Human Embryoid Body Differentiation Research Articles

Bioinformatic analysis predicts that ethanol exposure during early development causes alternative splicing alterations of genes involved in RNA post-transcriptional regulation.

Prenatal ethanol exposure is associated with neurodevelopmental defects and long-lasting cognitive deficits, which are grouped as fetal alcohol spectrum disorders (FASD). The molecular mechanisms underlying FASD are incompletely characterized. Alternative splicing, including the insertion of microexons (exons of less than 30 nucleotides in length), is highly prevalent in the nervous system. However, whether ethanol exposure can have acute or chronic deleterious effects in this process is poorly understood. In this work, we used the bioinformatic tools VAST-TOOLS, rMATS, MAJIQ, and MicroExonator to predict alternative splicing events affected by ethanol from available RNA sequencing data. Experimental protocols of ethanol exposure included human cortical tissue development, human embryoid body differentiation, and mouse development. We found common genes with predicted differential alternative splicing using distinct bioinformatic tools in different experimental designs. Notably, Gene Ontology and KEGG analysis revealed that the alternative splicing of genes related to RNA processing and protein synthesis was commonly affected in the different ethanol exposure schemes. In addition, the inclusion of microexons was also affected by ethanol. This bioinformatic analysis provides a reliable list of candidate genes whose splicing is affected by ethanol during nervous system development. Furthermore, our results suggest that ethanol particularly modifies the alternative splicing of genes related to post-transcriptional regulation, which probably affects neuronal proteome complexity and brain function.

Effects of DMSO on the Pluripotency of Cultured Mouse Embryonic Stem Cells (mESCs).

DMSO is a commonly used solvent in biological studies, as it is an amphipathic molecule soluble in both aqueous and organic media. For that reason, it is the vehicle of choice for several water-insoluble substances used in research. At the molecular and cellular level, DMSO is a hydrogen-bound disrupter, an intercellular electrical uncoupler, and a cryoprotectant, among other properties. Importantly, DMSO often has overlooked side effects. In stem cell research, the literature is scarce, but there are reports on the effect of DMSO in human embryoid body differentiation and on human pluripotent stem cell priming towards differentiation, via modulation of cell cycle. However, in mouse embryonic stem cell (mESC) culture, there is almost no available information. Taking into consideration the almost ubiquitous use of DMSO in experiments involving mESCs, we aimed to understand the effect of very low doses of DMSO (0.0001%-0.2%), usually used to introduce pharmacological inhibitors/modulators, in mESCs cultured in two different media (2i and FBS-based media). Our results show that in the E14Tg2a mESC line used in this study, even the smallest concentration of DMSO had minor effects on the total number of cells in serum-cultured mESCs. However, these effects could not be explained by alterations in cell cycle or apoptosis. Furthermore, DMSO did not affect pluripotency or differentiation potential. All things considered, and although control experiments should be carried out in each cell line that is used, it is reasonable to conclude that DMSO at the concentrations used here has a minimal effect on this particular mESC line.

Hyperthermia Disturbs and Delays Spontaneous Differentiation of Human Embryoid Bodies.

Various types of stress stimuli have been shown to threaten the normal development of embryos during embryogenesis. Prolonged heat exposure is the most common stressor that poses a threat to embryo development. Despite the extensive investigation of heat stress control mechanisms in the cytosol, the endoplasmic reticulum (ER) heat stress response remains unclear. In this study, we used human embryonic stem cells (hESCs) to examine the effect of heat stress on early embryonic development, specifically alterations in the ER stress response. In a hyperthermic (42 °C) culture, ER stress response genes involved in hESC differentiation were induced within 1 h of exposure, which resulted in disturbed and delayed differentiation. In addition, hyperthermia increased the expression levels of activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) genes, which are associated with the protein kinase RNA-like endoplasmic reticulum kinase (PERK) signaling pathway. Furthermore, we demonstrated that tauroursodeoxycholic acid, a chemical chaperone, mitigated the delayed differentiation under hyperthermia. Our study identified novel gene markers in response to hyperthermia-induced ER stress on hESCs, thereby providing further insight into the mechanisms that regulate human embryogenesis.

Inspiration from heart development: Biomimetic development of functional human cardiac organoids.

Recent progress in human organoids has provided 3D tissue systems to model human development, diseases, as well as develop cell delivery systems for regenerative therapies. While direct differentiation of human embryoid bodies holds great promise for cardiac organoid production, intramyocardial cell organization during heart development provides biological foundation to fabricate human cardiac organoids with defined cell types. Inspired by the intramyocardial organization events in coronary vasculogenesis, where a diverse, yet defined, mixture of cardiac cell types self-organizes into functional myocardium in the absence of blood flow, we have developed a defined method to produce scaffold-free human cardiac organoids that structurally and functionally resembled the lumenized vascular network in the developing myocardium, supported hiPSC-CM development and possessed fundamental cardiac tissue-level functions. In particular, this development-driven strategy offers a robust, tunable system to examine the contributions of individual cell types, matrix materials and additional factors for developmental insight, biomimetic matrix composition to advance biomaterial design, tissue/organ-level drug screening, and cell therapy for heart repair.

In Vivo Osteogenic Differentiation of Human Embryoid Bodies in an Injectable in Situ-Forming Hydrogel.

