Uterine Stem Cells and Benign Gynecological Disorders: Role in Pathobiology and Therapeutic Implications

Abstract

Stem cells in the endometrium and myometrium possess an immense regenerative potential which is necessary to maintain the menstrual cycle and support pregnancy. These cells, as well as bone marrow stem cells, have also been implicated in the development of common benign gynecological disorders including leiomyomas, endometriosis and adenomyosis. Current evidence suggests the conversion of uterine stem cells to tumor initiating stem cells in leiomyomas, endometriosis stem cells, and adenomyosis stem cells, acquiring genetic and epigenetic alterations for the progression of each benign condition. In this comprehensive review, we aim to summarize the progress that has been made to characterize the involvement of stem cells in the pathogenesis of benign gynecologic conditions that, despite their enormous burden, are not yet fully understood. We focus on the stem cell characteristics and aberrations that contribute to the development of benign gynecological disorders and the possible clinical implications of what is known so far. Lastly, we discuss the role of uterine stem cells in the setting of regenerative medicine, particularly in the treatment of Asherman syndrome.

Graphical Abstract

INTRODUCTION

Stem cells are undifferentiated cells that are present in many tissues and are capable of reproducing themselves and differentiating into different cell types. They proliferate, through asymmetric division, to replace themselves and give rise to transient amplifying progenitors [1, 2]. Progenitor cells multiply in large numbers, differentiate into tissue-specific cells, and have a lesser ability to self-renew [2]. The terms stem cells and progenitor cells have been used interchangeably to describe cells with proliferating potential [3]. Stem cells are usually present in the G₀ phase (quiescent state) of the cell cycle and are located in an environment known as the niche [4]. Embryonic stem cells were first identified in 1981 in the mouse embryo [5], and it was not until more than 20 years later that colony forming cells were identified in the endometrium [6]. Since then, studies have looked into stem cells in the uterus as well as stem cell contributions from the bone marrow. These cells are studied in the context of the normal physiology of the reproductive system as they are involved in the monthly regeneration of the endometrium during menses and sustaining pregnancy. They were also studied in the context of the development of benign and malignant gynecologic conditions. Understanding the aberrations that lead to the transformation of endometrial and myometrial stem cells that drive the development of leiomyomas, endometriosis, and adenomyosis are crucial to establish targeted therapeutic options for these conditions.

In this comprehensive review, we aim to summarize the involvement of stem cells in the pathogenesis of benign gynecologic conditions that, despite their enormous burden, are not yet clearly understood. We focus on the stem cell characteristics and aberrations that contribute to the development of these conditions and the potential clinical implications of stem cell involvement. Lastly, we discuss the role of uterine stem cells in the setting of regenerative medicine, highlighting the studies where stem cells were used for the treatment of Asherman syndrome.

SEARCH METHODS

A comprehensive search of PubMed, Embase, Scopus, and Cochrane databases up to September 2020 was conducted to identify peer-reviewed literature. We used the following keywords: uterine fibroid/leiomyoma, endometriosis, adenomyosis, stem cells, and progenitor cells. Screening of studies was done by two authors (ME and SA). Results were further reviewed by the senior author (MAB). Articles were initially screened based on title and abstract, and a second round of screening was done after reading the full-text articles. We only included articles in the English language. This review included studies in both humans and animals.

STEM CELLS IN UTERINE LEIOMYOMAS

Myometrial Stem Cells

While the myometrium is relatively quiescent during the reproductive cycle, it is able to substantially grow during pregnancy and regress after delivery [7, 8]. This significant remodeling suggests a possible role of stem cells.

Several groups have demonstrated that myometrial Hoechst-stained cells contain a small percentage of side population (SP) cells, suspended in the G₀ phase of the cell cycle [9, 10]. SP cells were first identified in the bone marrow as cells that rapidly efflux the Hoechst 33342 DNA binding dye [11]. Uptake occurs universally in all cells, but efflux through ATP binding cassette protein (ABC) transporters only occurs in a specific population of cells [12]. SP cells are considered a stem cell population and have been identified in several tissues [12]. Myometrial SP cells represent around 3% of the total myometrial cells and have lower expression levels of estrogen receptor α and progesterone receptor, as well as smooth muscle cell-specific markers such as calponin and smoothelin [9]. These cells were able to generate functional human myometrial tissues and were shown to be multipotent, differentiating into osteocytes and adipocytes in vitro [9]. The myometrial SP cells also showed upregulation of the mRNA level of octamer-binding transcription factor 4 (OCT4), a transcription factor expressed in embryonic, germ and adult stem cells [13].

Myometrial SP cells only proliferate at low oxygen tension (2% oxygen) and not the normoxic environment (20% oxygen), suggesting they survive and grow in a hypoxic environment, consistent with the stem cell niche [9, 14]. Interestingly, Shynlova et al. showed in vivo studies on murine models that mechanical stretching of the uterine wall during pregnancy induces hypoxia [15]. This observation could explain how these cells contribute to the enlargement and remodeling seen during pregnancy.

Stem Cell Markers of Myometrial Stem Cells

Using the Hoechst dye for stem cell isolation has some limitations since the dye is toxic and might affect clonogenicity, and this assay is particularly sensitive to slight variations in staining conditions including dye concentrations, temperature, light conditions and duration of staining [9, 12]. Hence, other methods were developed to isolate myometrial stem cells based on cell surface markers. CD49f and CD34, known stem/progenitor cell markers [16, 17], were found to be preferentially expressed by myometrial SP cells [18]. After purification, myometrial cells that expressed both of these cell surface markers displayed high colony forming ability, had the capacity to differentiate into multiple lineages, preferentially proliferated under hypoxic conditions, and expressed lower levels of estrogen and progesterone receptors [18]. These double positive cells were also capable of forming human myometrial tissues that expressed vimentin and α-smooth muscle actin when xeno-transplanted into the uteri of immunodeficient mice [18]. In the murine model, CD44⁺ myometrial cells were also recognized as somatic stem cells, expressing typical undifferentiated markers [19].

