Presupuestos teóricos fundamentales

The human endometrium exhibits an extraordinary ability to cyclically regenerate during each menstrual cycle. As mentioned, endometrial tissue undergoes menstrual shedding in response to estrogen and progesterone withdrawal in the absence of pregnancy (Gellersen & Brosens, 2014). Menstruation is a rare event occurring only in a few species all characterised by spontaneous decidualization, such as higher primates, elephant shrews, fruit bats and the common (Cairo) spiny mouse (Bellofiore et al., 2017; Emera et al., 2012). One of the theories about the evolutionary purpose of menstruation infers its unique role in restarting the next reproductive cycle (Emera et al., 2012), through cyclic activation of stem/progenitor cells (Evans et al., 2016).

Human endometrium harbours two stem cell populations, such as epithelial stem/progenitor cells and mesenchymal stem cells (MSCs) (Chan et al., 2004; Garget et al., 2009; Masuda et al., 2012). The latter represents the major fraction of stem cells within the endometrial cell population (about 1-5%) (Gargett et al., 2009). Endometrial MSCs (eMSCs) exhibit high proliferative potential, self- renewal capacity in vitro, and ability to differentiate into more mature progeny in vivo (Gargett et al., 2016;Miyazaki et al., 2012; Wolff et al., 2007). Bone marrow- derived cells have been shown to migrate into the endometrium of both human and mice, although at low levels. These observations suggest that human endometrium regeneration mainly depends on the endogenous stem cell population (Gargett et al., 2012).

Several approaches have been employed to isolate endometrial stromal populations enriched in MSCs, including flow activated cell sorting of cells that co-express cluster of differentiation 140b (CD140b), also known as platelet- derived growth factor receptor β (PDGFRβ) and CD146 (also known as melanoma cell adhesion molecule, MCAM) (Schwab & Gargett, 2007). Recently, a monoclonal antibody (W5C5) has been identified to selectively isolate clonogenic perivascular eMSCs using magnetic-activated cell sorting (Masuda et al., 2012). Subsequent studies demonstrated that W5C5 antibody binds the type 1 integral membrane protein Sushi domain containing 2 (SUSD2)

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(Sivasubramaniyan et al., 2013). This method makes it possible to overcome damaging effects on cell viability associated with the use of flow cytometry (Schwab & Gargett, 2007).

Identity and gene profile of eMSCs

eMSCs exhibit the ability to both self-renew and differentiate. Cell fate decisions are determined by signals emanating from the cellular niche (Li & Xie, 2005). Understanding of the gene signature defining eMSC identity provides insights into their role in the endometrial regeneration. A recent genome-wide expression profiling study compared freshly isolated CD146+ PDGFR-β+ eMSCs to endometrial fibroblasts and endothelial cells. The study showed increased expression in eMSCs of genes associated with Notch, insulin-like growth factor (IGF),epidermal growth factor (EGF), Hedgehog, transforming growth factor β (TGF-β), WNT and G-protein-coupled receptor signalling pathways, which have been associated with both self-renewal and differentiation, highlighting the role of eMSCs in endometrial regeneration and remodelling (Spitzer et al., 2012). CD146+ PDGFR-β+ eMSCs express high levels of SUSD2, the integral protein used as single marker for eMSC prospective isolation (Spitzer et al., 2012; Masuda et al., 2012).

eMSCs locate in the perivascular niche of the endometrium and express pericyte markers, genes associated with angiogenesis, immunomodulation and responses to hypoxia (Spitzer et al, 2012). A gene profiling study comparing SUSD2+ and SUSD- cells showed that cells derived from SUSD2+ cells are characterised by a perivascular gene signature. Interestingly, SUSD2+ cells become the major source of cytokines and chemokine production during decidualization. This suggests a role for eMSCs in promoting trophoblast migration towards maternal vessels and mediating maternal immune response in pregnancy (Murakami et al., 2014).

