The perivascular niche of endometrial mesenchymal stromal/stem-like cells : Reproductive and Developmental Medicine

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Special Issue: Updates on Research in Endometrium

The perivascular niche of endometrial mesenchymal stromal/stem-like cells

Chan, Rachel W. S.1,2,*; Li, Tian-Qi1; Zhang, Si-Si1; Fang, Yuan1; Xu, Jing-Wen1,2

Author Information
Reproductive and Developmental Medicine 6(4):p 208-214, December 2022. | DOI: 10.1097/RD9.0000000000000038
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Abstract

Introduction

The endometrium is the innermost mucosal layer in the uterus. The tissue is required for the implantation of a competent blastocyst. If implantation fails, the endometrium degenerates, sheds, and regenerates during the subsequent menstrual cycle. Thus, the endometrium is a dynamic tissue that undergoes ~400 cycles of proliferation, differentiation, and shedding during the reproductive years of a woman’s lifespan. During each menstrual cycle, the cellular components of the endometrium undergo changes under the regulation of sex steroids. The cellular behaviors of the human endometrium, which include epithelial cells (ciliated and unciliated), stromal cells, endothelium, immune cells, and smooth muscle cells, were profiled in a recent single-cell RNA sequencing study using endometrial tissues from different stages of the menstrual cycle[1]. The bilayer structure of the human endometrium, in which the upper functionalis is shed at menses and regenerates from the remaining basalis in the subsequent cycle, has motivated researchers to search for putative stem cells.

In the past 2 decades, great progress has been made in the field of endometrial stem/progenitor cells. In addition to the identification and characterization of various subtypes of stem/progenitor cells within the epithelial and stromal compartments, progress has been made in the development of clinical and therapeutic applications of endometrial stem/progenitor cells. In this review, we summarize the advancements made in the field of endometrial stem cells with a primary focus on the identity and location of perivascular endometrial mesenchymal stromal/stem cells (eMSCs) from human and mouse studies.

Origin of eMSCs

Many years ago, proliferation kinetic studies introduced the concept that endometrial regeneration is mediated by stem cells located in the basalis endometrium rather than the functionalis or myometrium[2–5]. In 2004, 2 different research groups provided the first direct evidence demonstrating the existence of clonogenic cells in hysterectomy samples[6] and the specific role of bone marrow-derived mesenchymal stromal/stem cells (BMSCs) in endometrial regeneration[7]. The bone marrow is considered an exogenous source of stem cells contributing to the endometrial microenvironment. Since then, emerging evidence has revealed the contribution of BMSCs to endometrial regeneration and remodeling[8–12]. A combination of endogenous (endometrial-derived) and exogenous (bone marrow-derived) BMSCs is likely to coexist in the endometrium and be responsible for cellular turnover.

Multi-dynamic properties of endometrial stromal cells

The initial approaches to identify MSCs in the human endometrium primarily used the stemness properties of somatic stem cells: extensive proliferative potential, self-renewal capacity, and differentiation. The first direct evidence was reported in 2004, when clonogenic stromal cells were identified, comprising 1.3% of stromal fibroblasts harvested from hysterectomy tissues[6]. Endometrial stromal clonogenic cells are present throughout the menstrual cycle and in the inactive endometrium[13]. In a serial replating experiment of the large colony forming units, the stromal cells could undergo four rounds of passage, and their population doubled more than 30 times, an indicator of their remarkable self-renewal capacity and proliferative potential[14]. They also display classical markers of cells of mesenchymal origin, as defined by the International Society for Cellular Therapies[15]. Stromal clonogenic cells can differentiate into mesodermal[14,16] and ectodermal[17] lineages under specific in vitro conditions.

Another common property of somatic stem cells is their quiescence. This infrequent division prevents exhaustion during tissue regeneration and repair. Using fluorescent nanoparticles to trace slow-cycling cells in a live heterogeneous population of endometrial stromal cells, slow-cycling stromal cells were identified in vitro[18]. The fluorescent persistent cells displayed high colony forming ability, underwent more rounds of self-renewal, and had greater enrichment of phenotypic eMSC markers compared with the non-labeled cells (see below). These findings indicated that the human endometrium contains a small population of MSCs.