Moreover, Mas et al. showed that CD44 and Stro-1 can also be used as cell surface markers for myometrial stem cells [20]. Isolated cells positive for these markers also expressed ABCG2 transporter as well as other stem cell markers such as OCT4, Nanog homeobox (NANOG) and GDB3 [20]. Similar to myometrial SP cells, they had a low expression of estrogen and progesterone receptors [20]. These cells were also capable of forming fibroid-like tissues when xeno-transplanted in immunodeficient mice and were demonstrated to have regenerative potential in vivo [20]. More recently, CD146⁺CD140⁺ and/or sushi domain containing-2 (SUSD2⁺) myometrial cells were found to be located in the perivascular region and were capable of forming colonies and differentiating into adipogenic and osteogenic lineages in vitro [21].

Leiomyoma Stem Cells

Uterine leiomyomas are benign smooth muscle tumors and are the most common benign gynecological tumor in women of reproductive age, affecting an estimated 70% of women [22, 23]. Symptoms include excessive uterine bleeding, pelvic discomfort, infertility, urinary frequency and spontaneous abortion [24]. These tumors are characterized by smooth muscle proliferation and extensive extracellular matrix deposition and remodeling. Despite the high prevalence and burden of uterine leiomyoma, their precise pathogenesis is still largely unknown. In addition to aberrations of signaling pathways, particularly estrogen and progesterone signaling pathways [25, 26], studies suggest epigenetic mechanisms such as DNA methylation, micro-RNA as well as histone modification [27–32]. Several genetic aberrations, such as deletions in 7q, trisomy of chromosome 12, and gene mutations like mediator complex subunit 12 (MED12), fumarate hydratase, and high mobility AT-hook 2 (HMGA2) have also been associated with leiomyoma development [33–37]. Studies using X chromosome-linked clonality suggest that leiomyomas are monoclonal tumors [38], and an increasing body of evidence supports that these tumors originate from stem cells in the myometrium, perhaps in combination with all previously stated aberrations [39]. For example, it was shown that African American women, who are at the highest risk of developing leiomyomas, have a higher level of myometrial stem cells compared to Caucasian women [40].

Several groups have isolated undifferentiated somatic stem cells or tumor progenitor cells from leiomyomas [41, 42]. These SP cells were present in the G₀ phase and expressed stemness markers such as OCT4, NANOG, DNA (cystosine-5-)-methyltransferase 3 beta (DNMT3B), and growth differentiation factor-3 (GDF-3) [42]. Stem cells derived from leiomyomas, unlike those derived from the myometrium, carry the MED12 mutations, a mutation present in up to 70% of leiomyomas [41, 37]. Additionally, expression of HMGA2 in myometrial cells induced leiomyoma-like tissue formation through abnormal proliferation [43]. Orciani et al. showed that leiomyoma progenitor cells had a shorter doubling time and higher expression of stemness genes compared to myometrium progenitor cells [44]. Moreover, using c-KIT immunostaining, telocytes, involved in tissue repair and renewal, were detected in higher number and activity in leiomyoma stem cells compared to myometrial stem cells and normal myometrium cells [45]. Similar to myometrial stem cells, leiomyoma stem cells were shown to express CD34⁺/CD49b. They also expressed low levels of estrogen receptor, progesterone receptor, smooth muscle actin and high levels of stem cell markers such as Krüppel-like factor 4 (KLF4), NANOG, sex-determining region Y-box 2 (SOX2), and OCT4, asserting their undifferentiated status [46].

Epigenetic changes have also been observed in leiomyoma stem cells. A recent study highlighted dysregulation in DNA methylation in leiomyoma stem cells, consistent with global hypermethylation, compared to differentiated leiomyoma cells [47]. Hypermethylation suppressed genes involved in the function of myometrium such as contraction and hormone response. Furthermore, 5’-Aza, the hypomethylating agent, stimulated the stem cells’ differentiation, reducing the stem cell population and preventing tumor formation [47]. These cells were also shown to have increased DNA damage and altered DNA repair gene expression and signaling [48].

Leiomyomas possess a much smaller stem cell population compared to myometrium [10]. Leiomyoma stem progenitor cells also appear to be deficient in estrogen and progesterone receptors, but their growth is stimulated by estrogen and progesterone hormones [41, 42]. Given that leiomyoma stem progenitor cells require the presence of mature leiomyoma or myometrial cells to proliferate, it is hypothesized that these cells respond to steroid hormones through the surrounding differentiated cells via paracrine signaling [41, 49]. There is some evidence that bone marrow derived cells (BMDCs) can contribute to leiomyoma growth, with the migration of these cells being dependent on CXCL12 expression [50].

Transformation of Myometrial Stem Cells into Tumor Initiating Cells

The origin of leiomyoma stem cells is less understood. In the murine model for leiomyoma, the primary site for leiomyoma development is the cervix, which was shown to be a hypoxic niche, linking back to the necessity of a low oxygen environment for stem cell survival [51]. In the same study, it was noted that myometrial stem cells responded to environmental cues, with their count decreasing with age and absence of steroid hormones [51].

Several studies have shown that estrogen allows the propagation and differentiation of stem cells. As mentioned earlier, uterine leiomyoma growth is stimulated by estrogen and progesterone, although the exact mechanism is not entirely elucidated. Early life exposure to endocrine disrupting chemicals such as diethylstilbestrol (DES) reprogramed the epigenome of myometrial stem cells to a pro-leiomyoma condition in a murine model by increasing the expression of estrogen responsive genes [52, 53].

