Las cooperativas no agropecuarias en Villa Clara

In a previous study in our laboratory, RNA-sequencing was used to determine the transcriptome profiles of clonal eMSCs and time-matched unselected perivascular cells (PVCs). Briefly, total mRNA was isolated of eMSCs subjected to clonal assay for 12 days and matched to primary PVCs also cultured for 12 days. To determine if A83-01 maintains cultured eMSCs in a less differentiated state, I examined the overlap in differentially expressed genes in both datasets. Figure 4.14 shows a graphic representation of the two datasets compared in this part of the study.

Figure 4.14. ‘Untreated vs A83-01 treated eMSCs’ vs ‘Clonal eMSCs vs short-term cultured PVCs’. Graphic representation of the two datasets compared. (A) shows dataset one where freshly isolated eMSCs were cultured in the presence or absence of TGF-β-R inhibitor for 36 days and then subjected to ATAC-seq and RNA-seq. (B) represent dataset two where clonal eMSCs were subjected to CFU assay for 12 days and compared to primary PVCs cultured for 12 days. At day 12 clonal eMSCs and PVCs were subjected to RNA-seq.

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Comparison between the two dataset showed that A83-01 treated cells and clonal eMSCs share 38 commonly upregulated genes, including SUSD3 (q = 1.36 x 10- 11_{), LPAR3 (q = 2.67 x 10}-5_{) and ITGA8 (q = 7.07 x 10}-6_{) (Figure 4.15A-B). The}

Human Protein Atlas was used to determine the spatial expression of these genes in the endometrial tissue (Uhlén et al, 2015), particularly in the perivascular niche. As shown in Figure 4.16 (left panel), immunohistochemistry analysis revealed that SUSD3(sushi domain containing 3), LPAR3(lysophosphatidic acid receptor 3), and ITGA8 (integrin subunit alpha 8) are indeed expressed in cells surrounding the terminal spiral arteries, in keeping that eMSCs reside predominantly in the perivascular niche (Masuda et al., 2012).

A total of 120 genes were commonly down-regulated in both A83-01 and clonal eMSCs and included several genes coding for extracellular matrix (ECM) proteins such as MFAP5 (q = 1.07 x 10-5_{), FN1 (q = 2.35 x 10}-8_{), and VCAN (q = 2.72 x}

10-7_{) (Figure 4.17). Again, the Human Protein Atlas was used to annotate the}

cellular distribution of these genes in the perivascular regions of the endometrial tissue. As shown in Figure 4.16B, MFAP5 (microfibrillar associated protein 5),

FN1 (fibronectin) and VCAN (versican) were abundantly expressed in the stromal compartment but less prominently in perivascular cells around the terminal spiral arteries.

Taken together, these results suggest that A83-01-induced TGF-β-R blockade upon prolonged eMSC culture only partly recapitulates the gene signature of clonal eMSCs. In keeping with the known role of TGF- in regulating ECM (Verrecchia & Mauviel, 2002), the effect of A83-01 on eMSCs in prolonged culture may equally relate to the inhibition of ECM components in prolonged cultures.

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Figure 4.15. A83-01 treated and clonogenic eMSCs share a set of commonly upregulated genes. (A) Venn diagram showing overlap of 38 genes commonly upregulated both in A83-01 treated and clonogenic eMSCs (Log2-fold change ≥ 1; q < 0.05). (B) Heatmap representing the gene expression level of the 38 commonly upregulated genes, which are listed on the right. The colour key is represented above the heat map, the most highly enriched genes are indicated in red.

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Figure 4.16. Cellular distribution of A83-01 responsive genes in cycling human endometrium. (A) The Human Protein Atlas was mined to determine the tissue distribution of 3 proteins (SUSD3, LPAR3, and ITGA8) associated with clonal eMSCs and induced at mRNA level in prolonged cultures in response to A83-01. (B) The same resource was also mined to determine the tissue distribution of 3 proteins (MFAP5, FN1, and VCAN) down-regulated in clonal eMSCs as well as in response to A83-01 treatment of prolonged eMSC cultures. Overall, the selected A83-01 induced genes were more prominent expressed at protein level around the terminal spiral arteries whereas the opposite pattern of expression was apparent for A83-01 repressed genes.

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Figure 4.17. A83-01 treated and clonogenic eMSCs share a set of commonly downregulated genes. (A) Venn diagram showing overlap of 120 genes commonly downregulated both in A83-01 treated and clonogenic eMSCs (log2- fold change ≤ -1; q < 0.05). (B) Heatmap showing gene expression level of 120 commonly downregulated genes, which are listed on the right. The colour key is represented above the heat map, the most highly enriched genes are indicated in red. The heatmap was divided into two parts because of the size.

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GO analysis of eMSC-associated genes induced by A83-01

GO enrichment analysis of the 38 commonly upregulated genes, i.e. eMSCs versus PVCs and A83-01 treated versus untreated cultures, was performed to identify relevant biological processes.

Based on p < 0.05, GO analysis showed enrichment for eight GO terms, including ‘positive regulation of collateral sprouting’ (p = 1.56 x 10-2_{), ‘oxidation-reduction}

process’ (p =2.79 x 10-2_{) and ‘negative regulation of endothelial cell migration’ (p}

= 3.48 x 10-2_{). GO term associated to ‘positive regulation of collateral sprouting’}

included EFNA5 (ephrin A5) and LPAR3 (lysophosphatidic acid receptor 3); ‘oxidation-reduction process’ included PTGS2 (prostaglandin-endoperoxide synthase 2; also known as cyclooxygenase-2 or COX-2) and VATL1 (vesicle amine transport 1 like); and ‘negative regulation of endothelial cell migration’ included DLL4 (delta-like canonical Notch ligand 4) and SLIT2 (slit guidance ligand 2). Interestingly, enrichment analysis of the 38 commonly up-regulated genes for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways revealed enrichment of the ‘vascular endothelial growth factor (VEGF) signalling pathway’ (p = 9.57 x 10-3_{), that included RAC2 (Rac family small GTPase 2),} PTGS2 (prostaglandin-endoperoxide synthase 2) and PLA2G4A (phospholipase A2 group IVA) (Figure 4.18A).

GO analysis of the 120 commonly downregulated genes showed depletion of ten biological processes. The most significantly (p < 0.05)down-regulated were ‘ECM organization’ (p = 2.95 x 10-5_{) and ‘cell adhesion’ (p = 1.28 x 10}-4_{). ‘ECM}

organization’ category included ELN (elastin) and FN1; ‘cell adhesion’ included TGFBI (transforming growth factor beta induced) and CDH2 (cadherin 2). Enrichment analysis of KEGG pathway showed depletion for seven categories, for example ‘signalling pathways regulating pluripotency of stem cells’ (p = 1.17 x 10-2_{) and ‘cell adhesion molecules (CAMs)’ (p = 1.23 x 10}-2_{) (Figure 18B).}

‘Signalling pathways regulating pluripotency stem cells’ included FGFR2

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adhesion molecules (CAMs)’ included JAM2 (junctional adhesion molecule 2) and CLDN1 (claudin 1).

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Figure 4.18. A83-01 treatment partly recapitulates stem cell features.

Enrichment analysis of biological processes and KEGG pathways associated with the 38 and 120 commonly up- (A-B) and down-regulated (C-D) genes, respectively, in both RNA-sequencing datasets: A83-01 treated versus untreated cultured eMSCs and clonal eMSCs versus PVCs. The pie charts showed the

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significantly enriched (A-B) or depleted categories (C-D), and the numbers indicate the number of genes contained in each identified biological process or KEGG pathway.

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Divergent transcriptomic profiles between clonal and A83-01 treated eMSCs in prolonged culture

Inhibition of TGF-β-R signalling differentially regulated genes that partly recapitulated gene signature of clonal eMSCs, but also resulted in a unique gene signature. Specifically, A83-01 treatment induced 160 genes that were downregulated in the clonal eMSCs when compared to PVCs (Figure 4.19). Also, TGF-β-R blockade negatively regulated 58 genes, which were enriched in clonal eMSCs when compared to PVCs (Figure 4.20). To identify biological processes associated with these discordant genes, I performed further GO analysis. The top three most significant (p < 0.05) GO terms uniquely induced in the A83-01 treated eMSCs were ‘negative regulation of substrate adhesion-dependent cell spreading’ (p = 1.43 x 10-4_{), ‘response to retinoic acid’ (p = 3.4 x 10}-4_{) and}

‘regulation of systemic arterial blood pressure by renin-angiotensin’ (p = 9.07 x 10-4_{). The top three most negatively downregulated by A83-01 whereas were}

‘collagen catabolic process’ (p = 7.13 x 10-4_{), ‘extracellular matrix disassembly’}

(p = 0.02), and ‘cellular response to vitamin D’ (p = 0.02). It is notable that GO terms related to ECM were down-regulated in prolonged eMSC cultures treated with A83-01.

Taken together, the comparison of the two RNA-seq data sets revealed that TGF- β-R blockade upon prolonged culturing of eMSCs partly recapitulates the identity of clonal eMSCs, but also modulates a distinct gene network, consisting of 218 genes. A preponderance of genes selectively inhibited by A83-01 in prolonged eMSC culture relate to ECM proteins and ECM turnover.

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Figure 4.19. Discordant genes between A83-01 treated and clonogenic eMSCs. (A) Venn diagram showing overlap of 160 genes differentially regulated in A83-01 treated and clonogenic eMSCs (q < 0.05). (B) Heatmap representing expression level of the 160 discordant genes, which are listed on the right. Again, the heatmap was divided into two parts because of its size.

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Figure 4.20. Discordant genes between A83-01 treated and clonogenic eMSCs. (A) Venn diagram showing overlap of 58 genes differentially regulated in A83-01 treated and clonogenic eMSCs (q < 0.05). (B) Heatmap representing expression level of 58 discordant genes, which are listed on the right.

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