Menstrual blood-derived stem cells (MenSCs) represent a novel population of mesenchymal stromal cells obtained through non-invasive collection. Since their first description in 2007, MenSCs have emerged as a promising source for regenerative medicine applications due to their high proliferative capacity, multipotent differentiation potential, and immunomodulatory properties. This comprehensive review synthesizes current knowledge on MenSC isolation, characterization, therapeutic mechanisms, and clinical applications. We examine comparative data with other MSC sources, explore exosome-mediated effects, discuss regulatory considerations, and identify future research directions. The evidence supports MenSCs as a viable alternative to bone marrow and adipose-derived MSCs, with particular advantages in accessibility and ethical acceptability.
Keywords: menstrual blood, mesenchymal stem cells, MenSCs, regenerative medicine, cell therapy, exosomes, immunomodulation
The field of menstrual blood-derived stem cells began with a groundbreaking study by Meng et al. in 2007, who first identified and characterized a population of multipotent cells from menstrual blood[1]. This discovery opened a new avenue in regenerative medicine, providing an ethically uncomplicated, non-invasive source of stem cells.
Menstrual blood collection represents one of the most accessible methods for obtaining stem cells. Unlike bone marrow aspiration or adipose tissue liposuction, menstrual blood can be collected non-invasively using menstrual cups or specialized collection devices.
Optimal results are obtained when samples are processed within 24-48 hours of collection. Studies by Gargett et al. (2014) showed that viability decreases by approximately 15% per day at room temperature. Transport at 4°C with appropriate anticoagulants (ACD, EDTA, or heparin) maintains viability for up to 72 hours.
The most widely used method employs Ficoll-Paque density gradient media (1.077 g/mL) to separate mononuclear cells from red blood cells and other components. This method yields high purity but requires careful technique to avoid disturbing the buffy coat interface.
| Parameter | Ficoll Gradient | RBC Lysis | Direct Plating |
|---|---|---|---|
| Cell yield (per 10mL) | 1-5 × 10⁶ | 2-8 × 10⁶ | Variable |
| Purity (% MSCs) | 85-95% | 70-85% | 40-60% |
| Processing time | 2-3 hours | 30-45 min | 15 min |
| Viability | High (>90%) | Moderate (80-90%) | Variable |
| Best application | Research, clinical | Screening, banking | Not recommended |
Ammonium chloride-based lysis buffers provide a rapid alternative. While yields are higher, purity is lower due to retention of other blood cell types. This method is suitable for applications where downstream purification steps are employed.
Standard culture employs DMEM or α-MEM supplemented with 10-20% fetal bovine serum (FBS). However, serum-free and xeno-free formulations are increasingly used for clinical applications. Key supplements include:
MenSCs are cultured at 37°C, 5% CO₂, and 95% humidity. Standard tissue culture-treated plastic is sufficient, though some studies suggest enhanced attachment on collagen-coated surfaces. Initial plating density of 1-2 × 10⁵ cells/cm² is optimal for rapid expansion.
MenSCs can be expanded for 10-15 passages while maintaining multipotency. Beyond passage 15, cells enter replicative senescence characterized by enlarged morphology, decreased proliferation, and altered differentiation potential. For clinical applications, early passages (P2-P5) are preferred.
Standard cryopreservation uses 90% FBS + 10% DMSO with controlled-rate freezing (-1°C/min) to -80°C, followed by transfer to liquid nitrogen. Post-thaw viability typically exceeds 85%. Serum-free alternatives using 5-10% DMSO with methylcellulose or albumin are available for clinical applications.
MenSCs display typical fibroblast-like morphology with a spindle-shaped appearance. At low density, cells exhibit a scattered, stellate configuration. At confluence, they form a uniform monolayer with characteristic swirling patterns. Cell size ranges from 15-30 μm in diameter.
MenSCs meet the minimal criteria for MSC definition established by the International Society for Cell & Gene Therapy (ISCT)[2]:
Beyond ISCT criteria, MenSCs express several markers that distinguish them from other MSC sources:
Osteogenic: MenSCs readily differentiate into osteoblasts when cultured with dexamethasone, β-glycerophosphate, and ascorbic acid. Mineralization is confirmed by Alizarin Red S staining for calcium deposition and expression of osteocalcin, osteopontin, and Runx2.
Adipogenic: Adipogenic differentiation is induced with IBMX, dexamethasone, insulin, and indomethacin. Lipid droplet accumulation is visualized with Oil Red O staining. Adiponectin and PPAR-γ expression confirm differentiation.
Chondrogenic: Chondrogenic differentiation requires TGF-β3, dexamethasone, and ascorbate in pellet culture. Proteoglycan production is detected with Alcian Blue or Safranin O staining. Collagen II and aggrecan expression confirm chondrocyte phenotype.
MenSCs have demonstrated differentiation capacity beyond the traditional MSC lineages:
The mechanism of apparent transdifferentiation remains debated. Some studies suggest true lineage conversion, while others propose cell fusion with resident cells or paracrine-mediated effects. Current evidence supports a combination of direct differentiation and trophic support mechanisms.
