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

1Almost all human tissues contain a small resident population of perivascular mesenchymal stem/stromal cells (MSC). MSC have also been identified in a wide variety of small and large mammalian species, although these have been studied to a lesser degree. Here we discuss a novel source of MSC from the highly regenerative endometrial lining of the uterus (Figure 1A), accessible by biopsy in an office-based procedure without an anesthetic, or non-invasively from menstrual blood (Figure 1B). This review will focus on the biological properties and recent functional characterization of endometrial MSC (eMSC) and menstrual blood MSC (MenSC). The reader is referred to a recent comprehensive review providing greater details on earlier studies describing the identification and early characterization of endometrial MSC, MenSC and endometrial stromal fibroblasts. Both epithelial progenitors and MSC have been identified in human and mouse endometrium, however, it is the endometrial and menstrual blood MSC that comprise the topic of this review.

2Recently the MSC field has been criticized, due to the poor characterization of MSC from various sources, which has resulted in underwhelming outcomes of many clinical trials using MSC. Counter arguments reiterate the importance of using the appropriate definition of the cells under study (i.e., perivascular MSC versus fibroblasts). Integrated transcriptomic profiling of human MSC from different tissues show their segregation by tissue of origin and distinct tissue-specific MSC immune signatures that are similar between fresh and cultured MSC from the same tissue source. Herein we will distinguish between perivascular eMSC and endometrial stromal fibroblasts. We will also differentiate between potential regenerative “stem cell” properties and immunomodulatory function of both eMSC and MenSC. It has become clear that the regenerative properties of MSC are mainly due to the trophic factors they secrete. These stimulate endogenous cells to repair damaged tissues, rather than MSC functioning as true stem cells. MSC are reparative rather than regenerative. It is also recognized that MSC have profound immunomodulatory effects on innate and adaptive immune cells that promote healing by reducing inflammation and immune responses. In this review, we recognize these important developments in the MSC field and our review on endometrial and menstrual blood MSC and fibroblasts has been structured around these themes.

3Human endometrium is a dynamic remodeling tissue, undergoing cycles of growth, differentiation and shedding on a monthly basis as part of the menstrual cycle. These dynamic processes occur about 400 times in women until menopause. During menstruation, the upper functional layer of endometrial tissue sloughs off and the tissue fragments exit the body in menstrual blood, leaving a residual 1–2 mm of endometrial tissue (basalis) overlying the myometrium (uterine muscle) (Figures 1A–C). The raw surface rapidly reepithelializes and the new functional layer (functionalis), comprising epithelial-lined glands and an extensive vascularized stroma regenerates under the influence of rising, circulating estrogen levels in the next cycle. Atrophic postmenopausal endometrium, which transcriptionally resembles cycling basalis endometrium, also regenerates a functionalis-like layer when women take estrogen-only hormone replacement therapy. MSC can be harvested from this regenerated tissue.

4Endometrial MSC were first identified as clonogenic stromal cells, comprising 1.3% of stromal fibroblasts harvested from hysterectomy tissue, which contains both functionalis and basalis endometrium. The large stromal colonies, comprised densely packed small cells with a fibroblast-like morphology and distinguishable from self-limiting small, sparsely packed colonies, likely originated from colony forming units-fibroblast (CFU-F) and do not vary in frequency during the menstrual cycle. This and their presence in postmenopausal endometrium indicate hormone independence of endometrial CFU-F. In comparison to single small colonies, individual large CFU-F showed high proliferative capacity, undergoing 30 population doublings (PD) and producing > 600 billion cells. Single large endometrial CFU-F underwent self-renewal in vitro by serial cloning at very low seeding densities (5–10 cells/cm2) and differentiated into adipocytes, chondrocytes, myocytes and osteocytes. They also expressed the classic pattern of International Society for Cellular Therapies (ISCT) markers (Table 1). These properties indicate that human endometrium contains a small population of MSC.

5eMSC were identified as perivascular cells by comparing cloning efficiencies of endometrial stromal cells purified using flow cytometry for several surface markers used for bone marrow MSC (Figure 2A). Most CFU-F were from cells co-expressing CD140b and CD146. CD140b+CD146+ cells, comprising 1.5% of endometrial stromal cells, were enriched eightfold for CFU-F over unsorted stromal cells and fulfilled the ISCT MSC criteria. Their perivascular identity was revealed in both the basalis and functionalis (Figures 2B,J), indicating that CD140b+CD146+ eMSC could be isolated from endometrial biopsies and would be shed in menstrual blood. Gene profiling CD14b+CD146+ eMSC versus CD140b+CD146– endometrial fibroblasts showed 762 differentially expressed genes, indicating that perivascular eMSC are distinct from endometrial fibroblasts as for other MSC types.

6Similar to bone marrow MSC, perivascular eMSC are also MSCA-1+, a marker antibody that identifies tissue non-specific aminopeptidase (TNAP), however, this marker is not useful for sorting eMSC as it also marks glandular epithelial cells (Figures 2G,H).

7To identify a single marker of perivascular eMSC, endometrial stromal cell suspensions were screened with a panel of perivascular and other novel antibodies by flow cytometry and immunohistochemistry of human endometrium. Using this strategy, the W5C5 antibody identified a robust marker of stromal cells from pre- and postmenopausal endometrium with MSC properties, enriching clonogenic cells 18-fold over W5C5– fibroblasts. Sushi Domain-containing 2 (SUSD2) was the antigen identified by the W5C5 antibody. SUSD2+ cells comprise 4.1% of endometrial stromal cells and in addition to satisfying the ISCT criteria, they reconstituted stromal tissue in vivo under the kidney capsule of NOD-Scid γ (NSG) mice. Non-ISCT markers also expressed by freshly isolated SUSD2+ eMSC include CD117, CD140b, CD146, and STRO-1 (Figure 2E). More clonogenic cells were present in the SUSD2+CD146+ and SUSD2hi subpopulations than in the CD140b+CD146+ co-expressing population. SUSD2 enables prospective isolation of eMSC from freshly isolated cell suspensions using magnetic bead sorting, providing a more clonogenic population than obtained by flow cytometry sorting, which adversely affects cell viability. This is an important consideration for clinical translation.

