Abstract 14565: Comparative Transcriptomic Analysis of Multiple Cardiovascular Fates From Embryonic Stem Cells Predicts Regulatory Genes, Alternative Splicing and Long Non-coding RNAs in Human Cardiogenesis

Introduction: Dissecting the gene expression programs which control the early stage cardiovascular development is essential for understanding the molecular mechanisms of human heart development and heart disease. Many lineage-specific genes are involved in the differentiation into specific cell fate. Hypothesis: The lineage-specificity of the genes may predict regulatory potential during human cardiovascular differentiation. Methods: Here, we performed transcriptome sequencing (RNA-seq) of highly purified human Embryonic Stem Cells (hESCs), hESC-derived Multipotential Cardiovascular Progenitors (MCPs) and MCP-specified three cardiovascular lineages. A novel algorithm, named as Gene Expression Pattern Analyzer (GEPA), was developed to obtain a refined lineage-specificity map of all sequenced genes, which reveals dynamic changes of transcriptional factor networks underlying early human cardiovascular development. Results: The GEPA predictions captured ∼90% of top-ranked regulatory cardiac genes that were previously predicted based on chromatin signature changes in hESCs, and further defined their cardiovascular lineage-specificities, indicating that our multi-fate comparison analysis could predict novel regulatory genes. Furthermore, GEPA analysis revealed the MCP-specific expressions of genes in ephrin signaling pathway, positive role of which in cardiomyocyte differentiation was further validated experimentally. By using RNA-seq plus GEPA workflow, we also identified stage-specific RNA splicing switch and lineage-enriched long noncoding RNAs during human cardiovascular differentiation. Conclusions: Overall, our study utilized multi-cell-fate transcriptomic comparison analysis to establish a lineage-specific gene expression map for predicting and validating novel regulatory mechanisms underlying early human cardiovascular development.

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Genetic control of pluripotency epigenome determines differentiation bias in mouse embryonic stem cells

10.1101/2021.01.15.426861 ◽

2021 ◽

Author(s):

Candice Byers ◽

Catrina Spruce ◽

Haley J. Fortin ◽

Anne Czechanski ◽

Steven C. Munger ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Fate ◽

Genetic Regulation ◽

Embryonic Stem ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Embryoid Bodies ◽

Cell Fate Decisions

AbstractGenetically diverse pluripotent stem cells (PSCs) display varied, heritable responses to differentiation cues in the culture environment. By harnessing these disparities through derivation of embryonic stem cells (ESCs) from the BXD mouse genetic reference panel, along with C57BL/6J (B6) and DBA/2J (D2) parental strains, we demonstrate genetically determined biases in lineage commitment and identify major regulators of the pluripotency epigenome. Upon transition to formative pluripotency using epiblast-like cells (EpiLCs), B6 quickly dissolves naïve networks adopting gene expression modules indicative of neuroectoderm lineages; whereas D2 retains aspects of naïve pluripotency with little bias in differentiation. Genetic mapping identifies 6 major trans-acting loci co-regulating chromatin accessibility and gene expression in ESCs and EpiLCs, indicating a common regulatory system impacting cell state transition. These loci distally modulate occupancy of pluripotency factors, including TRIM28, P300, and POU5F1, at hundreds of regulatory elements. One trans-acting locus on Chr 12 primarily impacts chromatin accessibility in ESCs; while in EpiLCs the same locus subsequently influences gene expression, suggesting early chromatin priming. Consequently, the distal gene targets of this locus are enriched for neurogenesis genes and were more highly expressed when cells carried B6 haplotypes at this Chr 12 locus, supporting genetic regulation of biases in cell fate. Spontaneous formation of embryoid bodies validated this with B6 showing a propensity towards neuroectoderm differentiation and D2 towards definitive endoderm, confirming the fundamental importance of genetic variation influencing cell fate decisions.

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Large-Scale Liquid Culture Production of Erythroid Cells from Human Embryonic Stem Cells.

