Placental growth factor exerts a dual function for cardiomyogenesis and vasculogenesis during heart development – Nature Communications

Embryonic heart single-cell RNA-seq analyses define atlases of cardiogenic growth factors

To clarify the roles and associations of each of the growth factors in heart development, we first utilized a single-cell RNA-seq dataset, previously obtained from human embryonic heart samples (4.5 to 10 weeks of fetal ages)^6,18. A total of the 458 individual cardiac cells derived from micro-dissected heart regions, i.e., outflow tract (OFT), atria, and ventricles were segregated into 10 clusters including a cono-ventricular region-specific heart progenitor (CVP; cluster #1) that appeared predominantly in OFT at earlier stages (4.5 to 5.5 weeks of fetal age), by a dimensionally reduction method such as t-distributed stochastic neighbor embedding (tSNE) based on the profiles of differentially expressed genes (Supplementary Fig. 1a, b). Expression of the early cardiogenic and SHF progenitor-specific genes, such as ISL1²², BMP4²³, MEIS2²⁴, PDGFRA²⁵, and LGR5⁶ were enriched in the cells of the CVP cluster (Supplementary Fig. 1c, d), suggesting that the CVPs would represent more immature heart progenitors through the stages. To assess the significance of the co-expression of genes in cells, we performed Guilt-by-Association and correlation analysis²⁶. Among representative 52 growth factors (Supplementary Data 1), we identified the top 12 growth factors that were highly co-expressed in the typical SHF/OFT progenitor ISL1⁺ cells, indicating the specific roles of these factors in the human early heart progenitors (Supplementary Fig. 1e).

To gain further insights regarding the roles and associations of growth factors in cardiogenesis, we next analyzed single-cell RNA-seq datasets of the obtained non-human primate embryonic hearts (Methods). Similarly, the harvested hearts were micro-dissected as a whole heart (4 weeks of fetal age) or into OFT, atria, and right and left ventricle (RV and LV) (7 weeks of fetal age), and single cardiac cells dissociated from each of the compartments were obtained using a fluorescence-activated cell sorter. The gating strategies for sorting the live cells are shown in Supplementary Fig. 2a. Then, through single-cell RNA-seq analysis, a total of 1786 individual cardiac cells derived from the primate embryonic hearts were divided into 13 clusters, including the SHF/OFT progenitors (cluster #5), by tSNE using the Seurat program²⁷ (Fig. 1a, b). Differential gene expression analysis revealed that the pan-cardiac and FHF-related markers, such as NKX2-5, HAND1, IRX4, and TNNT2^28,29 were enriched in clusters #2, #9, and #10, which were considered as late CMs, FHF progenitors, and proliferative CMs, respectively, whereas the SHF/OFT progenitor markers ISL1, FGF10, NKX2-6, and HOXA1 were enriched in a cluster #5 (Fig. 1c, d). The EC markers such as PECAM1 and CDH5 were enriched in both clusters #8 and #11, while an endocardium marker NPR3 was enriched only in a cluster #8, indicating that clusters #8 and #11 were considered as endocardium and ECs, respectively (Supplementary Fig. 3a, b). The neural crest cell (NCC) and SMC markers such as NGFR and MYH11 were enriched in clusters #6 and #13, which were considered as NCCs and SMCs, respectively (Supplementary Fig. 3b). Expression patterns of genes specific to other clusters are also shown in Supplementary Fig. 3b.

a The tSNE analysis segregated a total of 1786 single cardiac cells, obtained from micro-dissected heart regions of primate embryos at 4 and 7 weeks of fetal age, into 13 clusters, including SHF/OFT progenitors (cluster #5). b Heatmap image depicting the representative differentially expressed genes in each of the 13 clusters in a. c Feature plots of the pan-cardiac and first heart filed (FHF)-related genes, as well as the SHF marker genes on the tSNE plots in a. d Violin plots of the same genes as in c in the segregated 13 clusters of the primate embryonic heart-derived single cells. e The rankings of the growth factor genes correlated with expression of the SHF-specific gene ISL1 (left) and the pan-cardiac/FHF-specific gene NKX2-5 (right) in single-cell RNA-seq data of primate embryonic hearts. The top 8 growth factors are highlighted in red shades, respectively. The corrected P-value for each gene was calculated by Guilt-by-Association and correlation analysis²⁶ (with Pearson correlation coefficient test). Source data are provided as a Source Data file. f Atlas of growth factor expression from multiple heart cell types in developing hearts (i.e., SHF, FHF, CM, pacemaker cell [PM], EC, SMC, and cardiac fibroblasts [CFB]), which was analyzed and defined by the Seurat and Guilt-by-Association and correlation analyses using single-cell RNA-seq data of primate embryonic hearts. In the Seurat program, each cluster (e.g., SHF: cluster #5, FHF: cluster #9, etc. [Supplementary Fig. 2a, b])-specific growth factors were identified, while in the latter, the growth factors correlated with each of the cell type-specific markers (i.e., SHF: ISL1, FHF: NKX2-5, CM: TNNT2; PM: SHOX2; EC: PECAM1, SMC: ACTG2, and CFB: DCN) were identified. The red, yellow, blue, and sky-blue colors indicate that each growth factor is ranked within the top 300 (red), top 301–600 (yellow), top 601–1200 (blue), or top 1201–2000 (sky-blue) genes correlated with each of the clusters and/or the cell type-specific markers. Thus, a red color in the chart indicates the strongest association between each of the growth factors and each of the heart cell types.