Liu et al. recently demonstrated that receptor activator of nuclear factor κB ligand (RANKL), a key factor in tumorigenesis, upregulated leiomyoma stem cell proliferation through Cyclin D1 activation [54, 55]. Progesterone agonist, R5020, induced RANKL gene transcription in leiomyoma tissue through DNA methylation upstream of the RANKL transcription start site [55]. Moreover, RANKL gene expression was higher in leiomyoma tissue containing a MED12 mutation [55]. Taken together, this study highlights the important role of progesterone-mediated RANKL expression, linking RANKL epigenetic changes and MED12 mutations, in activating stem cell proliferation and leiomyoma development. Paracrine pathways are also involved in leiomyoma stem cell proliferation. Moravek et al. showed that insulin growth factor 2 (IGF2) increases cell number through extracellular signal-regulated kinase (ERK) phosphorylation [56]. Leiomyoma stem cells also demonstrates higher expression of Insulin receptor A (IR-A) [56]. More studies are needed to identify the exact mechanism for the conversion of myometrial stem cells to leiomyoma stem cells.

Role of the Wnt/β-Catenin Pathway

The wingless integration site family (Wnt) signaling pathways is one of the main signaling pathways involved in several cellular functions such as cell proliferation, migration, development, and apoptosis [57]. Among the several pathways involved in leiomyoma development, the Wnt/β-catenin pathway is one of the best understood and seems to play a vital role in somatic-stem cell function. The activation of this pathway results in the inhibition of β-catenin degradation, ultimately increasing the amount of this protein in the nucleus, allowing it to interact with chromatin and the family of transcription factors that regulate the expression of a large set of dependent genes [57].

In leiomyoma SP cells cocultured with mature myometrial cells, estrogen and progesterone hormones induced nuclear translocation of β-catenin and the transcriptional activity of its heterodimeric partner T-cell factor and their target gene axis inhibition protein 2 (AXIN2), resulting in the proliferation of these SP cells [58]. Moreover, ectopic expression of β-catenin inhibitor in these cells blocked the hormone dependent growth of human tumors in vivo [58], highlighting the role of estrogen and progesterone on the Wnt/β-catenin pathway in the growth of leiomyomas.

Selective deletion of β-catenin in uterine mesenchyme during embryonic development reduces uterine size and replaces these cells with adipocytes in mice [59]. This study suggests that β-catenin is involved in the differentiation of stem cells into smooth muscle phenotype. This was confirmed in another study in human leiomyoma cells where a β-catenin antagonist resulted in the detection of intracytoplasmic lipid droplets, confirming adipocytic trans-differentiation or metaplasia of leiomyoma cells [60]. Moreover, constitutive overexpression of β-catenin during embryonic development resulted in myometrial hyperplasia that developed into mesenchymal tumors in the uterus that histologically resembled human leiomyomas [61].

A recent study showed that the Wnt/β-catenin pathway’s expression varied depending on the differentiation of leiomyoma cells [62]. WNT4 was highly expressed in intermediately differentiated leiomyoma cells while Wnt receptor, FZD6, was highly expressed in leiomyoma stem cells [62]. WNT4 expression increased proliferation through upregulation of the expression of pro-proliferative gene such as cyclin D1 and c-Myc and resulted in the activation of Akt signaling pathway and β-catenin [62]. Activated β-catenin was shown to induce the expression of transforming growth factor-beta 3 (TGFβ3), which stimulated proliferation and extracellular matrix (ECM) formation in leiomyoma tissue [63, 61]. Chen et al. showed that fucoidan, an anti-fibrotic polysaccharide, reduced leiomyoma stem cell proliferation in the Eker rat model with inhibition of TGFβ3-induced cell growth as well as reduction in β-catenin translocation into the nucleus [64]. MED12 has also been implicated in the regulation of Wnt/β-catenin signaling in leiomyoma pathogenesis. Markoski et al. showed that MED12 mutations increased the expression of Wnt ligand, Wnt4, in leiomyoma cells compared to the cells that did not carry the mutation [65]. Figure 1 shows proposed stem cell theory for the development of leiomyomas. More studies are needed to clarify the exact mechanism of interplay between the different genes involved in the development of leiomyoma.

Figure 1:

Flow chart demonstrating the search strategy and number of articles screened and included in the study.

Clinical Implications and Future Research

Despite significant leiomyoma research and the array of new and emerging therapeutic options[66], the available medications are limited with a potential of recurrence after discontinuation. This reinforces the need to recognize the specific pathogenesis of these tumors to develop better therapies. From what is currently known about leiomyoma stem cells, it seems plausible that any of the previously discussed pathways could be a potential therapeutic target. For example, Wnt antagonists block the estrogen/progesterone-induced effect on β-catenin and its target genes, consequently inhibiting the proliferation in leiomyoma SP cells [58]. Moreover, RANK-Fc, an inhibitor of the RANKL pathway, significantly decreases proliferation and tumor growth [67].

Tumor-initiating leiomyoma stem cells have been targeted for gene therapy through magnetic nanoparticles complexed to adenovirus resulting in apoptosis and suppressed proliferation [68]. In addition, Simvastatin, known to possess anti-leiomyoma properties [69–74], also modulates stem cells [75, 76]. Therefore, it appears intriguing to examine if some of these anti-leiomyoma effects are mediated through leiomyoma stem cells. Moreover, Elkafas et al. recently demonstrated that vitamin D3 protects myometrial stem cells against DNA damage induced by DES in the same Eker rate model discussed above [77]. Vitamin D3 results in DNA repair via the MRN complex/ATM axis, returns the DNA repair signaling network, and augments DNA damage response [77]. These results suggest that vitamin D3, a readily available and safe supplement, can be a possible therapeutic approach for the prevention of DNA damage during leiomyoma development. Another possible treatment target could be DNA methylation since hypermethylation resulted in maintenance of the stem cell phenotype.

Further research on the drivers of myometrial stem cell conversion to tumor initiating cells will result in a better understanding of the pathogenesis of leiomyomas. This will ultimately lead to the development of new endocrine disruptors, effective prevention, and treatment options for leiomyomas with lower recurrence rates and long-lasting effects, reducing the burden of these common tumors.

