Modeling of Alpha-1 Antitrypsin Deficiency with Syngeneic Human iPSC-Hepatocytes Reveals Metabolic Dysfunction and Cellular Heterogeneity in PiMZ and PiZZ Hepatic Cells (scRNA-Seq)

Individuals homozygous for the "Z" mutation in alpha-1 antitrypsin deficiency are known to be at increased risk for liver disease. It has also become clear that some degree of risk is similarly conferred by the heterozygous state. A lack of model systems that recapitulate heterozygosity in human hepatocytes has limited the ability to study the impact of a single Z alpha-1 antitrypsin (ZAAT) allele on hepatocyte biology. Here, we describe the derivation of syngeneic induced pluripotent stem cells (iPSCs) engineered to determine the effects of ZAAT heterozygosity in iPSC-hepatocytes (iHeps). We find that heterozygous MZ iHeps exhibit an intermediate disease phenotype and share with ZZ iHeps alterations in AAT protein processing and downstream perturbations including altered endoplasmic reticulum (ER) and mitochondrial morphology, reduced mitochondrial respiration, and branch-specific activation of the unfolded protein response in cell subpopulations. Our model of MZ heterozygosity thus provides evidence that a single Z allele is sufficient to disrupt hepatocyte homeostatic function. 10X: We profiled the transcriptome of 11,284 individual iHeps derived from PiZZ1 syngeneic (ZZ, MZ, MM) and PiZZ6 ZZ iPSCs that underwent directed differentiation to the hepatic lineage using 10x Chromium controller. For scRNA-seq iHeps were disassociated using 0.25% Trypsin and sorted for live cells using Calcein Blue (ThermoFisher Scientific) on a MoFlo Astrios EQ (Beckman Coulter). Single live cells were then captured, and library preparation performed using the Chromium Single Cell 3’ v3 user protocol and Chromium Controller instrument per manufacturer instructions (10X Genomics). Each library was then sequenced using the Illumina NextSeq 500 to obtain sequencing depths of between 25-50K reads/cell. Fastq and count matrix files were generated using Cell Ranger v 3.0.2 and the transcriptome mapped again to the ENSEMBL human reference genome GRCh38. We then used Seurat v3 to further process and analyze the data. Data was normalized using the regularized negative binomial regression method with cell degradation regressed out. Fluidigm_C1: We profiled the transcriptome of 475 individual iHeps derived from PiZZ100 syngeneic ZZ and MM iPSCs that underwent directed differentiation to the hepatic lineage using Fluidigm C1 platform. Additionally day 12 progenitor cells from PiZZ100 ZZ were also sequenced but not used in this manuscript For scRNA-seq iHeps were disassociated using 0.25% Trypsin and sorted for live cells using Calcein Blue (ThermoFisher Scientific) on a MoFlo Astrios EQ (Beckman Coulter). Single live cells were then captured using a large (15-25mm cell diameter) 800 cell capacity microfluidic chip for mRNA-seq (Fluidigm) was utilized for single cell capture. After a live-dead sort cells were loaded onto the chip per manufactures instructions using the Fluidigm C1 HT workflow to capture, lyse, reverse transcribe RNA and for library preparation.