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A preliminary analysis of volatile metabolites of human induced pluripotent stem cells along the in vitro differentiation.

ABSTRACT: Cellular metabolism of stem cell biology is still an unexplored field. However, considering the amount of information carried by metabolomes, this is a promising field for a fast identification of stem cells itself and during the differentiation process. One of the goals of such application is the identification of residual pluripotent cells before cell transplantation to avoid the occurrence of teratomas. In this paper, we investigated in vitro the volatile compounds (VOCs) released during human induced pluripotent stem cells (hiPSCs) reprogramming. In particular, we studied hiPSCs differentiation to floating and adherent embryoid bodies until early neural progenitor cells. A preliminary Gas Chromatographic/Mass Spectrometer (GC/MS) analysis, based on a single extraction method and chromatographic separation, indicated 17 volatile compounds whose relative abundance is altered in each step of the differentiation process. The pattern of VOCs shown by hiPSCs is well distinct and makes these cells sharply separated from the other steps of differentiations. Similar behaviour has also been observed with an array of metalloporphyrins based gas sensors. The use of electronic sensors to control the process of differentiation of pluripotent stem cells might suggest a novel perspective for a fast and on-line control of differentiation processes.

SUBMITTER: Capuano R

PROVIDER: S-EPMC5431616 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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A preliminary analysis of volatile metabolites of human induced pluripotent stem cells along the in vitro differentiation.

Capuano Rosamaria R Spitalieri Paola P Talarico Rosa Valentina RV Domakoski Ana Carolina AC Catini Alexandro A Paolesse Roberto R Martinelli Eugenio E Novelli Giuseppe G Sangiuolo Federica F Di Natale Corrado C

Scientific reports 20170509 1

Cellular metabolism of stem cell biology is still an unexplored field. However, considering the amount of information carried by metabolomes, this is a promising field for a fast identification of stem cells itself and during the differentiation process. One of the goals of such application is the identification of residual pluripotent cells before cell transplantation to avoid the occurrence of teratomas. In this paper, we investigated in vitro the volatile compounds (VOCs) released during huma ...[more]

PMID: 28487523

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Project description:Efficient and effective methods for converting human induced pluripotent stem cells into differentiated derivatives are critical for performing robust, large-scale studies of development and disease modelling, and for providing a source of cells for regenerative medicine. Here, we describe a 14-day neural differentiation protocol which allows for the scalable, simultaneous differentiation of multiple iPSC lines into cortical neural stem cells We currently employ this protocol to differentiate and compare sets of engineered iPSC lines carrying loss of function alleles in developmental disorder associated genes, alongside isogenic wildtype controls. Using RNA sequencing (RNA-Seq), we can examine the changes in gene expression brought about by each disease gene knockout, to determine its impact on neural development and explore mechanisms of disease. The 10-day Neural Induction period uses the well established dual-SMAD inhibition approach combined with Wnt/β-Catenin inhibition to selectively induce formation of cortical NSCs. This is followed by a 4-day Neural Maintenance period facilitating NSC expansion and rosette formation, and NSC cryopreservation. We also describe methods for thawing and passaging the cryopreserved NSCs, which are useful in confirming their viability for further culture. Routine implementation of immunocytochemistry Quality Control confirms the presence of PAX6-positive and/or FOXG1-positive NSCs and the absence of OCT4-positive iPSCs after differentiation. RNA-Seq, flow cytometry, immunocytochemistry (ICC) and RT-qPCR provide additional confirmation of robust presence of NSC markers in the differentiated cells. The broader utility and application of our protocol is demonstrated by the successful differentiation of wildtype iPSC lines from five additional independent donors. This paper thereby describes an efficient method for the production of large numbers of high purity cortical NSCs, which are widely applicable for downstream research into developmental mechanisms, further differentiation into postmitotic cortical neurons, or other applications such as large-scale drug screening experiments.

Project description:Studies in embryonic development have guided successful efforts to direct the differentiation of human embryonic and induced pluripotent stem cells (PSCs) into specific organ cell types in vitro. For example, human PSCs have been differentiated into monolayer cultures of liver hepatocytes and pancreatic endocrine cells that have therapeutic efficacy in animal models of liver disease and diabetes, respectively. However, the generation of complex three-dimensional organ tissues in vitro remains a major challenge for translational studies. Here we establish a robust and efficient process to direct the differentiation of human PSCs into intestinal tissue in vitro using a temporal series of growth factor manipulations to mimic embryonic intestinal development. This involved activin-induced definitive endoderm formation, FGF/Wnt-induced posterior endoderm pattering, hindgut specification and morphogenesis, and a pro-intestinal culture system to promote intestinal growth, morphogenesis and cytodifferentiation. The resulting three-dimensional intestinal 'organoids' consisted of a polarized, columnar epithelium that was patterned into villus-like structures and crypt-like proliferative zones that expressed intestinal stem cell markers. The epithelium contained functional enterocytes, as well as goblet, Paneth and enteroendocrine cells. Using this culture system as a model to study human intestinal development, we identified that the combined activity of WNT3A and FGF4 is required for hindgut specification whereas FGF4 alone is sufficient to promote hindgut morphogenesis. Our data indicate that human intestinal stem cells form de novo during development. We also determined that NEUROG3, a pro-endocrine transcription factor that is mutated in enteric anendocrinosis, is both necessary and sufficient for human enteroendocrine cell development in vitro. PSC-derived human intestinal tissue should allow for unprecedented studies of human intestinal development and disease.

Dataset Information

A preliminary analysis of volatile metabolites of human induced pluripotent stem cells along the in vitro differentiation.

Publications

A preliminary analysis of volatile metabolites of human induced pluripotent stem cells along the in vitro differentiation.

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