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eMSCs in regenerative medicine

eMSCs hold great promise for cell-based therapy for women with reproductive disorders. eMSCs are clonogenic, self-renewing, highly proliferative, immunomodulatory and multipotent, hence they are attractive candidates of cell- based therapy in regenerative medicine (Darzi et al., 2016).

eMSCs represent an easily accessible source of cells. They can be selectively isolated from endometrial biopsy, which can be obtained in a routine office procedure without anaesthesia (Garget et al., 2016). They have been detected also in post-menopausal endometrium. This enables an autologous use of eMSCs for reproductive disorders, including pelvic organ prolapse (POP) in post- menopausal women (Ulrich et al., 2014). Synthetic polypropylene meshes have mainly been used for POP treatment. However, adverse side effects, including contraction and pain, raised warnings on their clinical use (Darzi et al., 2016). Ulrich et al. (2012) developed a new kind of mesh, composed of polyamide and gelatin, with enhanced mechanical properties. eMSCs seeded with these new meshes promoted angiogenesis and collagen deposition, compared with mesh alone, when implanted into an immunocompromised rat model. Furthermore, an early inflammatory response was observed and characterized by an influx of M1 macrophages, that switched to M2 would healing phenotype and then gradually decreased (Edwards et al., 2015). In vitro, eMSCs seeded with polyamide/gelatin meshes have been shown to differentiate into smooth muscle cells and fibroblasts, suitable to regenerate vaginal tissue structure and ultimately restore its function. Further studies have been undertaken to determine if delivered eMSCs function through paracrine effect or they differentiate themselves and reconstitute the vaginal wall (Gargett et al., 2016).

Altered endometrial stem/progenitor cell function may potentially hinder endometrial regeneration and ultimately compromise the ability of the endometrium to support embryo implantation (Yu et al., 2008). Women with a thin endometrial layer unable to respond to estrogen stimulation are particularly challaging in IVF treatment. Asherman’s syndrome is a reproductive disorder characterized by a thin dysfunctional endometrium and it has been hypothesized

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that it might be related to lack of eMSCs or compromised eMSC function (Gargett et al., 2016). In a rat model of Asherman’s syndrome, adipose-derived MSCs administrated in the uterine horn promoted angiogenesis and cell proliferation, and together with estrogen, reduced fibrosis. Even though, a local delivery of MSCs resulted in an apparent improved endometrial regeneration, results in terms of endometrial receptivity were not assessed (Kilic et al., 2014; Gargett et al., 2016). In several case studies, cultured autologous bone-marrow cells were injected into the uterine cavity or the sub-endometrial zone, together with estrogen administration. This cell-based approach resulted in a modest increase of thickness of the endometrium, no sufficient to guarantee a successful pregnancy outcome. However, because of lack of controls, data need to be interpreated with caution (Singh et al., 2014; Gargett & Healy, 2011).

Endometrium is a promising, alternative source of MSCs for autologous and allogenic cell-based therapy, that might be exploited to treating gynaecological disorders, including POP and Asherman’s syndrome. However, because of their rarity the use of eMSCs for clinical trials first requires their expansion in culture (Ulrich et al., 2013). As in the case of MSCs from other cell systems (Baxter et al., 2004), cultured eMSCs undergo spontaneous differentiation (Gurung et al., 2015). Further, the telomeres of eMSCs become shortened due to replicative stress. Consequently, eMSCs lose their proliferative capacity as well as the ability to reconstitute tissue in vivo (Baxter et al., 2004; Banfi et al., 2002). These observations predate the realization that prolonged culture of eMSCs results in reduced clinical efficacy (Darzi et al., 2016). Gurung et al., (2015) showed that culturing eMSCs in medium containing TGF-β Receptor (TGF-β-R) inhibitor, A83- 01, in serum free conditions, maintains eMSCs in an undifferentiated state in prolonged culture. Their findings describe the use of small molecules, to selectively inhibit signalling pathways associated to differentiation and improve the purity of cells during culture expansion for therapeutical applications.

Nevertheless, for clinical trials it is of paramount importance to understand molecular ‘programming’ underlying pharmacological approaches. Development of novel techniques, such as Assay for Transposase-Accessible Chromatin with High-Throughput Sequencing (ATAC-seq), for fast and sensitive epigenomic

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profiling of chromatin structure of defined cell populations provides a powerful strategy to address this question (Buenrostro et al., 2013).

