Caracterización de la propuesta

CD146+/PDGFR-β+ cells have been isolated from menstrual blood (Schwab and Gargett, 2007), and are designated MenSCs. MenSCs also express MSC markers such as CD9, CD44, CD29, CD73, CD90 and CD105 along with pluripotency markers OCT-4, C-KIT and Stage-specific embryonic antigen-4 (SSEA-4) but lack expression of STRO-1, a marker which is expressed in perivascular cells isolated from endometrial biopsies. MenSCs undergo 68 passages when cultured in vitro, demonstrating its proliferative property. These cells showed tri-lineage differentiation potential of differentiating into osteoblasts, chondrocytes and adipocytes (Meng et al., 2007a, Musina et al., 2008, Patel et al., 2008). Menstrual blood perivascular cells produce high amounts of MMPs and factors promoting angiogenesis (Gargett et al., 2014). Menstrual blood perivascular cells showed varied differentiation potential by differentiating into cardiomyocytes, hepatocytes, and neural and pancreatic lineages from all three germ layers (Meng et al., 2007b, Patel et al., 2008). MenSCs are considered to be an easily available source for cardiac stem cell therapy (Bockeria et al., 2013).

1.25 Clonogenicity

A unique property of stem/progenitor cells is that they can form a clone from a single cell. Studies proved that the endometrial stromal stem/progenitor cells and epithelial cells could form colonies when seeded in a low cell concentration. The clonogenic capacity is lower in epithelial cells compared to stromal cells. Growth factors play a major role in the formation of colonies. Basic fibroblast growth factor (bFGF) is required for endometrial stromal progenitor cells to form colonies, whereas PDGFβ, tumour growth factor alpha (TGFα) and epidermal growth factor (EGF) are important in colony formation from epithelial progenitor cells (Chan et al., 2004).

Clonogenicity varies between epithelial and stromal progenitor cells during the various stages of the menstrual cycle. High clonogenicity was elicited by stromal progenitor

cells during proliferative phase, and by epithelial progenitor cells during the secretory phase. Chan et al. (2004) could show that within 15 days, 0.22% of endometrial epithelial cells and 1.25% of stromal cells were capable of forming individual colonies. Clonogenicity was higher in both ePCs and SP cells compared to EnSCs. ePCs also demonstrated differentiation potential like that of mesenchymal stem cells from any other source. The differentiation potential of clonogenic endometrial stromal stem/progenitor cells by inducing the cells into adipogenic lineage (Dimitrov et al., 2008). A study analysing endometrial tissue biopsies showed that clonogenic potential is enriched in ePCs population compared to EnSCs. Interestingly, the body mass index (BMI) negatively correlated with the proportion of ePCs, suggesting that the plasticity of the endometrium is compromised in obese individuals, which may potentially result in adverse pregnancy outcomes. (Murakami et al. 2013).

1.26 Decidualization effect on ePCs

Preliminary studies from our lab suggest that the decidual markers PRL and IGFBP1 are expressed at similar levels in both ePCs EnSCs. However, upon decidualization ePCs express a significantly different secretome when compared to EnSCs. Analysis of the cytokine profile at transcription levels shows that ePCs cells elicit a pro- inflammatory response on decidualization, in comparison to the anti-inflammatory responses mounted by EnSCs. Secretome analysis of conditioned medium from differentiating and non-differentiating ePCs show that decidualization induced perivascular cells to secrete high levels of immunomodulatory factors (Murakami et al., 2014).

1.27 Trophoblast invasion into decidua

In normal human pregnancy, the fetal trophoblast cells invade into the wall of the uterus in a complex and tightly controlled process. From tips of anchoring villi, cytotrophoblast cell columns are formed and a shell develops through which extravillous trophoblasts invade decidua. By the eighth week of gestation, interstitial cells from extravillous trophoblasts reach the superficial myometrium (Pijnenborg et al., 1981). In the uterus, the blood vessel diameter decreases sequentially as it progresses through myometrium and endometrium. Spiral arteries supply blood to the endometrial layer and span inner myometrium and for decidua in the case of a pregnant uterus. During pregnancy, placental bed spiral arteries undergo vascular remodelling from high resistance low flow vessels into low resistance high flow vessels (Whitley and Cartwright, 2010).

Perivascular cells which are terminally differentiated and are capable of switching between contractile and proliferative phenotype surround the spiral arteries in the endometrium (Kaplan-Albuquerque et al., 2005). The contractile phenotype is due to expression of contractile proteins such as α-actin, smooth muscle myosin heavy chain, calponin, and smooth muscle 22α. Perivascular cells do not proliferate, migrate or synthesise extracellular proteins (Owens et al., 2004). In response to changes in extracellular cues, perivascular cells adopt a more synthetic phenotype leading to proliferation and migration.

During spiral artery remodelling, a change in phenotype of perivascular cells might lead to increased sensitivity to apoptotic stimuli (Su et al., 2006) or increased migration (Newby, 2006). Defects in the phenotype switching might lead to altered vessel structure, loss of layered organisation of cells or migration away from lumen as evidenced in atherosclerosis (Newby, 2006).

Decidualization is associated with the influx of maternal natural killer cells that are present during spiral artery remodelling and eventually decrease during mid- gestation. In mice, it was shown that uNK cells are involved in spiral artery remodelling stimulated with interferon gamma (IFNγ) (Ashkar et al., 2000). In humans, it was demonstrated that uNK cells along with IFNγ produce TNFα, LIF, CSF-1and IL-8 (Ashkar and Croy, 2001, Hanna et al., 2006). IFNγ has been shown to antagonise decidual PRL expression (Christian et al., 2001).IFNγ can cause apoptosis as well as the proliferation of perivascular cells. IFNγ may prime the perivascular cells for death receptor-induced apoptosis (Bai et al., 2008). Interferon γ can increase TRAIL production in trophoblasts (Phillips et al., 1999). Immunohistochemical studies show that uNK cells and macrophages are localised at the site of spiral artery remodelling suggesting that immune cells may play a role in the reorganisation of vascular cells and smooth muscle layer separation (Smith et al., 2009). uNK cell also produces a myriad of angiogenic factors including VEGF, PLGF and Ang-2 (Hanna et al., 2006).A defect in vascular remodelling leading to uteroplacental hemodynamics has been considered to be a causative factor for pre-eclampsia (Hanna et al., 2006).

1.28 Endometrial scratch

Intentional injury to the endometrium of a woman wanting to conceive is termed as endometrial scratching. This insult to the endometrium is considered to increase the chance of an embryo implanting in turn increasing the chance of pregnancy. Various instruments are utilized to perform the biopsy. The most used instrument for performing endometrial scratch is a thin flexible plastic tube, 3mm wide, called a Pipelle catheter. The Pipelle is inserted through the cervix into the uterus and moved back and forth and rotated to cause some disruption. It is a simple, inexpensive outpatient procedure without anaesthetic and less time consuming.