8The specific markers of eMSC show that these cells are located around blood vessels in both the functionalis (Figures 1, 2) indicating they are shed into menstrual fluid as the functionalis breaks down during menstruation (Figure 1B). Similarly, stromal fibroblasts are shed into menstrual fluid. Both eMSC and stromal fibroblasts (MenSC) are shed in numbers proportionate to their composition in endometrial functionalis tissue, with eMSC comprising a minority subpopulation. The adult stem cell properties of human eMSC suggest that stromal fibroblasts are their progeny, and to date the only evidence comes from xenografting SUSD2+ eMSC into immunocompromised mice where stromal tissue was generated.

9Physiologically, eMSC around spiral arterioles differentiate into decidual cells under influence of the pregnancy hormone, progesterone, during the secretory stage of the menstrual cycle. This decidual differentiation spreads to the stromal fibroblasts beneath the luminal epithelium. Decidual cells are specialized secretory cells that provide an immunoprivileged environment for an implanting embryo to establish the materno-fetal interface. Subpopulations of eMSC and stromal fibroblasts undergo senescence during the differentiation process and when no embryo implants, progesterone levels fall and menstruation ensues (Figure 1).

10Transcriptional profiling of endometrial SUSD2+ eMSC and SUSD2– stromal fibroblasts revealed a distinct gene signature for both cell types following decidual differentiation in vitro. Known and novel perivascular genes were upregulated in SUSD2+ eMSC, which produced lower levels of inflammatory mediators and chemokines in vitro compared to SUSD2– stromal fibroblasts. Similarly, the inflammatory gene signature of freshly isolated and cultured CD140b+CD146+ eMSC had fewer transcripts than CD140b+CD146– endometrial stromal fibroblasts. Upon decidualization (differentiation) induction SUSD2+ eMSC and SUSD2– stromal fibroblasts showed greater divergence of their respective secretomes, with the eMSC producing much higher levels of leukemia inhibitory factor and the chemokine CCL7 than stromal fibroblasts. These varying features highlight differences between perivascular eMSC and stromal fibroblasts.

11Embryologically, endometrium derives from the mesoderm. Thus, it is not unexpected that endometrial MSC and stromal fibroblasts can be induced to differentiate into mesodermal lineages. Differentiation of eMSC into classic mesodermal lineages in vitro as recommended by the ISCT has been shown for clonogenic endometrial stromal cells, SUSD2+ and CD140b+CD146+ cells. Endometrial stromal fibroblasts also show similar mesodermal lineage differentiation, as do bone marrow-derived stromal cells. The reader is referred to table III in for a comprehensive list of studies describing mesodermal lineage differentiation of eMSC and endometrial stromal fibroblasts. Differentiation into endodermal lineages include hepatocytes from clonogenic eMSC in vitro and insulin-and glucagon-secreting pancreatic lineages both in vitro and in vivo from endometrial stromal fibroblasts. Similarly, cultured endometrial fibroblasts have been differentiated into ectodermal lineages such as dopamine-secreting neurons in vitro and in vivo, where they may have also induced endogenous neural cell secretion. Oligodendrocyte progenitor cells have been differentiated from endometrial stromal fibroblasts. It is not known if eMSC differentiate into ectodermal lineages.

12The dynamic nature of the endometrium is not limited to humans. Endometrium of other species also undergo proliferation and differentiation, and endure high levels of regeneration during their reproductive life. No animal models can replace humans, however, they provide preclinical models bridging the gap between the in vitro potential of MSC and future clinical use in humans. The identification of endometrial stromal/stem cells in non-human species has made this possible. Mammalian MSC have been identified in the endometrium of mice, guinea pig, pig, sheep, cow, goat, horse and non-human primates (Table 1). Label-retention, plastic adherence or specific surface markers have been used to isolate endometrial MSC and demonstrate properties similar to human eMSC and bmMSC.

13Quiescent stem-like endometrial stromal cells were first identified in mice as bromodeoxyuridine label-retaining cells (LRC), which accounted for 6% of the population after 12 weeks of chase in normal cycling mice. They were localized adjacent to luminal epithelium, near blood vessels and at the endometrial-myometrial junction, similar to their basalis location in human endometrium. Stromal LRCs were both CD31– and CD45–, indicating they were neither endothelial cells or leukocytes, and perivascular LRCs were αSMA+. Approximately 16% expressed estrogen receptor-α (ER-α) and were recruited into cell-cycle following estrogen stimulation indicating their involvement in cyclical endometrial regeneration. LRCs were also positive for stem cell markers c-Kit and Oct4. However, stem-cell antigen-1 (Sca-1) was not expressed in stromal LRCs.

14Recently, CD34+KLF+ endometrial stromal stem/progenitor cells were identified in the perivascular region in the endometrium of a menstruating mouse model. They expressed the smooth muscle marker, SM22α, and vimentin and upon estrogen stimulation trans-differentiated into gland-like structures lined with E-cadherin-expressing epithelial cells. SUMO-endopeptidase-1 (SENP1)-deletion in SM22α+cells induced SUMOylation and activation of ERα which promoted SM22α+CD34+KLF4+ cell proliferation and transdifferentiation into endometrial epithelium via cyclin D1. Mice share ∼85% of protein-coding regions with humans and have 99% genetic similarity, their small size and the development of transgenic humanized mice allow their utility as cost-effective models. More research on mouse eMSC isolation and characterizationis warranted.

