MenSCs possess a typical MSC phenotypic profile (surface marker expression) compared to other MSCs, although they also express unique, pluripotency-related surface markers. The phenotypic analysis of in vitro expanded MenSCs revealed a positive expression for the surface markers CD44, CD73, CD90, and CD105 and negative for CD14, CD34, CD45, CD80, and HLA-DR, while endometrial MSCs have positive expression for CD73, CD90, CD105, CD13, CD29, CD44 markers and the absence of expression of the hematopoietic cell surface antigens CD19, CD34, CD45, CD117, CD130 and HLA-DR (class II) (Zemelko et al., 2012). Moreover, MenSCs possess pluripotency markers, such as Oct-4, SOX2, NANOG, and SALL-4, which make them a unique, MSC type, as compared to other sources MSCs (Borlongan et al., 2010). However, the expression of some pluripotency-associated markers in MenSCs does not equate to the full functional capacity of embryonic stem cells or induced pluripotent stem cells (iPSCs). To date, no definitive evidence has demonstrated the ability of MenSCs to differentiate into all three germ layers in vivo, which is a critical hallmark of true pluripotency. Thus, more comprehensive studies, including comparative transcriptomic and functional analyses are needed to validate MenSCs pluripotency, while currently MenSCs remain classified as multipotent.

A MenSC surface marker panel, including positive and negative markers (expressed and non-expressed) as well as differentiation capabilities, is provided in Table 1 and summarized in Figure 1, comparing them to embryonic stem cells, BMMSCs, umbilical cord (UC)/Wharton’s jelly, adipose tissue (ATMSCs), amniotic fluid and placental MSCs.

Beside phenotypical differences with other types of stem cells, MenSCs also differ in their differentiation capabilities. It is known that MenSCs differentiate into a wide range of cell types, and are even able to differentiate into cardiomyocytes with the functions of spontaneously beating cells after induction, resulting in the decreased myocardial infarction area in a rat model (Hida et al., 2008; Ikegami et al., 2010). Furthermore, it has been shown that MenSCs are capable of differentiation into neural, epidermal-like cells (Azedi et al., 2017; Faramarzi et al., 2016; Chen et al., 2015; Toyoda et al., 2007), functional hepatocytes (Mou et al., 2013) and even oocyte-like cells (Asgari et al., 2017) which suggest a superior spectrum of their differentiation potential compared to other tissue MSCs.

MenSCs secrete large amounts of paracrine factors, including growth factors responsible for endometrium regeneration, which may be a potential co-stimulant for other tissue regeneration purposes (Chen et al., 2019; Liu et al., 2018). MenSCs also secrete angiogenic factors VEGF, HGF, ANG and MMP-1 and different cytokines (IL-6, IL-8 and IFN-gamma), and most importantly, MenSCs were shown to be safe to transplant due to their low tumorigenicity (Liu et al., 2018). The secretome of MenSCs also includes EVs, containing proteins or miRNAs (more information in section 2). Different studies reported secretion of various growth factors by MenSCs. Table 2 summarizes all current findings on MenSCs secretome, including protein family, functions and comparison to other types of MSCs.

Factors secreted by MenSCs already showed positive immunomodulatory, cardioprotective, angiogenic and regenerative effects. Paracrine effects of MenSCs were analysed in numerous studies and their effects were proposed being more superior to BMMSCs (Alcayaga-Miranda et al., 2015). For instance, MenSC paracrine factors possessed promising results in rat model of myocardial infarction by reducing apoptosis of cells and stimulating endogenous regeneration, while transplantation of MenSCs achieved significantly better cardiac performance than BMMSCs or ATMSCs (Jiang et al., 2013; Wang et al., 2017). Important to note, BMMSCs were shown to secrete higher concentrations of IL-1β (Jiang and Wang, 2012; Manshori et al., 2022). Also, it was revealed that MenSC secrete higher amounts of EGF, FGF and HIF-1α, as compared to BMMCS, while no differences were observed in VEGF or angiopoietin secretion, which were higher in the MenSC secretome compared to UC and dental pulp MSCs. Moreover, MenSC secrete higher levels of HGF than dental pulp MSCs and higher levels of GM-CSF compared to UCMSCs.