Full size image

Next, using Guilt-by-Association and correlation analysis for single-cell RNA-seq data of primate embryonic hearts, we examined the co-expression of each of growth factors with each of the typical markers for multiple heart cell types (i.e., SHF, FHF, CM, pacemaker cell, EC, SMC, and cardiac fibroblasts) and identified the growth factors specific to each of the cell types (Fig. 1e). Finally, we merged both differential gene expression analysis in the clusters defined by tSNE on the Seurat program (Fig. 1a, b) and Guilt-by-Association and correlation analysis (Fig. 1e), which established the atlas of growth factor expression profiles of multiple heart cell types in developing hearts (Fig. 1f). For example, expression of bone morphogenetic protein 5 (BMP5) and epidermal growth factor-like protein 7 (EGFL7) were strongly distributed to FHF/CM and ECs/endocardial cells, respectively, highlighting the functional roles of these factors in multiple heart cell types (Fig. 1f; Supplementary Fig. 3c, d).

Biphasic expression patterns of placental growth factor in SHF progenitors and vascular cells

Based on the results in single-cell RNA-seq analyses of human and primate embryonic hearts (Fig. 1; Supplementary Figs. 1 and 3), we selected the 24 growth factors that appear to promote cardiomyogenesis and/or vasculogenesis via enhancing the function of heart progenitors among the representative 52 growth factors, and generated modRNAs of these 24 growth factors, which involve: (1) the top 12 growth factors that are highly co-expressed in the human ISL1⁺ SHF/OFT progenitor cells (Supplementary Fig. 1e); (2) 6 growth factors (CLEC11A [SCGF], HGF, IGFBP4, NGF, NRG1, and PDGFA) that exhibit high association with CM and/or SMC/EC in Fig. 1f; and (3) 6 growth factors (CSF3 [GCSF], FGF2, FGF4, FGF17, RLN2, and TNFα) that showed cardiogenic effects in the in vitro hESC-CM differentiation^18,30 pilot studies, using each of recombinant proteins (Supplementary Data 1).

We then tested the potential cardiomyogenic and/or vasculogenic effects of these modRNAs in the in vitro hESC-CM differentiation. In the cardiomyogenic assays, the cells were treated with the modRNA for 5 h on either day 3 or 6 and harvested 3 days later for flow cytometry analysis to detect differentiated CMs. The gating strategies on flow cytometry experiments are shown in Supplementary Fig. 2b. When modRNAs were added to cell culture on day 3, only PLGF modRNA significantly increased the number of differentiated TNNT2⁺ CMs on day 6 among the 24 growth factor modRNAs (Fig. 2a, b). The number of totally cultured cells or a cell-proliferation marker Ki67⁺ cells was not different between the PLGF modRNA-treated cells and control on day 6. Therefore, PLGF appeared to enhance CM differentiation among the hESC-derived cells, rather than promoting CM proliferation. Interestingly, when PLGF or other growth factors’ modRNA was added on day 6, the number of differentiated TNNT2⁺ CMs on day 9 was not increased compared to control (Fig. 2c, d), suggesting PLGF modRNA may promote the early-staged (but not late-staged) heart progenitors to differentiate into more CMs in vitro.

a, b The cardiomyogenic assays were analyzed on day 6. For the in vitro hESC-CM differentiation^18,30, the cells were treated with each of the 24 selected growth factors’ modRNAs for 5 h on day 3 and analyzed for a CM marker TNNT2 by flow cytometry on day 6. The cell numbers of TNNT2⁺ CMs were then calculated. The panels in a show selected representative images on flow cytometry analysis, and the chart in b shows relative ratios of the cell numbers of TNNT2⁺ CMs, obtained with treatment with the representative 10 growth factors’ modRNAs, as well as control (no modRNA transfection [TF]) and LacZ modRNA-transfected cells. Of note, only PLGF modRNA significantly increased the number of TNNT2⁺ CMs on day 6 among all growth factors tested. c, d The cardiomyogenic assays were analyzed on day 9. The cells were treated with modRNAs encoding each of the 24 selected growth factors for 5 h on day 6 and analyzed for a CM marker TNNT2 by flow cytometry on day 9. e, f The vasculogenic assays for ECs analyzed on day 8. In the hESC-CM differentiation, the cells were treated with each of the 24 selected modRNAs encoding growth factors for 5 h on day 5 and analyzed for an EC marker PECAM1 by flow cytometry on day 8. VEGF-A modRNA significantly increased the number of PECAM1⁺ ECs, as expected. Albeit to a lesser degree than VEGF-A, PLGF modRNA also showed a significant increase in the number of ECs compared to the control. g, h The vasculogenic assays for SMCs analyzed on day 8. The same cells as in e and f were also analyzed for an SMC marker PDGFRB by flow cytometry. Data in b, d, f, and h are presented as mean ± SD (n = 5 independent experiments). Differences between groups were examined with one-way ANOVA followed by Tukey multiple comparisons test. *P < 0.05, **P < 0.01, and ***P < 0.0001 vs. control. Source data are provided as a Source Data file.

