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Comparative transcriptomic analysis of multiple cardiovascular fates from embryonic stem cells predicts novel regulators in human cardiogenesis.

Li Y, Lin B, Yang L - Sci Rep (2015)

Bottom Line: 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 non-coding RNAs during human cardiovascular differentiation.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental Biology, University of Pittsburgh School of Medicine, 530 45th Street, Rangos Research Center, Pittsburgh, PA 15201.

ABSTRACT
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. 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. Moreover, our 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 non-coding RNAs during human cardiovascular differentiation. 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.

No MeSH data available.


Related in: MedlinePlus

Validation of GEPA predicted regulatory pathways using in vitro cardiovascular differentiation from human iPS cells. (a-d) Validating the roles of well-known signaling pathways in human cardiovascular differentiation. (a) A scheme of modulating BMP and Wnt pathways from day1 to day4. No factor was added from day 0 to day 1 and ActivinA (3 ng/ml) was added into all conditions through day 1 to day 4. (b) Representative images showing EBs of day 20. Scale bar, 100 μm. (c) Quantitative analysis of EB numbers at day 20. Statistical analysis were performed with unpaired t-test, *p < 0.01 (d) Quantitative analysis of ratio of CM at day 20 EBs. EBs were dissociated, immunostained with anti-CTNT antibody and FACS analyzed. CTNT+ cells represent CMs. Statistical analysis were performed with unpaired t test, *p < 0.1 (e) A scheme of inhibiting ephrin receptor signaling pathway using lithocholic acid (LA), which is a small molecule inhibitor of ephrin receptors, from day 4 to day 20 of differentiation in human iPS cells. DMSO treatment served as control. (f) Representative images showing day 20 EBs in DMSO and LA treated wells. Scale bar, 100 μm. (g) FACS analysis of ratios of CTNT+ CMs from day 20 EBs. Results were represented as mean ± S.D., from 3 independent experiments, statistical analysis were performed with unpaired t test, *p < 0.01.
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f6: Validation of GEPA predicted regulatory pathways using in vitro cardiovascular differentiation from human iPS cells. (a-d) Validating the roles of well-known signaling pathways in human cardiovascular differentiation. (a) A scheme of modulating BMP and Wnt pathways from day1 to day4. No factor was added from day 0 to day 1 and ActivinA (3 ng/ml) was added into all conditions through day 1 to day 4. (b) Representative images showing EBs of day 20. Scale bar, 100 μm. (c) Quantitative analysis of EB numbers at day 20. Statistical analysis were performed with unpaired t-test, *p < 0.01 (d) Quantitative analysis of ratio of CM at day 20 EBs. EBs were dissociated, immunostained with anti-CTNT antibody and FACS analyzed. CTNT+ cells represent CMs. Statistical analysis were performed with unpaired t test, *p < 0.1 (e) A scheme of inhibiting ephrin receptor signaling pathway using lithocholic acid (LA), which is a small molecule inhibitor of ephrin receptors, from day 4 to day 20 of differentiation in human iPS cells. DMSO treatment served as control. (f) Representative images showing day 20 EBs in DMSO and LA treated wells. Scale bar, 100 μm. (g) FACS analysis of ratios of CTNT+ CMs from day 20 EBs. Results were represented as mean ± S.D., from 3 independent experiments, statistical analysis were performed with unpaired t test, *p < 0.01.