STEM CELLS AND ENDOMETRIOSIS

Endometrial Stem Cells

The human endometrium exhibits an immense regenerative capacity, undergoing over 400 cycles of growth, alluding to the presence of endometrial stem cells [78]. In 2004, Chan et al. published the first report of purified human endometrial epithelial and stromal stem cells isolated from hysterectomy tissues [6]. Since then, progress has been made to identify and characterize the endometrial stem/progenitor cells involved in endometrial physiology as well as their involvement in the pathogenesis of endometriosis.

Epithelial stem cells

During the menstrual cycle, the functionalis layer of the endometrium is shed while the basal component is conserved. It was hypothesized that the basalis layer contains epithelial progenitor cells that allow the functionalis to re-epithelialize under the influence of increasing estrogen levels [79]. Epithelial progenitor cells have been identified as colony-forming units (CFUs) in freshly isolated and purified epithelial and stromal cells. The single cell-derived large CFU, comprising 0.08% of epithelial cells, exhibited adult stem cell properties as they had self-renewal activity, high proliferative potential, and differentiated into large gland-like structures when grown in three-dimensional (3D) cultures [80].

There are several studies looking into identifying specific markers to isolate endometrial epithelial stem cells. One of the proposed markers is stage-specific embryonic antigen 1 (SSEA-1), which is highly expressed in the basalis layer. Cultured SSEA-1(+) endometrial cells had greater telomerase activity and longer telomeres and lower proliferation rates, a feature of progenitor cells, when compared to SSEA(−) epithelial cells. They also express lower levels of estrogen (ESR1) and progesterone (PR) receptors, indicating a less differentiated phenotype [81]. Another proposed marker is the leucine repeat-containing G protein-coupled receptor 5 (LGR5), located on epithelial cells in the lower functionalis layer closest to the basalis layer [82]. N-cadherin was also studied as a potential marker of epithelial progenitor cells. N-cadherin(+) basalis epithelial cells displayed high clonogenicity, proliferative potential, and self-renewing capacity compared to N-cadherin(−) cells [83]. These cells, predominantly located in the basalis endometrium, were capable of differentiating into cytokeratin(+) gland-like organoids in-vitro [83].

Mesenchymal stem cells (MSCs)

In contrast to endometrial epithelial stem cells, MSCs can be readily isolated and enriched. These cells are located in both functionalis and basalis layers as perivascular cells, and they are able to regenerate functionalis stroma [84]. Schwab et al. showed that CD146(+) PDGF-Rbeta(+) stromal cells made up 1.5% of the sorted endometrial tissue and were enriched with CFUs that were able to differentiate into adipogenic, osteogenic, myogenic and chondrogenic lineages [84]. Another studied marker is the SUSD2 antigen, sometimes referred to as W5C5. W5C5(+) cells comprised 4.2% of endometrial stromal cells and also resided in the perivascular area.

These cells were capable of producing endometrial stromal-like tissue in vivo [85]. Endometrial MSCs also were shown to express genes involved in angiogenesis, steroid hormone/ hypoxia responses, inflammation, immunomodulation, cell communication, and proteolysis/inhibition [86]. They exhibited characteristic properties of self-renewal and multipotency of adult tissue MSC through increased expression of Notch, TGFβ, IGF, Hedgehog, and G-protein-coupled receptor (GPCR) signaling pathways [86]. These cells exhibit considerable plasticity and were shown to differentiate along mesodermal, ectodermal and endodermal lineages into adipocytes [87], smooth muscle [88], urinary bladder [89], hepatocytes [90], and megakaryocytes [91]. MSCs were also driven to differentiate for therapeutic options such as endodermal insulin secreting pancreatic lineages [92, 93] and dopaminergic neuron-like cells that were able to restore dopamine levels in a mouse Parkinson model [94].

Side population (SP) cells

Human endometrium contains up to 5% of SP cells in freshly isolated tissue [95]. The percentage of SP cells differed during the menstrual cycle, with studies showing conflicting results [95, 96]. Masuda et al. showed that the proportion of endometrial SP cells was highest in early proliferative phase and declined gradually until the end of the cycle, attributing the decrease to the dilution of SP cells as the functionalis layer grows and increases in thickness [97]. SP cells are located in both the functionalis and basalis layers, particularly in the perivascular area, suggesting similarities between these cells and MSCs [97]. These cells display telomerase activity level between that of embryonic stem cells and mature cells, and they do not express ESR1 or PR, similar to MSCs, but show ESR2 expression [98]. Freshly isolated endometrial SPs gave rise to three distinct types of colonies in culture: fibroblastic stromal cells CD13(+), endothelial CD31(+) cells, and epithelial cells that express cytokeratin [97]. SP cells also reconstituted endometrial-like tissues containing glandular structures, stromal tissue and blood vessels in vivo when transplanted underneath the kidney capsule of immunocompromised mice [97, 98].

Menstrual blood endometrial stem cells (MenSCs)

The presence of endometrial mesenchymal stem cells in both functionalis and basalis layer indicated that endometrial stem cells could be shed in menstrual blood. Endometrial stem cells were first characterized in 2007; they were maintained in tissue culture with more than 60 doublings and exhibited a high-proliferative rate [99]. At the time, they were named endometrial regenerative cells, as they were capable of differentiating into 9 lineages [99]. These cells express CD9, CD29, CD44, CD75, CD90, CD105, CD166, OCT-4, SSEA-4, and C-X-C chemokine receptor type 4 (CXCR4) [99–102]. They can be induced to differentiate to endothelial, respiratory epithelial, pancreatic, hepatic, adipocytic, cardiomyocytic, myocytic, neurocytic, and cartilaginous using appropriate differentiation techniques [99, 103, 101, 104–106]. These cells are considered a promising treatment for many diseases in the field of regenerative medicine since they are easily accessible through non-invasive collection.