Side population and expression of stem cell markers in human endometrium

Using the side population assay, several groups have identified a discrete population of endometrial stromal cells, known as SP cells, with the ability to efflux Hoechst 33,342 DNA-binding dye. SP cells can be isolated from fresh human endometrium[19–21] and short-term cultured endometrial cells[22]. The SP marker ABCG2 is evenly distributed across the functionalis and basalis layers of the endometrium and colocalizes with CD31+ endothelial cells[20]. The stromal SP cells have typical characteristics of MSCs; they are decidualized when cultured in the presence of estrogen and progesterone, and quiescent in vivo. When transplanted underneath the kidney capsule of immunocompromised mice, SP cells contributed mainly toward the vascular and perivascular lineages of the re-constituted tissue[23,24]. These features of the stromal SP cells suggest that the vascular and perivascular cells are enriched with this cell population.

Several studies have used the expression of well-established stem cell markers to determine the distribution of stem/progenitor cells in endometrial tissue[25–28]. However, most of these studies did not examine endometrial cells from the basalis or performed functional validation of the identified cells, making the results less informative.

Surface markers for human endometrial mesenchymal stromal/stem-like cells

CD140b+CD146+ eMSCs

The first set of markers proposed to identify eMSCs included co-expression of the perivascular marker CD146 and platelet-derived growth factor receptor β (PDGFR-β/CD140b)[29]. Co-expressing cells display higher clonogenic activity and multipotency. CD140b+CD146+ cells express typical mesenchymal surface markers such as CD29, CD44, CD73, CD90, and CD105, and are negative for hematopoietic (CD34, CD45) and endothelial (CD31) markers. Pericytes surrounding endothelial cells in capillaries and microvessels of all human organs co-express CD140b and CD146[30]. Immunodetection confirms the presence of these cells in the capillaries and microvessels in the basalis and functional layers of the human endometrium (Fig. 1)[31]. The proportion of CD140b+CD146+ eMSCs remained the same throughout the menstrual cycle. However, the cells obtained from the menstruation phase exhibited better self-renewal activity than those from the secretory phase, indicating the importance of the microenvironment in stem cell activities[32]. When cultured in DMEM/serum-free medium supplemented with fibroblast growth factor and epidermal growth factor on fibronectin-coated culture plates, these cells adhered faster and displayed better proliferation[33]. Further analysis revealed that hypoxic conditions can enhance the proliferation and long-term viability of eMSCs compared with normoxic conditions. Hypoxia occurs during breakdown and repair of the endometrium during menstruation[34]. Therefore, co-culture with ssmenstrual-phase endometrial cells enhances the proliferation and self-renewal activities of CD140b+CD146+ eMSCs[35].

F1
Fig. 1.:
Schematic diagram of endometrial mesenchymal stromal/stem cells in human endometrium. EMSCs that express surface markers such as SUSD2, CD146, and CD140b are located around blood vessels throughout the human endometrial tissue. Pericytes surrounding the endothelial cells in microvessels are located in the functionalis and the basalis, while the CD34-expressing adventitial cells reside in the outermost layer of large vessels, mainly in the basalis region.

Gene profiling of CD140b+CD146+ eMSCs showed that these cells display pathways of self-renewal, lineage specification, and capacity to react to environmental changes during endometrial regeneration and differentiation[36]. Extensive cultivation leads to the downregulation of eMSC-related genes and upregulation of fibroblast-associated genes, indicating a gradual loss of functionality and spontaneous differentiation[37]. The discrepancies in transcriptome from single-cell sequencing data between cultured CD140b+CD146+ eMSCs and primary endometrial perivascular cells reveal the importance of the in vivo microenvironment in the gene expression of eMSCs[38].

