**5.1 Stem cells**

298 Endometriosis - Basic Concepts and Current Research Trends

underlying smooth muscle. The endometrium is composed of single columnar epithelial layer, resting on a layer of connective tissue, the stroma. Tubular glands reach through the endometrial surface to the base of stroma. In a woman of reproductive age, the endometrium itself is structurally and functionally divided into two relatively distinct layers of upper functionalis and lower basalis. The functionalis zone is adjacent to uterine cavity and contains glands and supportive stroma for optimal implantation and growth condition of embryo. Basalis (basal layer), adjacent to myometrium, provides basal region of glands, dense stroma and lymphoid aggregates and from it functional layer develops. During menstrual cycle only the functional layer is regenerated and the basal layer is not affected. Both endometrium and subendometrial myometrium originate from the Müllerian ducts during embryonic life. However, outer myometrial layer is formed during fetal life and from

The cellular components of human endometrium can be primarily divided into two cell types; the epithelial cells (luminal and glandular) and the supporting mesenchymal cells

It is acknowledged that about 11% of all women population during reproductive age is affected by endometriosis (Buck Louis, Hediger et al. 2011). Endometriosis is a chronic benign gynecological disease which is characterized by the ectopic formation of endometrial stroma and glands mostly seen in pelvic peritoneum. However, it might be seen outside the pelvic peritoneum such as, pelvic viscera(Vercellini, Meschia et al. 1996), rectovaginal septum (Nisolle and Donnez 1997), pleura, abdominal wall, and even sometime in brain (Thibodeau, Prioleau et al. 1987). Briefly, the underlay mechanism is attachment of endometrial cells to the pelvic peritoneum, invasion into the mesothelium, and survival and proliferation of the ectopic endometrial cells. Endometriosis is usually diagnosed after symptoms such as pelvic pain, which might correlate with menstrual cycle, or infertility.

The source of endometrial implants is not fully understood. However, there are evidences for competence of some women over others (Di and Guo 2007). The reasons for susceptibility of some patients for development of ectopic lesions are not clear. Comparative microarray analysis of gene expression in patients with ectopic endometrial cells and that of eutopic endometrium has demonstrated an alternative pattern of gene expression in two groups (Taylor, Lundeen et al. 2002; Giudice 2003; Giudice 2006). This is also seen between patient and non-affected women (Gogusev, de Joliniere et al. 1999; Taylor, Lundeen et al.

Although etiology of disease has not been clearly understood, a number of theories have been proposed in correlation with the pathogenesis of endometriosis which includes: genetic and environmental factors, immune system, retrograde menstruation, coelomic metaplasia, embryonic rest theory, lymphovascular metastasis and stem cell-based theory. Clinical manifestation of endometriosis and ectopic formation of endometriotic lesions usually

non- Müllerian origin (Ferenczy and Bergeron 1991).

**3. Endometriosis** 

(stromal cells) as well as vascular (endothelial) cells and leukocytes.

**4. Etiology and pathophysiology of endometriosis** 

2002; Giudice 2003; Giudice 2006; Wu, Strawn et al. 2006).

considered as end point result of several aberrant biological process.

Stem cells are undifferentiated cells which are defined by their functional properties such as; high proliferative potential, substantial self-renewal capacity and ability to differentiate to other organ/tissue-specific cell types. Cellular self- renewal is a capacity that parental stem cells are able to divide into two daughter cells which can happen through symmetrical or asymmetrical division. The symmetrical division produces two identical daughter stem cells or transit amplifying (TA) cells. TA cells undergo repetitive cell division cycles and progressively acquire differentiation marker and lose their self-renewal capacity. Asymmetrical division leads to an identical daughter cell as well as a more differentiated cell.

Although stem cells are potentially capable to proliferation, they remain mostly in a quiescent state until an inductive factor to induce them to proliferation.

Adult stem cells present in all organs and tissues and are responsible for tissue regeneration and repair after damage and trauma encountered during life time.

Rarity and lack of distinguishable morphological features and specific molecular markers of stem cells have hindered their isolation, purification and studies for several years.

Broadly they can be divided into two categories, embryonic and adult stem cells. Stem cells can also be divided into various groups according to their differentiation capacity. For example, the zygote can be considered as a totipotent stem cell, which means it is able to differentiate into all other cell types in embryo and extraembryonic tissue. Other stem cells with decreased levels of potency can be named as; pluripotent, which can differentiate into nearly all cell types (e.g., inner cell mass of the blastocyst), multipotent, which can differentiate into a number of cells that are closely related family of cell (e.g., Hematopoetic stem cells), oligopotent which differentiate into a few cell types (e.g., myeloid or lymphoid stem cells) and unipotent, which differentiate into only one cell type with self-renewal capacity(e.g., muscle stem cell ).