Full size image

In the vasculogenic assays, the cells were treated with the modRNA on day 5 in CM differentiation and employed for flow cytometry analysis to detect differentiated vascular cells on day 8. As expected from the previous literature^15,16,17, VEGF-A modRNA significantly increased the number of differentiated PECAM1⁺ ECs (Fig. 2e, f). Of note, albeit to a lesser degree than VEGF-A, PLGF modRNA also increased the number of differentiated ECs compared to control (Fig. 2e, f). This suggests that PLGF modRNA may promote the late-staged heart progenitors to differentiate into more ECs in vitro. In contrast, no modRNAs increased the number of differentiated PDGFRB⁺ SMCs in the vasculogenic assays (Fig. 2g, h).

Next, we examined expression patterns of PLGF mRNA in single-cell RNA-seq data of primate embryonic hearts. Intriguingly, in consistency with the findings in the in vitro hESC cardiogenesis assay, PLGF was specifically expressed in the SHF/OFT progenitors (cluster #5) in the early stage and expressed in the ECs (cluster #11) and SMCs (cluster #13) in the late stage (Fig. 3a–c). To corroborate these findings, we then employed immunostaining of human embryonic sectioned hearts. We observed that PLGF⁺ cells appeared predominantly in the OFT region of the early-staged heart (5.5 weeks of fetal age), often co-expressing an SHF/OFT progenitor marker ISL1 and/or a CM marker TNNT2 (Fig. 3d). In contrast, in the late-staged heart (≥8 weeks), PLGF expression was often seen in VE-cadherin (VEC)⁺ ECs/endocardial cells (Fig. 3e) and SM22⁺ SMCs (Fig. 3f) in the heart, irrespective of their anatomical locations. Collectively, these indicate that PLGF shows biphasic and specific expression patterns in the SHF heart progenitors and CM intermediates at the earlier stage and in the vascular cells at the later stage during heart development, and may thereby exert a dual effect for both cardiomyogenesis and vasculogenesis.

a The 13 cell populations were segregated by the Seurat/tSNE analysis using a total of 1786 single cardiac cells of primate embryonic hearts. b Feature plots of PLGF, a SHF marker ISL1, a SMC marker ACTG2, and an EC marker PECAM1 on the tSNE plots in a. c Violin plots of the same genes as in b, in the segregated 13 clusters of the primate embryonic heart-derived single cells. d Immunohistochemistry of the sectioned human embryonic heart at 5.5 weeks of fetal age. Coronal view. The confocal microscopic images highlight the PLGF⁺ cells (green) co-expressing an SHF marker ISL1 (red) and/or a CM marker TNNT2 (light gray) in the outflow tract (OFT) region. Vent, ventricle. e Immunohistochemistry of the sectioned human embryonic heart at 8 weeks of fetal age. Coronal view. The confocal microscopic images highlight the PLGF⁺ ECs (top; arrowheads) in the ventricular wall and the PLGF⁺ endocardial cells (bottom; arrowheads) in the atrium, both of which co-expressed an endothelial marker VE-cadherin (VEC; red). f Immunohistochemistry of the sectioned human embryonic heart at 8 weeks of fetal age. Coronal view. The confocal microscopic images highlight the PLGF⁺ SMCs, which co-expressed a SMC marker SM22 (arrowheads). Representative images in each of d, e, and f were obtained from the repeated experiments (n = 2 [d] or 3 [e and f] biologically independent samples) with similar results.

Full size image

PLGF deletion attenuates the induction of both cardiomyocytes and vascular cells

To further explore the functional role of PLGF in human cardiomyogenesis and vasculogenesis, we generated PLGF-knockout (PLGF-KO) hESC lines for the loss-of-function experiments. Through CRISPR/Cas9 technology³¹, we established the two PLGF-KO hESC clones, which had frameshift mutations in the third exon of the PLGF gene in both alleles, respectively (Supplementary Fig. 4). During the in vitro hESC-CM differentiation, expression of PLGF protein in wild-type (WT) cells peaked at day 3 and was detectable until day 12 (Supplementary Fig. 5a, b). In contrast, very little or no expression of PLGF protein could be detected in PLGF-KO hESC-derived cells during CM differentiation (Supplementary Fig. 5c).