Mentions: Since the findings from human ESCs could benefit the generation of patient-specific cardiac cells from human iPSCs, we next validated the functions of predicted signaling pathways using human iPSCs. Firstly, we examined the roles of several key signaling pathways in vertebrate heart development, including BMP, Wnt and FGF, during cardiovascular differentiation from human Y1-iPSCs (Fig. 6). We generated embryoid bodies (EBs) from Y1 iPS cells at day 0 using our established cardiac differentiation method (Fig. 6a) , followed with modulations of BMP and Wnt pathways before the formation of MCPs, which was from day 1 to day 4 of differentiation. Adding BMP4 (10 ng/ml) significantly increased the total number of EBs from day 4, when compared with the absence of BMP4 or with the adding of BMP antagonist, Dorsomorphin (2 μM). Activating Wnt pathway by adding Wnt3a (100 ng/ml) from day 1 to day 4 increased the number of EBs, whereas blocking Wnt pathway by adding a Wnt inhibitor, DKK1, substantially decreased the EB formation efficiency (Fig. 6b,c). Next, we changed all the day 4 media, which contained various pathway modulators (Fig. 6a), to basal medium without any factor until day 20. The day 20 EBs were dissociated, followed by immunostaining of CTNT, which is a marker for CM. Ratio of CMs (CTNT+ cells) was examined using fluorescence-activated cell sorting (FACS). Approximately 20% CTNT+ CMs were generated with presence of BMP from day 1 to day 4, which was significantly higher than that from the control condition containing no BMP/Wnt modulators from day 1 (Fig. 6d). Although early presence of Wnt3a increased the number of total EBs at day 4 (Fig. 6b,c), only 5% CM was found at day 20, which was similar to the control condition containing no pathway modulators (Fig. 6d). This indicates that Wnt pathway could play different roles in early vs. late stages of human cardiovascular development. Additionally, these data demonstrate that our cardiac differentiation system could provide an in vitro model for testing the functionality of signaling pathways in early human cardiovascular development. Therefore, we next sought to examine the role of ephrin receptor signaling pathway, which was highly enriched in the MCP formation stage by GEPA and IPA analyses (Fig. 3b and Fig. 5d) with unknown function during early human heart formation. The expressions of ephrin and ephrin receptor genes were highly enriched in “MCP” and “MCP&CM” (Fig. 5e), suggesting an essential role of ephrin receptor signaling during early human CM differentiation. A previous study indicated that ephrin activity could be completely blocked by adding lithocholic acid (LA) at 200 μM, which is a small molecule to inhibit binding of ephrin ligands to EphA/B receptors23. We thus added LA into the differentiating EBs from day 4 to 20 with increased doses (Fig. 6e) to examine the impact of blocking Ephrin pathway on human MCP formation and CM commitment. Treating with LA at 100 and 200 μM did not affect the growth of EBs, whereas the loading of LA at 300 μM showed negative effect in EB growth from day 6 (Fig. 6f). To determine the efficiency of CM differentiation from human iPS cell post modulating of ephrin signaling, we used FACS to measure the ratios of CTNT positive CMs in day 20 EBs and observed a dose-dependent suppressing role of LA in CM-derivation from iPS cells (Fig. 6g). Consistently, a lower ratio of beating EBs was found in the LA treated EBs when compared with the control DMSO treated EBs (Supplementary Video 1 and 2). These results, together with the finding that EphB4-knockout mouse ES cells were deficient in CM differentiation24, demonstrate that ephrin signaling plays a key role in mammalian cardiogenesis. Moreover, this demonstrates that the GEPA analysis of comparative RNA-seq data could successfully predict novel functional signaling pathways in early human cardiovascular development, and possibly in other biological processes.


Comparative transcriptomic analysis of multiple cardiovascular fates from embryonic stem cells predicts novel regulators in human cardiogenesis.

Li Y, Lin B, Yang L - Sci Rep (2015)

