Identification of small molecules for human hepatocyte expansion and iPS differentiation

Abstract

Cell-based therapies hold the potential to alleviate the growing burden of liver diseases. Such therapies require human hepatocytes, which, within the stromal context of the liver, are capable of many rounds of replication. However, this ability is lost ex vivo, and human hepatocyte sourcing has limited many fields of research for decades. Here we developed a high-throughput screening platform for primary human hepatocytes to identify small molecules in two different classes that can be used to generate renewable sources of functional human hepatocytes. The first class induced functional proliferation of primary human hepatocytes in vitro. The second class enhanced hepatocyte functions and promoted the differentiation of induced pluripotent stem cell-derived hepatocytes toward a more mature phenotype than what was previously obtainable. The identification of these small molecules can help address a major challenge affecting many facets of liver research and may lead to the development of new therapeutics for liver diseases.

Fig. 1. High-throughput identification of small molecules that induce proliferation and enhance functions of primary human hepatocytes

(a) High-throughput liver platform. Primary human hepatocytes (green) were seeded on a feeder layer of confluent J2-3T3 fibroblasts (red) in 384-well plates. Line graph shows representative rate of albumin secretion in screening co-cultures and hepatocyte-only cultures (green) over time. Bar graph displays albumin secretion as a function of hepatocyte density in screening cultures. Upper inset phase-contrast imaging shows morphology of feeder-layer co-cultures (scale bar = 100 µm). Far right, Hoechst staining of screening co-cultures shows that hepatocyte nuclei (green *) have uniform texture while fibroblast nuclei (red *) are punctate (scale bar = 50 µm). Automated high-content imaging assay identifies and classifies individual nuclei. (b) Chemical screening. Bar graph depicts categories of screened and hit compounds. Scatter plots display replicates of the screen, shown separately for the image-based proliferation and competitive-ELISA functional readouts. Blue and red data points represent DMSO and experimental small molecules, respectively. Boxed regions indicate hit zones. (c) Hit validation. All primary hits were retested in an 8-point dose-response curve. Active hits had increasing curves of hepatocyte nuclei counts and/or decreasing curves of competitive [Albumin] with flat curves of cell-free [Albumin]. (d) Chemical structures of hit compounds FPH1, FPH2 and FH1. All data presented as mean ± s.d.

Fig. 2. Expansion of primary human hepatocytes

(a) Primary screening data for FPH1 and FPH2. Data presented as mean ± s.d. (b) Ki67 (red) and albumin (green) immunofluorescent staining after 6 days of culture. Bar graphs show CellProfiler quantification of displayed images. (c) FACS (right) and Cellometer (left) Automated Cell Counter analysis. Fibroblasts were labeled with CM-DiI prior to initiation of culture in order to allow identification of hepatocytes via negative selection. FACS cell counting was further enabled by fluorescent counting beads. Control cultures were treated with empty vehicle (DMSO). Data presented as mean ± SEM.

Fig. 3. Functional enhancement of human primary hepatocytes and iHeps

(a) Primary screening data of FH1. (b) Primary screening data of FPH1. Control cultures (Ctrl) were treated with empty vehicle (DMSO). All data presented as mean ± s.d. (c) Morphology and colony size of FPH1- and FH1-treated iHeps in 6-well plates 9 days post-initiation-of-treatment. Untreated iHeps are shown for comparison (scale bar = 100 µm). Yellow arrows point to select bile canaliculi. (d) Albumin, CYP3A and AFP staining of iHeps after 9 days of culture (scale bar = 100um). Bar graphs represent quantifications of displayed images.

Fig. 4. Maturation of human iHeps

Gene expression profiling of FPH1-treated (a) and FH1-treated (b) iHeps. Heat map displays of Luminex analysis for 83 liver-specific genes, shown separately for independent experiments. Columns of the heatmap are averaged values of replicate (n≥3) loadings of mRNA extracted from various populations of iPS cells (250ng total RNA per replicate). mRNA expression was determined relative to the average of control gene transferrin, and heat maps are row-normalized. Bar graphs are select gene sets comparing the relative mRNA expression of small molecule-treated hepatocytes (colored bars) to iPS (light gray), untreated iHeps (gray bars) and fetal human hepatocytes (dark gray), normalized to control (black) for nuclear receptors, phase I, phase II and phase III drug metabolism genes. Control refers to adult cryopreserved human primary hepatocytes stabilized by micropatterned co-culture (Adult Control, more details in Supplementary Note 1). Data represent the mean ± SEM of Luminex-loaded replicates. (c) ELISA assays for secreted albumin and AFP, and quantitative CYP3A4 and CYP2A6 activity assays. For all analyses, iHeps were cultured for 9 days post-differentiation, in 6-well plates (n=3). All data presented as mean ± SEM.