Model Of Early Human Development Research Articles

Time-integrated BMP signaling determines fate in a stem cell model for early human development

How paracrine signals are interpreted to yield multiple cell fate decisions in a dynamic context during human development in vivo and in vitro remains poorly understood. Here we report an automated tracking method to follow signaling histories linked to cell fate in large numbers of human pluripotent stem cells (hPSCs). Using an unbiased statistical approach, we discover that measured BMP signaling history correlates strongly with fate in individual cells. We find that BMP response in hPSCs varies more strongly in the duration of signaling than the level. However, both the level and duration of signaling activity control cell fate choices only by changing the time integral. Therefore, signaling duration and level are interchangeable in this context. In a stem cell model for patterning of the human embryo, we show that signaling histories predict the fate pattern and that the integral model correctly predicts changes in cell fate domains when signaling is perturbed. Our data suggest that mechanistically, BMP signaling is integrated by SOX2.

A p53-Dependent Checkpoint Induced upon DNA Damage Alters Cell Fate during hiPSC Differentiation.

SummaryThe ability of human induced pluripotent stem cells (hiPSCs) to differentiate in vitro to each of the three germ layer lineages has made them an important model of early human development and a tool for tissue engineering. However, the factors that disturb the intricate transcriptional choreography of differentiation remain incompletely understood. Here, we uncover a critical time window during which DNA damage significantly reduces the efficiency and fidelity with which hiPSCs differentiate to definitive endoderm. DNA damage prevents the normal reduction of p53 levels as cells pass through the epithelial-to-mesenchymal transition, diverting the transcriptional program toward mesoderm without induction of an apoptotic response. In contrast, TP53-deficient cells differentiate to endoderm with high efficiency after DNA damage, suggesting that p53 enforces a “differentiation checkpoint” in early endoderm differentiation that alters cell fate in response to DNA damage.

Self-organizing neuruloids model developmental aspects of Huntington's disease in the ectodermal compartment.

Harnessing the potential of human embryonic stem cells to mimic normal and aberrant development with standardized models is a pressing challenge. Here we use micropattern technology to recapitulate early human neurulation in large numbers of nearly identical structures called neuruloids. Dual-SMAD inhibition followed by bone morphogenic protein 4 stimulation induced self-organization of neuruloids harboring neural progenitors, neural crest, sensory placode and epidermis. Single-cell transcriptomics unveiled the precise identities and timing of fate specification. Investigation of the molecular mechanism of neuruloid self-organization revealed a pulse of pSMAD1 at the edge that induced epidermis, whose juxtaposition to central neural fates specifies neural crest and placodes, modulated by fibroblast growth factor and Wnt. Neuruloids provide a unique opportunity to study the developmental aspects of human diseases. Using isogenic Huntington's disease human embryonic stem cells and deep neural network analysis, we show how specific phenotypic signatures arise in our model of early human development as a consequence of mutant huntingtin protein, outlining an approach for phenotypic drug screening.

X chromosome inactivation in human pluripotent stem cells as a model for human development: back to the drawing board?

Human pluripotent stem cells (hPSC), both embryonic and induced (hESC and hiPSC), are regarded as a valuable in vitro model for early human development. In order to fulfil this promise, it is important that these cells mimic as closely as possible the in vivo molecular events, both at the genetic and epigenetic level. One of the most important epigenetic events during early human development is X chromosome inactivation (XCI), the transcriptional silencing of one of the two X chromosomes in female cells. XCI is important for proper development and aberrant XCI has been linked to several pathologies. Recently, novel data obtained using high throughput single-cell technology during human preimplantation development have suggested that the XCI mechanism is substantially different from XCI in mouse. It has also been suggested that hPSC show higher complexity in XCI than the mouse. Here we compare the available recent data to understand whether XCI during human preimplantation can be properly recapitulated using hPSC. We will summarize what is known on the timing and mechanisms of XCI during human preimplantation development. We will compare this to the XCI patterns that are observed during hPSC derivation, culture and differentiation, and comment on the cause of the aberrant XCI patterns observed in hPSC. Finally, we will discuss the implications of the aberrant XCI patterns on the applicability of hPSC as an in vitro model for human development and as cell source for regenerative medicine. Combinations of the following keywords were applied as search criteria in the PubMed database: X chromosome inactivation, preimplantation development, embryonic stem cells, induced pluripotent stem cells, primordial germ cells, differentiation. Recent single-cell RNASeq data have shed new light on the XCI process during human preimplantation development. These indicate a gradual inactivation on both XX chromosomes, starting from Day 4 of development and followed by a random choice to inactivate one of them, instead of the mechanism in mice where imprinted XCI is followed by random XCI. We have put these new findings in perspective using previous data obtained in human (and mouse) embryos. In addition, there is an ongoing discussion whether or not hPSC lines show X chromosome reactivation upon derivation, mimicking the earliest embryonic cells, and the XCI states observed during culture of hPSC are highly variable. Recent studies have shown that hPSC rapidly progress to highly aberrant XCI patterns and that this process is probably driven by suboptimal culture conditions. Importantly, these aberrant XCI states seem to be inherited by the differentiated hPSC-progeny. The aberrant XCI states (and epigenetic instability) observed in hPSC throw a shadow on their applicability as an in vitro model for development and disease modelling. Moreover, as the aberrant XCI states observed in hPSC seem to shift to a more malignant phenotype, this may also have important consequences for the safety aspect of using hPSC in the clinic.

