Fig. 4: Elevating AA levels induces time-dependent maturation of HLC and HC3X progeny.

a–c Relative CYP3A4 expression in (a) HLC D20 and HC3X D20 cultured in control medium and in medium supplemented with AA3, and an increasing amount of glycine; (b) in HLCs and HC3X cultured in control and in AAGLY medium at D20, D40, and D50 of differentiation; (c) in HLCs cultured in medium supplemented with AA3 with or without 2% of either glycine (GLY), serine (SER), alanine (ALA), leucine (LEU), proline (PRO), isoleucine (iLEU), aspartate (ASP), valine (VAL), or a mix of all of the above. N = 3 independent differentiations. Significance was calculated compared with HLC or HC3X LDM D20 with 2% DMSO by unpaired two-tailed Student’s t-test. d Relative CYP3A4/CYP2E1-dependent BFC metabolization for HLCs and HC3X grown in different media conditions at different times of differentiation and compared with PHHs. N = 3 independent differentiations. N = 4 donors for PHH 0 h and PHH 12 h. Significance was compared with PHH 0 h by unpaired two-tailed Student’s t-test. e, f IPA analysis showing major differentially expressed pathways when comparing HLC LDM D20 with (e) PHH 0 h and (f) HLC AAGLY D40. g PCA plot showing the distribution of HLCs and HC3X differentiated in different media and compared with PHH 0 h. h Quadrant analysis showing pathways that are normalized (Q1 and Q3), overexpressed (Q2), or underexpressed (Q4) when comparing HLC LDM D20 with PHH 0 h and HLC AAGLY D40. Color intensity correlates with −log(p). Data in all panels represent mean ± SEM with P-values indicated when significant. Source data are provided as a Source Data file.