Noteworthy, MenSC secretome can be modulated by different environmental conditions. For instance, under hypoxic conditions MenSCs secreted significantly higher levels of VEGF, while EGF and TGF-β secretion was not affected (Jiang and Wang, 2012; Alcayaga-Miranda et al., 2015). Hypoxia can also enhance the release of EVs, as previously shown in UCMSCs (Zhang et al., 2012). Moreover, it was demonstrated that endometrial MSC MiRNAs: miR-148a-3p, hsa-miR-378a-3p (related to angiogenesis, wound healing), hsa-miR-424-5p (associated with angiogenesis), hsa-miR-23a-3p, and hsa-miR-let-7a-5p (related to immune modulation) were the most widely expressed in acute hypoxic conditions (0.1%-1%), while hsa-miR-34a-5p (reduces expression of VEGF), hsa-miR-532-5p, hsa-miR-221-3p, hsa-miR-93-5p (regulating cell cycle and proliferation) were detected only under normoxic conditions (de Pedro et al., 2023). These results are directly associated with MenSC physiological behavior in vivo and differences obtained in vitro.

In order to stimulate MenSC immunomodulator or regenerative properties, MenSCs can be additionally stimulated by external factors using different cultivation conditions. MenSCs increase IDO1 secretion and EVs release under treatment with IFN-γ and TNF-α (de Pedro et al., 2021). bFGF and 5-aza increased the levels of VEGF, SDF-1, HIF-1α, IL-1β, and ANG-1 secretion from MenSCs (Manshori et al., 2022). Moreover, MenSCs may help protect insulin-producing pancreatic β-cells from autoimmune attack in type 1 diabetic mice. By modulating immune responses, these cells could potentially slow disease progression and preserve insulin production (Wu et al., 2014).

Mainly, MenSC-EVs carry proteins related to processes such as cellular transport, including vesicle-mediated transport or cell adhesion and migration. An additional group of proteins is related to cellular components, including extracellular organelles, membrane components and parts of the cytosol. MenSC-EVs are also enriched with different binding proteins. Upon evaluation of the functional properties of the most abundant proteins in MenSC-EVs, it was determined that the majority are associated with immune system processes and extracellular matrix (ECM) organization (de Pedro et al., 2023). Additionally, MenSC-EVs contain various bioactive molecules, including cytokines. A comparative analysis of MenSCs and MenSC-EVs revealed that the latter contain higher concentrations of IL-6 and IL-8, intercellular cell adhesion molecule-1 (ICAM-1), angiopoietin-2, Axl, angiogenin, insulin-like growth factor-binding protein 6 (IGFBP-6), and osteoprotegerin (Chen et al., 2017). Moreover, it was reported that MenSC-EV are enriched with E3 ubiquitin ligase (UBR4), which inhibited fibrosis of rat endometrial stromal cells by affecting YAP activity (Qi et al., 2023).

The culturing conditions of MenSCs significantly alter the cargo and the EV-associated proteome. Proinflammatory conditions were found to downregulate proteins related to wound healing, adhesion and migration processes and upregulate proteins involved in angiogenesis and inflammatory responses. As an example, MenSCs cultured under physioxic conditions (1%–2% O₂) secreted EVs enriched with proteins related to cell adhesion and intracellular transport. Acute hypoxia (<1% O₂) had different effects on EV cargo–it upregulated proteins associated with cell adhesion, cell migration and angiogenesis pathways (de Pedro et al., 2023).

At present, the available information regarding MenSC-EV cargo is relatively limited in relation to MSC-derived EVs from alternative sources, such as ATMSCs, BMMSCs, and UCMSCs. BMMSC-EVs contain proteins involved in ion and other protein transport. In addition, proteins associated with cell cycle regulation, transcription and translation regulation, cell adhesion and lipid metabolism, apoptosis and inflammation were identified in BMMSCs. Upon classification of proteins according to cellular components, the majority of proteins were found to be associated with the cell membrane, nucleus, cytoplasm, mitochondria and endoplasmic reticulum (McBride et al., 2021).

ATMSC-EVs encompass a multitude of proteins which play crucial roles in various biological processes. These processes include cellular migration, modulation of immune responses, proliferation of cells, formation of new blood vessels, metabolism of osteocytes, and regeneration of nerve tissue (Alonso-Alonso et al., 2022).

Human UCMSC exosomes are enriched with proteins related to different mechanisms and signaling pathways. The majority of proteins detected in UCMSC exosomes play roles in modulating various biological processes, including complement response, HIF-1, MAPK signaling, metabolic pathways, NF-κB pathway, and microbial infection. Additionally, proteins related to PI3K-AKT, cholesterol metabolism, IgA production, VEGF, and B-cell receptor signaling pathways were detected (Bi et al., 2022). Table 3 presents a more detailed categorization of cargo proteins in different MSC-derived EVs and their respective functions.