3 SIRT1—A POSSIBLE MARKER FOR REPRODUCTIVE AGING OF IN VIVO-DERIVED BOVINE OOCYTES?

Maternal aging-associated reduction of oocyte viability is a common feature in mammals. Effective measures to counteract this process have not yet been developed. Cows are commonly used as a model of early human development, including maternal aging, because both species share a very high degree of similarity, including follicle selection, cleavage and blastocyst formation and a long reproductive lifespan. SIRT1, a member of the Sirtuin family, deacetylates transcriptional regulators localised in the nucleus and cytoplasm by a NAD+-dependent mechanism. Resveratrol (3,4′,5-trihydroxystilbene) is an antioxidant identified in various plant species and red wine which enhances SIRT1 activity. Based on these observations, the goal of the present study was to examine, if SIRT1 gene and protein expression is either affected by maternal age and/or can be modulated by resveratrol. Cumulus-oocyte-complexes of prepubertal (5–6 months old) and adult/aged (2 to 8 lactation) cows were collected by ovum pick-up twice a week. Medium for in vitro maturation (TCM 199) and in vitro fertilization (FertTalp) was supplemented with 20 µL of Resveratrol® (Sigma-Aldrich, Buchs, Switzerland) to get a final concentration of 2 µM Resveratrol respectively. Standard (TCM 199 and FertTalp) media without Resveratol were used as control. Cleavage rates and blastocyst formation were evaluated. Comprehensive gene expression assays of germinal vesicle and metaphase II (MII)-stage oocytes and blastocyst were conducted using next-generation sequencing technology. Finally, SIRT1 protein expression in oocytes and blastocysts were analysed by fluorescence immunostaining under a confocal microscope (LSM510, Zeiss, Germany) and relative fluorescent intensity was calculated. The cleavage rates of adult and prepubertal donors did not differ significantly among the treatments (standard protocol: 56.5 ± 5.4% for adult and 53.0 ± 4.7% for prepubertal donors, Resveratrol supplemented protocol: 62.1 ± 4.3% for cows and 63.6 ± 3.9% for calves). The blastocyst rates were slightly enhanced in the Resveratrol supplemented groups (cows: 34.2 ± 3.8% and calves: 33.1 ± 4.2%) compared to those of standard protocol (cows: 27.5 ± 4.8% and calves: 26.4 ± 3.3%). Relative mRNA abundance levels of SIRT1 were lower in oocytes and blastocysts derived from cows than in those derived from their younger counterparts (2.8-fold change; P = 0.05), but did not differ significantly among treatment groups. Protein expression profiles revealed that bovine SIRT1 was localised in the nucleus. The relative fluorescence levels of SIRT1 were significantly lower (221 ± 34 FIU) in control groups compared to the resveratrol treated groups (865 ± 45 FIU, respectively; P = 0.05). Additionally, SIRT1 protein levels were significantly higher in MII-oocytes (1255 ± 56 FIU) and blastocysts (984 ± 26 FIU) derived from calves compared with their older counterparts (442 ± 37 FIU and 310 ± 23 FIU, respectively, P = 0.05). In conclusion, these results indicate that resveratrol affects SIRT1 protein expression in oocytes and blastocysts of donors in different age. Thus, we hypothesise that SIRT1 is a reliable marker for reproductive aging, which could also be useful for better understanding of human infertility caused by aging.