SUSD2+ eMSCs

Sushi domain containing-2+ (SUSD2+) was the next endometrial mesenchymal stromal/stem cell marker. Using this single perivascular marker, SUSD2+ eMSCs displayed better clonogenic and self-renewal activity than SUSD2- and unselected cells[39]. These cells are multipotent and can induce differentiation into osteocytes, adipocytes, myocytes, chondrocytes, and endothelial cells. When transplanted under the kidney capsule of mice, stromal-like connective tissues were observed 10 weeks post-transplantation. Genomic profiling revealed a distinct gene signature between SUSD2+ eMSCs and SUSD2- stromal fibroblasts following decidual differentiation[40]. The effect of decidualization on the stem cell potential of SUSD2+ eMSCs has been reported[41]. The biophysical and functional properties of SUSD2+ eMSCs are not only distinct from those of stromal cells but also critically dependent on VAP-1 expression[42]. The SUSD2+ eMSCs co-cultured with myometrial smooth muscle cells support the early development of mouse blastocysts[43]. The co-cultured eMSCs could potentially be used to study implantation. The potential cellular therapy and tissue engineering applications of the SUSD2+ population in women with pelvic organ prolapse have been evaluated[44–47]. A lack of endometrial SUSD2+ eMSCs is associated with recurrent pregnancy loss, indicating that stem cell deficiency contributes to consecutive miscarriages[48,49]. The therapeutic potential of SUSD2+ eMSCs has been examined using a T-cell-mediated skin inflammation mouse model. Lymphocyte proliferation is an important part of the immune response, and the ability of MSCs to suppress lymphocyte proliferation is a measure of their immunosuppressive capacity. Yang et al.[50] demonstrated that SUSD2+ eMSCs suppressed murine T lymphocyte proliferation.

The MSC markers CD146 and SUSD2 have been proposed to serve as superior markers for monitoring MSC stemness because they are more specific for perivascular cells and not stromal fibroblasts[33]. Thus, both CD140b+CD146+ and SUSD2+ perivascular eMSCs are likely to be responsible for the regeneration and differentiation of the stromal vascular components of the endometrium. They are located around large and small blood vessels in close proximity to endothelial cells and have pericyte identity (Fig. 1). Other perivascular MSC marker-expressing cells are localized to the adventitia of the blood vessels[51]. Recently, CD34+CD146-CD45-CD56-CD144- cells were localized to the outermost layer of endometrial large vessels, while CD146+ pericytes were localized to the surrounding inner endothelial cells of microvessels[52]. Although the number of adventitial cells in the endometrium was ~40% higher than that of pericytes, the CD34+ population failed to regenerate the endometrium in functional studies. Further investigation is required to understand the hierarchical association between pericytes and adventitial cells.

Endometrial mesenchymal stromal/stem cells in mouse endometrium

Because there is no consensus on mouse endometrial stem cell markers, researchers have utilized the label retention method to identify, characterize, and localize these stem cell populations. The label-retaining cell (LRC) approach is a well-established method for identifying putative stem/progenitor cells based on their quiescent nature. Using the DNA analog bromodeoxyuridine (BrdU), a small population of stromal LRCs was localized around blood vessels, adjacent to the luminal epithelium and near the endometrial-myometrial junction[53,54]. These stromal LRCs continue to be present during gestation, proliferate at postpartum, and return to a quiescence state after postpartum repair[55]. They express a panel of surface markers, such as c-kit, Oct4, αSMA, CD44, CD90, CD140b, and CD146, but are negative for SCA-1 or CD45. Approximately 16% of stromal LRCs express ERα and are recruited into the cell cycle for endometrial regeneration[56]. Using the GFP-H2B system, it has been demonstrated that stromal LRCs contribute to epithelial[57] and myometrial repair during uterine involution[58].

