Abstract
MODY1 is a maturity-onset monogenic diabetes, caused by heterozygous mutations of the HNF4A gene. To date the cellular and molecular mechanisms leading to disease onset remain largely unknown. In this study, we demonstrate that insulin-positive cells can be generated in vitro from human induced pluripotent stem cells (hiPSCs) derived from patients carrying a non-sense HNF4A mutation, proving for the first time, that a human HNF4A mutation is neither blocking the expression of the insulin genes nor the development of insulin-producing cells in vitro. However, regardless of the mutation or diabetes status, these insulin-producing cells are immature, a common downfall off most current β-cell differentiation protocols. To further address the immature state of the cells, in vitro differentiated cells and adult human islets were compared by global proteomic analysis. We report the predicted upstream regulators and signalling pathways characterizing the proteome landscape of each entity. Subsequently, we focused on the molecular components absent or misregulated in the in vitro differentiated cells, to probe the components involved in the deficient in vitro maturation towards fully functional β-cells. This analysis identified the modulation of key developmental signalling pathways representing potential targets for improving the efficiency of the current differentiation protocols.
Highlights
Monogenic disorders are caused by germline single gene defects where different mutations in the causal gene usually trigger a defined disorder with characteristic clinical features
The synchronous differentiation of the four human pluripotent stem cells (hiPSCs) lines towards insulin-producing cells (n = 3 repeated rounds of differentiation) was done following the seven step differentiation protocol established by Rezania et al (Fig. S1) and resulted in the successful generation of insulin+ cells in all four samples regardless of the mutation or diabetes status (Fig. 1b)
The global proteomic comparison of the final stage (S7) cells derived from MODY1-mutation carriers and their family control detected in all samples similar levels of insulin and most key β-cell specific markers, regardless of the mutation or diabetes status (Fig. 1d), confirming the initial immunofluorescence (IF) observations
Summary
Monogenic disorders are caused by germline single gene defects where different mutations in the causal gene usually trigger a defined disorder with characteristic clinical features. The identification of the genes and molecular networks underlining monogenic disorders allows for unbiased characterization of the basic mechanisms regulating cell-fate decisions during development and disease onset This approach facilitates the understanding of the aetiology of the more prevalent corresponding multifactorial diseases as well as general developmental aspects. The protocols reported in 2014 by Rezania et al and Pagliuca et al shared a similar stepwise directed differentiation strategy attempting to mimic different stages of the embryonic development of β-cells by modulating similar developmental signalling pathways Both protocols achieved to generate β-like cells on a comparable www.nature.com/scientificreports/. As the current knowledge of β-cell differentiation process during development is incomplete, a common problem of the available in vitro differentiation protocols is the production of mostly immature “β-like cells”[13] unable to perform accurate glucose-stimulated insulin secretion unless they are transplanted into mice and allowed to mature in vivo[14]. In any of these cases, transcriptomics tools will fail to detect correctly the changes in gene product abundance or signalling patterns
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