SummaryDifferentiated derivatives of human pluripotent stem cells in culture are generally phenotypically immature compared to their adult counterparts. Their identity is often difficult to determine with certainty because little is known about their human fetal equivalents in vivo. Cellular identity and signaling pathways directing differentiation are usually determined by extrapolating information from either human adult tissue or model organisms, assuming conservation with humans. To resolve this, we generated a collection of human fetal transcriptional profiles at different developmental stages. Moreover, we developed an algorithm, KeyGenes, which uses this dataset to quantify the extent to which next-generation sequencing or microarray data resemble specific cell or tissue types in the human fetus. Using KeyGenes combined with the human fetal atlas, we identified multiple cell and tissue samples unambiguously on a limited set of features. We thus provide a flexible and expandable platform to monitor and evaluate the efficiency of differentiation in vitro.

Transforming growth factor-beta (Tgf␤) is essential for normal embryogenesis. The cardiac phenotypes obtained after knockout of each of the three mammalian isoforms suggest different roles during morphogenesis. We studied cardiovascular expression of Tgf␤1-3 in parallel tissue sections of normal mouse embryos from 9.5 to 15.5 days post coitum (dpc) by using radioactive in situ hybridisation. The Tgf␤ isoforms are differentially expressed in unique and in overlapping patterns during cardiovascular development. In the vessels, Tgf␤1 is found in the intima, whereas Tgf␤2 and -␤3 are mainly present in the media and adventitia of the great arteries. Tgf␤1 is present in the endocardium at all stages examined. The Tgf␤2 signal in the endocardium of the atrioventricular canal and outflow tract (9.5 dpc) shifts during epithelial-mesenchymal transformation toward the mesenchymal cushions (10.5-11.5 dpc) after which it exhibits a marked spatiotemporal expression pattern as the cushion differentiation progresses (11.5-15.5 dpc). The myocardium underlying the endocardial cushions and the atrial muscular septum are intensely positive for Tgf␤2 at early stages (9.5-11.5 dpc) and expression decreases at 12.5 days. In contrast to earlier reports, we find marked overlap of Tgf␤2 and -␤3 expression. Tgf␤3 expression shows a characteristic distribution in the mesenchymal cushions, suggesting a role in cushion differentiation, possibly additional to Tgf␤2. From 14.5 dpc onward, a strong Tgf␤3 signal is found in the fibrous septum primum of the atrium and in the fibrous skeleton of the heart. Special attention was paid to the proepicardial organ and its derivatives. The proepicardial organ strongly expresses Tgf␤2 as early as 9.5 days, and all isoforms are present in the epicardium from 12.5 dpc onward. The spatiotemporal cardiovascular expression of Tgf␤1-3 supports both specific and complementary functions during cardiovascular development that might explain functional redundancy between the Tgf␤-isoforms. The information provided favors novel roles of Tgf␤1-3 in epicardial development, of Tgf␤2 in myocardialisation, and of Tgf␤3 in differentiation of the fibrous structures of the heart. Developmental Dynamics 227: 431-444, 2003.

Differentiated derivatives of human pluripotent stem cells (hPSCs) are often considered immature because they resemble foetal cells more than adult, with hPSC-derived cardiomyocytes (hPSC-CMs) being no exception. Many functional features of these cardiomyocytes, such as their cell morphology, electrophysiological characteristics, sarcomere organization and contraction force, are underdeveloped compared with adult cardiomyocytes. However, relatively little is known about how their gene expression profiles compare with the human foetal heart, in part because of the paucity of data on the human foetal heart at different stages of development. Here, we collected samples of matched ventricles and atria from human foetuses during the first and second trimester of development. This presented a rare opportunity to perform gene expression analysis on the individual chambers of the heart at various stages of development, allowing us to identify not only genes involved in the formation of the heart, but also specific genes upregulated in each of the four chambers and at different stages of development. The data showed that hPSC-CMs had a gene expression profile similar to first trimester foetal heart, but after culture in conditions shown previously to induce maturation, they cluster closer to the second trimester foetal heart samples. In summary, we demonstrate how the gene expression profiles of human foetal heart samples can be used for benchmarking hPSC-CMs and also contribute to determining their equivalent stage of development.