15Endometrial stromal fibroblasts have been isolated from Fars native sheep uteri as plastic adherent cells or clonogenic cells from Border-Leicester-Marino ewes. Plastic adherent cells expressed CD73 but not CD34, with osteogenic and adipogenic differentiation potential and karyotype stability over four passages. Population doubling times were directly related to the cell seeding density and inversely to age of the ewe (Table 2). Clonogenic stromal cells were enriched using CD271+CD49f– surface markers, showing properties similar to bmMSC and human eMSC, with greater clonogenicity, self-renewal by serial cloning compared to CD271–CD49f– fibroblasts, and differentiated into adipogenic, myogenic, osteogenic and chondrogenic lineages. Apart from CD271, human MSC markers CD44, CD90, CD140b, and CD146 did not cross react with ovine eMSC. The lack of specific ovine marker antibodies has hampered further surface phenotype characterization. Although in a perivascular location, ovine eMSC unlike their human counterpart, were not located in close apposition to vWF+ endothelial cells. Neither did they co-localize with αSMA suggesting they were not pericytes, but rather perivascular adventitial cells, a cell population with similar properties to MSC. The ability to purify characterized ovine eMSC has enabled the investigation of their use in uro-gynecological disorders such as pelvic organ prolapse (POP). The domestic ewe has proven a good large animal model because they develop spontaneous POP after vaginal delivery with incidence increasing with parity, similar to women. Autologous ovine eMSC in a gelatin hydrogel applied onto a polyamide scaffold and implanted into the vagina of the ovine POP model survived for at least 30 days, modulated the inflammatory response, promoting good tissue integration with no postoperative mesh-exposure, one of the main complications associated with human transvaginal mesh.

16Endometrial-derived stromal cells from the non-human primate, green monkey engrafted and differentiated into neuron-like cells when injected into the striatum of males with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced Parkinson’s disease. The expanded cells used expressed CD140b, CD146, and CD90, but were otherwise not characterized, possibly due to lack of suitable antibodies. The differentiated cells expressed tyrosine hydrolase, a rate limiting enzyme for L-DOPA synthesis, proliferated and produced dopamine metabolites indicating their potential use in cell-based therapies. Although non-human primates are ideal for simulating human diseases due to their phylogenetic similarity, their utility in research raises ethical, practical and financial issues.

17Menstrual blood is a readily accessible, non-invasive source of large numbers of endometrial stem/stromal cells (MenSC), easily collected using a menstrual cup (Figure 1B). Menstrual blood contains fragments of shedding endometrial tissue (Figure 1C) which is cultured directly onto plastic similar to bmMSC. Adherent MenSC have typical stromal fibroblast morphology and rapidly propagate with a doubling time of 18–36 h. MenSC have higher proliferative capacity, 30–47 PD before senescence, compared to bmMSC, which are generally limited to ∼20 PD. MenSC yield is 2–4-fold higher compared to bmMSC. As for other adult stem cells, the lifespan of MenSC is relatively short in comparison with human embryonic stem cells (hESC). MenSC only maintain 50% of their telomerase activity at passage 12 compared to hESCs. However, MenSC have more telomerase activity than bmMSC, perhaps due to the regenerative capacity of the endometrial stroma.

18Besides shed endometrial tissue fragments, menstrual fluid also contains peripheral blood. The source of MSC in menstrual fluid could be from bone marrow as well as endometrium. However, peripheral blood contains exceedingly rare bmMSC, as demonstrated by CFU-F analysis. Just 2 CFU-F were found in peripheral blood from 10 patient samples at a frequency of 0–1 CFU-F per 4 × 107 nucleated cells. In contrast, menstrual fluid from 18 healthy women contains 600 CFU-F per ml, suggesting that these CFU-F are derived from the endometrial fragments rather than circulating bone-marrow-derived MSC. Rigorous lineage tracing in chimeric mouse models has also shown that bone marrow stem cells do not contribute to endometrial stromal lineages. Thus MenSC, comprising mainly endometrial stromal fibroblasts and a small proportion of eMSC found in menstrual fluid are derived from sloughing endometrial tissue rather than non-shedding bone marrow stroma.

19MenSC possess the classic ISCT bmMSC markers and are positive for HLA-ABC, negative for HLA-DR and do not express hematopoietic lineage markers. MenSCs, similar to clonogenic eMSC, differ from bmMSC as they do not express STRO-1. Another difference is that MenSCs highly express cytoplasmic OCT-4 which is not expressed by eMSC or conventional bmMSC. Several inconsistencies in MenSC phenotype have been observed for pluripotency markers c-KIT and SSEA-4. Some have reported these markers in isolated MenSCs, while others were negative. Others observed a different pattern of these markers in isolated MenSC including expression of SSEA-4 and/or NANOG in some c-KIT+ cells from cultured menstrual blood. Heterogeneity of MenSs cultures may explain these disparities, resulting from differences in menstrual blood sampling day, collection technique and enrichment protocol.

20MenSC have been induced to differentiate into mesodermal lineages characteristic of bmMSC (Table 3) to satisfy the minimal criteria for MSC. Similar to marker expression, the degree of MenSC differentiation into mesodermal lineages varies and is dependent on the isolation method. MenSCs isolated by density gradient centrifugation and plastic adherence have lower capacity to differentiate toward osteoblasts compared to bmMSC using both cytochemical and molecular analyses. In contrast, c-KIT+ MenSC possessed similar differentiation capacity to bmMSC, although, Alizarin red alone without quantification was used. It is unknown whether c-KIT-sorted MenSC have osteogenic differentiation ability.

