Modeling inherited metabolic disorders of the liver using human induced pluripotent stem cells

Abstract

Human induced pluripotent stem (iPS) cells hold great promise for advancements in developmental biology, cell-based therapy, and modeling of human disease. Here, we examined the use of human iPS cells for modeling inherited metabolic disorders of the liver. Dermal fibroblasts from patients with various inherited metabolic diseases of the liver were used to generate a library of patient-specific human iPS cell lines. Each line was differentiated into hepatocytes using what we believe to be a novel 3-step differentiation protocol in chemically defined conditions. The resulting cells exhibited properties of mature hepatocytes, such as albumin secretion and cytochrome P450 metabolism. Moreover, cells generated from patients with 3 of the inherited metabolic conditions studied in further detail (alpha1-antitrypsin deficiency, familial hypercholesterolemia, and glycogen storage disease type 1a) were found to recapitulate key pathological features of the diseases affecting the patients from which they were derived, such as aggregation of misfolded alpha1-antitrypsin in the endoplasmic reticulum, deficient LDL receptor-mediated cholesterol uptake, and elevated lipid and glycogen accumulation. Therefore, we report a simple and effective platform for hepatocyte generation from patient-specific human iPS cells. These patient-derived hepatocytes demonstrate that it is possible to model diseases whose phenotypes are caused by pathological dysregulation of key processes within adult cells.

Figure 1. Generation of hepatocytes from disease-specific human iPS cells.

(A) Protocol used to differentiate the disease-specific human iPS cell library into hepatocytes. (B) Immunostaining analyses for expression of the indicated proteins marking key stages of hepatocyte development (day 4, endoderm; day 20, hepatic progenitor; day 25, fetal hepatocyte). (C) Real-time PCR analysis for expression of genes marking key stages of disease-specific human iPS cell (hIPSC) differentiation to hepatocytes. Error bars denote SEM. (D) Fraction of cells expressing albumin after 25 days of hepatic differentiation, as shown by FACS analyses. (E) Morphologic analysis of disease-specific human iPS cell–derived hepatocytes (day 25) by transmission EM, showing the presence of apical microvilli and glycogen rosettes (numerals 1 and 2, respectively). Original magnification, ×20 (A); ×40 (B); ×3,000 (E). The data shown were taken from 1 line (patient 1; line 1), but are representative of all lines similarly characterized (Table 1).

Figure 2. In vitro modeling of A1ATD using disease-specific human iPS cells.

(A) A1ATD disease-specific human iPS cells (dhIPSCs) differentiated to hepatocytes displayed functional activity characteristic of primary human hepatocytes, including the presence of intracellular albumin, glycogen storage (shown by PAS staining), LDL cholesterol uptake (shown by fluoresceinated LDL incorporation), albumin secretion, and active CytP450 metabolism. Error bars denote SEM. (B) Immunostaining analyses for expression of misfolded polymeric α₁-antitrypsin using the polymer-specific 2C1 antibody (green) or an antibody that detects all forms of α₁-antitrypsin (red) in A1ATD patient-specific and control human iPS cell–derived hepatocytes. Merged images are shown at right. (C) Endoglycosidase H (EndoH) digestion of A1ATD disease-specific human iPS cell–derived hepatocyte microsomal subcellular fraction, confirming retention of misfolded polymeric α₁-antitrypsin protein within the endoplasmic reticulum. n = 3. (D) ELISA to assess the intracellular expression (cells) and secretion (medium) of all α₁-antitrypsin and polymeric α₁-antitrypsin protein in A1ATD patient-specific and control human iPS cell–derived hepatocytes after overnight proteasomal inhibition by MG132. Error bars denote SEM. n = 3. Original magnification, ×20 (A); ×40 (B).

Figure 3. In vitro modeling of FH using disease-specific human iPS cells.

(A) FH disease-specific human iPS cells differentiated to hepatocytes displayed functional activity characteristic of primary human hepatocytes, including intracellular presence of albumin, glycogen storage, albumin secretion, and active CytP450 metabolism. Error bars denote SEM. (B) FACS analysis for fluoresceinated LDL incorporation confirmed FH disease-specific human iPS cell–derived hepatocytes (red curve) lacked the ability to efficiently take up LDL compared with the positive control (blue curve). Human iPS cells grown in the absence of LDL were used as a negative control (unstained; black curve). Original magnification, ×40 (A); ×20 (B).

Figure 4. In vitro modeling of GSD1a using disease-specific human iPS cells.

(A) GSD1a disease-specific human iPS cells differentiated to hepatocytes displayed functional activity characteristic of primary human hepatocytes, including intracellular presence of albumin, albumin secretion, and active CytP450 metabolism. Error bars denote SEM. (B) PAS staining showing excessive accumulation of intracellular glycogen in GSD1a disease-specific human iPS cell–derived hepatocytes compared with human iPS cell–derived hepatocytes from control subjects. n = 3. (C) BODIPY staining showed excessive accumulation of intracellular lipid in GSD1a disease-specific human iPS cell–derived hepatocytes compared with human iPS cell–derived hepatocytes from control subjects. n = 3. (D) Disease-specific human iPS cell–derived hepatocytes appropriately upregulated transcriptional targets of glucagon, as shown by quantitative RT-PCR analysis of PEPCK, glucose-6-phosphatase (G6P), and IGFBP1 expression analyzed 0, 1, 2, and 3 hours after stimulation with 100 nM glucagon hydrochloride. Error bars denote SEM. n = 3. (E) GSD1a disease-specific human iPS cell–derived hepatocytes secreted more lactate than did human iPS cell–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. Original magnification, ×40 (A; B, bottom; and C).