Next, to detect any cardiogenic phenotype in regard to PLGF deletion, the PLGF-KO hESC clones underwent in vitro CM differentiation using previously published protocols^18,30 and we measured the ratios of cells that were positive for a cell-proliferation marker Ki67³², a SHF heart progenitor marker ISL1, and a differentiated CM marker TNNT2 on days 6 and 15 by flow cytometry (Fig. 4a–c). The gating strategies are shown in Supplementary Fig. 2b. On day 6 when ISL1 expression reaches a peak, the PLGF-KO hESC-derived cells exhibited lower ratios of both ISL1⁺ and TNNT2⁺ cells compared to WT control ([%ISL1⁺] WT 86.0 ± 7.3% vs. KO 49.4 ± 22.9%, P < 0.05; [%TNNT2⁺] WT 31.5 ± 7.3% vs. KO 2.7 ± 3.7%, P < 0.01), although %Ki67⁺ did not change between WT and PLGF-KO cells (Fig. 4a, c). Likewise, on day 15, the PLGF-KO hESC-derived cells exhibited a significantly lower ratio of TNNT2⁺ cells compared to WT (WT 77.0 ± 17.7% vs. KO 19.1 ± 14.6%; P < 0.01) (Fig. 4b, c). The number of the beating CMs (TNNT2⁺) generated from PLGF-KO hESCs was also much lower than those generated from WT hESCs (WT 3.6 ± 0.5 × 10⁶ per well vs. KO 0.9 ± 0.5 × 10⁶ per well; P < 0.01). These results indicate that PLGF would be important for proper induction of CMs from hESCs in vitro.

a, b Representative images on flow cytometry analysis showing the ratios of a cell-proliferation marker Ki67⁺ (left), a SHF/heart progenitor marker ISL1⁺ (middle), and a differentiated CM marker TNNT2⁺ (right) in WT (top) and PLGF-KO (bottom) cells at days 6 (a) and 15 (b) in hESC-CM differentiation^18,30. c Statistical data of the ratios of %Ki67⁺ (left), %ISL1⁺ (middle), and %TNNT2⁺ (right) in a and b. d Flow cytometry analysis and statistical data showing the ratios of vascular SMCs (PDGFRB⁺) at day 6 in hESC-SMC differentiation³³ of WT and PLGF-KO hESCs. e Flow cytometry analysis and statistical data showing the ratios of vascular ECs (VE-cadherin [VEC]⁺) at day 6 in hESC-EC differentiation³⁴ of WT and PLGF-KO hESCs. Data in c, d, and e are presented as mean ± SD (n = 5 independent experiments). Differences between groups were examined with one-way ANOVA followed by Tukey multiple comparisons test. *P < 0.05, **P < 0.01, and ***P < 0.0001. Source data are provided as a Source Data file.

Full size image

To evaluate capabilities to differentiate into vascular cells, we then employed the PLGF-KO hESC clones in the in vitro SMC³³ and EC³⁴ differentiation protocols. In line with the previous reports, the mesodermal lineage-derived vascular SMCs (PDGFRB⁺) and ECs (VEC⁺) were obtained from WT hESCs on day 6 in SMC and EC differentiation, occupying around 70% (SMC) and 50% (EC) of the cultured cells, respectively (Fig. 4d, e). Of particular interest, PLGF-KO hESC-derived cells exhibited much lower efficacies for induction into both vascular SMCs and ECs ([SMC] WT 72.6 ± 18.7% vs. KO 10.8 ± 8.8%, P < 0.01; [EC] WT 45.5 ± 7.6% vs. KO 2.8 ± 0.9%, P < 0.0001) (Fig. 4d, e). These results strongly support the notion that PLGF would be essential for induction into both vascular cells, i.e., SMCs and ECs from hESCs in vitro.

Finally, we performed rescue experiments for the PLGF-KO hESC clones using recombinant human PLGF protein (Peprotech) in in vitro differentiation assays. Treatment with 50 or 100 ng/mL of PLGF protein during days 3–7 in CM differentiation and days 4-6 in SMC and EC differentiation improved the efficacies for induction of TNNT2⁺ CMs, PDGFRB⁺ SMCs, and VEC⁺ ECs in PLGF-KO hESC-derived cells, respectively (Supplementary Fig. 6).

PLGF modRNA promotes the upregulation of heart and vasculature development-related genes

To clarify molecular signatures in human PLGF-associated cardiac development, we analyzed population RNA-seq data obtained from both WT and PLGF-KO hESC-derived cells on days 4 and 6 of the hESC-CM differentiation protocol. WT hESC-derived cells that were transfected with PLGF or LacZ (control) modRNA on day 3 or 5 and then harvested on day 4 or 6, respectively, were also involved in the analysis. Principal component analysis and differential gene expression analysis clearly segregated the four cell groups in a stage-dependent manner (Fig. 5a, b). We then compared directly the transcriptomes between WT and PLGF modRNA-transfected cells, as well as between WT and PLGF-KO cells on the same differentiation day with the limma package³⁵ in R/Bioconductor, and performed the gene set enrichment analysis (GSEA) using the GSEA software (Broad Institute; http://www.gsea-msigdb.org/gsea/) (Fig. 5c–j).