Deletion of the Polycomb-Group Protein EZH2 Leads to Compromised Self-Renewal and Differentiation Defects in Human Embryonic Stem Cells

SummaryThrough the histone methyltransferase EZH2, the Polycomb complex PRC2 mediates H3K27me3 and is associated with transcriptional repression. PRC2 regulates cell-fate decisions in model organisms; however, its role in regulating cell differentiation during human embryogenesis is unknown. Here, we report the characterization of EZH2-deficient human embryonic stem cells (hESCs). H3K27me3 was lost upon EZH2 deletion, identifying an essential requirement for EZH2 in methylating H3K27 in hESCs, in contrast to its non-essential role in mouse ESCs. Developmental regulators were derepressed in EZH2-deficient hESCs, and single-cell analysis revealed an unexpected acquisition of lineage-restricted transcriptional programs. EZH2-deficient hESCs show strongly reduced self-renewal and proliferation, thereby identifying a more severe phenotype compared to mouse ESCs. EZH2-deficient hESCs can initiate differentiation toward developmental lineages; however, they cannot fully differentiate into mature specialized tissues. Thus, EZH2 is required for stable ESC self-renewal, regulation of transcriptional programs, and for late-stage differentiation in this model of early human development.

Low ethanol concentration alters CHRNA5 RNA levels during early human development

Alcohol use is common and consumption during pregnancy has been shown to lead to a myriad of physical and neurologic abnormalities commonly referred to as fetal alcohol spectrum disorder. Substance addiction, which includes alcohol, has been shown to involve the major nicotinic acetylcholine receptor subunit CHRNA5. Using human embryonic stem cells as a model of early human development, we show that low concentrations of ethanol (20 mM) can alter the expression of CHRNA5. Changes in CHRNA5 expression is linked to altered GABA and NMDA receptor expression, as well as abnormal development of the frontal cortex. These results suggest that alcohol exposure can alter early neurologic development, which may favor addiction and other developmental abnormalities in unborn children.

A Modified Culture Medium Increases Blastocyst Formation and the Efficiency of Human Embryonic Stem Cell Derivation from Poor-Quality Embryos

Human embryonic stem cells (HESCs) are defined as self-renewing cells that retain their ability to differentiate into all cell types of the body. They have enormous potential in medical applications and as a model for early human development. There is a need for derivation of new HESC lines to meet emerging requirements for their use in cell replacement therapies, disease modeling, and basic research. Here, we describe a modified culture medium containing human recombinant leukemia inhibitory factor and human basic fibroblast growth factor that significantly increases the number of human blastocysts formed and their quality, as well as the efficiency of HESC derivation from poor-quality embryos. Culturing poor-quality embryos in modified medium resulted in a two-fold increase in the blastocyst formation rate and a seven-fold increase over the derivation efficiency in conventional medium. We derived 15 HESC lines from poor-quality embryos cultured in modified culture medium and two HESC lines from quality embryos cultured in conventional culture medium. All cell lines shared typical human pluripotent stem cell features including similar morphology, normal karyotypes, expression of alkaline phosphatase, pluripotency genes, such as Oct4, and cell surface markers (SSEA-4, TRA-1-60, TRA-1-81), the ability to form teratomas in SCID mice, and the ability to differentiate into cells of three embryonic germ layers in vitro. Our data suggest that poor-quality embryos that have reached the blastocyst stage in our modified culture medium are a robust source for normal HESC line derivation.

Differentiation of human embryonic stem cells into hepatocytes in 2D and 3D culture systems in vitro

Human embryonic stem cells (hESCs) have enormous potential as a source of cells for cell replacement therapies and as a model for early human development. In this study we examined the differentiating potential of hESCs into hepatocytes in two- and three-dimensional (2D and 3D) culture systems. Embryoid bodies (EBs) were inserted into a collagen scaffold 3D culture system or cultured on collagen-coated dishes and stimulated with exogenous growth factors to induce hepatic histogenesis. Immunofluorescence analysis revealed the expression of albumin (ALB) and cytokeratin-18 (CK-18). The differentiated cells in 2D and 3D culture system displayed several characteristics of hepatocytes, including expression of transthyretin, alpha-1-antitrypsin, cytokeratin 8, 18, 19, tryptophan-2,3-dioxygenase, tyrosine aminotransferase, glucose-6-phosphatase (G6P), cytochrome P450 subunits 7a1 and secretion of alpha-fetoprotein (AFP) and ALB and production of urea. In 3D culture, ALB and G6P were detected earlier and higher levels of urea and AFP were produced, when compared with 2D culture. Electron microscopy of differentiated hESCs showed hepatocyte-like ultrastructure, including glycogon granules, well-developed Golgi apparatuses, rough and smooth endoplasmic reticuli and intercellular canaliculi. The differentiation of hESCs into hepatocyte-like cells within 3D collagen scaffolds containing exogenous growth factors, gives rise to cells displaying morphological features, gene expression patterns and metabolic activities characteristic of hepatocytes and may provide a source of differentiated cells for treatment of liver diseases.