1.7 ATAC-seq

The human genome contains approximately two metres of DNA, which is hierarchically packed into chromatin within a five-micron nucleus. DNA is wrapped around a core of proteins, called histones, to form nucleosomes, and nucleosomes are compacted into chromatin (Figure 1.3) (Kornberg, 1974; Sha & Boyer, 2009). This hierarchical packaging plays a central role in the regulation of gene expression (Gross & Grarrard, 1988; Bell et al., 2011). In the genome, two different varieties of chromatin are distinguishable, heterochromatin and euchromatin. The former identifies inactive genomic regions, whereas the latter refers to regions where DNA is lightly packed and hence accessible to transcription factors, and therefore is potentially biologically active (Buenrostro et al., 2015). Chromatin states are regulated by a dynamic epigenetic code which include DNA methylation, histone modification, chromatin remodellers, nucleosome positioning, non-coding RNAs, as well as changes due to the interaction with transcriptions factors (Kouzarides, 2007). Epigenetic mechanisms manipulate chromatin structure and its compaction level, regulating chromatin accessibility. Hence, their role in determining cellular phenotypes is obvious (Chen & Dent, 2014).

Open chromatin profiling methods are highly effective means to understand the epigenetic code governing chromatin remodelling. They interrogate chromatin accessibility and probe regions of nucleosome positioning and TF binding. Mapping of differential chromatin opening enables identification of the cis- regulatory landscape responsible for different cellular functions, including cell proliferation and differentiation (Tsompana & Buck, 2014). ATAC-seq is a recently developed method for genome-wide analysis of the chromatin structure. By contrast to earlier techniques, including DNase-seq (Song & Crawford, 2010), ATAC-seq does not require many cells as starting material. The number of cells

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required to successfully map chromatin accessibility by ATAC-seq ranges between 500 and 50,000. Hence, ATAC-seq provides a powerful new method for genome-wide chromatin analysis of rare cell types, including stem cells (Buenrostro et al., 2013). It uses hyperactive Tn5 transposase to probe DNA accessibility by inserting sequencing adapters into relatively open regions of chromatin (Figure 1.4).

ATAC-seq simultaneously reveals information on chromatin compaction, allows mapping of nucleosome positions and genomic locations of transcription-factor binding. Finally, the whole assay requires less complex and time-consuming protocols when compared to previous genome-wide analysis methods (Buenrostro et al., 2013).

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Sha and Boyer, StemBook 2009

Figure 1.3. Chromatin organization. The DNA is wrapped around a core of eight proteins, called histones, to form nucleosome (light blue). The latter is the basic unit in which the chromatin is organized. Nucleosome packaging into higher order structures generates two varieties of chromatin organization, such as euchromatin and heterchromatin. The level of the packaging regulates chromatin accessibility and consequently gene expression.

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Buenrostro JD et. al, Nat Methods 2013

Figure 1.4. Schematic representation of ATAC-seq reaction. Tn5 transposase (green) simultaneously cuts and tagments accessible genomic regions (mapped between nucleosomes, in gray) with unique sequencing adapters (blue and red). This reaction generates genomic fragments that can be amplified via PCR and subjected to high-throughput sequencing.

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Nucleosome positions

ATAC-seq discloses detailed genome-wide information on chromatin compaction. In particular, paired-end reads generated by high-throughput sequencing reveal nucleosome positioning in regulatory elements.

Specifically, the DNA winds 1.7 turns around the nucleosome wrapping a total length of 147 bp (McGinty & Tan, 2015). Buenrostro et al., (2013) showed that the fragment size distribution of sequenced paired-end reads from human chromatin has a periodicity of 200 bp, suggesting that some regions in the DNA are protected by steric hindrance which makes the transposition less probable (Buenrostro et al., 2013). Hence, fragments with a length of 200 bp may include a single nucleosome and larger fragments may be protected by multiple nucleosomes. Therefore, high molecular weight fragments may contain chromatin in a more compact state compared to short fragments, hence refractory to the transcription machinery that drives differential gene expression.