In a menstruation mouse model, stromal LRCs were associated with endometrial vasculature, and a distinctly lower proportion was observed during endometrial repair than before decidualization[59]. In a separate study, the SM22α+-derived CD34+KLF+ stem/progenitor cells located in the stroma of the menstruation mouse model proliferated upon estrogen stimulation and migrated to the injured epithelial region[60]. Overall, stromal LRCs are heterogeneous. Recently, a comprehensive study using scRNA-seq revealed an equivalent population of perivascular CD140b+CD146+ cells in the mouse endometrium and demonstrated the exclusive expression of NG2[61]. Finding these markers provides hope for better understanding their role in endometrial repair and regeneration.

Bone marrow MSCs contribute to the endometrial stem cell niche

In addition to the existence of resident stem cells in the endometrial stem cell niche, BMSCs serve as an exogenous source of stem cells. Human and mouse BMSCs can differentiate into endometrial cells such as epithelial, stromal, and endothelial cells[9,62–64]. Furthermore, the BMSCs can circulate to the decidual stroma and contribute to the formation and function of the decidua during pregnancy[65]. On the contrary, a study using transplantation of BMSCs carrying an mTERt-green fluorescent protein into irradiated recipient mice to produce chimeric mouse endometrial tissue showed that BMSCs did not transdifferentiate and contributed to endometrial repair[66]. Therefore, the extent to which BMSCs and/or resident endogenous eMSCs contribute toward endometrial regeneration and pregnancy remains largely debatable. Elucidating the identity of MSCs that contribute to the regeneration process will require further in-depth research.

Signaling pathways regulating eMSCs

Considering the cyclical phenomena of the human endometrium, a well-orchestrated network of signaling pathways is required to regulate menstrual cycle events. In the past few years, researchers have begun to gain knowledge on the interplay between endometrial stem/progenitor cells and their niche. Understanding the intrinsic signaling pathways within the uterine microenvironment enables the generation of homogeneous potent stem cells for cell-based therapies.

Aging endometria display distinct age-associated morphological and physiological changes that are associated with reduced reproductive potential. A decline in Hedgehog signaling has been reported to be related to the aging and senescence of human eMSCs[67]. The same research group extended their investigation and described the role of a Hedgehog pathway ligand in promoting the regenerative properties of human eMSCs[68]. Although both studies used eMSC cultures from unsorted human endometrial stromal cells, they provide insight into the role of Hh signaling components in the function of endometrial biology.

Several studies have assessed the influence of the microenvironment on eMSC fate. Gurung et al. investigated the role of the transforming growth factor-β (TGF-β) signaling pathway in endometrial SUSD2+ eMSCs[69,70]. Their data suggested that A83-01, a TGF-β receptor inhibitor, could promote culture expansion of the SUSD2+ population and maintain its stemness. Furthermore, the A83-01-treated SUSD2+ eMSCs exhibited a gene expression profile indicating enhanced anti-inflammatory, anti-fibrotic, and immunoregulatory properties when compared with the untreated cells[71]. More recently, A83-01 treatment was shown to promote the expansion of SUSD2+ eMSCs in vitro by blocking their differentiation and senescence[72]. The treatment upregulated RARB expression and other genes implicated in retinoic acid signaling. These findings suggest that induction of certain retinoic acid target genes may provide new opportunities for clinical translation.

Myometrial cells modulate CD140b+CD146+ eMSC self-renewal via Wnt5a/β-catenin signaling[73]; Wnt5a interacts with frizzled receptor 5 and low-density lipoprotein receptor-related protein-5 to regulate proliferation and self-renewal of CD140b+CD146+ eMSCs[74]. During endometrial repair, endometrial epithelial or stromal cells promote the proliferation and self-renewal of CD140b+CD146+ eMSCs upon activation of the Wnt/β-catenin signaling pathway[35]. Consistently, there is transient expression of active β-catenin in mouse endometrial stem cells (LRCs) during postpartum repair[55]. These findings highlight the importance of Wnt/β-catenin signaling in the modulation of stem cell activity in the endometrium. Further studies on regulatory molecules can aid in the development of an efficient protocol for the culture expansion of eMSCs for cell-based therapy.

