A Highly Phenotyped Open Access Repository of Alpha-1 Antitrypsin Deficiency Pluripotent Stem Cells

Abstract

Summary Individuals with the genetic disorder alpha-1 antitrypsin deficiency (AATD) are at risk of developing lung and liver disease. Patient induced pluripotent stem cells (iPSCs) have been found to model features of AATD pathogenesis but only a handful of AATD patient iPSC lines have been published. To capture the significant phenotypic diversity of the patient population, we describe here the establishment and characterization of a curated repository of AATD iPSCs with associated disease-relevant clinical data. To highlight the utility of the repository, we selected a subset of iPSC lines for functional characterization. Selected lines were differentiated to generate both hepatic and lung cell lineages and analyzed by RNA sequencing. In addition, two iPSC lines were targeted using CRISPR/Cas9 editing to accomplish scarless repair. Repository iPSCs are available to investigators for studies of disease pathogenesis and therapeutic discovery.

Recombinant Lloviu virus as a model to study inaccessible zoonotic viruses

“…To gain insight into LLOV host cell tropism, we compared the ability of rLLOVcomp and rEBOV to infect two key target cell types during filovirus infections in humans, macrophages and hepatocytes 17 . Infection of both monocyte-derived macrophages (MDMs) and induced pluripotent stem cell (iPSC)-derived, primary-like hepatocytes (iHeps) 18 with rLLOV and rEBOV revealed similar infection rates, with higher infection rates with rEBOV in the iHeps at later time points (Fig. 3a and b).…”

“…BU3 was confirmed as mycoplasma free by PCR analysis of gDNA using the following mycoplasma specific primers: 5' CTT CWT CGA CTT YCA GAC CCA AGG CAT 3' and 5' ACA CCA TGG GAG YTG GTA AT 3' and verified as karyotypically normal as determined by G-band karyotyping analysis from 20 metaphases. iPSC directed differentiation towards the hepatic lineage was performed using a previously published protocol 18,39,40 . Briefly, undifferentiated iPSCs were cultured until confluent and then passaged on Day 0 using gentle cell dissociation reagent (GCDR, StemCell Technologies) to achieve single cell suspensions and replated at 1x10 6 cells per well of a Matrigel coated 6-well plate.…”

Adenine base editing reduces misfolded protein accumulation and toxicity in alpha-1 antitrypsin deficient patient iPSC-hepatocytes

“…Base-edited MZ and MM iPSC-derived hepatocytes are protected from a Z-AAT-driven disease signature After generating MZ and MM iPSCs, we next derived iHeps to test the effects of editing in SERPINA1-expressing cells. Using an established protocol that we have found to generate hepatic cells with a transcriptional profile overlapping significantly with primary hepatocyte controls, 12,17,23 we differentiated parental ZZ and edited MZ and MM daughter iPSCs in biological triplicate. All three syngeneic lines efficiently generated iHeps, quantified by expression of alpha-fetoprotein (AFP), a marker of fetal hepatocytes (Figures 2A and 2B; Figure S3A).…”

Derivation of Thyroid Follicular Cells From Pluripotent Stem Cells: Insights From Development and Implications for Regenerative Medicine

“…In the years since their discovery, iPSCs have been generated by reprogramming easily accessible cells, including skin fibroblasts (9,11) or peripheral blood, taken from patients with a variety of disease affecting nearly every organ system or lineage (12,13), including patients with congenital hypothyroidism (14). While the generation and banking of normal or disease-specific iPSCs is no longer a hurdle (15)(16)(17)(18) a variety of iPSC bio-repositories having been established for researchit has become clear that the main hurdle to applying these cells for in vitro disease modeling or in vivo regenerative transplantation therapies is the limited capacity to effectively or reliably differentiate these PSCs into stable, functional, or mature downstream lineages.…”