Buenrostro et al. (2013) also demonstrated that ATAC-seq provides information about the different functional states of chromatin, showing that short fragments of DNA were enriched in CCCTC-binding factor (CTCF)-bound regions, whereas transcription start sites (TSSs) were differentially reduced to fragments associated with one, two and three nucleosomes. Furthermore, this study showed that transcribed sequences of DNA and regions mapped to promoters had longer multi-nucleosomal fragments, and were hence less accessible compared to TSSs. These large fragments of the DNA were enriched in heterochromatin that is inaccessible to nuclease digestion (Ghirlando et al.,

2004). This body of evidence suggests that ATAC-seq reveals functional states of chromatin that are differentially accessible.

In order to show the efficacy of ATAC-seq to map nucleosomes within the genome, Buenrostro et al. (2013) separated sequencing datasets into shorter reads generated from nucleosome-free regions and reads generated by nucleosome-associated chromatin, based on the observations related the periodicity of the insert size distributions (about 200 bp). Then, a data track was calculated and used to map nucleosomes within accessible regions of chromatin

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using a simple heuristic model, which measures positively nucleosome- associated fragments and negatively weights nucleosome-free fragments. The nucleosome-free regions were massively enriched in TSSs when compared to distant elements, which tended to be enriched in nucleosomes.

Thus, ATAC-seq allows high-resolution readouts of regions protected by nucleosomes and nucleosome-free regions in the regulatory chromatin landscape (Buenrostro et al., 2013).

ATAC-seq discloses the position of DNA-binding proteins and the interplay between their genomic location and nucleosome positioning

According to Buenostro et al. (2013), transposition is less probable in chromatin regions bound by DNA-binding proteins. Footprints generated from ATAC-seq inferred the presence of DNA-binding proteins at specific sites in the genome, similar to data obtained from DNase digestion footprints; thus providing information on transcription factor occupancy. In other words, ATAC-seq promises to be a powerful technique to delineate genome regulatory frameworks (Hesselberth et al., 2009).

As aforementioned, ATAC-seq allows detailed mapping of nucleosome positions. With respect to the nucleosome locations mapped through ATAC-seq data, Buenrostro et al. (2013) identified the position of four major classes of DNA- binding proteins. A class of proteins has been shown to strongly avoid nucleosomes, with binding sites approximately at 180 nucleotides from the closest nucleosome positions (NFYA, C-FOS and IRF3 included). A gradation of different behaviours, resulting in avoiding or overlapping nucleosome, has been mainly ascribed to transcription factors (TFs). The other two groups include cohesin-complex subunits RAD21 and SMC3 and chromatin looping factors, which belong to a group of factors whose binding sites locate next to nucleosome boundary, and proteins which tend to bind DNA associated to nucleosomes (Buenrostro et al., 2013).

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Epigenomic profiling on clinical timescales

ATAC-seq is a rapid and information-rich genome-wide analysis method and thus might provide a powerful tool to generate epigenomic analysis on a diagnostic timescale. In order to show that clinically remarkable information might be inferred by ATAC-seq, Buenrostro et al. (2013) applied the assay to a healthy volunteer’s

T cells to profile their regulatory chromatin landscape. In particular, IL2 locus was the subject of this study. IL-2 is a cytokine which plays a key role in autoimmune and inflammatory diseases and is responsible of T-cell growth (Fraser et al.,

1991). ATAC-seq data revealed the drug target involved in the therapeutic IL-2 inhibition in proband T cells. Through footprints the method enabled the profiling of personalised gene regulatory networks and demonstrated that ATAC-seq might be compatible with future diagnostic applications.

Taken together, ATAC-seq might have a wide applicability to understand genome regulatory networks and thus gene expression and offer the possibility to be applicable to the translational research for mapping an individual’s epigenome. It may potentially be applied to rare, relevant cellular subtypes during the different stages of differentiation to improve the understanding of the development of human diseases (Buenrostro et al., 2013).

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