Single-cell sequencing to understand the endometrial microenvironment

The development of scRNA-seq, which combines single-cell isolation techniques with RNA sequencing, creates an opportunity to study the transcriptomes of individual cells, enabling clear distinctions between sub-populations and thorough assessment of gene transcripts in an unbiased manner. Human endometrial biopsies submitted for scRNA-seq revealed that CD140b-, CD146-, and SUSD2-expressing cells are likely smooth muscle cells[1]. More recently, single-cell sequencing and Visium spatial transcriptomics of human endometrial biopsies and full-thickness endometrium have provided new information on cell signatures in the basal layer of the endometrium[75]. A novel population of fibroblasts restricted to the basal layer of the endometrium was identified in proliferative and secretory phase samples. Whether this population of fibroblasts is associated with putative stromal/stem cells is of great interest. In a separate study, a systematic map of endometrial stromal cells from the proliferative phase showed that the markers used to identify eMSCs were expressed by all cells in the perivascular environment[76]. Their bioinformatic data demonstrated the complexity of endometrial stromal cells with multiple stromal populations, and the expression of one or two genes might not be sufficient to specify a specific cell population. Comparison of human endometrial cell composition in the proliferative phase at single-cell resolution between normal and thin endometrium revealed cellular senescence in the stroma and epithelium, accompanied by collagen deposition around blood vessels in the thin endometrium[77]. Undoubtedly, integrative maps of cellular profiles of the human endometrium serve as an essential reference for endometrial physiology and disorders. However, the challenges and limitations of this technology should be considered because a lack of information other than transcript levels can lead to inaccurate analysis. In the future, more efforts are needed to include the simultaneous profiling of multiple omics within a single cell, such as scRNA-seq coupled with DNA sequencing or proteomic analysis.

Extracellular matrix of the endometrial stem cells niche

As a key component of the stem cell niche, the extracellular matrix (ECM) is important because it provides structural support. The matrix also actively and constantly interacts with resident cells and participates in determining cell fate. The ECM plays a role in cell adhesion and signals cells through adhesion receptors[78]. The mechanical properties of the ECM, such as stiffness and deformability, also affect the cellular behavior[79]. In the past years, mounting evidence has demonstrated the importance of ECM in the differentiation, proliferation, survival, polarity, and migration of stem cells[80]. As an outstanding example, Magalhaes et al. transplanted a polyglycolic acid (PGA)/PLGA scaffold seeded with autologous endometrial cells to replace the excised portion of the rabbit uterus. At 6 months post-implantation, the transplanted uterus developed a native tissue-like structure and supported the pregnancy. The animals transplanted with the scaffold without the cells did not show normal uterine development. These findings provided evidence that scaffolds and cell sources are essential for tissue growth.

As the ECM varies in composition across different tissues, investigations into tissue-specific matrix composition and function have been conducted. It has been argued that a tissue-specific matrix can lead to more efficient regeneration than a non-homologous matrix[81,82]. The cyclical change of the human endometrium naturally leads to a constant change in ECM components throughout the menstrual cycle. Ex vivo investigation into the stiffness (elastic modulus) of the human endometrium and decidua basalis showed significant differences, suggesting a reciprocal relationship between ECM remodeling and physiological events[83]. However, the exact temporal and spatial profiles of the ECM in endometrial tissue remain unknown.

The first study using a decellularized matrix from a rat uterus was published in 2014[84]. The decellularized scaffold can act as a supportive material to regenerate functional uterine tissue in vitro and in vivo. In a separate study, primary uterine cells and BMSCs were used to reconstruct the decellularized rat uterine tissue and supported pregnancy[85]. Uterus decellularization protocols for larger mammals have been successfully optimized. Campo et al. decellularized porcine uterine scaffolds and re-cellularized them using a mixture of human endometrial stromal and epithelial SP cells[86]. Their finding demonstrated preservation of a functional ECM and in vitro re-organization of the seeded cells onto the scaffold in the presence of vimentin+ and cytokeratin+ cells after 2 weeks. Tiemann et al. compared different decellularization protocols of uteri to support re-cellularization of sheep pluripotent stem cells[87].

