GSD1a disease modelled hepatocytes

DefiniGEN provide disease modelled glycogen storage disease type 1a hepatocytes which are highly functional patient-derived human hepatocytes. This genetic disease results from a deficiency in the glucose-6-phosphatase (G6P) enzyme which impairs the ability of the liver to produce free glucose from glycogen and from gluconeogenesis. The cells are generated using the dermal fibroblasts from a glycogen storage disease patient with mutations at the G6P gene positions G222R and Q347X. These cells can offer disease modelling and drug discovery researchers unprecedented tool for elucidating the underlying mechanisms of this and similar lysosomal storage diseases.

• Highly standardized cell product containing >98% human hepatocyte cells with consistent performance and biologically relevant data
• Disease circuit verified two heterozygous mutations G222R and Q347X
• Phenotypic analysis of the cells using periodic acid/diastase staining has demonstrated that the cells hyper accumulate glycogen as the glucose-6-phosphatase enzyme is not functional.

Disease circuit verification

Figure 1. DNA sequencing chromatogram illustrating the two heterozygous mutations found in Def-HEP GSD1a.

Detection of Glycogen Storage via PAS staining

Period acid-Schiff (PAS) staining revealed Def-HEP GSD1a hepatocytes accumulated substantially greater amounts of intracellular glycogen compared with iPSC derived hepatocytes from control subjects n=3 (figure 2a); BODIPY staining showed excessive production of intracellular lipid in Def-HEP GSD1a hepatocytes (figure 2b); and Def-HEP GSD1a hepatocytes secrete more lactate compared with iPSC derived hepatocytes from control subjects, as assessed by ELISA analysis of a 24-hour collection of cell culture medium. Error bars denote SEM. N=3 (Figure 2C).

Figure 2. Def-HEP GSD1a cell characterization. A) Period acid-Schiff (PAS) staining revealed Def-HEP GSD1a hepatocytes accumulated substantially greater amounts of intracellular glycogen than did those of controls (figure 2A) and showed excessive production of lipid (figure 2B) and lactic acid (figure 2C) confirming the cellular disease phenotype. (Courtesy; Rashid et. al 2010).

PAS/Diastase functional test

Period acid-Schiff (PAS) is a staining method used to detect glycogen storage in tissue and in the diagnosis of glycogen storage diseases. Diastase is an alpha-amylase enzyme that breaks down glycogen and is used in combination with a PAS stain to specifically identify glycogen granules. PAS staining revealed that Def-HEP GSD1a hepatocytes accumulated significantly higher amounts of intracellular glycogen than the negative control (Figure 3).

Figure 3. PAS/Diastase staining of cryopreserved Def-HEP GSD1a. (A) PAS/Diastase staining showing breakdown of accumulated glycogen in the cells. (B) PAS staining showing the accumulation of glycogen in the cells. Magnification level: x400.