MenSC-derived EVs contain a broad range of microRNAs (miRNAs). The identified mi RNAs in MenSC-EVs included let-7a-5p, miR-143-3p, miR-21-5p, let-7b-5p, let-7f-5p, miR-16-5p, miR-199a-3p, miR-199b-3p, miR-126-3p, let-7i-5p, miR-26a-5p, which are involved in the regulation of cell cycle, proliferation, differentiation, apoptosis and angiogenesis (Marinaro et al., 2019). Let-7 and miR-21 play crucial roles in controlling mitochondrial-DNA damage, promoting cell survival and proposed to provide superior cardioprotection and alleviate pulmonary fibrosis (Wang et al., 2017; Sun et al., 2019). Cargo of MenSC-EVs also contain information related to certain diseases and their predispositions to them. The elevated levels of miR-4443 found in MenSC-EVs were discovered to play a role in the progression of endometriosis. This specific miRNA was found to suppress ACSS2 expression and as a result activate the PI3K/AKT signaling pathway. This activation resulted in enhanced migration and proliferation of endometrial stem cells (Ji et al., 2024).

An analysis of EVs from MenSCs, BMMSCs, and ATMSCs revealed that MenSC-EVs exhibited the highest levels of miR-21 among the three sources of EVs. Additionally, the paracrine effect of MenSCs on rat myocardial infarction was compared to that of BMMSCs and ATMSCs. MenSCs were found to enhance cardioprotection through the transfer of miR-21 via EVs. miR-21 from menstrual blood EVs downregulated phosphatase and tensin homolog (PTEN), enhancing Akt survival kinase activity, resulting in reduced apoptosis in cardiomyocytes and improved angiogenesis in endothelial cells (Wang et al., 2017). This finding demonstrates the superior cardioprotective effect of MenSC-EV cargo compared to BMMSC or ATMSC EVs. Additionally, EVs derived from MenSCs attenuate severe pulmonary inflammation and damage through the transmission of miRNA-671. This miRNA is known to target the kinase AAK1 for post-transcriptional degradation. AAK1 positively regulates the NF-κB signaling pathway (Lian et al., 2023).

MiRNAs highly expressed in BMMSC-EVs were found to be associated with cellular proliferation, death, metabolism and immune regulation (miRs-21, miR-22, miR-26a, miR-10b, miR-99b, miR-125b, and miR-148a) (Vaka et al., 2023; Baglio et al., 2015). In comparison with BMMSC-EVs, UCMSC-EVs are enriched with miRNA related to regenerative processes, aging and cell proliferation (Vaka et al., 2023). Several studies reported, that the most abundant miRNA in UCMSC-EVs are miR-16, miR-21, miR-23, miR-34, miR-146a and miR-222, which are associated with cell proliferation and immune regulation (Jothimani et al., 2022). MenSC-EV and other MSC EV miRNAs are presented in Table 4.

In a rat model of POI induced by chemotherapeutic agents, MenSC-EVs restored ovarian function by increasing ovarian weight, follicle numbers at various developmental stages, and serum estrogen levels. The therapeutic effects were associated with activation of the PI3K/AKT signaling pathway, inhibition of apoptosis, and overall enhancement of ovarian microenvironment stability (Robalo Cordeiro et al., 2024). MenSC-EVs contribute to endometrial repair by enhancing cell proliferation and stimulating VEGF production, which promotes angiogenesis (Marinaro et al., 2018). A study by Zhang et al. demonstrated that MenSC-EVs had the effect of promoting ovarian cell proliferation, inhibiting apoptosis and regulating the ovarian extracellular matrix, while increasing the expression of follicle markers DAZL and FOXL2 in rat ovaries. Also, MenSC exosome injections restored the female rat estrous cycle and increased fertility, as treated subjects exhibited increased endometrial thickness, improved glandular formation and reduced fibrosis. Notably, repeated EV administration enhanced endometrial receptivity and improved embryo implantation rates, suggesting potential clinical applications in infertility treatment (Zhang et al., 2021b).

