Figure 1.
Generation of the Hepatocyte Likeness Index
(A) Schematic representation of work-flow that established morphological parameters and protein signatures for the Hepatocyte Likeness Index (HLI) and its validation across different cell types. AH, adult hepatocytes (n = 17 donors); F, fibroblasts (n = 3 donors).
(B) Image-based algorithm enables the automated identification of hepatocytes by morphology and albumin. (Left column) DAPI and albumin staining in adult hepatocytes (AH), i-Heps (IH), and fibroblasts (F). (Right column) Cells highlighted green represent identified hepatocytes; gray or red highlighted cells are recognized as non-hepatocytes. Scale bar, 200 μm.
(C) (Top) Receiver-operating characteristic curve demonstrating high diagnostic ability of albumin as a surrogate marker of hepatocyte likeness and maturity. (Bottom) Pearson correlation analysis of showing that high albumin expression is closely related with metabolic (cytochrome 2A6) function (Pearson coefficient r2 = 0.9503, 95% confidence interval, p < 0.01).
(D) Mean fluorescence intensity (MFI) of albumin is validated by ELISA and qPCR in adult hepatocytes (AH), fetal hepatocytes (FH), i-Heps (IH), and fibroblasts (F). n = 9 donors (AH), n = 4 donors (FH), n = 3 donors (IH), and n = 3 donors (F). Error bars show mean ± SD.
(E) HLI discriminates freshly isolated adult hepatocytes from cryopreserved adult, cultured adult, freshly isolated fetal hepatocytes, and i-Heps in a manner independent of cell viability (n = 3 different biological samples in all groups, all independent experiments).
(F) HLI discriminates hepatocytes from other cells in vivo. (Left panel) Human liver section (10×) stained for albumin (red) demonstrating albumin-positive and albumin-negative (blue) cells. (Right panel) Application of HLI algorithm enables automated detection of hepatocytes (green) from non-hepatocytes (red) by machine learning. Enlarged square: accurate distribution of hepatocytes and non-hepatocyte cells around periportal area. Scale bar, 100 μm.
(G) HLI discriminates hepatocytes from other cells in vitro. Co-culture of GFP-labeled HUVECs (green) and hepatocytes (red) (left panel) were discriminated by HLI (right panel) better than by flow cytometry (right). Scale bar, 100 μm.