Blood ◽

10.1182/blood.v106.11.3631.3631 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3631-3631

Author(s):

Emmanuel N. Olivier ◽

Caihong Qiu ◽

Eric E. Bouhassira

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Red Blood Cells ◽

Human Embryonic Stem Cells ◽

Blood Cells ◽

Liquid Culture ◽

Embryonic Stem ◽

Erythroid Cells ◽

Early Human

Abstract Early human erythropoiesis is difficult to study because the material is hard to access experimentally. Hence, relatively little is known about the gene expression profiles or the mechanism of globin gene expression in these early cells. We report here a system to produce large quantities in liquid culture of virtually pure erythroid cells starting from H1 human embryonic stem cells (hESCs). The system is adapted from methods to produce enucleated red blood cells from cord blood and consist of five steps. During the first step, hESCs are differentiated by co-culture on immortalized human fetal hepatocytes (FH-B-hTERT) for two weeks to produce hematopoietic cells. CD34 positive cells are then magnetically sorted and placed in step 2 for seven days in serum free medium in the presence of SCF, Epo, hydro-cortisone, flt-3 ligand, BMP-4 and IL3. In step 3, the cells are incubated for seven days in the same medium and cytokine cocktail but with IGF-1 and without flt-3-ligand. In step 4, the cells are incubated with Epo for 3 days, and in step 5 the cells are incubated without cytokine on a feeder layer of MS-5 cells. In a typical experiment, 2 millions hESCs (two 10cm2 wells) yield 50,000 sorted CD34 positive cells. Culture of these cells for about three weeks yields about 5 millions erythroid cells. This corresponds to a 5 to 10,000-fold amplification of the sorted hematopoietic cells since we estimate that only a few percent of the cells recovered with the CD34 magnetic beads are hematopoietic. Flow cytometry analysis revealed that at the beginning of the second step the CD34+ cells are CD45−, CD71low and CD235a−. After 7 days in liquid culture CD34 expression is less than 10%, CD45 and CD71 expressions are more than 95% and CD235a is less than 20%. Eight days later the cells are 95% CD34− CD45− CD71high and CD235a+. Finally at the end of the culture the cells become CD34−, CD45−, CD71− and CD235a+. Morphological analysis by Wright-Giemsa staining revealed that the differentiation process in the liquid culture is relatively synchronous and that at the end of the culture the majority of the cells are orthochromatic erythroblasts. In contrast to cord blood derived cells placed in similar differentiation conditions, very few enucleated red blood cells could be obtained from hESCs. Hemoglobin can first be detected spectrophotometrically after day 10 of liquid culture and reach a concentration of 20 pmol/106 cells at the end of the culture. Globin chain analysis by PCR and HPLC reveals that ξ, α, ε, and γ globin chains are synthesized by these cells but not β-globin could be detected. A detailed analysis of globin expression in early human erythroid cells will be presented in an accompanying abstract. This experimental system will be useful to study early erythropoiesis, to test gene therapy vectors, and to create genetically modified red blood cells.

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Gene expression variability in mammalian embryonic stem cells using single cell RNA-seq data

Computational Biology and Chemistry ◽

10.1016/j.compbiolchem.2016.02.004 ◽

2016 ◽

Vol 63 ◽

pp. 52-61 ◽

Cited By ~ 19

Author(s):

Anna Mantsoki ◽

Guillaume Devailly ◽

Anagha Joshi

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Single Cell ◽

Embryonic Stem ◽

Rna Seq ◽

Expression Variability

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TINC - a method to dissect transcriptional complexes at single locus resolution - reveals novel Nanog regulators in mouse embryonic stem cells

10.1101/2020.04.03.023200 ◽

2020 ◽

Author(s):

AS Knaupp ◽

M Mohenska ◽

MR Larcombe ◽

E Ford ◽

SM Lim ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Fate ◽

Protein Complex ◽

Affinity Purification ◽

Embryonic Stem ◽

Regulatory Elements ◽

Genomic Region ◽

Profiling Techniques

AbstractCellular identity is ultimately controlled by transcription factors (TFs), which bind to specific regulatory elements (REs) within the genome to regulate gene expression and cell fate changes. While recent advances in genome-wide epigenetic profiling techniques have significantly increased our understanding of which REs are utilized in which cell type, it remains largely unknown which TFs and cofactors interact with these REs to modulate gene expression. A major hurdle in dissecting the whole composition of a multi-protein complex formed at a specific RE is the shortage of appropriate techniques. We have developed a novel method termed TALE-mediated Isolation of Nuclear Chromatin (TINC). TINC utilizes epitope-tagged TALEs to isolate a specific genomic region from the mammalian genome and includes a nuclei isolation and chromatin enrichment step for increased specificity. Upon cross-linking of the cells and isolation of the chromatin, the target region is purified based on affinity purification of the TALE and associated nucleic acid and protein molecules can be subjected to further analyses. A key TF in the pluripotency network and therefore in embryonic stem cells (ESCs) is NANOG. It is currently not fully understood how Nanog expression is regulated and consequently it remains unclear how the ESC state is maintained. Using TINC we dissected the protein complex formed at the Nanog promoter in mouse ESCs and identified many known and numerous novel factors.