In 2017, Olalekan et al. were the first to use decellularized human endometrial tissue as a 3D scaffold for culturing human endometrial cells[88]. Their study argues for the superiority of a decellularized matrix over other 3D models utilizing a mixture of proteins and that the decellularized scaffolds closely mimic normal tissue physiology. When re-cellularizing native scaffolds with primary cells, the cells proliferated and remained viable for an extended period. With hormonal supplementation mimicking the human menstrual cycle, the seeded endometrial cells expressed both estrogen and progesterone receptors and responded actively to hormonal treatment[88].

Decellularized tissue can also be used to establish the temporal and spatial profiles of endometrial ECM. As various 3D human endometrium bioengineering models utilize either biomaterial or decellularized tissue, the optimization of scaffold biocompatibility and biomechanical properties requires further investigation. The potential clinical regeneration of the human endometrium with stem cell therapy also requires a thorough investigation of the stem cell niche that supports and regulates the biological activities of the cells. Investigation into the ECM components of the endometrium can be carried out using decellularized human endometrial tissue throughout the menstrual cycle. Using mass spectrometry analysis, researchers can break down the decellularized tissue and acquire information on the composition of each sample to establish changes in the chemical composition of the endometrial ECM. With the invention of more advanced tools, it is now possible to use matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) to visualize the spatial location of biomolecules in tissues[89]. The specific ECM protein localization patterns across different stages of the menstrual cycle can thus be visualized and analyzed. The ECM contains a complex combination of biochemical cues, in which it is difficult to determine which specific components or their combination cause specific cell behaviors. To achieve mechanistic studies, a combination of thorough quantitative analysis of the ECM components along with high-throughput cell-biomaterial interaction screening is needed.

Conclusion

Here, we provide an overview of the endometrial stem cell niche, focusing on endometrial mesenchymal stromal/stem cells. The cell source responsible for endometrial regeneration has been well defined, and the way stem cell function relies on well-timed cues from the microenvironment is slowly being explored. New advances in technology have changed the way research is being conducted and introduced possibilities for exploration into the complexities of eMSC functions and regulation by the niche. Although MSCs from a variety of sources are now being used for cell therapy, some limitations exist. These include a limited lifespan in culture, low viability of the transplanted cells, and lack of integration in the engrafted tissue[90]. Since eMSCs originate from tissues with high regenerative capacity, they are excellent candidates for therapeutic use. The more we understand the complex milieu that supports repair and regeneration, the faster eMSCs can be used in clinical applications. The potential use of perivascular eMSCs in regenerative medicine for the treatment of women with endometrial proliferation disorders is promising. Undoubtedly, advances in basic science go hand-in-hand with the progression of clinical use in treating women with inadequate endometrium, such as patients with intrauterine adhesions and Asherman’s syndrome.

Acknowledgments

We sincerely acknowledge Professor William Yeung for the critical discussions and proof reading of the manuscript.

Author contributions

R.W.S.C. contributed to the review concept, writing, and editing of the manuscript. T.Q.L. contributed to the work related to signaling pathways. S.S.Z. contributed to interpretation of the studies related on stem cells in the mouse endometrium. Y.F. contributed to the work related to single-cell sequencing. J.W.X. contributed to interpretation of the studies on extracellular matrix.

Funding(s)

The Hong Kong University Shenzhen Hospital Scientific Research Training Plan (HKUSZH20192003) and the National Natural Science Foundation of China/Research Grants Council Joint Research Scheme (N_HKU 732/20).

Conflicts of interest

All authors declare no conflict of interest.

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Keywords:

Endometrium; Regeneration; Stem/progenitor cells; Stem cell niche

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