Endometrial and Menstrual Blood Mesenchymal Stem/Stromal Cells: Biological Properties and Clinical Application

Abstract

A highly proliferative mesenchymal stem/stromal cell (MSC) population was recently discovered in the dynamic, cyclically regenerating human endometrium as clonogenic stromal cells that fulfilled the International Society for Cellular Therapy (ISCT) criteria. Specific surface markers enriching for clonogenic endometrial MSC (eMSC), CD140b and CD146 co-expression, and the single marker SUSD2, showed their perivascular identity in the endometrium, including the layer which sheds during menstruation. Indeed, cells with MSC properties have been identified in menstrual fluid and commonly termed menstrual blood stem/stromal cells (MenSC). MenSC are generally retrieved from menstrual fluid as plastic adherent cells, similar to bone marrow MSC (bmMSC). While eMSC and MenSC share several biological features with bmMSC, they also show some differences in immunophenotype, proliferation and differentiation capacities. Here we review the phenotype and functions of eMSC and MenSC, with a focus on recent studies. Similar to other MSC, eMSC and MenSC exert immunomodulatory and anti-inflammatory impacts on key cells of the innate and adaptive immune system. These include macrophages, T cells and NK cells, both in vitro and in small and large animal models. These properties suggest eMSC and MenSC as additional sources of MSC for cell therapies in regenerative medicine as well as immune-mediated disorders and inflammatory diseases. Their easy acquisition via an office-based biopsy or collected from menstrual effluent makes eMSC and MenSC attractive sources of MSC for clinical applications. In preparation for clinical translation, a serum-free culture protocol was established for eMSC which includes a small molecule TGFβ receptor inhibitor that prevents spontaneous differentiation, apoptosis, senescence, maintains the clonogenic SUSD2⁺ population and enhances their potency, suggesting potential for cell-therapies and regenerative medicine. However, standardization of MenSC isolation protocols and culture conditions are major issues requiring further research to maximize their potential for clinical application. Future research will also address crucial safety aspects of eMSC and MenSC to ensure these protocols produce cell products free from tumorigenicity and toxicity. Although a wealth of data on the biological properties of eMSC and MenSC has recently been published, it will be important to address their mechanism of action in preclinical models of human disease.

Keywords: MenSC; cell therapy; culture expansion; eMSC; endometrium; immunomodulation; menstrual blood; perivascular MSC.

FIGURE 1

Ovarian and Menstrual cycle, structure of human endometrium and its shedding during menstruation. (A) Schematic showing human ovarian hormonal changes corresponding with endometrial growth, differentiation and shedding, during the menstruation, proliferative and secretory phases, respectively, of a menstrual cycle. (B) Schematic showing endometrial tissue collection from human endometrium as an office-based procedure using a Pipelle endometrial suction catheter and menstrual fluid using a menstrual cup. (C) Histological sections stained with H&E of pre-menopausal human endometrium during the growth (proliferative), differentiation (secretory) and menses stages of the menstrual cycle showing the functionalis, basalis and myometrial layers of the endometrium. *Endometrial tissue breakdown early in the menstrual stage, leaving behind an intact basalis layer. Menstrual blood showing endometrial tissue fragments comprising endometrial stroma, glands and blood cells. Dotted lines define the layers of the endometrium. g, glands. Reproduced with permission from Figure 1A from Elsevier (Gargett et al., 2008) and Figure 1C from Oxford University Press (Nguyen et al., 2012).

FIGURE 2

Clonogenic endometrial MSC identified using various surface markers. Surface markers (A) CD146, (B) co-expression of CD140b/CD146, SUSD2 (red) in (C) pre-menopausal and (D) postmenopausal endometrium, showing no colocalization with ERα (green), (E) STRO-1 and (F) EphA3 (red, black arrows), mark clonogenic perivascular eMSC. (G) MSCA-1 (TNAP) detected by the W8B2 antibody is expressed by both perivascular and (H) epithelial cells in the human endometrium. (I) CD271 (red) adventitial perivascular cells mark clonogenic cells in ovine endometrium. (J) Schematic of endometrium showing location of endometrial MSC around the vessels. (K) Schematic enlargement of blood vessels from (J) showing different vascular cell types in the endometrium. Human CD140b⁺CD146⁺ cells are pericytes (pink cytoplasm), and SUSD2, STRO-1, and EphA3 cells are perivascular cells (yellow cytoplasm). Ovine CD27⁺ cells are adventitial cells (violet cytoplasm). Mouse CD34⁺KLF4⁺ and LRC cells are also represented by perivascular cells (yellow cytoplasm). Images reprinted with permission from: (A,E) Figures 1E, 4C from Oxford University Press (Schwab et al., 2008), (B) Figure 4I from Oxford University Press (Schwab and Gargett, 2007), (C) Figure 1A from Bioscientifica (Yang et al., 2019), (D) Figure 4A from Oxford University Press (Ulrich et al., 2014b), (F) Figure 1A from Public Library of Science (To et al., 2014), (G,H) Figures 5C,D from Mary Ann Liebert (Sobiesiak et al., 2010), (I) Figure 5D from Public Library of Science (Letouzey et al., 2015) and (K) Figure 2 from Wiley Online Library (Darzi et al., 2016).

FIGURE 3

Properties of MenSCs in vitro and in vivo. (A) Similar to their well-known counterparts (bmMSC), MenSCs have the ability to differentiate to cells of different lineages and express typical MSC-associated markers. (B) Impact of MenSCs on the adaptive and innate immune systems. MenSCs secrete IL-6 and IL-10 that inhibit optimal maturation of human monocyte-derived dendritic cells (DC). MenSCs shift macrophages toward the M2 (anti-inflammatory) profile. MenSCs either inhibit or support T cell proliferation depending on MenSC/T cell (PBMC) ratio. MenSCs increase Treg frequency, decrease the number of TH17 and IFNγ⁺ CD4⁺/CD8⁺ T cells and cytotoxic capacity of NK cells. MenSCs decrease antigen-specific antibody secretion and proliferation of B cells. (C) Utilization of MenSCs in preclinical settings ranges from immune-mediate disorders: arthritis and GVHD to regenerative and reparative applications: cardio protection, liver and lung fibrosis and neuroprotection.