a The principal component analysis using the population RNA-seq data of WT, modRNA (LacZ or PLGF)-transfected WT, and PLGF-KO cells harvested at days 4 and 6 in hESC-CM differentiation. ModRNA transfection was conducted 24 h before cell harvesting (i.e., at day 3 or 5). b Differential gene expression analysis of the 8 cell groups in a. Heatmap image depicting the representative differentially expressed genes (partly listed in the left column) in each of the 8 groups. c, e, g, i Volcano plots visualizing differentially expressed gene analysis with the limma package³⁵ between WT and PLGF modRNA-transfected cells (c [day 4] and g [day 6]), as well as between WT and PLGF-KO cells (e [day 4] and i [day 6]) in hESC-CM differentiation, respectively. For each gene, the average difference (log₂[Fold change]) between the cell groups on the same day was plotted against the power to discriminate between groups (-log₁₀[p.value]), in which p.values were obtained from a two-tailed unpaired t-test. Top-scoring genes for both metrics are indicated as red dots, and representative differentially expressed genes’ names are labeled. d, h The gene set enrichment analysis (GSEA) was performed using the top 250 WT or PLGF modRNA-transfected cells-enriched genes with the GSEA software (Broad Institute; http://www.gsea-msigdb.org/gsea/). Bar graphs showing the representative gene ontology (GO) terms specific to WT (right) or PLGF modRNA-transfected cells (left) at days 4 (d) and 6 (h), respectively. f, j The GSEA was performed using the top 250 WT or PLGF-KO cells-enriched genes with the GSEA software. Bar graphs showing the representative GO terms specific to WT (left) or PLGF-KO cells (right) at days 4 (f) and 6 (j), respectively. Source data are provided as a Source Data file.

Full size image

On day 4, the genes upregulated in PLGF modRNA-transfected cells compared to WT cells were enriched for gene ontology (GO) terms such as heart development, muscle tissue development, vasculature development, and mesenchyme development, which contained genes such as HAND2, MEF2C, HOXA1, HEY1, TBX5, MEIS2, PDGFRA, FGF10, MYOCD, TGFB2, etc. (Fig. 5c, d; Supplementary Data 2). In contrast, the genes upregulated in WT cells compared to PLGF modRNA-transfected cells on day 4 were enriched for GO terms such as neurogenesis, endoderm differentiation, mesodermal commitment pathway, and ectoderm differentiation, which contained genes such as SOX2, FOXA2, SOX17, POU5F1, EOMES, LHX1, HHEX, TBXT, etc. (Fig. 5c, d; Supplementary Data 2). In analogy with the relationship between the PLGF modRNA-transfected and WT cells, the genes upregulated in WT cells compared to PLGF-KO cells on day 4 were enriched for GO terms such as heart development, mesenchyme development, muscle structure development, and vasculature development, which contained genes such as BMP4, NKX2-5, TBX20, TBX5, KDR, APLNR, PDGFRA, HAND1, HAND2, HEY1, MEIS2, MYOCD, TGFB2, PDGFRB, etc. (Fig. 5e, f; Supplementary Data 2). In contrast, the genes upregulated in PLGF-KO cells compared to WT cells on day 4 were enriched for GO terms such as cell migration, apoptotic process, regulation of cell death, and neurogenesis, which contained genes such as ANXA1, JUN, CFLAR, CCL2, ID1, FOS, RGCC, POU5F1, FOXA1, SOX2, TDGF1, etc. (Fig. 5e, f; Supplementary Data 2).

On day 6, the genes upregulated in the PLGF modRNA-transfected cell group compared to WT cells were also enriched for GO terms such as heart development, striated muscle contraction, cardiac muscle contraction, and muscle tissue development, which contained genes such as MEF2C, NKX2-5, MYOCD, ACTN2, TNNT2, MYH6, MYH7, ACTA2, PLN, etc. (Fig. 5g, h; Supplementary Data 3). The upregulation of these sarcomere proteins and cardiac/smooth muscle actins indicated a more differentiated status of the in vitro cardiac cells on day 6 than on day 4. In contrast, the genes upregulated in WT cells compared to PLGF modRNA-transfected cells on day 6 were enriched for GO terms such as epithelium development, regulation of locomotion, morphogenesis of an epithelium and mesodermal commitment pathway, which contained genes such as GATA3, FOXA2, SOX17, POU5F1, ZFP42, TBX3, HHEX, BMP4, etc. (Fig. 5g, h; Supplementary Data 3). Again, in analogy with the relationship between the PLGF modRNA-transfected and WT cells, the genes upregulated in WT cells compared to PLGF-KO cells on day 6 were enriched for GO terms such as heart development, muscle structure development, circulatory system development, and regulation of heart contraction, which contained genes such as NKX2-5, MEF2C, TPM1, TNNT2, MYH6, TBX5, ACTN2, PLN, CACNA1C, MYOCD, ACTA1, etc. (Fig. 5i, j; Supplementary Data 3). In contrast, the genes upregulated in PLGF-KO cells compared to WT cells on day 6 were enriched for GO terms such as ectoderm differentiation, embryonic morphogenesis, negative regulation of multicellular organismal process, and negative regulation of cell population proliferation, which contained genes such as HNF4A, MLXIPL, CFLAR, OVOL2, OTX1, ZIC3, FOXA2, SOX17, etc. (Fig. 5i, j; Supplementary Data 3).