A Molecular Basis for Human Embryonic Stem Cell Pluripotency

Embryonic stem cells (ESCs) are able to generate a wide array of differentiated cell fates while maintaining self-renewal. Understanding the biology of these choices may be central to the use of human embryonic stem cells (HESCs), both as a model for early human development as well as a resource for cell based therapies. Efforts to dissect the molecular mechanisms that mediate stem cell identity are underway, and in this review we summarize recent progress in defining the markers and pathways involved in these decisions. We discuss recent efforts to assess the molecular signature of pluripotent HESCs and highlight work demonstrating a set of genes, including representatives from the FGF, TGFbeta, and Wnt signaling pathways, that consistently mark the undifferentiated state. In addition, we describe experiments in which signaling of HESCs is augmented by chemical probing with small molecule compounds. Using these compounds, we have demonstrated an important role for Wnt signaling in HESC pluripotency and shown a requirement for TGFbeta signaling in the maintenance of the undifferentiated state. These experiments have revealed some molecular aspects of the pluripotent state and demonstrated clear differences between mouse and human ESCs in the maintenance of this identity.

Trophoblast Differentiation in Embryoid Bodies Derived from Human Embryonic Stem Cells

Trophoblast differentiation and early placental development are essential for the establishment of pregnancy, yet these critical events are not readily investigated in human pregnancy. We used embryoid bodies (EBs) prepared from human embryonic stem (hES) cells as an in vitro model of early human development. The levels of human chorionic gonadotropin (hCG), progesterone, and estradiol-17beta in medium from hES cell-derived EBs grown in suspension culture for 1 wk were higher than unconditioned culture medium or medium from undifferentiated hES cells or spontaneously differentiated hES cell colonies. EBs were explanted into Matrigel (MG) "rafts" and cultured for up to 53 d. During the first 7-10 d of three-dimensional growth in MG, small protrusions appeared on the outer surface of EBs, some of which subsequently extended into multicellular outgrowths. The secretion of hCG, progesterone, and estradiol-17beta began to increase on approximately d 20 of MG culture and remained dramatically elevated over the next 30 d. EBs maintained in suspension culture failed to demonstrate this elevation in hormone secretion. Suspension-cultured and MG-embedded EBs exhibited widespread expression of cytokeratins 7/8, demonstrating extensive epithelial differentiation as well as consistent hCG expression. We propose that hES cell-derived EBs may be a useful model for investigation of human trophoblast differentiation and placental morphogenesis.

Retinoid Target Gene Activation during Induced Tumor Cell Differentiation: Human Embryonal Carcinoma as a Model

Many agents that exhibit chemopreventive activity are able to mediate a differentiation response in premalignant and malignant tissues. One of the most widely studied classes of tumor differentiation agents is the retinoids. There is rapidly evolving evidence for beneficial retinoid actions in the prevention or treatment of clinical tumors. However, the use of retinoids in the clinic is limited by acquired resistance and toxicity, especially when administered chronically in preventive strategies. Although retinoids are known to regulate gene transcription by activating retinoid receptors, the identity of the target genes that mediate the beneficial effects of retinoids are largely unknown. Here we review a useful model of retinoid-induced tumor cell differentiation: human embryonal carcinoma. The pluripotent nature and ease of use make human embryonal carcinoma cells a valuable and practical complement to human embryonic stem cells as an in vitro model of early human development. In addition, retinoid treatment of human embryonal carcinoma is an important model of induced tumor cell differentiation because retinoids cause the reversal of the malignant phenotype coincident with terminal neuronal differentiation. We have used both de novo and candidate approaches with this system in an effort to uncover critical downstream targets of retinoid receptors during differentiation induction.

Attachment and the regulation of the right brain

It has been three decades since John Bowlby first presented an over-arching model of early human development in his groundbreaking volume, Attachment. In the present paper I refer back to Bowlby's original charting of the attachment landscape in order to suggest that current research and clinical models need to return to the integration of the psychological and biological underpinnings of the theory. Towards that end, recent contributions from neuroscience are offered to support Bowlby's assertions that attachment is instinctive behavior with a biological function, that emotional processes lie at the foundation of a model of instinctive behavior, and that a biological control system in the brain regulates affectively driven instinctive behavior. This control system can now be identified as the orbitofrontal system and its cortical and subcortical connections. This ‘senior executive of the emotional brain’ acts as a regulatory system, and is expanded in the right hemisphere, which is dominant in human infancy and centrally involved in inhibitory control. Attachment theory is essentially a regulatory theory, and attachment can be defined as the interactive regulation of biological synchronicity between organisms. This model suggests that future directions of attachment research should focus upon the early-forming psychoneurobiological mechanisms that mediate both adaptive and maladaptive regulatory processes. Such studies will have direct applications to the creation of more effective preventive and treatment methodologies.