21Adipogenic differentiation of MenSC isolated by density gradient centrifugation and plastic adherence is limited when assessed by Oil-red O staining and significantly lower than umbilical cord MSC and bmMSC. Fortification of adipogenic induction medium with rosiglitazone promoted MenSC differentiation into adipocytes as analyzed by molecular and cytochemical techniques. Purifying cultured MenSC using the c-KIT (CD117) marker resulted in high adipogenic differentiation capacity (60–70%), likely due to a more homogeneous MSC population, although it is unknown if CFU-F are also purified in the c-KIT+ subpopulation of cultured MenSC.

22Differentiation of MenSC into the chondrocytic lineage has been demonstrated for CD117 purified cells in 2D cultures yielding 45% positive cells, similar to bmMSC. Unfractionated MenSCs cultured in a 3D nanofibrous scaffold enhanced chondrogenic commitment compared to a 2D culture system, with extensive cartilage-like extracellular matrix containing ∼50% more glycosaminoglycan than control MenSCs differentiated in 2D. Chondrogenic differentiation requires very low O2 tension, which is better established in 3D compared to 2D cultures. MenSCs produce high levels of Activin A, IGF-1, and FGF2, key growth factors involved in chondrogenesis.

23Co-culture of MenSC with murine cardiomyocytes generated spontaneously beating human cells expressing cardiac specific markers, indicating their differentiation into troponin T-expressing cardiomyocytes. In vitro cardiac differentiation of MenSC was compared with bmMSC using two differentiation protocols and showed that continuous FGF2 was superior to 5-aza-2′-deoxycytidine alone, as shown by increased levels of late-stage cardiac markers (connexin 43 and Troponin T) (Table 3). This suggests that FGF2 has a key role in differentiating cardiac cells from MSC sources. MenSC had greater capacity than bmMSC to differentiate toward cardiomyocytes regardless of the differentiation protocol.

24Collectively, the differentiation capability of unfractionated MenSC isolated using conventional methods into two mesodermal lineages (osteoblasts and adipocytes) is lower than bmMSC isolated in a similar manner. This suggests differences in tissue specific MSC properties likely reflecting their tissue of origin. Purification of the rarer perivascular MSC with specific markers appears to improve MenSC differentiation, although c-KIT is not expressed on perivascular cells of human endometrium in vivo (Figure 2), but whether induced in cultured perivascular eMSC is unknown.

25Endometrial stromal fibroblasts naturally differentiate into decidualized stromal cells in vivo. Decidualization is mediated by estrogen and progesterone, and supports the establishment and continuation of pregnancy. MenSC have been differentiated into decidual-like cells with 8-Br-cAMP and progesterone, showing morphological changes and increased expression of the decidualization markers prolactin and insulin-like growth factor binding protein-1 (IGFBP-1), and attenuated expression of MSC surface markers. Greater secretion of these decidual proteins was observed for MenSC compared to bmMSC and amnion MSC. It is possible that easily obtained MenSC could be differentiated into decidual-like cells as a potential strategy to mitigate infertility associated with insufficient endometrial decidualization.

26Cultured MenSC transdifferentiate across lineage boundaries into ectodermal (neurons and glia), and endodermal (hepatocyte) lineages (Table 3). c-KIT sorted MenSCs express neuronal phenotypic markers when grown in appropriately conditioned medium. MenSC formed neurosphere-like cells and then differentiated into neural and glial-like cells comparable to bmMSC, and shown by increased expression of classic neural markers (nestin, microtubule-associated protein 2, gamma-aminobutyric acid type B receptor subunit 1 and 2, and tubulin β3 class III) (Table 3).

27Differentiation of MenSC into hepatocytes is dependent on the concentrations of hepatocyte growth factor (HGF), oncostatin M (OSM) and removal of serum from the induction medium. Up-regulation of albumin and CYP7A1 expression was higher in MenSC- compared to bmMSC-derived hepatocyte-like cells, although cytokeratin-18 expression, albumin production and glycogen accumulation were either lower or not different, indicating similarity between MenSCs and bmMSC hepatocyte-like cell differentiation potential.

28Unfractionated MenSC have potential to differentiate into keratinocyte-like cells, generating epidermal lineage markers via co-culturing with keratinocytes derived from the foreskin of healthy newborns. MenSC were also induced to differentiate into keratinocyte-like cells in 3D culture with human foreskin-derived keratinocytes on a bilayer scaffold composed of amniotic membrane and silk fibroin. The MenSC-derived keratinocytes expressed keratinocyte-specific markers K14, p63 and IVL (involucrin). Generating keratinocytes from MenSC on an efficient natural construct has potential applicability for MSC-based skin wound healing and regeneration, although these tissue engineering constructs have yet to be evaluated in an animal skin wound repair model.

29MenSC show considerable capacity to differentiate into numerous lineages in vitro, although have lesser ability for several bmMSC lineages. It would now be important to demonstrate the potential of MenSC to undergo differentiation into these lineages in vivo in animal models. Genomic sequencing (e.g., RNAseq) would also shed light on how far down the various lineages MenSC differentiate.

30Since the first report on the immunomodulatory properties of MSC in 2002, numerous studies have shown significant impact of MSC on key cells of the innate and adaptive immune systems. These non-stem cell properties have been exemplified in bmMSC. MSC regulate monocyte infiltration to the site of injury, macrophage polarization and dendritic cell maturation. MSC also inhibit NK cell proliferation and target cell killing, and secrete IFN-γ. In the adaptive immune system, MSC suppress T cell proliferation, shifting Th17 cells toward T regulatory cells (Tregs). MSC also impede B cell proliferation. Mechanism of interaction relies on direct cell-cell contact and/or MSC secretion of immunosuppressive factors including Indoleamine 2,3 deoxygenase (IDO), prostaglandin E2 (PGE2), nitric oxide (NO), human leukocyte antigen G5 (HLA-G5), IL-10, IL-6 and TGF-β. MSC have also been used to successfully treat patients with severe immune disorders, including Graft-Versus-Host Disease (GVHD) and Crohn’s disease. This extensive literature indicates that MSC have immunosuppressive rather than immunostimulatory functions. Here we review the literature on the immunomodulatory properties of eMSC and MenSCs.