Collectively, these transcriptional analyses revealed that PLGF modRNA would be a positive enhancer for both heart and vasculature development (i.e., cardiomyogenesis and vasculogenesis) when administered at the heart progenitor stage during in vitro hESC cardiogenesis, which was consistent with the findings in the in vitro differentiation assays (Fig. 2). In fact, expression of a great number of the cardiomyogenesis drivers, such as typical transcription factors (e.g., HAND2, MEF2C, MYOCD, etc.) and chemical mediators (e.g., WNT, FGF, etc.), was significantly upregulated by treatment with PLGF modRNA while reversely downregulated by PLGF deletion (Supplementary Fig. 7a). This enhancement of the cardiomyogenesis programs was more predominant when the differentiating cells were treated with PLGF modRNA earlier (day 3 > 5). On the other hand, a number of vasculogenesis drivers (e.g, ETV1, APLNR, ANGPT1, VCAM1, etc.) were also significantly upregulated by treatment with PLGF modRNA while reversely downregulated by PLGF deletion, which was more predominant when the differentiating cells were treated with PLGF modRNA later (day 3 < 5) (Supplementary Fig. 7b). These results suggest that PLGF would activate both the cardiomyogenesis and vasculogenesis programs in a stage-dependent fashion. In contrast, PLGF deletion would induce cell migration, lower cell viabilities, cell apoptosis, and/or shifting of the differentiating cell trajectories towards the ectoderm (e.g., neurons, epithelium) or endoderm lineages (e.g., hepatocytes) from the originally destined mesoderm lineages during the in vitro hESC-CM differentiation.

PLGF modRNA exerts in vivo cardiomyogenic and vasculogenic effects

To elucidate the effects of PLGF in the context of in vivo differentiation of human heart progenitors (HPs) for cardiac muscle formation, we transplanted hESC-derived HPs that were transfected with or without PLGF modRNA into kidney capsules of immunocompromised mice¹⁹. We employed the same in vitro CM differentiation protocol¹⁸, and cardiac differentiating cells at day 3 were electroporated with 5 µg of GFP (control) or PLGF modRNA and harvested at day 5 (termed early HPs). Separately, cardiac differentiating cells at day 5 were electroporated with 5 µg of GFP or PLGF modRNA and harvested at day 6 (termed late HPs). From a pilot study, we observed that injection of the combined early and late HPs could generate bigger heart muscle grafts on a kidney capsule than injection of only the single early or late HPs (relative increase ratios of the graft areas: 1.95 ± 0.64; P < 0.01).

We then injected the following groups of cells into murine kidney capsules, respectively: (1) 1.5 million of intact early HPs plus 1.5 million of intact late HPs, which were harvested at day 5 and 6 without any modRNA transfection (No TF control; 3 million cells in total); (2) 1.5 million of the GFP modRNA-transfected early HPs plus 1.5 million of the GFP modRNA-transfected late HPs (GFP control; 3 million cells in total); (3) 1.5 million of the PLGF modRNA-transfected early HPs plus 1.5 million of the PLGF modRNA-transfected late HPs (3 million cells in total). The engrafted kidneys were harvested at 1 month after surgery (Fig. 6a). Interestingly, the PLGF modRNA-transfected HP-engrafted kidneys were heavier in weight than controls (Fig. 6b), likely reflecting the bigger sizes of their generated cardiac muscle grafts in vivo.

a In vivo human heart progenitor (HP)-derived cardiac muscle grafts on murine kidney capsules (arrow) were generated by transplantation of hESC-derived HPs. b Comparison of the weights of the non-transfected (NoTF) HP-, GFP modRNA (modGFP)-transfected HP-, and PLGF modRNA (modPLGF)-transfected HP-engrafted kidneys. c Immunohistochemistry of the sectioned human HP-derived cardiac muscle grafts on murine kidney capsules, generated by NoTF HPs (left) and modPLGF-transfected HPs (right). The grafts (Gr) were indicated by white dotted lines in the left images, respectively. The right image in each is the enlarged one of a yellow square in the left image, respectively. Scale bars, 500 μm (left in each) and 100 μm (right in each). Kid, kidney; VIM, vimentin. d Quantitative data of the entire graft areas (left) and TNNT2⁺ areas in grafts (right) in the three groups, i.e., NoTF-, modGFP-, and modPLGF-HPs. Quantitative analyses were conducted using ImageJ/FIJI software (NIH, USA). e Immunohistochemistry of the sectioned human HP-derived cardiac muscle grafts on murine kidney capsules generated by NoTF HPs (left) and modPLGF-transfected HPs (right), highlighting a CM maturation marker MLC2V and a proliferation marker Ki67. Scale bars, 100 μm. f Quantitative data of MLC2V⁺ areas in grafts (left) and Ki67⁺ density in grafts (right) in the three groups. g Immunohistochemistry of the sectioned human HP-derived cardiac muscle grafts on murine kidney capsules generated by NoTF HPs (left) and modPLGF-transfected HPs (right), highlighting an EC marker VE-cadherin (VEC) and a SMC marker α-smooth muscle actin (αSMA). The right image in each is the enlarged one of a yellow square in the left image, respectively. Scale bars, 100 μm (left in each) and 50 μm (right in each). h Quantitative data of VEC⁺ areas in grafts in the three groups. Data in b, d, f, and h are presented as mean ± SD (n = 4–5 biologically independent samples). Differences between groups were examined with one-way ANOVA followed by Tukey multiple comparisons test. *P < 0.05 and **P < 0.01 between modPLGF-transfected HP-engrafted kidneys vs. NoTF HP- or modGFP-transfected HP-engrafted kidneys. Source data are provided as a Source Data file.

