A1ATD disease modelled hepatocytes
DefiniGEN provide disease modelled alpha-1 antitrypsin deficiency hepatocytes which are highly functional patient-derived human hepatocytes.
DefiniGEN provide disease modelled alpha-1 antitrypsin deficiency hepatocytes which are highly functional patient-derived human hepatocytes. The cells are generated using the dermal fibroblasts from an alpha-1 antitrypsin (A1ATD) disorder patient carrying the ‘Z’ (E342K) point mutation in the A1ATD gene SERPINA1. This mutation leads to the formation of mutant A1ATD polymers that ultimately cause liver and lung damage. DefiniGEN’s A1ATD patient derived hepatocytes represent an optimized disease model for drug discovery applications and are an effective tool for elucidating the underlying mechanisms of the disease.
Hepatocyte maturation markers
Def-HEP A1ATD cells display the functional characteristics of primary human hepatocytes (PHH) including albumin and A1ATD production (Figure 2). Accordingly, they should be handled in a similar fashion to primary human hepatocytes.
Figure 2. Functional analysis of hepatocyte-specific markers in Def-HEP A1ATD cells. Immunostaining of Def-HEP A1ATD cells overviewing the expression of general hepatocyte maturation markers.
Detection of disease markers via ELISA
ELISA for polymer and secreted A1ATD using antibodies specific for A1ATD polymers (2C1mAb, top) or all conformers of A1ATD (bottom).
Figure 3. Detection of disease specific markers in Def-HEP A1ATD cells. Quantification of intracellular A1ATD polymers and intercellular total A1ATD secretion in Def-HEP A1ATD cells. Primary human hepatocytes (PHH) and hepatocellular liver carcinoma cells (Hep G2) are used as internal controls.
Immunocytochemistry analysis of Def-HEP A1ATD mutant polymer
Previous studies have shown that the Z allele (Glu342Lys) results in the formation of ordered polymers of a1-antitrypsin that are retained within the ER. This pathway of a1-antitrypsin polymerization is central to the clinical phenotype. We therefore used the 2C1 polymer specific monoclonal antibody to detect polymers within Def-HEP A1ATD hepatocytes. Polymers were detected by immunostaining (Figure 4).
Figure 4. Accumulation of a1-antitrypsin polymers in Def-HEP A1ATD cells. Immunostaining analyses for expression of misfolded polymeric a1-antitrypsin using the polymer-specific 2C1 antibody (green) or an antibody that detects all forms of a1-antitrpysin (red) in Def-HEP A1ATD disease modelled cells and control Def-HEP WT. Merged images are shown at right.
Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells.