Aspectos generales del municipio de Manicaragua

Differentially expressed genes in A83-01 treated and untreated cultures were further interrogated to identify transcription factors (TFs) regulated in response to TGF-β-R inhibition.

Based on q < 0.05, a robust list of 104 differentially expressed TFs in response to A83-01 treatment was identified. RARB (retinoic acid receptor beta; q =1.06 x 10-33_{); WT1 (Wilms tumor 1; q = 4.94 x 10}-16_{), and HEYL (hes related family bHLH}

transcription factor with YRPW motif-like; q = 2.21 x 10-10_{) were amongst the most}

highly enriched TFs. Highly repressed TFs included MEOX1 (mesenchyme homeobox 1; q = 1.06 x 10-12_{), ESR1 (estrogen receptor 1; q = 7.04 x 10}-29_{), and} EGR2 (early growth response 2; q = 6.59 x 10-11_{) (Figure 4.23).}

Out of the 104 A83-01 responsive TFs, only 8 were concordantly regulated between clonal eMSCs and matched PVSCs. A83-01-responsive TFs enriched in clonal eMSCs included TFAP2C (transcription factor AP-2 gamma), HOXB3

(homeobox B3) and TOX2 (TOX high mobility group box family member 2). Conversely, A83-01-responsive TFs downregulated upon differentiation of clonal to PVSCs included DLX5 (distal-less homeobox 5), ZFHX2 (zinc finger homeobox 2) and ZNF853 (zinc finger protein 853).

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Figure 4.23. Changes in TF expression upon A83-01 treatment. Inhibition of TGF-β-R signalling pathways alters expression of genes encoding TFs. Graph shows expression of selected significantly up- and down-regulated TFs (log2-fold change ≥ 1 and ≤ -1).

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4.3 Discussion

Cell reprogramming via the use of small molecules that modulates stem cell specification and function offers significant opportunities to study the cell biology and enhance our knowledge of the therapeutic potential of stem cells (Yu et al., 2014; Li et al., 2013). Compared to genetic tools, the use of small molecules for cell reprogramming provide several advantages, for example their effect is reversible, that means that they can temporally regulate cellular function. This allows to yield sufficient amounts of homogeneous cells for clinical applications. Also, small molecules can be used in synergy with other factors, and this may improve their efficacy (Li et al., 2012). Small molecules can act as activators or repressors of signalling pathways. In this way, they regulate downstream gene transcription (Yun et al., 2014). The use of a glycogen synthase kinase (GSK) 3 inhibitor to maintains the pluripotency of induced pluripotent stem (iPS) cells exemplified such approach (Takahashi & Yamanaka, 2006; Li et al., 2009; Ying

et al., 2008; Silva et al., 2008).

A recent study showed that inhibition of TGF-β-R signalling, through TGF-β-R inhibitor A83-01, promotes eMSC proliferation, maintaining their clonogenic phenotype and preventing spontaneous differentiation (Gurung et al., 2015). The use of A83-01 as a signalling pathway modulator has provided a robust tool to generate amount in the large scale manufacture of homogenous eMSC populations for clinical applications. Understanding gene expression programs and mapping of altered chromatin accessibility in response to A83-01 treatment is required to realize regulatory mechanisms that maintains eMSCs in an undifferentiated state.

Data confirmed that TGF-β-R inhibition enhances proliferative ability of eMSCs as shown by increased cumulative cell population and number of PDs upon treatment. Furthermore, data revealed that TGF-β-R blockade maintains clonogenic phenotype of MSCs. Specifically, A83-01 positively regulated the number of CD140b- and SUSD2- positive cells in prolonged culture and increase for the same markers. Also, results confirmed that TGF-β-R blockade improves

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the ability of cultured eMSCs to form colonies, preventing loss of clonogenicity. Notably, results showed marked variation in the responsiveness of primary cultured eMSCs to treatment, underlying the intrinsic variability between primary cultures.

Gene profiling of cultured eMSCs upon A83-01 treatment revealed that TGF-β-R blockade involves wholesale reprograming of the transcriptome. The resulting differentially expressed genes are implicated a broad range of functions. For example, A83-01 treatment resulted in upregulation of genes involved in intracellular receptor signalling pathways and regulation of growth, and downregulation of genes encoded for collagen catabolism and cell fate commitment. Notably, RNA-seq revealed upregulation of SUSD2, consistent with flow cytometry data showing increase in the percentage of SUSD2- positive cells and MFI for the same marker. Conversely, transcriptomic profiling revealed depletion of perivascular markers, such as MCAM, MYH11 and ELN.

Mining of the data revealed that TGF-β-R inhibition repressed the expression of numerous genes encoding for ECM components, including COL1A1, COL1A2

and SPARC. ECM production is a well characterized response to injury and TGF- β signalling is master regulator. More accurately, in the case of tissue damage, fibroblasts transit from a quiescent to an activated state, where TGF-β signalling, amongst others signalling pathways, activates genes encoding for ECM proteins and cytoskeletal remodelling (Nakerakanti and Trojanowska, 2012; Biernacka, A., et al, 2011). This suggests that the effect of TGF-β-R blockade in maintaining

eMSCs in a less differentiated state might be mediated by preventing fibroblast activation and limiting ECM deposition in prolonged culture. Hence, it would be interesting to validate the induction of ECM genes at a mRNA and protein level through RT-qPCR and western blot, although time limitations prevented me of pursuing this line of investigation any further.

To determine if A83-01 induces a stem cell signature, I compared the transcriptome profile of A83-01 treated and untreated eMSCs with the RNA-seq data obtained from another dataset, comparing clonal eMSCs to time-matched unselected PVCs.

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This analysis revealed a dataset of shared genes accounting for 38 concordant up- and 120 concordant down- regulated loci. Interestingly, functional annotation of the commonly down-regulated genes highlighted an abundance of genes encoding for ECM components or associated to ECM organization.

For therapeutic application, it is of paramount importance to ensure that A83-01 treatment does not alter expression level of transcripts associated to clinical disorders. Functional annotation revealed a majority to be associated with metabolic and vascular disorders, which might alert in terms of clinical use. On the other hand, analysis of the data showed that TGF-β-R blockade correlates with downregulation of genes associated to cancer, reassuring for a safe use of these cells in regenerative medicine.

Taken together, the current findings suggest that A83-01 effect might be related to prevention of fibroblast activation.

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Chapter 5

Analysis of dynamic chromatin