Full size image

In histological analyses, as expected, the PLGF modRNA-transfected HP-engrafted cardiac grafts presented with larger cross-sectional areas and TNNT2⁺ areas when compared to controls (Fig. 6c, d), suggesting that the PLGF modRNA-transfected HPs would differentiate into more CMs than No TF and GFP controls upon in vivo transplantation. Similarly, areas that were positive for the CM maturation marker MLC2V were larger in the PLGF modRNA-transfected HP-engrafted group when compared to controls, indicating that PLGF modRNA also promoted cardiac maturation in the in vivo muscle grafts (Fig. 6e, f). Next, we examined the expression of a proliferation marker Ki67 in the grafts and found the number of Ki67⁺ cells in the PLGF modRNA-transfected HP-engrafted group was higher than those in No TF and GFP controls (Fig. 6e, f), suggesting that PLGF modRNA could enhance proliferation of the transplanted HP-derived cardiac cells upon in vivo transplantation, likely leading to the larger sizes of the grafts. We then assessed to what extent vascular structures formed within the grafts through histological examination of an EC marker (VEC) and an SMC marker (α-smooth muscle actin [αSMA]), respectively. Of note, the PLGF modRNA-transfected HP-engrafted cardiac grafts exhibited significantly higher vascular density (VEC⁺) than controls (Fig. 6g, h), which most likely contributed to the bigger sizes of the in vivo generated cardiac grafts. Overall, these findings demonstrated the in vivo cardiomyogenic and vasculogenic effects of PLGF modRNA upon HP transplantation.

PLGF is a target of both EOMES and SOX17

To clarify molecular machinery in human PLGF-associated cardiomyogenesis and vasculogenesis, we explored a previously unrecognized transcription network behind PLGF. We focused on an early cardiogenic TCF EOMES²⁰ and a vasculogenic TCF SOX17²¹, as both genes were significantly upregulated in WT cells compared to PLGF modRNA-transfected cells, as well as upregulated in PLGF-KO cells compared to WT cells, on day 4 (EOMES) and on days 4 and 6 (SOX17) (Fig. 5). This evidence may imply that both genes function upstream of PLGF during the in vitro cardiac differentiation. Through the TCF motif analysis with the Jaspar database (http://jaspar.genereg.net/) and the MatInspector software (Genomatix, http://www.genomatix.de/), we found the putative EOMES-binding motif sites (5’-(AG)GTGTGA-3’) on the PLGF promoter region, which is 1934, 1408, 790, and 724 bp upstream of the transcription start site (TSS) of the human PLGF gene (Fig. 7a, top). We also found the putative SOX17-binding motif sites (5’-(C/T)ATTGT(C/G)−3’) on the PLGF promoter region, which are 3901, 1638, 1542, and 1384 bp upstream of the TSS of the human PLGF gene (Fig. 7a, bottom).

a Schematic showing the putative binding sites of a cardiogenic transcription factor EOMES (5’-[AG]GTGTGA-3’; top) and a vasculogenic transcription factor SOX17 (5’-[C/T]ATTGT[C/G]−3’; bottom) on the human PLGF promoter region. b Chromatin immunoprecipitation (ChIP) assays demonstrated that recruitment of EOMES protein onto one of the putative EOMES-binding motif sites of the human PLGF promoter (−1934 bp upstream from the transcription start site [TSS]) was significantly augmented at days 3 (D3) and 6 (D6) in CM differentiation of WT hESCs. The degree of fold enrichment was larger at day 3 than at day 6. Albeit to a lesser degree, recruitment of EOMES was also detected onto another putative EOMES-binding motif site (−790/−724 bp upstream from the TSS) at D3 and D6. c The ChIP assays demonstrated that recruitment of SOX17 protein was significantly augmented onto one of the putative SOX17-binding motif sites of the human PLGF promoter (−3901 bp upstream from the TSS) at D3 and onto all of the four putative SOX17-binding motif sites (−3901, −1638, −1542, and −1384 bp upstream from the TSS) at D6. Data in b and c are presented as mean ± SD (n = 5 independent experiments). Differences between groups were examined with one-way ANOVA followed by Tukey multiple comparisons test. *P < 0.01 and **P < 0.0001 vs. IgG (negative control). Source data are provided as a Source Data file. d Schematic highlighting the EOMES-PLGF-mediated cardiomyogenesis in the early embryonic stage and the SOX17-PLGF-mediated vasculogenesis in the middle/late embryonic stage. e Schematic highlighting the stage-dependent PLGF’s roles in cardiogenesis. In the early stage, the second heart field (SHF) heart progenitors play a dual role, such as an autocrine role in the SHF and a paracrine role to function to the vascular progenitors (VP). In the late stage, smooth muscle cells (SMCs) play a paracrine role in functioning to endothelial cells (ECs), while ECs play an autocrine role. Black dashed arrows indicate putative differentiation paths. CM cardiomyocyte.