Very Small Embryonic Like Stem Cells (VSELs)

VSELs are a population of small, nonhematopoietic, pluripotent cells that do not express lineage markers and are hypothesized to serve as a backup pool for adult stem cells that are mobilized under stress conditions such as hypoxic injury [107]. They express pluripotency markers such as OCT4 and NANOG, have the ability to give rise to derivatives of the three germ layers in vitro both in mice and humans, and are able to form embryoid-like bodies [108]. These cells are quiescent cells, do not readily divide in culture, and are able to survive various kinds of toxic insults [108]. Despite their presence being controversial, VSELs have been identified in several mouse and human tissue and were isolated from adult mouse uterus [108, 109]. James et al. showed that gonadotropin and steroid hormones activated VSELs in the endometrium. Estrogen treatment resulted in hypertrophy, progesterone treatment resulted in hyperplasia and crowding, while follicular stimulating hormone (FSH) treatment stimulated these stem cells [110]. OCT-4 positive VSELs have also been isolated in the perimetrium, are steroid hormone dependent, and have been hypothesized to be tumor initiating cells [111].

Bone marrow-derived stem cells involvement in endometrial regeneration

Bone marrow has been proposed as a source of stem cells with tremendous differentiative capacity to produce diverse cell lineages [112]. Several reports suggest that bone marrow can give rise to endometrial stromal, epithelial and endothelial cells, but others have questioned the nature of the recruited bone marrow cells since the endometrium is an immunologically active tissue that recruits immune cells.

The first study that reported bone marrow-derived endometrial cells, which was done on patients that had received HLA-mismatched bone marrow transplants, was published in 2004 [113]. Donor-derived endometrial cells were identified in endometrial biopsy samples from those who received the bone marrow transplant. Remarkably, these cells comprised up to 48% of the epithelial cells and up to 52% of the stromal cells. Ikoma et al. subsequently showed similar results using the Y-chromosome as a marker in patients who had received sex-mismatched bone marrow transplants. However, the reported contribution to the endometrium was substantially less, accounting up to 8.4% epithelial cells and 9.8% of stromal cells [114]. Mints et al. also reported the presence of bone marrow-derived endothelial stem cells in the human endometrium and in mice [115]. However, more recently, Ong et al. reported that the bone marrow cells detected in the mouse endometrium after transplant were immune cells, expressing the pan leukocyte marker CD45, including T cells and macrophages, and there was no evidence of bone marrow-derived stromal, epithelial or endothelial cells [116]. The authors suggested that the previous studies were likely detecting immune cells that infiltrated the epithelial and vascular endometrial compartments which could be mistaken for endometrial cells. More studies are needed to clear this discrepancy.

Role of Endometrial Stem or Progenitor Cells in Development of Endometriosis

Endometriosis is a gynecological condition characterized by the development and growth of endometrial tissue outside the uterine cavity, affecting the ovaries, rectovaginal space, bladder, bowel, and rarely, more distal organs such as pleura, lungs, pericardium and the brain [117]. It is estimated to affect 6–10% of women in their reproductive age and up to 60% of women with infertility [118]. It presents as a variety of symptoms including dysmenorrhea and dyspareunia, and it is the most common cause of chronic pelvic pain in women in their reproductive age [119, 120].

Several mechanisms have been proposed to explain the pathophysiology of endometriosis with a unifying theory that explains its origin remaining elusive. The most widely accepted theory is retrograde menstruation through the fallopian tubes which results in the implantation of endometrial tissue in the pelvis [121]. Other theories include the spread of menstrual tissue to distant body sites through veins and lymphatics, celomic metaplasia, and the embryonic cell rests of Mullerian origin differentiating under a specific stimulus [121]. However, none of these theories can fully explain the etiology of all types of endometriosis, and accumulating evidence suggests the involvement of endometrial stem cells in the pathogenesis of this disorder, perhaps in parallel with all of the proposed theories. The dysregulation of endometrial stem cells has been proposed as a possible means for seeding ectopic endometriotic lesions, in accordance with the most accepted theory of retrograde menstruation [122]. Women who are affected with endometriosis have increased shedding of the basalis portion in the menstrual flow [123], which might explain why not all women develop endometriosis despite all of them exhibiting retrograde flow during the menstrual cycle. Moreover, Hapangama et al. showed that women with endometriosis exhibited higher number of basalis-like cells, which are SSEA1(+) and SOX9(+), in the functionalis layer of the endometrium compared to healthy controls [124].

The peritoneal fluid of patients with endometriosis showed increased levels of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), and angiogenin as well as a decrease in the inhibitors of angiogenesis such as adiponectin and IP-10 compared to healthy population [125, 126]. It is also hypothesized that the endometrial stem cells of women who have endometriosis differ from those in the healthy population. Barragan et al. showed that endometrial stromal fibroblasts obtained from endometriosis patients’ eutopic MSCs were resistant to progesterone and exhibited pro-inflammatory phenotype [127]. It was also shown that the expression of stem cell markers, such as Notch-1 and Numb, was significantly upregulated in the endometrium of patients known to have endometriosis, and this was positively correlated with disease severity [128]. Several markers associated with self-renewal of embryonic stem cells have been identified in ectopic endometrial tissue such as undifferentiated embryonic cell transcription factor 1 (UTF1) [129], NANOG [130], OCT-4 [131], sex-determining region Y-box 2 (SOX2) [132], and importin 13 (IPO13) [133]. Endometriosis epithelial cells are also associated with aberrant expression of telomerase and increased telomere length [134], and peripheral blood telomere content was shown to be greater in patients known to have endometriosis compared to controls [135].

Endometrial stem cells were also isolated from biopsies of ectopic endometrium. Stem cells isolated from endometriosis tissue exhibited increased migration and proliferative capability during in vitro assays compared to eutopic endometrium stem cells, and transplanted MSCs isolated from ectopic endometrial tissue demonstrated higher levels of invasiveness and vasculogenic potential [136]. Intriguingly, cells from ectopic endometrial tissue can possibly migrate to the eutopic endometrium since they express higher levels of cytokeratin, Wnt, and proteins involved in epithelial-mesenchymal transition (EMT)[137].

