide), to enable iPSC-HHs metabolism of primaquine (PQ), a prototypical anti-malaria drug. Small molecule treatment potentially enabled PQ metabolism and subsequent inhibition of P. falciparum propagation in iPSC-HHs. Adapted from (Ng et al., 2015) under the Creative Commons Attribution 4.0 license.