Integration of metabolomic and transcriptomic profiling to compare two protocols of differentiation of human induced pluripotent stem cells into hepatocytes

Abstract

Human hepatocyte-like cells derived from human induced pluripotent stem cells (hiPSC) may provide an unlimited supply of cells for in vitro liver models. However, hiPSC differentiation remains a major challenge due to immaturity of the hepatocytes obtained and the high cost of differentiation protocols currently proposed. Here, we studied the efficacy of new protocol, with reduction of growth factors, for the generation of hepatocyte-like cells from hiPSC. We performed metabolomic and mRNA analysis by RTqPCR and nanoCAGE processing to identify and understand key metabolisms during differentiation. By reducing the change frequency of the culture medium in the new protocol, we successfully generated hepatocyte-like cells producing albumin, urea, and CYP3A4 positive. The metabolomic analysis successfully extracted both signatures, common and specific, for each differentiation step. Integrating the metabolomic data with transcriptomic contributed to explaining the kinetics of carbohydrate, lipid and nitrogen metabolism throughout differentiation.The information extracted during differentiation showed that the cells moved from an aerobic-like respiration pattern to a mitochondrial oxidative respiration pattern in both protocols. Reducing culture medium renewal led to reduced glucose consumption, followed by fructose production and significant extracellular lipogenesis throughout differentiation. We believe that the overall dataset can provide information on the sequence of process.

A Novel UPLC-MS Metabolomic Analysis-Based Strategy to Monitor the Course and Extent of iPSC Differentiation to Hepatocytes

“…In spite of its potential Journal of Proteome Research pubs.acs.org/jpr Article applicability, this approach has been rarely used to get further insights into the differentiation process of iPSCs. 6,16,17 To validate our approach, we examined the changes in the metabolome of three iPSC progenies (transfected with/ without key transcription factors), cultured in three differentiation media, and compared them to PHHs. We recently described that, by an overexpression of a combination of three transcription factors (TFs), namely, HNF1A, PROX1, and FOXA3 (cells termed HC3x), or six TFs, namely, HNF1A, PROX1, FOXA3, PGC1A, SIRT1, and activated AMPK (cells termed HC6x), and by complementing a basal liver differentiation medium (LDM) with high levels (8×) of nonessential amino acids (media named LDM-AA) or by a combination of 8× nonessential amino acids and extra glycine concentration (media named LDM-AAGly), it was possible to drive the transcriptional and metabolic maturation of HLCs, with a concomitant expression of CYP450 enzymes.…”

“…A metabolomic analysis allows one to measure changes in the concentration of a wide range of cell metabolites and to exploit that information to infer the performance of the different hepatic metabolic pathways. In spite of its potential applicability, this approach has been rarely used to get further insights into the differentiation process of iPSCs. ,, To validate our approach, we examined the changes in the metabolome of three iPSC progenies (transfected with/without key transcription factors), cultured in three differentiation media, and compared them to PHHs.…”