It is also hypothesized that bone marrow stem cells can contribute to the pathogenesis of endometriosis, particularly in the rare cases of endometriosis in men and very distal endometriosis present in the brain and lung, which cannot be explained by retrograde menstrual flow [138–140]. Du and Taylor described the contribution of donor bone marrow-derived stem cells (BMDSCs) to experimentally-induced ectopic endometrial tissue in a mouse model [141]. They showed that ischemia and reperfusion injury promote the recruitment of bone marrow stem cells to the endometrium, and it was hypothesized that since endometriosis is an inflammatory condition, bone marrow-stem cells could also be recruited to ectopic endometriotic tissue [142]. The recruitment factors that induce the migration of BMDSCs remain unclear. The CXCL12/CXCR4 axis has been proposed to be implicated. CXCR4 is a chemokine receptor expressed on the surface of stem cells, and CXCL12 is its ligand. In cancer cells, their activation results in upregulation of metalloproteinases, increased angiogenesis, and endothelial cell recruitment [143]. In an in vitro study, physiological level of 17β-estradiol (E₂) was shown to increase the bone marrow stem cell expression of CXCL12 and CXCR4 while progesterone (P₄) increased their expression by human endometrial cells [144, 145]. Endometriotic epithelial cells also exhibited higher expression of both CXCR4 and CXCL12 compared to normal uterine endometrium [144, 146]. It was shown that 17β-estradiol could promote the chemotaxis and migration of BMDSCs though the enhanced secretion of chemokines such as SDF-1α [147]. In endometrial stromal cells from women with endometriosis, BMDSCs enhanced cell proliferation and activation of cyclin dependent kinase-1 (CDK1), a key regulator of cell cycle regulation and division [148]. It is still unclear whether bone marrow stem cells induce the formation of new endometriosis lesions or seed in already formed ectopic tissue. Figure 2 depicts proposed stem cell theory for the development of endometriosis.

Figure 2: Proposed stem cell concept for the development of leiomyomas.

Myometrial stem cells are essential for normal myometrial development and growth of the uterus during pregnancy. These cells divide to give rise to progenitor cells that subsequently differentiate to mature myometrial cells. In response to genetic mutations, epigenetic aberrations, and chromosomal abnormalities, these cells can undergo tumorigenesis, transforming to leiomyoma stem cells. These stem cells interact with mature myometrial cells through the Wnt/β-catenin pathway to promote proliferation and ECM formation, characteristic of leiomyoma development.

Conversion of Endometrial Stem Cells to Endometriosis Stem Cells

The involved pathways in the maintenance and progression of endometrial stem cells to endometriosis stem cells are less understood. Diao et al. showed that women with endometriosis had higher levels of the CCL19 and CCR7, inflammatory chemokine and its ligand, in the endometrium compared to healthy controls [149]. They also showed that CCL19/CCR7 increased endometrial stem cell proliferation and invasion through activating PI3K/Act signal pathways, involved in cell proliferation and survival, as well as increased phosphorylation of Bcl2, involved in regulating apoptosis, and matrix metalloproteinases (MMP2 and MMP9), involved in cell migration [149]. Activin A, which is involved in a wide spectrum of physiologic and pathogenic events, was also implicated in endometriosis-associated fibrosis. Activin A levels were higher in endometriotic peritoneal fluid, and its specific receptor, ALK4, was enhanced in ectopic endometrial MSCs compared to eutopic endometrial MSCs. Activin A induced myofibroblast differentiation of endometrial MSCs with increasing the expression of connective tissue growth factor (CTGF) [150]. This increased expression required the activation of Smad2/3 through phosphorylation, nuclear translocation, and binding to the CTGF promotor [150].

Molecular evidence from micro-RNA expression have also suggested their involvement in the development of endometriosis. A recent study showed miR-200b, miR-145, and let-7b deregulation in endometriosis; their expression has been implicated in modulating proliferation and differentiation of stem cells, and these changes can potentially increase self-renewal and migration [151]. miR-15a-5p, miR-34a-5p, and miR-199q-5p were also shown to be down-regulated in patients with endometriosis [152–154]. Abdel-Rasheed et al. found thirty-two miRNAs that are significantly dysregulated in endometriosis; these miRNAs are involved in cell cycle processes such as apoptosis, proliferation, differentiation, epithelium development, angiogenesis, and many other functions [155].

Clinical Implications and Future Research

Currently, laparoscopy is the gold standard for the diagnosis of endometriosis to visualize ectopic lesions and examine pathological tissue biopsies. This modality has its shortcomings, including cost, anesthesia exposure, and possible injury during the surgery. Having a better understanding of endometriosis can aid in developing sensitive diagnostic modalities for early diagnosis and intervention for such a debilitating disease. Multiple studies have looked into the value of peripheral blood markers and have identified promising indicators for early endometriosis detection [156–158].

Having a better understanding of the development of endometriosis can guide targeted therapy, aiming at inhibiting stemness, preventing retrograde flow of cells, or inhibiting the involvement of bone marrow stem cells. For example, in a recent study, notch pathway was inhibited by γ-secretase inhibitor (GSI), resulting in reduced cell viability, increased apoptosis, and stimulated transcriptional downregulation of stemness-associated factors, introducing the application of GSI as a possible approach for treatment [159]. Moreover, lovastatin was shown to increase the differentiation of endometrial MSCs toward osteogenic and adipogenic lineages and decreased expression of stemness markers through epigenetic reprograming [160]. Statins are readily available drugs with a good safety profile, allowing them to be a good treatment candidate.

When it comes to the involvement of bone marrow stem cells, Hufnagel et al. showed that bazedoxifene (BZA), which is a selective estrogen receptor modulator, decreased bone marrow stem cell seeding in ectopic endometrial sites while it increased their recruitment in eutopic endometrium in a murine model of endometriosis [161]. Moreover, small molecule receptor antagonists AMD3100 and CCX771, which target the CXCR4 and CXCR7, reduced BMDSCs recruitment into endometriosis implants and decreased pro-inflammatory cytokine production and vascularization [162].