Validation of GEPA predicted regulatory pathways using in vitro cardiovascular differentiation from human iPS cells. (a-d) Validating the roles of well-known signaling pathways in human cardiovascular differentiation. (a) A scheme of modulating BMP and Wnt pathways from day1 to day4. No factor was added from day 0 to day 1 and ActivinA (3 ng/ml) was added into all conditions through day 1 to day 4. (b) Representative images showing EBs of day 20. Scale bar, 100 μm. (c) Quantitative analysis of EB numbers at day 20. Statistical analysis were performed with unpaired t-test, *p < 0.01 (d) Quantitative analysis of ratio of CM at day 20 EBs. EBs were dissociated, immunostained with anti-CTNT antibody and FACS analyzed. CTNT+ cells represent CMs. Statistical analysis were performed with unpaired t test, *p < 0.1 (e) A scheme of inhibiting ephrin receptor signaling pathway using lithocholic acid (LA), which is a small molecule inhibitor of ephrin receptors, from day 4 to day 20 of differentiation in human iPS cells. DMSO treatment served as control. (f) Representative images showing day 20 EBs in DMSO and LA treated wells. Scale bar, 100 μm. (g) FACS analysis of ratios of CTNT+ CMs from day 20 EBs. Results were represented as mean ± S.D., from 3 independent experiments, statistical analysis were performed with unpaired t test, *p < 0.01.
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f6: Validation of GEPA predicted regulatory pathways using in vitro cardiovascular differentiation from human iPS cells. (a-d) Validating the roles of well-known signaling pathways in human cardiovascular differentiation. (a) A scheme of modulating BMP and Wnt pathways from day1 to day4. No factor was added from day 0 to day 1 and ActivinA (3 ng/ml) was added into all conditions through day 1 to day 4. (b) Representative images showing EBs of day 20. Scale bar, 100 μm. (c) Quantitative analysis of EB numbers at day 20. Statistical analysis were performed with unpaired t-test, *p < 0.01 (d) Quantitative analysis of ratio of CM at day 20 EBs. EBs were dissociated, immunostained with anti-CTNT antibody and FACS analyzed. CTNT+ cells represent CMs. Statistical analysis were performed with unpaired t test, *p < 0.1 (e) A scheme of inhibiting ephrin receptor signaling pathway using lithocholic acid (LA), which is a small molecule inhibitor of ephrin receptors, from day 4 to day 20 of differentiation in human iPS cells. DMSO treatment served as control. (f) Representative images showing day 20 EBs in DMSO and LA treated wells. Scale bar, 100 μm. (g) FACS analysis of ratios of CTNT+ CMs from day 20 EBs. Results were represented as mean ± S.D., from 3 independent experiments, statistical analysis were performed with unpaired t test, *p < 0.01.
Mentions: Since the findings from human ESCs could benefit the generation of patient-specific cardiac cells from human iPSCs, we next validated the functions of predicted signaling pathways using human iPSCs. Firstly, we examined the roles of several key signaling pathways in vertebrate heart development, including BMP, Wnt and FGF, during cardiovascular differentiation from human Y1-iPSCs (Fig. 6). We generated embryoid bodies (EBs) from Y1 iPS cells at day 0 using our established cardiac differentiation method (Fig. 6a) , followed with modulations of BMP and Wnt pathways before the formation of MCPs, which was from day 1 to day 4 of differentiation. Adding BMP4 (10 ng/ml) significantly increased the total number of EBs from day 4, when compared with the absence of BMP4 or with the adding of BMP antagonist, Dorsomorphin (2 μM). Activating Wnt pathway by adding Wnt3a (100 ng/ml) from day 1 to day 4 increased the number of EBs, whereas blocking Wnt pathway by adding a Wnt inhibitor, DKK1, substantially decreased the EB formation efficiency (Fig. 6b,c). Next, we changed all the day 4 media, which contained various pathway modulators (Fig. 6a), to basal medium without any factor until day 20. The day 20 EBs were dissociated, followed by immunostaining of CTNT, which is a marker for CM. Ratio of CMs (CTNT+ cells) was examined using fluorescence-activated cell sorting (FACS). Approximately 20% CTNT+ CMs were generated with presence of BMP from day 1 to day 4, which was significantly higher than that from the control condition containing no BMP/Wnt modulators from day 1 (Fig. 6d). Although early presence of Wnt3a increased the number of total EBs at day 4 (Fig. 6b,c), only 5% CM was found at day 20, which was similar to the control condition containing no pathway modulators (Fig. 6d). This indicates that Wnt pathway could play different roles in early vs. late stages of human cardiovascular development. Additionally, these data demonstrate that our cardiac differentiation system could provide an in vitro model for testing the functionality of signaling pathways in early human cardiovascular development. Therefore, we next sought to examine the role of ephrin receptor signaling pathway, which was highly enriched in the MCP formation stage by GEPA and IPA analyses (Fig. 3b and Fig. 5d) with unknown function during early human heart formation. The expressions of ephrin and ephrin receptor genes were highly enriched in “MCP” and “MCP&CM” (Fig. 5e), suggesting an essential role of ephrin receptor signaling during early human CM differentiation. A previous study indicated that ephrin activity could be completely blocked by adding lithocholic acid (LA) at 200 μM, which is a small molecule to inhibit binding of ephrin ligands to EphA/B receptors23. We thus added LA into the differentiating EBs from day 4 to 20 with increased doses (Fig. 6e) to examine the impact of blocking Ephrin pathway on human MCP formation and CM commitment. Treating with LA at 100 and 200 μM did not affect the growth of EBs, whereas the loading of LA at 300 μM showed negative effect in EB growth from day 6 (Fig. 6f). To determine the efficiency of CM differentiation from human iPS cell post modulating of ephrin signaling, we used FACS to measure the ratios of CTNT positive CMs in day 20 EBs and observed a dose-dependent suppressing role of LA in CM-derivation from iPS cells (Fig. 6g). Consistently, a lower ratio of beating EBs was found in the LA treated EBs when compared with the control DMSO treated EBs (Supplementary Video 1 and 2). These results, together with the finding that EphB4-knockout mouse ES cells were deficient in CM differentiation24, demonstrate that ephrin signaling plays a key role in mammalian cardiogenesis. Moreover, this demonstrates that the GEPA analysis of comparative RNA-seq data could successfully predict novel functional signaling pathways in early human cardiovascular development, and possibly in other biological processes.

Bottom Line: 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 non-coding RNAs during human cardiovascular differentiation.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental Biology, University of Pittsburgh School of Medicine, 530 45th Street, Rangos Research Center, Pittsburgh, PA 15201.

ABSTRACT
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. 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. Moreover, our 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 non-coding RNAs during human cardiovascular differentiation. 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.

No MeSH data available.


Related in: MedlinePlus