31The immunomodulatory properties of perivascular eMSC have only recently been investigated. A transcriptional study on CD140b+CD146+ eMSC revealed that perivascular eMSC differentially expressed several immunomodulatory genes compared to endometrial stromal fibroblasts (Table 4). A similar gene profile of differentially expressed genes was observed between SUSD2+ perivascular eMSC and SUSD2– stromal fibroblasts (Table 4), including anti-inflammatory IL10. The gene profile of perivascular eMSC treated and non-treated with the TGFβ receptor (TGFβR) inhibitor, A83-01 to prevent apoptosis and senescence during culture expansion, revealed upregulation of many immune response genes in treated cells, including interleukins (IL15, IL33, IL6ST), TNF and IFNγ related genes, PGE2 synthesis genes (PLA2G4A, PTGS2/COX-2, PTGES), and Toll-like receptors, TLR2 and TLR3. Together, these finding suggest that eMSC have the ability to interact with the immune system in a similar manner to bmMSC, exerting immunosuppressive functions through paracrine mechanisms and by direct contact with the target cell.

32eMSC also influence T cell function by suppressing ConA-stimulated murine T lymphocyte proliferation in a dose-dependent manner. Blocking the TGFβR in eMSC reversed the immunosuppressive effects of eMSC on T cell proliferation, indicating a role for the TGFβ signaling pathway. Of the various mechanisms assessed, neither IL-10, PGE2, TGFβ or Tregs mediated the eMSC anti-proliferative effects on T cells, however, IL-17A and Dickkopf-1 (DKK-1) were secreted and may be candidates involved. Systemic eMSC failed to inhibit swelling in a T cell-mediated mouse model of skin inflammation, suggesting that eMSC have distinct immunomodulatory properties that may not be sufficient to restrain some T cell-mediated events.

33The immunomodulatory role of perivascular eMSC has been explored in animal models of foreign body response to the implanted biomaterials (Table 4). In a nude rat wound healing model, human eMSC seeded on a gelatin-coated polyamide mesh initially increased inflammatory M1 macrophages at the mesh-tissue interface, followed by reduced inflammation around the mesh filaments by switching macrophages from an M1 to a wound healing M2 phenotype. Long term, there were reduced CD68+macrophages at the mesh tissue interface. Human eMSC seeded on the same polyamide/gelatin mesh inhibited secretion of the inflammatory cytokines IL1β and TNF in early stages of the host response, whereas induced gene expression of anti-inflammatory M2 markers was observed later. These responses occurred earlier and were more marked in the immunocompetent than immunocompromised mice. This favorable immunosuppression by eMSC was confirmed subsequently by eMSC-seeded degradable poly(l -lactic acid)-co-poly(ε-caprolactone) (PLACL) nanofibers and 3D bio-printed tissue engineering constructs in the same wound healing immunocompetent mouse models.

34The immunomodulatory properties of MenSC have been also been investigated, often in comparison with bmMSC. MenSC effects have been mainly studied on T cell responses. In mixed lymphocyte reactions (MLR), comprising MenSCs mixed with allogenic human peripheral blood mononuclear cells (PBMCs), cellular proliferation, IFN-γ and TNF levels were suppressed, while IL-4 production increased. The MenSC suppressive effect on the allogenic MLR was dose-dependent and biphasic, with a high MenSC:PBMC ratio (1/2) suppressing PBMC proliferation and a low ratio (1/32) supporting proliferation. MenSC effects on cytokine levels were similarly concentration dependent with reduced anti-inflammatory IL4+IL10+CD4+ T cells at low MenSC:PBMC ratio compared to bmMSC. These effects suggest distinct immunosuppressive activity of MenSC, similar to eMSC and appear less than bmMSC. This may be due to more HLA-DR molecules on the MenSC surface and fewer IFN-γ receptors, and that MenSC produce less IDO, COX2, and Activin A compared with bmMSC.

35Pretreatment (licensing) of bmMSC with an inflammatory stimulus enhances their immunosuppressive properties. In contrast to the anti-proliferative effect of IFN-γ-pre-treated bmMSC on CD4+ T cells, IFN-γ-IFN-g-pre-treated MenSC show a milder response. Untreated MenSC cocultured with CD4+ T cells at ratios of 1:2–1:8 had a pro-inflammatory effect, increasing the proliferation of anti-CD3/CD28-acitivated T cells, and IFN-γ pretreatment only partially reduced this effect, suggesting that IFN-γ responsiveness of MenSC is comparatively lower than bmMSC (Figure 3). The impact of MenSC on human T cell proliferation in vitro is complex and depends on the cytokine milieu, T cell stimulating factors, MenSC/T cell ratio and the co-culture system. MenSC have a weaker and distinct in vitro effect on T cell proliferation compared to bmMSC, possibly similar to the lesser characterized eMSC responses.

