FT-IR spectroscopy supported by PCA–LDA analysis for the study of embryonic stem cell differentiation

As recently pointed out in the literature, Fourier transform infrared (FT-IR) spectroscopy is emerging as a powerful tool in stem cell research. In this work we characterizedin situby FT-IR microspectroscopy the differentiation of murine embryonic stem cells (ES) to monitor possible changes in the cell macromolecular content during the early stages of differentiation. Undifferentiated and differentiating cells at 4, 7, 9 and 14 days were measured. Data were analyzed by the principal component and subsequent linear discriminant analyses (PCA–LDA) that enabled us to segregate ES cell spectra into well separate clusters and to identify the most significant spectral changes. Important changes in the lipid (3050–2800 cm–1), protein (1700–1600 cm–1) and in the nucleic acid (1050–850 cm–1) absorption regions were observed between days 4 to 7 of differentiation, indicating the appearance – at day 7 – of the new phenotype into cardiomyocyte precursors. Also the presence of DNA/RNA hybrid bands (954 cm–1and 899 cm–1) suggests that the transcriptional switch of the genome started at this stage of differentiation. Particularly noteworthy, we suggest that the 2936 cm–1shoulder we observed could reflect methyl group vibrations thus allowing the detection of variations in methylation levels of the stem cell during differentiation. These infrared results were found to be in agreement with the biochemical characterization of these differentiating cells, underlying the great potential of FT-IR spectroscopy in stem cell research

FT-IR spectroscopy supported by PCA–LDA analysis for the study of embryonic stem cell differentiation

As recently pointed out in the literature, Fourier transform infrared (FT-IR) spectroscopy is emerging as a powerful tool in stem cell research. In this work we characterizedin situby FT-IR microspectroscopy the differentiation of murine embryonic stem cells (ES) to monitor possible changes in the cell macromolecular content during the early stages of differentiation. Undifferentiated and differentiating cells at 4, 7, 9 and 14 days were measured. Data were analyzed by the principal component and subsequent linear discriminant analyses (PCA–LDA) that enabled us to segregate ES cell spectra into well separate clusters and to identify the most significant spectral changes. Important changes in the lipid (3050–2800 cm–1), protein (1700–1600 cm–1) and in the nucleic acid (1050–850 cm–1) absorption regions were observed between days 4 to 7 of differentiation, indicating the appearance – at day 7 – of the new phenotype into cardiomyocyte precursors. Also the presence of DNA/RNA hybrid bands (954 cm–1and 899 cm–1) suggests that the transcriptional switch of the genome started at this stage of differentiation. Particularly noteworthy, we suggest that the 2936 cm–1shoulder we observed could reflect methyl group vibrations thus allowing the detection of variations in methylation levels of the stem cell during differentiation. These infrared results were found to be in agreement with the biochemical characterization of these differentiating cells, underlying the great potential of FT-IR spectroscopy in stem cell research

Human pre-valvular endocardial cells derived from pluripotent stem cells recapitulate cardiac pathophysiological valvulogenesis