Full size image

Next, chromatin immunoprecipitation (ChIP) assays with antibodies specific to EOMES and SOX17 (Abcam) were performed using extracts derived from WT hESC-derived cells on days 3 and 6 in the in vitro CM differentiation. We first confirmed successful and specific protein immunoprecipitation with anti-EOMES and anti-SOX17 antibodies by western blotting analysis. We then found that recruitment of EOMES protein was significantly augmented onto one of the putative EOMES-binding motifs on the PLGF promoter (−1934 bp upstream from the TSS) on day 3 and 6 (day 3 > 6) (Fig. 7b). On the other hand, recruitment of SOX17 protein was augmented onto one of the putative SOX17-binding motifs on the PLGF promoter (−3901 bp upstream from the TSS) on day 3 and onto all of the four putative SOX17-binding motifs on day 6 (Fig. 7c). These suggest that the identified EOMES-PLGF transcriptional interaction would function at the earlier embryonic stage, while the identified SOX17-PLGF transcriptional interaction would function at and following the early embryonic stage.

In consistency with the findings on the ChIP assays, the Guild-by-Association and correlation analysis using the primate embryonic heart single-cell RNA-seq data (Fig. 1) revealed that SOX17 expression was highly positively co-related to PLGF expression (corrected P-value: 1.1E-4), especially in the late stage (7 weeks of fetal age), and SOX17 was strongly expressed in the EC population (cluster #11), showing overlap with PLGF expression (Supplementary Fig. 8a–c). Since the early cardiogenic TCF EOMES had little or no expression in both human and primate embryonic heart single-cell RNA-seq datasets, we also analyzed the previously obtained single-cell RNA-seq data of in vitro hESC-derived cells during CM differentiation (Supplementary Fig. 8d)^6,18. The 366 high-quality individual cells were segregated into 6 clusters, in which we identified that cluster #2 was occupied by cells on day 3 in CM differentiation and expressed EOMES globally (thus, considered as early cardiac precursors) as well as PLGF, as shown in the feature plots on the tSNE plots (Supplementary Fig. 8e, f). These also imply that the putative EOMES-PLGF and SOX17-PLGF transcriptional interactions would be sequentially vital in a time-dependent fashion, i.e., for cardiomyogenesis in the early embryonic stage (EOMES-PLGF) and for vasculogenesis in the middle/late embryonic stage (SOX17-PLGF), respectively (Fig. 7d).

Lastly, we investigated expression patterns of PLGF and FLT1 (VEGFR1), a PLGF-specific receptor gene, in the single-cell RNA-seq datasets of the primate embryonic hearts and the in vitro hESC-derived cardiac cells (Supplementary Fig. 8g–j). Of interest, the in vitro early cardiac precursors (cluster #2 in Supplementary Fig. 8d) often co-expressed PLGF and FLT1, indicating the autocrine signaling and a cell-autonomous role of PLGF in these cells (Supplementary Fig. 8i, j). In the primate heart cells at 4 weeks when the SHF cells (cluster #5 in Supplementary Fig. 8a) expressed PLGF specifically, while some of the SHF cells co-expressed FLT1, many of the vascular progenitor cells (cluster #7) expressed FLT1 but not PLGF (Supplementary Fig. 8g, h). This suggests that at the early stage in vivo, the SHF-derived PLGF would play a dual role, i.e., an autocrine role in the SHF and a paracrine role in affecting the vascular progenitor cells for promoting intercellular vascular differentiation (Fig. 7e). In the primate heart cells at 7 weeks when the ECs and SMCs (cluster #11 and #13 in Supplementary Fig. 8a) expressed PLGF specifically, while ECs, as well as endocardium (cluster #8), expressed FLT1 strongly, SMCs showed little or no expression of FLT1 (Supplementary Fig. 8g, h). Thus, it is suggested that at the late stage in vivo, the EC-derived PLGF would play a cell-autonomous (autocrine) role, whereas the SMC-derived PLGF would play a paracrine role in affecting ECs and endocardium in promoting endothelial differentiation (Fig. 7e).

Collectively, we have newly identified the previously unrecognized gene regulatory network, involving interactions between EOMES and PLGF, as well as SOX17 and PLGF, and the stage-dependent autocrine and paracrine roles of PLGF, all of which would contribute to human PLGF-mediated cardiomyogenesis and vasculogenesis during heart development.

• SEO Powered Content & PR Distribution. Get Amplified Today.
• PlatoData.Network Vertical Generative Ai. Empower Yourself. Access Here.
• PlatoAiStream. Web3 Intelligence. Knowledge Amplified. Access Here.
• PlatoESG. Automotive / EVs, Carbon, CleanTech, Energy, Environment, Solar, Waste Management. Access Here.
• PlatoHealth. Biotech and Clinical Trials Intelligence. Access Here.
• ChartPrime. Elevate your Trading Game with ChartPrime. Access Here.
• BlockOffsets. Modernizing Environmental Offset Ownership. Access Here.
• Source: https://www.nature.com/articles/s41467-023-41305-7