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Mammalian SWI/SNF Chromatin Remodeling Complexes in Embryonic Stem Cells: Regulating the Balance Between Pluripotency and Differentiation

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.626383 ◽

2021 ◽

Vol 8 ◽

Author(s):

Ying Ye ◽

Xi Chen ◽

Wensheng Zhang

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Fate ◽

Chromatin Remodeling ◽

Molecular Mechanisms ◽

Embryonic Stem ◽

Self Renewal ◽

Baf Complex ◽

Chromatin Remodeling Complexes

The unique capability of embryonic stem cells (ESCs) to maintain and adjust the equilibrium between self-renewal and multi-lineage cellular differentiation contributes indispensably to the integrity of all developmental processes, leading to the advent of an organism in its adult form. The ESC fate decision to favor self-renewal or differentiation into specific cellular lineages largely depends on transcriptome modulations through gene expression regulations. Chromatin remodeling complexes play instrumental roles to promote chromatin structural changes resulting in gene expression changes that are key to the ESC fate choices governing the equilibrium between pluripotency and differentiation. BAF (Brg/Brahma-associated factors) or mammalian SWI/SNF complexes employ energy generated by ATP hydrolysis to change chromatin states, thereby governing the accessibility of transcriptional regulators that ultimately affect transcriptome and cell fate. Interestingly, the requirement of BAF complex in self-renewal and differentiation of ESCs has been recently shown by genetic studies through gene expression modulations of various BAF components in ESCs, although the precise molecular mechanisms by which BAF complex influences ESC fate choice remain largely underexplored. This review surveys these recent progresses of BAF complex on ESC functions, with a focus on its role of conditioning the pluripotency and differentiation balance of ESCs. A discussion of the mechanistic bases underlying the genetic requirements for BAF in ESC biology as well as the outcomes of its interplays with key transcription factors or other chromatin remodelers in ESCs will be highlighted.

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Transgenic Mice and Pluripotent Stem Cells Express EGFP under the Control of miR-302 Promoter

10.1101/450791 ◽

2018 ◽

Cited By ~ 1

Author(s):

Karim Rahimi ◽

Sara Parsa ◽

Mehrnoush Nikzaban ◽

Seyed Javad Mowla ◽

Fardin Fathi

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Transgenic Mice ◽

Cell Fate ◽

Core Promoter ◽

Expression Patterns ◽

Embryonic Stem ◽

Regulatory Sequences ◽

Somatic Tissues

MicroRNAs are a group of short non-coding RNAs that undertake various roles in different cell signaling pathways and developmental stages. They regulate gene expression levels at the post-transcriptional stage, which results in cleavage of mRNAs or repression of their translation. Some miRNAs, including the miR-302 cluster, are critical regulators for the stemness state of embryonic stem cells and cell fate patterning. The miR-302 cluster is located in the intron of a non-coding gene that has no other reported function, other than hosting miR-302, and grant a complex expression regulation through upstream its regulatory sequences. To date, analysis of the miR-302 expression pattern in a transgenic mouse model has not been reported. In this study, we generated transgenic mice that expressed EGFP driven by miR-302 upstream regulatory sequences that harbored the core promoter of its host gene. We examined the activity of the miR-302 promotor in somatic tissues of transgenic mice, transgenic blastocysts, and embryonic stem cells derived from transgenic blastocysts. Our results showed that miR-302 highly expressed in both blastocysts and the first passages of transgenic embryonic stem cells, and has low expression in the somatic tissues of transgenic mice. It could be concluded that different temporal and spatial gene expression patterns occur during the embryonic and adult stages in mice.

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Ethanol alters cell cycle gene expression in human embryonic stem cells

Journal of Pediatric Biochemistry ◽

10.1055/s-0036-1586381 ◽

2016 ◽

Vol 01 (03) ◽

pp. 201-208 ◽

Cited By ~ 2

Author(s):

Malini Krishnamoorthy ◽

Brian Gerwe ◽

Jamie Heimburg-Molinaro ◽

Rachel Nash ◽

Jagan Arumugham ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Embryonic Stem ◽

Cell Cycle Gene ◽

Cell Cycle Gene Expression

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Identification of New Transcription Factors that Can Promote Pluripotent Reprogramming

Stem Cell Reviews and Reports ◽

10.1007/s12015-021-10220-z ◽

2021 ◽

Author(s):

Ping Huang ◽

Jieying Zhu ◽

Yu Liu ◽

Guihuan Liu ◽

Ran Zhang ◽

...