In this study, we examined the in vivo osteogenic differentiation of human embryoid bodies (hEBs) by using an injectable in situ-forming hydrogel. A solution containing MPEG-b-(polycaprolactone-ran-polylactide) (MCL) and hEBs was easily prepared at room temperature. The MCL solution with hEBs and osteogenic factors was injected into nude mice and developed into in situ-forming hydrogels at the injection sites; these hydrogels maintained their shape even after 12 weeks in vivo, thereby indicating that the in situ-forming MCL hydrogel was a suitable scaffold for hEBs. The in vivo osteogenic differentiation was observed only in the in situ gel-forming MCL hydrogel in the presence of hEBs and osteogenic factors. In conclusion, this preliminary study suggests that hEBs and osteogenic factors embedded in an in situ-forming MCL hydrogel may provide numerous benefits as a noninvasive alternative for allogeneic tissue engineering applications.

Combinatorial Activin Receptor-Like Kinase/Smad and Basic Fibroblast Growth Factor Signals Stimulate the Differentiation of Human Embryonic Stem Cells into the Cardiac Lineage

The transforming growth factor beta/bone morphogenetic protein-activated Smad signaling pathway plays a complicated role in the maintenance of human embryonic stem cell (hESC) pluripotency and in the cell fate decision of hESCs. Here, we report that sustained inhibition of the transforming growth factor beta type I receptor (also termed activin receptor-like kinase or ALK) using a chemical inhibitor selective for ALK4/5/7 (ALKi) leads to the cardiac differentiation of hESCs under feeder-free and serum-free conditions. Treatment with ALKi reduced Smad2/3 phosphorylation and increased Smad1/5/8 phosphorylation in hESCs, suggesting a requirement for active Smad1/5/8 signaling for cardiac induction in these cells when ALK/Smad2/3 is inhibited. Importantly, active basic fibroblast growth factor (bFGF) signaling was also required for ALKi-mediated cardiac differentiation of monolayer-cultured hESCs. The FGF receptor inhibitor SU5402 blocked ALKi-mediated cardiac induction in hESCs, whereas bone morphogenetic protein-4 enhanced the ALKi-induced increase in phospho-Smad1/5/8 levels but failed to induce the cardiac differentiation of hESCs and instead promoted trophoblastic differentiation. We also confirmed that ALKi potentially enhanced the cardiac differentiation of human embryoid bodies, as determined by expression of cardiac-specific markers, increased beating areas, and action potential recorded from beating areas. These results demonstrate that an ALKi could be used as a potential cardiac-inducing agent and that the development of culture conditions that provide an appropriate balance between ALK/Smad and bFGF signaling is necessary to direct the fate of hESCs into the cardiac lineage.

Establishing a Dynamic Process for the Formation, Propagation, and Differentiation of Human Embryoid Bodies

The promise of human embryonic stem cells (hESCs) to provide an unlimited supply of cells for cell therapy depends on the availability of a controllable bioprocess for their expansion and differentiation. We describe here a robust and well-defined scale up platform for human embryoid body (EB) formation, propagation, and differentiation. The efficacy of the dynamic process as compared to the static cultivation in Petri dishes was analyzed. Our optimized conditions include specific bioreactor and impeller type, seeding and propagation parameters, and scale up. Quantitative analyses of viable cell concentrations, apoptosis percentages, and EB yield revealed 6.7-fold enhancement in the generation of hESC-derived cells after 10 cultivation days. Other metabolic indices such as glucose consumption, lactic acid production and pH all pointed to efficient cell expansion in the dynamic cultures. The hydrodynamic conditions during seeding and cultivation were found to be crucial for the EB formation and propagation. The EBs' prearrangement in the static system and EB cultivation in the Glass Ball Impeller spinner flask resulted in high EB yield, a round homogenous shape, and the fastest growth rate. The appearance of representative genes of the three germ layers as well as primitive neuronal tube organization and blood vessel formation indicated that the initial developmental events in the human EBs are not interfered by the dynamic system. Furthermore, well developed endothelial networks and contracting EBs with functional cardiac muscle were also obtained after two cultivation weeks. Collectively, our study defines the technological platform for the controlled large-scale generation of hESC-derived cells for clinical and industrial applications.

Formation and Hematopoietic Differentiation of Human Embryoid Bodies by Suspension and Hanging Drop Cultures

The in vitro aggregation of human embryonic stem cells (hESCs) into clusters termed embryoid bodies (EBs) allows for the spontaneous differentiation of cells representing endoderm, mesoderm, and ectoderm lineages. This stochastic process results however, in the generation of low numbers of differentiated cells, and can be enhanced to some extent by the addition of exogenous growth factors or overexpression of regulatory genes. In the authors' laboratory, the use of hematopoietic cytokines in combination with the mesoderm inducer bone morphogenetic protein-4 (BMP-4) was able to generate up to 90% of CD45(+) hematopoietic cells with colony-forming unit (CFU) activity. This unit describes two protocols that have been successfully applied in the authors' laboratory for the generation of EBs in (1) suspension and (2) hanging drop (HD) cultures from enzymatically digested clumps of undifferentiated hESC colonies.

Temporal gene expression during differentiation of human embryonic stem cells and embryoid bodies.

The aim of this study was to characterize human embryonic stem (ES) cells at the molecular level by performing large-scale complementary DNA (cDNA) analysis using DNA micro-arrays. The transcription profile of human ES cells was determined by comparing it to 2, 10 and 30-day old embryoid bodies (EBs) using Affymetrix Genechip human micro-arrays (U133). According to this analysis we demonstrate that two human ES cell lines are more close to each other than to their differentiated derivatives. We also show the spectrum of cytokine receptors that they express, and demonstrate the presence of five genes that are highly specific to human ES cells and to germ cells. Moreover, by profiling different stages in the differentiation of human embryoid bodies, we illustrate the clustering of five sets of temporally expressed genes, which could be related to the sequential stages of embryonic development. Among them are known genes that are involved in early pattern formation. The present study provides a molecular basis for the identity of human ES cells and demonstrates that during their in vitro differentiation they express embryonic specific genes in a stage specific manner.