BackgroundHearing depends on correct functioning of the cochlear hair cells, and their innervation by spiral ganglion neurons. Most of the insight into the embryological and molecular development of this sensory system has been derived from animal studies. In contrast, little is known about the molecular expression patterns and dynamics of signaling molecules during normal fetal development of the human cochlea. In this study, we investigated the onset of hair cell differentiation and innervation in the human fetal cochlea at various stages of development.ResultsAt 10 weeks of gestation, we observed a prosensory domain expressing SOX2 and SOX9/SOX10 within the cochlear duct epithelium. In this domain, hair cell differentiation was consistently present from 12 weeks, coinciding with downregulation of SOX9/SOX10, to be followed several weeks later by downregulation of SOX2. Outgrowing neurites from spiral ganglion neurons were found penetrating into the cochlear duct epithelium prior to hair cell differentiation, and directly targeted the hair cells as they developed. Ubiquitous Peripherin expression by spiral ganglion neurons gradually diminished and became restricted to the type II spiral ganglion neurons by 18 weeks. At 20 weeks, when the onset of human hearing is thought to take place, the expression profiles in hair cells and spiral ganglion neurons matched the expression patterns of the adult mammalian cochleae.ConclusionsOur study provides new insights into the fetal development of the human cochlea, contributing to our understanding of deafness and to the development of new therapeutic strategies to restore hearing.

Sensorineural hearing loss (SNHL) is one of the most common congenital disorders in humans, afflicting one in every thousand newborns. The majority is of heritable origin and can be divided in syndromic and nonsyndromic forms. Knowledge of the expression profile of affected genes in the human fetal cochlea is limited, and as many of the gene mutations causing SNHL likely affect the stria vascularis or cochlear potassium homeostasis (both essential to hearing), a better insight into the embryological development of this organ is needed to understand SNHL etiologies. We present an investigation on the development of the stria vascularis in the human fetal cochlea between 9 and 18 weeks of gestation (W9–W18) and show the cochlear expression dynamics of key potassium‐regulating proteins. At W12, MITF+/SOX10+/KIT+ neural‐crest‐derived melanocytes migrated into the cochlea and penetrated the basement membrane of the lateral wall epithelium, developing into the intermediate cells of the stria vascularis. These melanocytes tightly integrated with Na+/K+‐ATPase‐positive marginal cells, which started to express KCNQ1 in their apical membrane at W16. At W18, KCNJ10 and gap junction proteins GJB2/CX26 and GJB6/CX30 were expressed in the cells in the outer sulcus, but not in the spiral ligament. Finally, we investigated GJA1/CX43 and GJE1/CX23 expression, and suggest that GJE1 presents a potential new SNHL associated locus. Our study helps to better understand human cochlear development, provides more insight into multiple forms of hereditary SNHL, and suggests that human hearing does not commence before the third trimester of pregnancy. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1219–1240, 2015

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The beneficial effect of additional folic acid in the periconceptional period to prevent neural tube defects, orofacial clefts, and conotruncal heart defects in the offspring has been shown.

Remodelling the methylome is a hallmark of mammalian development and cell differentiation. However, current knowledge of DNA methylation dynamics in human tissue specification and organ development largely stems from the extrapolation of studies in vitro and animal models. Here, we report on the DNA methylation landscape using the 450k array of four human tissues (amnion, muscle, adrenal and pancreas) during the first and second trimester of gestation (9,18 and 22 weeks). We show that a tissue-specific signature, constituted by tissue-specific hypomethylated CpG sites, was already present at 9 weeks of gestation (W9). Furthermore, we report large-scale remodelling of DNA methylation from W9 to W22. Gain of DNA methylation preferentially occurred near genes involved in general developmental processes, whereas loss of DNA methylation mapped to genes with tissue-specific functions. Dynamic DNA methylation was associated with enhancers, but not promoters. Comparison of our data with external fetal adrenal, brain and liver revealed striking similarities in the trajectory of DNA methylation during fetal development. The analysis of gene expression data indicated that dynamic DNA methylation was associated with the progressive repression of developmental programs and the activation of genes involved in tissue-specific processes. The DNA methylation landscape of human fetal development provides insight into regulatory elements that guide tissue specification and lead to organ functionality.