36As expected, MenSC also have an impact on the innate immune system, including human blood monocyte-derived dendritic cells (MoDCs) (Table 4), NK cells and tissue macrophages. MenSC co-cultured with human monocytes interfered with MoDC differentiation at the phenotypic level. The immature DCs expressed suboptimal co-stimulatory molecules and had low levels of CD40, CD80, CD83 and CD86. MenSC secreted IL-6 and IL-10, which typically inhibit monocyte to DC differentiation. Uterine NK (uNK) cells constitute the main component of the endometrial innate immune system, comprising 50–70% of lymphoid cells in early pregnant endometrium. uNK cells function in maintaining a successful pregnancy by preventing allorejection of the embryo and regulating vascular remodeling. Dysregulation of uNK cell function may be involved in the pathogenesis of recurrent pregnancy loss indicating the importance of tightly regulating uNK cell cytotoxic function. Unstimulated MenSCs induced NK cell proliferation partly due to lower production of IGFBPs 1-4 compared to bmMSC. However, in a pro-inflammatory milieu involving IFN-γ/IL-1β, MenSCs substantially inhibited NK cell proliferation through production of IL-6 and TGF-β. IFN-γ/IL-1β-stimulated MenSC also curbed NK cell cytotoxicity by decreasing granzyme A, granzyme B, and perforin expression. NK cells also killed MenSC in a MHC- and time-dependent manner. These results suggest a critical role for MenSC in endometrial tissue homeostasis and induction of a pregnancy-friendly phenotype in decidual NK cells.

37Recently it was shown that MenSC influence the humoral immune responses in a mouse model of heart transplantation by attenuating antibody responses. MenSC injection 24 h following allograft heart transplantation prolonged graft survival in recipient mice by rapidly reducing intragraft deposition of donor-specific IgG and IgM antibodies and reducing donor-specific antibody secreting B cells. It remains to be investigated whether MenSC ameliorate autoantibody production in patients with antibody-mediated autoimmune diseases.

38In a murine model of colitis, intravenous MenSC injected 2–8 days after disease induction increased intra-colon IL-4 and IL-10 and decreased IL-2 and TNF, and splenic dendritic cells expressed lower levels of MHC-II compared to untreated controls. The percentage of CD3+CD25+ and CD3+CD8+ T cells was reduced and Tregs increased. MenSC also promoted F4/80+CD206+ M2 macrophage migration and reduced IgG deposition in the injured colon, decreased splenic plasma cells, increased regulatory B cells, and appeared to mediate these immunomodulatory effects via PD-L1.

39Not all studies have corroborated the immunomodulatory effects ofMenSC in disease models. MenSC failed to switch T cell-related immune responses toward an anti-inflammatory direction in a murine model of arthritis (Table 5). MenSC injection increased splenic TNF levels and did not reduce lymph node pro-inflammatory Th17 cells and in contrast, bmMSC did not exert any beneficial impacts on disease progression. MenSC injection into a humanized GVHD mouse model, where irradiated NOD-SCID mice received human PBMC, has been efficacious in reducing disease severity. In this acute inflammatory model, intra-peritoneal MenSC were superior to bmMSC, in contrast to the arthritis model. MenSC improved intestine structure and survival rate. However, immunosuppressive functions were not observed for MenSC, but rather their effect was attributed to their regenerative capacity. MenSCs expressed high levels of VEGF and FGF2, and induced greater vascularization compared with bmMSC. MenSC also had a greater migratory capacity to target organs due to their higher expression of surface CXCR4.

40MenSC also have anti-microbial activity. In a cecal ligation and puncture mouse model of sepsis, the combination of MenSC and antibiotic improved the survival rate of affected animals (up to 95%) compared with control animals receiving either treatment alone. The MenSC/antibiotic combination increased bacterial clearance from blood, and reduced the inflammatory cytokines IL-8, TNF, and MCP in peritoneal fluid, without loss of T and B lymphocytes.

41Taken together, eMSC and MenSC have a range of effects on both arms of the innate and specific immune responses (Figure 3), however, the discrepancies between in vitro and in vivo findings and considerable variation between experimental models necessitate further investigation to identify the underlying mechanisms that orchestrate the cross-talk that MenSC and eMSC utilize to modulate the immune system. Standardization of key variables of human disease and disease models including immune versus non-immune nature of the disease, local versus systemic administration of cells, chronic versus acute disease, and the dose and timing of injected MenSC as well as MSC cell type (perivascular versus unfractionated stromal fibroblast populations) and experimental models as has been promulgated for bmMSC will further increase understanding of the distinct properties of eMSC and MenSC.

42Hundreds of clinical trials of various MSC have overall shown underwhelming results in regenerative medicine applications. This is due in part to the use of preclinical mouse studies using syngeneic mice, MHC-matched cells and achievable dosing. Large animal preclinical models are particularly important to trial MSC before going to the clinic to resolve these issues. To date long term engraftment of substantial numbers of infused MSC has not been demonstrated, rather only a small population (2–10%) remain in the days following administration. Allogeneic MSC will be rapidly removed by the innate immune system, and while autologous cells may prevail, few appear to integrate into tissues. This lack of MSC integration is due to the ischemic or inflammatory environment of the diseased tissue/organ and loss of vascular niches which may be replaced with fibrosis. Aged tissues and chronic disease can also hinder MSC integration. Despite these challenges, MSC can have dramatic effects through their paracrine actions, appearing to reset the innate immune system and promoting endogenous cellular repair without substantial integration.

43Since perivascular eMSC originate from a cyclically regenerating tissue with accompanying angiogenesis they are excellent candidates with therapeutic potential for tissue repair and possibly tissue regeneration. Their pericyte and perivascular identity indicate their specific roles in regulating angiogenesis, inflammation and fibrosis, suggesting eMSC are a good candidate for regenerative or reparative medicine.