Keyword(s):

Stem Cells ◽

Transcription Factors ◽

Embryonic Stem Cells ◽

Rna Sequencing ◽

Human Urine ◽

Embryonic Stem ◽

Rna Seq ◽

Reprogramming Efficiency ◽

Yamanaka Factors ◽

Urine Cells

Abstract Background Four transcription factors, Oct4, Sox2, Klf4, and c-Myc (the Yamanka factors), can reprogram somatic cells to induced pluripotent stem cells (iPSCs). Many studies have provided a number of alternative combinations to the non-Yamanaka factors. However, it is clear that many additional transcription factors that can generate iPSCs remain to be discovered. Methods The chromatin accessibility and transcriptional level of human embryonic stem cells and human urine cells were compared by Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) and RNA sequencing (RNA-seq) to identify potential reprogramming factors. Selected transcription factors were employed to reprogram urine cells, and the reprogramming efficiency was measured. Urine-derived iPSCs were detected for pluripotency by Immunofluorescence, quantitative polymerase chain reaction, RNA sequencing and teratoma formation test. Finally, we assessed the differentiation potential of the new iPSCs to cardiomyocytes in vitro. Results ATAC-seq and RNA-seq datasets predicted TEAD2, TEAD4 and ZIC3 as potential factors involved in urine cell reprogramming. Transfection of TEAD2, TEAD4 and ZIC3 (in the presence of Yamanaka factors) significantly improved the reprogramming efficiency of urine cells. We confirmed that the newly generated iPSCs possessed pluripotency characteristics similar to normal H1 embryonic stem cells. We also confirmed that the new iPSCs could differentiate to functional cardiomyocytes. Conclusions In conclusion, TEAD2, TEAD4 and ZIC3 can increase the efficiency of reprogramming human urine cells into iPSCs, and provides a new stem cell sources for the clinical application and modeling of cardiovascular disease. Graphical abstract

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Nicotinamide promotes pancreatic differentiation through the dual inhibition of CK1 and ROCK kinases in human embryonic stem cells

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02426-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yumeng Zhang ◽

Jiaqi Xu ◽

Zhili Ren ◽

Ya Meng ◽

Weiwei Liu ◽

...

Keyword(s):

Stem Cells ◽

Cell Fate ◽

Human Embryonic Stem Cells ◽

Embryonic Stem ◽

Cell Fate Determination ◽

Rna Seq ◽

Pancreatic Cell ◽

Pancreatic Progenitor ◽

Pancreatic Progenitors ◽

Rock Inhibition

Abstract Background Vitamin B3 (nicotinamide) plays important roles in metabolism as well as in SIRT and PARP pathways. It is also recently reported as a novel kinase inhibitor with multiple targets. Nicotinamide promotes pancreatic cell differentiation from human embryonic stem cells (hESCs). However, its molecular mechanism is still unclear. In order to understand the molecular mechanism involved in pancreatic cell fate determination, we analyzed the downstream pathways of nicotinamide in the derivation of NKX6.1+ pancreatic progenitors from hESCs. Methods We applied downstream modulators of nicotinamide during the induction from posterior foregut to pancreatic progenitors, including niacin, PARP inhibitor, SIRT inhibitor, CK1 inhibitor and ROCK inhibitor. The impact of those treatments was evaluated by quantitative real-time PCR, flow cytometry and immunostaining of pancreatic markers. Furthermore, CK1 isoforms were knocked down to validate CK1 function in the induction of pancreatic progenitors. Finally, RNA-seq was used to demonstrate pancreatic induction on the transcriptomic level. Results First, we demonstrated that nicotinamide promoted pancreatic progenitor differentiation in chemically defined conditions, but it did not act through either niacin-associated metabolism or the inhibition of PARP and SIRT pathways. In contrast, nicotinamide modulated differentiation through CK1 and ROCK inhibition. We demonstrated that CK1 inhibitors promoted the generation of PDX1/NKX6.1 double-positive pancreatic progenitor cells. shRNA knockdown revealed that the inhibition of CK1α and CK1ε promoted pancreatic progenitor differentiation. We then showed that nicotinamide also improved pancreatic progenitor differentiation through ROCK inhibition. Finally, RNA-seq data showed that CK1 and ROCK inhibition led to pancreatic gene expression, similar to nicotinamide treatment. Conclusions In this report, we revealed that nicotinamide promotes generation of pancreatic progenitors from hESCs through CK1 and ROCK inhibition. Furthermore, we discovered the novel role of CK1 in pancreatic cell fate determination.

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