STEM CELLS IN ADENOMYOSIS

Uterine adenomyosis is a benign condition in which endometrial glands and stroma invade the uterine musculature resulting in hypertrophic and hyperplastic myometrium, producing a diffusely enlarged uterus [163, 164]. This condition is described as focal when a circumscribed nodular collection is identified or diffuse when different groups of glands are distributed throughout the myometrium [165, 166]. Adenomyosis can be asymptomatic, but the most common symptom is heavy menstrual bleeding; symptoms can also include dysmenorrhea and chronic pelvic pain [167, 168].

Sex steroid aberrations, neuro-angiogenesis, inflammation, and altered cell proliferation have been implicated in the development of adenomyosis, but the precise pathogenesis is still unknown [169]. Several hypotheses have been proposed, one of which is the invagination of the endometrial basalis layer in response to steroid hormones, hyperperistalsis, and the activation of the tissue injury and repair mechanism [170]. An alternative hypothesis is the metaplasia of displaced embryonic pluripotent Mullerian remnants or the differentiation of adult stem cells [170]. The Mullerian ducts share common embryological structures during fetal life, maturing to form the female uterine tract and differentiating into stroma and endometrial glands [171]. Metaplastic alterations of the intra-myometrial embryonic pluripotent Mullerian remnants form de novo ectopic endometrial tissue inside the myometrial wall, resulting in the development of adenomyotic lesions [163]. García-Solares et al. proposed that progenitor stem cells can be deposited in the peritoneal cavity after retrograde menstruation and differentiate into stroma and endometrial glands. These may then progress into de novo intra-myometrial endometrial implants and form an endometrial colony which leads to focal uterine adenomyosis [170]. Endometrial stem cells may also be activated by tissue injury, causing stem cell niche disruption and allowing them to differentiate into progenitor cells which subsequently invade the myometrium, progressing to adenomyosis [172].

Role of Adenomyosis-Derived and Endometrial Mesenchymal Stem Cells

Adenomyosis derived stem cells are not well understood. Adenomyosis-derived mesenchymal stem cells have previously been isolated from adenomyotic tissue [173]. These stem cells differentiated into mesodermal lineages and expressed MSCs surface markers and had a similar morphology, phenotype, and potential of multilineage differentiation compared to endometrial MSCs [173]. They exhibited high COX-2 expression compared to endometrial mesenchymal stem cells, highlighting the potential role of` COX-2 in the pathogenesis of adenomyosis [173]. Lupicka et al demonstrated increased mRNA expression of pluripotency markers, such as OCT4, NANOG, and SOX2 in bovine adenomyotic lesions compared to normal uteri [174]. Moreover, Musashi-1, an adult stem cell marker, was remarkably higher in eutopic and ectopic endometria from patients with adenomyosis in contrast to normal endometrium [175]. Further studies are needed to identify the functions and mechanisms of Musashi-1 in the development of adenomyosis. Ibrahim et al. identified intraepithelial pale cells at the interface of the endometrial-myometrial junction that actively migrated into the stroma in adenomyosis [176]. This group hypothesized that these cells may migrate into the stroma of the basal endometrium and into the myometrium, possibly facilitated by the high concentration of matrix metalloproteinases [176–178].

Clinical Implications and Future Research

Numerous advanced medical approaches have been developed for adenomyosis; however, more satisfactory treatment options are needed. More research is required to identify the properties of endometrial or progenitor stem cells that ultimately result in the migration and invasion of endometrial tissue into the myometrium to develop adenomyosis. Understanding the function and mechanism of stem cells in the initiation and progression of adenomyosis may generate new, targeted treatment strategies. For example, treatment with a COX-2 inhibitor suppressed migration and invasion and induced apoptotic capabilities of adenomyosis mesenchymal stem cells [173].

REGENERATIVE MEDICINE

Regenerative Potential in Asherman Syndrome

Asherman syndrome (AS) is a complex gynecological disorder defined by the presence of endometrial destruction and intrauterine adhesions resulting in hypomenorrhea or amenorrhea, infertility, recurrent miscarriages and abnormal placentation [179, 180]. The incidence of this condition ranges in different countries and is directly influenced by the number of abortions performed, the incidence of genital tuberculosis, as well as the criteria used to detect the adhesions [181]. A meta-analysis reporting on women evaluated with hysteroscopy within 12 months following spontaneous abortion (of which 86% underwent a curettage) showed that the prevalence of AS was 19.1% [182]. Treatment of AS is only suggested if the patient has infertility issues or clinical symptoms. Multiple hysteroscopic techniques, hormone therapy and intrauterine devices insertion are the most common treatments available for the AS patients, but the treatment efficacy is not consistent and may vary from patient to patient.

At present, there is immense attention to use stem cells as therapeutic options for regenerative medicine, and AS has appeared as a promising candidate for stem cell-driven regenerative treatment. It was shown that AS stem cells transplantation had a proliferative impact on the endometrium, resulting in an increased endometrium thickness, improved tissue construction, enhanced angiogenesis, and prevented gestation as well as pregnancy loss [183].

Stem cells as a treatment for AS has been explored in a limited number of animal experiments and clinical trials. On an experimental AS mouse model, stem cell recruitment and pregnancy rate were investigated after BMDCs transplantation [184]. BMDSC transplanted group had improved the fertility rate, with 90% of mice conceiving after the transplantation compared to the control group [184]. These results illustrated the functional role of BMDC in the uterine renovation. In another investigation, a combination of MSCs treatment and estrogen was tremendously effective in enhancing regeneration of endometrium in an AS rat model [185]. This therapy stimulated endometrial proliferation and angiogenesis by increasing PCNA, Ki-67 and VEGF expressions and decreasing fibrosis [185].

