Table 2.

High throughput platforms for drug discovery and toxicology assessment using iPSC derived hepatocytes.

Purpose of screen	Assay type	High throughput procedure	Outcome	Reference
Identification of drugs for treatment of Alpha-1-Antitrypsin (AAT) deficiency	Small Molecule Screen	AAT Patient iPSC- derived hepatocytes were treated with a drug library containing 3,131 clinical compounds and analyzed with immunofluorescence against cellular AAT. High Throughput microscopy was used to identify small molecules that reversed AAT accumulation.	5 drugs identified that consistently reduced AAT accumulation. One of the drugs identified was Carbamazepine which has previously been used to clear protein aggregates in vivo thus validating the screens application for drug discovery.	(61)
Prediction of Drug Induced Liver Injury using embryonic stem cell derived hepatocytes	Toxicity Screen	Twenty known hepatotoxins were screened using primary and stem cell-derived hepatocytes for 1, 4, or 7 days at concentration of 0.1, 1, 10, 25, 50, 100, or 200 μM. Cellular ATP was used to determine whether the drugs at each time point reduced cells to at least 50% viability (IC50).	At day 1 fewer compounds were toxic to stem cell derived hepatocytes (45%) compared to primary hepatocytes (60%), after 4 days of treatment both platforms showed similar sensitivity showing 65 and 60% sensitivity, respectively. After 7 days both platforms equally successfully identified 75% of the compounds as toxic.	(97)
Small molecule screen for hepatocyte proliferation and maturation.	Small Molecule Screen	12,480 small molecules were screened for their ability to enhance the expansion or function of cultured primary hepatocytes and then applied to iPSCs during hepatic differentiation.	2 small molecules assigned FH1 and FPH1 were identified that enhanced the maturation of cultured iPSC derived hepatocytes	(100)
iPSC-Human Hepatocyte-based micropatterned co-cultures platform for high throughput toxicity screening	Toxicity Screen	iPSC derived hepatocyte like cells were co-cultured with 3T3 fibroblasts and exposed to 47 compounds, 37 known to be toxic, 10 known non-toxic.	iPSC and primary human hepatocyte based co-culture platforms had sensitivities of 65 and 70%, respectively, for the 37 known hepatotoxic compounds tested. Neither model showed a false positive to the non-toxic compounds.	(33)
High-throughput confocal microscopy analysis of toxic compounds on the morphology and viability of iPSC derived 3D liver spheroids	Toxicity Screen	Representative set of 48 compounds 42 known to be cytotoxic or hepatotoxic and 6 with no known toxicity were analyzed for their effect on Human iPSC-derived hepatocyte spheroids.	36 of the toxic compounds resulted in toxicity effects in the spheroid assay (86%) with no false positives from the non-toxic compounds. 21 compounds showed a trend toward stronger toxicity effects in 3D culture when compared to 2D cultured cells. iPSC derived spheroids showed a markedly less toxic response to most anti-proliferative agents compared to HepG2 spheroids.	(98)
Identification of drugs for the treatment of Familial Hypercholesterolemia	Small Molecule Screen	2,320 small molecules were screened to identify compounds that could reduce the levels of apoB in hepatocytes derived from Familial Hypercholesterolemia iPSCs.	13 small molecules were identified which reliably reduced apoB. Five of the identified compounds were cardiac glycosides which reduced apoB via enhanced proteolytic turnover.	(70)
Small molecule screen to elucidate unknown cellular mechanisms that underly liver development	Small Molecule Screen	1,120 small molecules with well-defined molecular pathways were screened against iPSC during hepatic differentiation to determine which mechanisms were required for the maintenance of HNF4α	132 small molecules were identified that impacted HNF4α expression. Gene ontology analyses linking interactions between small molecules and proteins revealed heat shock protein 90 alpha family class B member 1 (HSP90β) played a role in HNF4α regulation. Disruption of HSP90β led to a reduction in HNF4α and co-immunoprecipitation indicated HSP90β plays a role in regulatingHNF4α protein folding.	(96)
Genetic and chemical high throughput screen to identify reagents that enhanced hepatic differentiation	Genetic and small molecule screen	A Genome wide CRISPR-Cas9-lentiviral screen along with an iPSC Albumin reporter line was used in a high throughput format to identify HDAC3 as a regulator of hepatic differentiation.	A high throughput small molecule screen found that treatment of iPSCs with the HDAC inhibitor CI-994 resulted in greater expression of several hepatic markers, as well as reduced expression of AFP, compared with control cells.	(99)
Small molecule screen to identify compounds that could reverse the disease phenotype of Mitochondrial DNA Depletion Syndrome (MTDPS3)	Small Molecule Screen	2,400 drugs, a majority of which had been approved for use in humans were screened against hepatocytes derived from iPSCs with a CRISPR loss of function mutation in DGUOK.	15 drugs were identified which enhanced endogenous ATP production in the diseased cells. The candidate drug NAD was selected for further assessment and successfully reversed the disease phenotype in DGUOK mutant rats	(79)