MenSCs have been extensively compared to the two most common MSC sources: bone marrow (BM-MSCs) and adipose tissue (AD-MSCs). Each source offers distinct advantages and limitations.
| Characteristic | MenSCs | BM-MSCs | AD-MSCs |
|---|---|---|---|
| Collection method | Non-invasive | Invasive (aspiration) | Minimally invasive (lipo) |
| Donor risk | None | Anesthesia, infection risk | Surgical complications |
| Collection frequency | Monthly (reproductive years) | One-time | Limited repeatability |
| Cell yield per volume | Moderate | Low | High |
| Proliferation rate | High | Moderate | Moderate |
| Maximum passages | 10-15 | 8-12 | 8-12 |
| CFU-F frequency | 1:100-500 | 1:10,000-100,000 | 1:100-2,500 |
| Angiogenic potential | High | Moderate | Moderate |
| Immunomodulation | Strong | Strong | Moderate |
| Ethical concerns | Minimal | Minimal | Minimal |
Genome-wide expression studies have revealed both shared and distinct features between MSC sources. Khanjani et al. (2021) demonstrated that MenSCs express higher levels of angiogenic genes (VEGFA, ANGPT1) and pluripotency markers compared to BM-MSCs. Proteomic analysis by Bortolomai et al. (2019) identified unique protein signatures in MenSC exosomes associated with tissue repair.
The primary therapeutic mechanism of MenSCs is paracrine signaling through secreted factors rather than direct differentiation and replacement of damaged cells. This "trophic" mechanism involves the secretion of multiple bioactive molecules that modulate the local microenvironment.
Growth Factors: VEGF, HGF, bFGF, IGF-1, EGF promote cell survival, proliferation, and angiogenesis. MenSCs secrete significantly higher levels of VEGF compared to BM-MSCs, contributing to their enhanced angiogenic potential.
Cytokines: IL-6, IL-8, IL-10, TGF-β modulate immune responses and tissue repair. The IL-6/IL-10 balance is particularly important for immunomodulatory effects.
Anti-inflammatory Molecules: TSG-6 (TNF-stimulated gene 6), PGE2, and IDO (indoleamine 2,3-dioxygenase) suppress inflammatory responses and promote resolution of inflammation.
Extracellular Matrix Components: Collagens, fibronectin, laminin, and proteoglycans support tissue structure and cell migration.
MenSCs modulate both innate and adaptive immune responses through multiple mechanisms:
MenSCs promote the shift from pro-inflammatory M1 macrophages to anti-inflammatory M2 macrophages. This occurs through secretion of IL-6, M-CSF, and PGE2. M2 macrophages support tissue repair through anti-inflammatory cytokine production and phagocytic clearance of apoptotic cells.
Exosomes are small extracellular vesicles (30-150 nm) that serve as critical mediators of intercellular communication. MenSC-derived exosomes carry therapeutic cargo including proteins, lipids, mRNA, and miRNA.
Cell-free exosome therapy offers several advantages over live cell transplantation: no risk of ectopic tissue formation, no microvascular obstruction, better stability, and potential for off-the-shelf availability. Preclinical studies demonstrate efficacy in myocardial infarction, stroke, wound healing, and inflammatory diseases.
Recent studies have identified mitochondrial transfer from MenSCs to injured cells as an additional therapeutic mechanism. This process, mediated through tunneling nanotubes and microvesicles, restores bioenergetic function in damaged cells and promotes survival.
Multiple preclinical studies demonstrate MenSC efficacy in myocardial infarction and heart failure models. Hida et al. (2008) first showed improved cardiac function following MenSC transplantation in immunodeficient mice. Subsequent studies confirmed reduced infarct size, enhanced neovascularization, and improved ejection fraction.
Mechanisms: Paracrine angiogenic factor secretion, anti-apoptotic effects on cardiomyocytes, modulation of cardiac fibroblasts, and promotion of endogenous cardiac progenitor cell activation.
Borlongan et al. (2010) demonstrated functional recovery in a stroke model following MenSC administration. Cells cross the blood-brain barrier and provide neuroprotection through anti-inflammatory and anti-apoptotic mechanisms. Studies in spinal cord injury, traumatic brain injury, and neurodegenerative diseases (Parkinson's, Alzheimer's) show promising results.
MenSCs improve glycemic control in type 1 and type 2 diabetes models through multiple mechanisms: pancreatic β-cell protection and regeneration, insulin sensitivity improvement in peripheral tissues, and modulation of autoimmune responses in type 1 diabetes.
In liver fibrosis and cirrhosis models, MenSCs reduce collagen deposition, promote hepatocyte regeneration, and modulate inflammatory responses. Studies suggest potential for treating non-alcoholic fatty liver disease (NAFLD) and acute liver failure.
The potent immunomodulatory properties of MenSCs make them candidates for treating multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, and inflammatory bowel disease. Clinical trials are ongoing for multiple sclerosis and Crohn's disease.
During the COVID-19 pandemic, MenSCs were rapidly deployed in clinical trials for acute respiratory distress syndrome (ARDS). Their anti-inflammatory properties and ability to modulate the cytokine storm showed promise in early-phase studies.
As of 2025, over 20 clinical trials have investigated MenSC-based therapies across multiple indications. Key completed trials include:
The safety profile of MenSCs is favorable based on clinical trial data. No serious adverse events directly attributed to cell therapy have been reported. Common observations include mild fever, transient injection site reactions, and self-limiting immune responses. No ectopic tissue formation or tumorigenicity has been observed.
Clinical-grade manufacturing requires adherence to Good Manufacturing Practice (GMP) standards:
MenSCs are regulated as somatic cell therapy products in most jurisdictions. In the United States, they fall under FDA regulation as 351 or 361 products depending on manufacturing and indication. The European Medicines Agency (EMA) classifies them as Advanced Therapy Medicinal Products (ATMPs).
MenSCs offer significant ethical advantages compared to embryonic stem cells. Collection is non-invasive, poses no risk to the donor, and avoids destruction of embryos. Informed consent protocols must address future use of cells, potential for commercialization, and data sharing.
Key challenges include standardization of isolation and characterization methods, development of predictive potency assays, scaling manufacturing for commercial viability, and navigating complex regulatory pathways. Opportunities exist in personalized medicine, combination therapies, and addressing unmet medical needs in regenerative medicine.
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