44As mentioned above, human eMSC differentiate into decidual cells. This property has been harnessed for generating a tissue engineered uterus through repopulating decellularized extracellular matrix (ECM) constructs with cells, including endometrial stromal fibroblasts. This bioengineering approach has been conducted in decellularized uterus from rat, pig and human. Allogeneic endometrial stromal cells and bmMSC seeded into decellularized rat uteri diffused into the matrix and expressed the stromal marker vimentin and endothelial marker CD31, demarcated by cytokeratin expressing epithelial cells. Transplantation of these uterine constructs into partially excised uteri, regenerated endometrial tissue which differentiated into desmin-expressing decidual-cells and achieved pregnancies. There was evidence of fetal development, however, no evidence of placentation at the graft site. In another approach to reconstitute native, functional endometrium, a rat GFP+ endometrial epithelial cell-sheet was layered on two layers of adherent GFP+ endometrial stroma cell-sheets and transplanted into a full thickness endometrial defect in a rat model. Full thickness endometrium with luminal and glandular epithelium and stromal compartments was generated and pregnancy established on the regenerated endometrium with placentation and fetal heart movements detected. Preliminary results using similar approaches in a larger pig model have commenced. Using human endometrial Side Population cells including stromal cells demonstrated integration of vimentin+ and cytokeratin+ cells in the decellularized pig uterus. In an in vitro model, endometrial stromal cells, which likely contained a small population of eMSC, repopulated decellularized human endometrial substrates and proliferated in the scaffold. Most importantly, the cells underwent decidual changes upon stimulation with an estrogen and progesterone treatment protocol mimicking the human 28-day menstrual cycle. The infiltrated cells expressed estrogen and progesterone receptors, and secreted prolactin and IGFBP-1, markers of decidualization. These recent approaches indicate the feasibility of using eMSC and endometrial stromal fibroblasts as cell sources for regenerative medicine in uterine biology.

45The potential of eMSC in regenerative or reparative medicine has been explored for a common women’s gynecological disorder, pelvic organ prolapse. An autologous approach has been investigated and tissue engineering constructs comprising eMSC and novel non-degradable mesh, degradable nanofiber mesh, and 3D bioprinted mesh/eMSC have been explored in both rodent and large animal ovine models. In these studies, xenogeneic human eMSC delivered on a mesh in a model of subcutaneous wound repair, exerted a paracrine effect. DiO- or mCherry lentiviral-labeled eMSC implanted on a non-degradable polyamide/gelatin composite mesh, were detectable for 1–2 weeks in immunocompromised rats and mice and up to 3 days in immune intact mice. Despite this, the eMSC exerted marked paracrine effects, promoting early neovascularization, an anti-inflammatory response and supporting the deposition of physiological, crimped collagen rather than scar. These changes induced by eMSC resulted in a clinically relevant outcome, a reduction in the stiffness of the mesh/tissue complex in the long term (90 days). In studies using degradable PLACL nanofiber/gelatin or 3D printed PCL scaffolds, m-Cherry-labeled eMSC again reduced the foreign body response, slowed mesh degradation and induced endogenous cell influx into the scaffold in vivo, thereby promoting tissue repair. These are outcomes of clinically desired responses, particularly for application in pelvic organ prolapse repair surgery.

46To more accurately model the translational approach to be used clinically, autologous, adventitial perivascular ovine eMSC (Table 2), labeled with paramagnetic nanoparticles conjugated to FITC were surgically delivered with non-degradable polyamide mesh in an ovine model of vaginal repair. A two-step procedure was required, firstly implanting the mesh followed by separate delivery of the eMSC in a collagen gel onto the mesh and crosslinking with blue light in situ, followed by suturing. Approximately 10–20% of the autologous eMSC survived 30 days following implantation. As observed in the rodent models, the eMSC modulated the inflammatory response and reduced myofibroblast accumulation around mesh filaments. Important lessons were learnt using this large animal model, particularly related to the separate delivery of eMSC and mesh. This modification to our protocol prevented one of the major adverse events associated with transvaginal mesh use, mesh exposure, that had resulted in the banning of polypropylene vaginal mesh by the FDA. This could not have been predicted from using mouse models of skin wound repair and shows the importance of closely recapitulating the clinical condition in large animal models before clinical translation of an MSC-based therapy.

47Human and monkey endometrial stromal fibroblasts transdifferentiated into cells with morphological, chemical and electrical activity of dopaminergic neurons have been investigated in several animal models. They engrafted and migrated to site of lesion in chemically induced mouse and green monkey (Table 2) models of Parkinson disease, differentiated to neuronal-like cells resulting in increased striatal dopamine and dopamine metabolite concentrations. However, further studies on clinical outcomes such as the behavioral studies are warranted. Type 1 diabetes mellitus is a clinical condition which can benefit from islet-based cell transplantation. The plasticity of human endometrial stromal fibroblasts has been utilized to generate insulin secreting cells. These differentiated cells produced human insulin, decreased blood glucose levels and minimized diabetes-associated complications such as weight loss, dehydration, cataracts, delayed wound healing and sedative behavior in a mouse model of diabetes mellitus. These differentiated cells were more efficient in a 3D construct, and were resistant to oxidative stress, normalized glucose levels and prolonged survival of the recipient mice.

48Both eMSC and endometrial fibroblasts show potential in regenerative and reparative medicine, with early studies suggesting capability in tissue engineering approaches using decellularized tissues or biomaterial scaffolds to deliver the cells to tissues requiring regeneration. Mode of action may be both cellular integration and paracrine.

49MenSC are also an attractive cell source with potential for clinical application. MenSC have been assessed in various preclinical animal models of disease and in regenerative and reparative medicine (Table 5). Here, we will briefly review these studies.

50One of the first studies proposing the prospective use of MenSC differentiation in the clinical setting assessed the ability of MenSC to improve cardiac function after myocardial infarction. MenSC transplanted into a rat model ameliorated left ventricular systolic function and diminished fibrotic areas. In vivo differentiation of MenSC to cardiomyocytes in infarcted areas was demonstrated in the absence of recipient and donor cell fusion. Similar outcomes were obtained following MenSC injection into the ischemic zones of an immunocompetent rat model of myocardial infarction. The therapy resulted in significant preservation of myocardial viability in the infarct zone and improvement in cardiac function, effects mostly attributed to the paracrine activity of MenSCs rather than transdifferentiation to myocardial cells.