This field of research is not only restricted to animal models as autologous stem cells were used in patients with AS. For example, a report was published on a patient with severe AS who failed to respond after the placement of intrauterine contraceptive device [186]. Adult autologous stem cells were isolated from patient’s own bone marrow, and endometrial angiogenic stem cells were separated from adult autologous stem cells. After the placement of the endometrial angiogenic stem cells in the endometrial cavity, the patient had increased endometrial thickness, improved vascularity, and was ultimately able to sustain a pregnancy [186]. Another pilot cohort study was conducted by using autologous CD133+ BMDSCs to treat endometrial atrophy and refractory AS [187]. In this study, stem cells were injected to the endometrial stem cell niche via intra-arterial catheterization. After stem cell implantation, all the patients had increased endometrial thickness and vascular density and improved endometrium and uterine cavity that were confirmed by hysteroscopic observations [187].

In another study, sub-endometrial autologous stem cells were implanted in patients with AS followed by the supplementing with oral estrogen therapy, and endometrial thickness were assessed after few months [188]. The autologous stem cell resulted in endometrial regeneration and restoration of menstruation in five out of six patients [188]. Menstrual blood-derived stromal cells have also been confirmed to be effective for the severe AS treatment. After the transvaginal transplantation of these stem cells, followed by hormonal stimulation, all the patients had remarkable endometria proliferation, and five of the seven patients gained more than 7mm endometrial thickness, which confirmed acceptability of embryo implantation [189]. Four patients went through frozen embryo transfer, after which two of them conceived, and one of them experienced spontaneous pregnancy after second transplantation [189].

Limitations and Future Research

Limitations are expected when using the stem cells for regenerative medicine. While stem cells enhanced the proliferation and renewal of normal or injured endometrium, they also have the ability to promote ectopic endometrial growth or endometriosis, as discussed above [141]. Stem cells and cancer cells have some common traits such as self-renewal capability, high proliferative and elasticity ability [190]. BMDCs may also result in endometriosis and cancer development [191].

Having said that, there is tremendous promise in stem cell therapy in regenerative medicine and in the restoration of injured tissues for which there are currently few therapeutic options. To increase the stem cells retention in the body, scientists have linked biocompatible and biodegradable materials such as hydrogel, scaffolds and nanostructure lipid carrier with stem cells or stem cell by-products. In a phase I clinical trial, collagen scaffold was used as an effective carrier for holding the umbilical cord-derived MSCs to prevent recurrent intrauterine adhesion after separation surgery [192]. Hormone replacement therapy was also used to stimulate a natural menstrual cycle. After the treatment, successful endometrial and angiogenic proliferation was evaluated, and ten out of the twenty-six patient became pregnant. The trial lacked a control group, making it difficult to identify whether the surgery, the stem cells loaded with collagen scaffold, the hormone treatment, or a combination made an alteration. Linking stem cells with biomaterials seems to be a very hopeful strategy to treat AS, but more research work is required. Moreover, secreted extracellular vesicles from umbilical cord-derived mesenchymal stem cells was used, with estrogen, to treat AS in an animal model [193]. Endometrial regeneration and decreased fibrotic appearance were observed after the treatment. More research is warranted to fill up the gaps in order for this research to be useful in fertility treatment.

CONCLUDING REMARKS

Our understanding of stem cells in the uterus and their role in the reproductive system has expanded considerably since their discovery. The developing knowledge on the stem cell niches and drivers of pathologic development in the different benign gynecologic conditions is opening doors for several therapeutic options. Moreover, the high regenerative capacity of these stem cells offers potential therapeutic promise for conditions such as Asherman syndrome and exciting prospects for other reproductive and non-reproductive pathologies, with some of these therapies being currently tested. However, many questions remain to be addressed in future research to make these advancements a therapeutic reality.

Figure 3: Proposed stem cell concept for the development of endometriosis.

Dysfunctional endometrial stem cells, due to multifactorial factors such as genetic predisposition and hormonal dysregulation, undergo retrograde menstruation reaching ectopic sites. Bone marrow stem cells, recruited by the inflammatory nature of this lesion, can also contribute to the endometriosis cell pool. These cells form ectopic endometrial lesions, acquiring genetic and epigenetic alterations to promote migration, proliferation, invasiveness and adhesion, ultimately resulting in inflammation, angiogenesis and increased growth and the subsequent clinical symptoms characteristic of endometriosis.

Table 1:

Cell marker expression of human uterine stem cell populations.

Cell type	Marker expression	References
Myometrial SP cells	CD49f, CD34, CD44, Stro-1, CD146, CD140, SUSD2	Ono et al. (2015), Mas et al. (2015), Patterson et al. (2020) [18, 20, 21]
Epithelial stem cells	SSEA-1 (CD15), LGR5	Valentijn et al. (2013), Gil-Sanchis C et al. (2013) [81, 82]
Mesenchymal stem cells	CD146, PDGF-Rβ, SUSD2, CD29, CD44, CD73, CD90, CD105, CD140b, CD166, HLA-ABC	Schwab and Gargett (2007), Masuda et al. (2012), Cheng et al. (2017) [84, 85, 194]
Endometrial SP cells	CD105, CD146, CD31, CD34, CD13, CD49f, EMA, CD73, CD90, CD9, CD10, VM, CD144, CD326, SUSD2	Tsuji et al. (2008), Carvello et al. (2010), Masuda et al. (2010), Miyazaki et al. (2012) [96, 98, 97, 195]
Menstrual blood stem cells	CD73, CD90, CD146, SSEA-4, CD44, CD105, OCT4, CD29, CD55, CD59, CD166, CD13, CD54, CD166, CD9, CD49f, C-KIT, CXCR4, MHC-1	Patel et al. (2008), Musina et al. (2008), Hida et al. (2008), Rossignoli et al. (2013), Sugawara et al. (2014) [100, 196, 104, 197, 198]

SUSD2: Sushi Domain Containing 2, SSEA: Stage-specific embryonic antigen, LGR5: Leucine-rich repeat-containing G-protein coupled receptor 5, PDGF-Rβ: platelet-derived growth factor receptor beta, HLA-ABC: major histocompatibility complex class I (MHC I), EMA: epithelial membrane antigen, VM: vimentin, Oct-4: octamer-binding transcription factor 4, CXCR4: C-X-C chemokine receptor type 4, MHC-1: The major histocompatibility complex class 1