The rate of disappearance of intracellular α-l-antitrypsin correlates with liver disease severity in iPScderived hepatocytes generated from PIZZ α-1-antitrypsin deficiency patients
Edgar N. Tafaleng1, 2, Bing Han1, 2, Pamela D. Hale2, 3, Souvik Chakraborty2, 3, Alejandro Soto-Gutierrez2, 4, Carol Feghali-Bostwick5, Darrell Kotton6, Masaki Nagaya1, Stephen A. Duncan7, Donna B. Stolz8, Stephen Strom9, Jayanta Roy-Chowdhury10, David H. Perlmutter2, 3, Ira J. Fox1, 11
1Department of Surgery,University of Pittsburgh School of Medicine, Pittsburgh, PA; 2Children's Hospital of Piffsburgn of UPMC, Pitstburgh, PA; 3Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA; 4Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA; 5Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA; 6Department of Medicine, Boston University School of Medicine, Boston, MA; 7Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI; 8Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA; 9Department of Laboratory Medicine, Division of Pathology, Karolinska Institutet, Stockholm, Sweden; 10Department of Medicine, Albert Einstein College of Medicine, New York, NY; 11 McGowan Institute for Regenerative Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA
Classical α-1-antitrypsin deficiency (ATD) is the most common cause of hereditary pediatric liver disease. The disorder results from the PIZZ mutation that alters the structure of a-1-antitrypsin (AT) and renders it aggregation-prone. This leads to mutant AT accumulation within hepatocytes, causing hepatic injury through a toxic gain-of-function mechanism. Although the PIZZ genotype is necessary for the development of ATD-mediated liver disease, there is variability in liver disease severity among PIZZ individuals. The prevailing hypothesis is that other yet to be identified genetic factors predispose PIZZ individuals to liver disease. Induced pluripotent stem cells (iPScs) from ATD patients with known clinical features could provide an unlimited source of hepatocytes for identifying these modifier genes. We generated 15 iPScs from PIZZ ATD patients with liver disease of varying severity. We differentiated an iPSc line from a severe liver disease ATD patient (severe LD), 3 iPSc clones from mild liver disease ATD patients (mild LD), and a wild-type control iPSc line into hepatocytes. Analysis of severe LD iPSc-derived hepatocytes by electron and confocal microscopy showed poorly organized and dilated rER and intense AT staining localized in the ER and Golgi similar to results seen in severe LD ATD 1° hepatocytes indicating that the cells model the disease. Similar to severe LD ATD 1° hepatocytes, severe LD iPSc-derived hepatocytes also have constitutively activated autophagy as measured by western blot of basal p62 and LC3I-II levels. Using pulse-chase analysis, we detected a slower rate of disappearance of intracellular AT in ATD iPSc-derived hepatocytes (t1/2 = 2.1 to 4 hr) compared to control cells (t1/2≈1.3 hr). More importantly, we found that the delayed disappearance of intracellular AT observed in ATD iPSc-derived hepatocytes is more apparent in cells obtained from the patient with severe LD (t1/2≈ 4 hr) compared to those from the patient with mild LD (t1/2≈ 2.1 hr). This result is consistent with results obtained from severe LD ATD 1° hepatocytes (t1/2≈ 4.2 hr). There was no significant difference between the rates of the two iPSc clones from the mild LD patient, suggesting that the results are not an artifact of reprogramming or differentiation. These results suggest that variation in the severity of liver disease is related to genetic modifiers that affect intracellular degradation pathways.
Stephen A. Duncan - Consulting: Primorigen Biosciences
Stephen Strom - Stock Shareholder: Stemnion, Yecuris
Ira J. Fox - Advisory Committees or Review Panels: Regenerative Medical Solutions
The following people have nothing to disclose: Edgar N. Tafaleng, Bing Han, Pamela D. Hale, Souvik Chakraborty, Alejandro Soto-Gutierrez, Carol FeghaliBostwick, Darrell Kotton, Masaki Nagaya, Donna B. Stolz, Jayanta Roy-Chowdhury, David H. Perlmutter