Anatomic structure surrounding stem cells which have profound effect on cell function is called niche. Signaling elements and various cellular and molecular interactions inside the niche can determine the fate of cell to stay in undifferentiated state until tissue regeneration or repair to motivate stem cell differentiation.

#### **5.2 Evidences for existence of endometrial stem cells**

#### **5.2.1 Indirect evidences**

Adult stem cells present throughout whole body and responsible for replenishment and regeneration of damaged tissues and contribute to the structural and functional maintenance of tissues and organs. Human endometrium undergoes periodical process of regeneration during menstrual cycle. The growth rate may vary between 0.5 -7mm in thickness. Menstruation is a phenomenon consisted of various cellular and tissue functions from cell proliferation and differentiation to shedding and regeneration. During each menstrual cycle, the functionalis and a part of basalis layer of endometrium undergo shedding. After shedding, the endometrium regrows under the influence of estrogen.

Endometrial Stem Cells and Endometriosis 301

study of lower fraction showed the capacity to differentiate into CD13+ stromal-like clusters. Additionally, to test whether endometrial SP cells reconstitute the endometrial tissue with stromal and glandular structure, they were xenotransplanted into NOG mice (Maruyama 2010). These studies not only demonstrated the presence of endometrial stem/progenitor cells, they have also provided an experimental animal model for endometriosis which is

Mouse could be used as an experimental model for endometriosis and endometrial stem cell studies. However, the structure and physiology of mouse endometrium is not exactly similar to that of human. The mouse lacks an endometrial basalies layer, and the endometrium does not shed during menstruation but rather it is reabsorbed after the cycle. In spite of these dissimilarities, the murine estrous cycle has characteristics similar to those of human menstrual cycle and then mouse uterus can provide data on the molecular and cellular information for the pathogenesis of endometriosis and normal activity of

On this basis, several studies have been conducted to demonstrate the presence of endometrial stem cells in mouse endometrium (Cervello, Martinez-Conejero et al. 2006; Chan and Gargett 2006). For example, label-retaining cells (LRCs) have been shown in the mouse uterus. Label retaining is a technique to identify a stem cell population *in vivo* on the basis of quiescent state of stem/progenitor cells. The cells in this state are able to retain DNA strand incorporated stains such as, 5-bromo-2´-deoxyuridine (BrdU) for a longer period than the active dividing cells. A study conducted by Chan and Gargett showed that 3% of mouse endometrial epithelium after a 56-day chase period and 6% of endometrial stromal cells after an 84-day chase period are LRCs. Also, in another study Cevello et al demonstrated that 9% of stromal cells after 49 days were LRCs and this decreased to 1.7% after 112-day chase period. However, no epithelial LRCs were identified even after 21 days (Cervello, Martinez-Conejero et al. 2006). Presence of LRCs have also been demonstrated in

Endometrial stem/progenitor cells have also been obtained from menstrual blood. These cells can be maintained in culture for more than 68 doubling and during this time they are able to retain their markers such as CD9, CD29, CD41a, CD44, CD59, CD73, CD90 and CD105 without karyotypic abnormalities (Musina, Belyavski et al. 2008). Proliferative rate of these cells are also significantly more than mesenchymal stem cells from the source of

Endometrial regenerative cells (EnRCs) from menstrual blood are different from endometrial stem cells at expression of STRO-1. These cells are also negative for hematopoetic markers such as, CD34, CD45, CD133, but express embryonic stem cell

Ease of collection could be considered as an important characteristic of EnRCs which make them more attractive. Karyotypic stability of EnRCs suggests a large scale expansion

valuable in endometriosis studies.

myometrium (Szotek, Chang et al. 2007).

**5.6 Endometrial regenerative cells (ERCs) from menstrual blood** 

**5.5 Evidence from mouse** 

endometrium tissue.

umbilical cord.

marker, Oct4.

capability.

Regeneration process is comprised of endometrial regrowth, angiogenesis and proliferation of endometrial stromal cells. Shedding and regeneration of the endometrial layer during menstruation and regeneration of functional layer may be considered as an indirect evidence for presence of progenitor/ stem cells. This concept was proposed in 1978 by Prianishnikov (Prianishnikov 1978), and then confirmed by following clinical observations (Wood and Rogers 1993), proliferation experiments and demonstration of gland monoclonality (Tanaka, Kyo et al. 2003; Chan, Schwab et al. 2004; Schwab, Chan et al. 2005). In some other species this process is carried out in the form of endometrial growth and apoptosis rather than menstrual cycle. Therefore, this is conceivable that endometrium consists of an active and regenerative population of cells which are known as endometrial progenitor/stem cells (EnSCs). Since endometrium consists of glands, surface epithelium and supportive stroma, the existence of both epithelial and stromal stem/progenitor cells are plausible.