MenSC-EVs have demonstrated efficacy in wound healing by promoting fibroblast proliferation, collagen synthesis, and reducing oxidative stress (Zhang et al., 2023). MiRNA cargo, including miR-21 and miR-29, facilitates keratinocyte migration and differentiation, accelerating skin repair. In models of skin injury, MenSC-EVs have been shown to accelerate wound by promoting the growth of new skin cells (keratinocytes and fibroblasts), increasing collagen production. MenSC-EV treatment promoted re-epithelialization, increased angiogenesis, and modulated inflammation, leading to improved healing outcomes in a diabetic mouse model (Dalirfardouei et al., 2019). These effects suggest their potential use in treating chronic wounds or burns.

MenSC-EVs have also shown ability to promote axonal regeneration and functional recovery following neural injury, further underscoring their broad therapeutic utility (Lopez-Verrilli et al., 2016). Moreover, MenSC-EVs can alleviate fulminant hepatic failure. In experimental models, these EVs reduced liver inflammation and promoted hepatocyte proliferation, leading to improved liver function (Chen et al., 2017). Additionally, MenSC-EVs have been shown to promote angiogenesis and reduce scarring in heart tissue, ultimately improving heart function and reducing long-term damage (Wang et al., 2017). And as mentioned before, MenSC-EVs may help slow down fibrosis by reducing the activity of fibroblasts (cells that contribute to scarring) and lowering levels of fibrotic markers, leading to improved lung function as well (Chen et al., 2021).

In cancer therapy, it was shown that MenSC-EVs block tumor associated angiogenesis and could be used as a tool for cancer treatment. MenSC-EVs reduced the secretion of VEGF and NF-κB activity in human prostate PC3 tumor cells (Alcayaga-Miranda et al., 2015). Other studies additionally emphasize the pro-angiogenic effect of MenSC-EVs (Chang et al., 2021a; Wang et al., 2017). Therefore, the precise mechanism of this targeted action of MenSC-EVs remains unclear.

MenSC-EVs also exhibit immunomodulatory properties by regulating T-cell proliferation, macrophage polarization, and inflammatory cytokine production (Song et al., 2023; Qi et al., 2023). This suggests potential therapeutic applications in autoimmune diseases, inflammatory disorders, and systemic tissue repair.

Despite the promising potential of MenSC-EVs in regenerative medicine and disease diagnostics, several limitations of the current source and EVs should be acknowledged. First of all, there is a lack of long-term safety and efficacy data in all of the published studies, as most of them focus on short-term outcomes. Long-term effect of MenSC-EVs on tissue homeostasis or potential off-target response remain largely unexplored, where rigous in vivo studies are essential to ensure translational relevance and clinical safety. Moreover, there is a significant variability in EV isolation and characterization protocols across studies, which is an important aspect to bear in mind working with various EV sources, not only MenSC. Differences in EV isolation, filtration methods, their parameters, quantification techniques, instruments used contribute to inconsistencies in EV purity, yield and functional content. Even if the protocols are normalized, refined according to the consensus guidelines as minimal information for studies of EVs (MISEV) (Welsh et al., 2024), the variability between batch-to-batch samples is also a significant issue adapting EVs for therapeutic purposes.

Also, the use of MenSC-EVs faces regulatory, manufacturing, and bioethical challenges that need to be addressed to ensure their safe and effective use in clinical applications. On the regulatory side, the absence of specific guidelines and the complexity of proving safety, efficacy, and pharmacokinetics make clinical approvals difficult (Stawarska et al., 2024; Wang et al., 2024). Manufacturing these EVs at scale while maintaining consistency, stability, and quality remains a major obstacle (Claridge et al., 2021; Wang et al., 2024). Ethically, while menstrual blood is a non-invasive source, it has a significant ethical advantage over other stem cell sources. Issues such as informed consent and donor privacy must be carefully managed (Achmad and Götte, 2014; Savary et al., 2023). Therefore, in order to ensure reproducibility and clinical applicability, future research should prioritize standardized methodologies, explore the mechanisms underlying MenSC-EV therapeuitc actions and conduct controlled in vivo studies with long-term follow ups to support their safety and integration into clinical therapies.

In conclusion, MenSCs and their EVs represent a potential tool for advancing diagnostics and therapies. Their ability to promote tissue regeneration, provide diagnostic insights, and enable personalized treatments holds immense potential for improving the quality of life for women of all ages. The development of biobanks for MenSCs could further enhance the accessibility and applicability of these cells and their EVs, offering a new hope for the development of innovative treatment strategies for different conditions.