Genetically modified mice have advanced our understanding of valve development and disease. Yet, human pathophysiological valvulogenesis remains poorly understood. Here we report that, by combining single cell sequencing and in vivo approaches, a population of human pre-valvular endocardial cells (HPVCs) can be derived from pluripotent stem cells. HPVCs express gene patterns conforming to the E9.0 mouse atrio-ventricular canal (AVC) endocardium signature. HPVCs treated with BMP2, cultured on mouse AVC cushions, or transplanted into the AVC of embryonic mouse hearts, undergo endothelial-to-mesenchymal transition and express markers of valve interstitial cells of different valvular layers, demonstrating cell specificity. Extending this model to patient-specific induced pluripotent stem cells recapitulates features of mitral valve prolapse and identified dysregulation of the SHH pathway. Concurrently increased ECM secretion can be rescued by SHH inhibition, thus providing a putative therapeutic target. In summary, we report a human cell model of valvulogenesis that faithfully recapitulates valve disease in a dish.We thank the Leducq Fondation for supporting Tui Neri, and funding this research under the framework of the MITRAL network and for generously awarding us for the equipment of our cell imaging facility in the frame of their program “Equipement de Recherche et Plateformes Technologiques” (ERPT to M.P.), the Genopole at Evry and the Fondation de la recherche Medicale (grant DEQ20100318280) for supporting the laboratory of Michel Puceat. Part of this work in South Carolina University was conducted in a facility constructed with support from the National Institutes of Health, Grant Number C06 RR018823 from the Extramural Research Facilities Program of the National Center for Research Resources. Other funding sources: National Heart Lung and Blood Institute: RO1-HL33756 (R.R.M.), COBRE P20RR016434–07 (R.R.M., R.A. N.), P20RR016434–09S1 (R.R.M. and R.A.N.); American Heart Association: 11SDG5270006 (R.A.N.); National Science Foundation: EPS-0902795 (R.R.M. and R.A. N.); American Heart Association: 10SDG2630130 (A.C.Z.), NIH: P01HD032573 (A.C. Z.), NIH: U54 HL108460 (A.C.Z), NCATS: UL1TR000100 (A.C.Z.); EH was supported by a fellowship of the Ministere de la recherche et de l’éducation in France.TM-M was supported by a fellowship from the Fondation Foulon Delalande and the Leducq Foundation. P.v.V. was sponsored by a UC San Diego Cardiovascular Scholarship Award and a Postdoctoral Fellowship from the California Institute for Regenerative Medicine (CIRM) Interdisciplinary Stem Cell Training Program II. S.M.E. was funded by a grant from the National Heart, Lung, and Blood Institute (HL-117649). A.T. is supported by the National Heart, Lung, and Blood Institute (R01-HL134664).S

Expression of phase I and phase II genes in mouse embryonic stem cells cultured in the presence of 2,3,7,8-tetrachlorodibenzo-para-dioxin.

Embryonic stem (ES) cells have features that resemble the pluripotent cells of peri-implantation embryos and have been used as an in vitro model to assess the effects of test substances on these stages of development. Here, for the first time, we report on the effects of the xenobiotic 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) on mouse ES cells cultured with TCDD at concentrations ranging from 0.0001 to 100 nM for 15 min to 48 h. TCDD effects were determined by analysing the induction of Cyp1A1, Cyp1A2, Cyp1B1 (phase I) and Nqo1, Gsta1, Ugt1a6 (phase II) genes. Cyp1A1 was the phase I gene most rapidly induced (4 h at 1 nM); Cyp1B1 was induced at 48 h (1 nM), whereas Cyp1A2 expression was not affected. TCDD did not alter phase II gene expression, which remained at basal levels throughout the 48 h of culture. We studied more accurately the expression of Cyp1A1, the earliest gene to respond to the presence of TCDD. We found that: 1) Cyp1A1 gene induction is dependent on the duration of exposure (precisely it is first induced after 3 h of culture at 1 nM, the minimum effective-dose); 2) Cyp1A1 induction requires the continuous presence of TCDD, being interrupted 4 h after removal of the xenobiotic; and 3) induced expression of CYP1A1 protein is dependent on TCDD concentration, the higher the concentration the earlier the production of the enzyme. Furthermore, after 48 h of treatment, TCDD did not promote either apoptosis or changes to the differentiation status of the ES cells. These results are the first important step to investigate the effects of dioxin on the very early stages of mammalian development

Embryonic stem cell differentiation studied by FT-IR spectroscopy

We propose, here, an FT-IR method to monitor the spontaneous differentiation of murine embryonic stem (ES) cells in their early development. Principal component analysis and subsequent linear discriminant analysis enabled us to segregate stem cell spectra into separate clusters – corresponding to different differentiation times – and to identify the most significant spectral changes during differentiation. Between days 4 to 7 of differentiation, these spectral changes in the protein amide I band (1700–1600 cm− 1) and in the nucleic acid absorption region (1050–850 cm− 1) indicated that mRNA translation was taking place and that specific proteins were produced, reflecting the appearance of a new phenotype. The DNA/RNA hybrid bands (954 cm− 1 and 899 cm− 1) were also observed, suggesting that the transcriptional switch of the genome started at this stage of differentiation. As confirmed by cytochemical assays, the FT-IR approach presented here allows to detect at molecular level the biological events of ES cell differentiation as they take place and to monitor in a rapid way the temporal evolution of the ES cell culture