No abstract

In recent years, protocols have been established to differentiate stem and precursor cells into more mature cell types. However, progress in this field has been hampered by difficulties to assess the differentiation status of stem cell-derived cells in an unbiased manner. Here, we present an analysis pipeline based on published data and methods to quantify the degree of differentiation and to identify transcriptional control factors explaining differences from the intended target cells or tissues. The pipeline requires RNA-Seq or gene array data of the stem cell starting population, derived 'mature' cells and primary target cells or tissue. It consists of a principal component analysis to represent global expression changes and to identify possible problems of the dataset that require special attention, such as: batch effects; clustering techniques to identify gene groups with similar features; over-representation analysis to characterize biological motifs and transcriptional control factors of the identified gene clusters; and metagenes as well as gene regulatory networks for quantitative cell-type assessment and identification of influential transcription factors. Possibilities and limitations of the analysis pipeline are illustrated using the example of human embryonic stem cell and human induced pluripotent cells to generate 'hepatocyte-like cells'. The pipeline quantifies the degree of incomplete differentiation as well as remaining stemness and identifies unwanted features, such as colon- and fibroblast-associated gene clusters that are absent in real hepatocytes but typically induced by currently available differentiation protocols. Finally, transcription factors responsible for incomplete and unwanted differentiation are identified. The proposed method is widely applicable and allows an unbiased and quantitative assessment of stem cell-derived cells.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'.

The basic helix-loop-helix (bHLH) transcription factor Math6 (Atonal homolog 8; Atoh8) plays a crucial role in a number of cellular processes during embryonic development, iron metabolism and tumorigenesis. We report here on its involvement in cellular reprogramming from fibroblasts to induced pluripotent stem cells, in the maintenance of pluripotency and in early fate decisions during murine development. Loss of Math6 disrupts mesenchymal-to-epithelial transition during reprogramming and primes pluripotent stem cells towards the mesendodermal fate. Math6 can thus be considered a regulator of reprogramming and pluripotent stem cell fate. Additionally, our results demonstrate the involvement of Math6 in SMAD-dependent TGF beta signalling. We furthermore monitor the presence of the Math6 protein during these developmental processes using a newly generated Math6Flag-tag mouse. Taken together, our results suggest that Math6 counteracts TGF beta signalling and, by this, affects the initiating step of cellular reprogramming, as well as the maintenance of pluripotency and early differentiation.