51MenSC’s ability to differentiate toward hepatic cells in vitro raised the possibility of MenSC application for treating liver disorders. Both MenSC and bmMSC, with the capacity to differentiate toward hepatocytes, prolonged the survival of mice with acute liver failure. MenSC localized in the damaged liver within 2 h following transplantation. This treatment also improved liver histology and architecture within weeks after MenSC injection. The MenSC therapy reduced serum levels of liver enzymes and metabolites (AST, ALT, urea, and total bilirubin) in the mice. Reduced hepatic degeneration, inflammatory cell infiltrate and collagen fiber deposition and improved glycogen storage were observed following MenSC administration, indicating the protective and reparative role of MenSC in acute liver damage.

52It is likely that MenSC improvement of liver function is due to paracrine activity rather than differentiating into hepatocyte-like cells in improving liver fibrosis. In a murine model, few MenSC differentiated into hepatocyte-like cells, suggesting the complexity of the in vivo milieu influencing MenSC differentiation. MenSC migrated to the fibrotic area, reduced already deposited collagen and significantly improved liver function. Nevertheless, end-stage liver fibrosis with few healthy hepatocytes may be a poor environment for survival of MenSC that also diminishes their ability to potentially differentiate into hepatocyte-like cells. This questions the utility of MenSC as a therapeutic modality to patients with cirrhosis. Further investigation in first reducing fibrosis in cirrhosis using MenSCs before attempting to regenerate hepatic tissue is warranted.

53The reparative capacities of MenSCs was exploited to treat premature ovarian failure (POF) in mouse/rat models. Intra-ovarian or intravenous injection of MenSCs reduced apoptosis and restored ovarian function, shown by expression of the ovarian markers, follicle-stimulating hormone receptor (Fshr), inhibin α/β, anti-Müllerian hormone (Amh), Ddx4 and Vegfa, and rising plasma levels of the ovarian hormones, estrogen and progesterone. Increased numbers of primary, mature and total ovarian follicle numbers were observed. DiI-labeled MenSC localized to GCs of immature ovarian follicles. Microarray analysis revealed greater similarity between mRNA expression patterns in the ovarian cells posttransplantation and human ovarian tissue than the pre-transplantation pattern in MenSCs, suggesting transdifferentiation into ovarian cells or fusion of MenSCs with murine cells. More importantly, mated mice grafted with MenSCs had more live births indicating the potential of MenSC to repair ovarian function. Mechanistic analyses revealed that mice treated with MenSCs improved ovarian microenvironment homeostasis through regulation of the ECM-dependent FAK/AKT signaling pathway. Similarly, in vitro, MenSC increased indices of mouse follicular growth, including survival rate, diameter and antrum formation, Bmp15 and Gdf9 expression and maturation rate in a 3D culture system. Secreted progesterone and estradiol increased in co-cultured murine preantral follicles and human MenSC implying a supportive role of MenSCs in follicle development, growth and maturation. In a recent clinical trial in 15 women, intraovarian injection of autologous MenSC in poor ovarian responders increased clinical pregnancy and live births.

54Collectively, MenSC impact on ovarian follicle development through paracrine signaling and potentially transdifferentiation highlights the possible utility of MenSC for ovarian function restoration in patients with POI and also fertility preservation approaches using in vitro follicle maturation. These pre-clinical findings and an early phase clinical trial warrant further investigation of the efficacy of MenSC for treatment of POI.

55MenSCs have been used to treat LPS-induced acute lung injury in a murine model. Intravenously administered MenSC localized to the injured area and demonstrated repair of lung tissue morphology, improved lung microvascular permeability, and reduced clinical symptoms. MenSC exerted an anti-inflammatory effect, reducing lung inflammatory cells and IL-1β, whereas IL-10 was increased in both lung tissue and broncho-alveolar lavage fluid. Keratinocyte growth factor, which plays a major part in repair of damaged lung was increased, and together with decreased caspase-3 expression further supported the protective impact of the infused MenSC. It is likely that the reparative effect of MenSC can be ascribed to cell-cell contact and/or their paracrine function. Similarly, MenSC injection improved acute lung injury scores through normalization of lung O2 pressure. MenSC reduced neutrophil frequency, myeloperoxidase activity, and pro-inflammatory cytokines levels in bronchoalveolar fluid. MenSC injection also reduced inflammation and collagen deposition in a mouse model of bleomycin-induced pulmonary fibrosis. In summary, in acute lung injury and pulmonary fibrosis models, MenSC suppressed innate immune cells, lowered pro-inflammatory cytokines and increased IL-10 in lung, and lung function and morphology were restored, effects likely mediated by paracrine mechanisms.

56The reparative potential of MenSCs in a murine excisional wound splinting model showed that intradermal injection of MenSCs into a persistent wound improved healing and increased angiogenesis. Two pro-angiogenic genes, Il-8 and Vegf were upregulated in the wounds and there was maturation of wound vasculature. The MenSC-injected group also showed high density, and well-organized collagen fibers, suggesting MenSC have potential application for wound healing and cutaneous regeneration. MenSC seeded on an amniotic membrane improved wound closure in a rat excisional wound defect model. In vitro, MenSCs differentiated into keratinocyte-like cells, an effect potentiated in 3D culture or in a biomimetic nanofibrous scaffold, two approaches that increased the wound healing capacity of MenSC. Such data imply the importance of ECM in the differentiation of MenSC into keratinocyte-like cells and further highlight the value of MenSCs as a potential therapeutic in clinical settings.

57The reparative potential of MenSC has been explored in a mouse model of endometrial injury and intrauterine adhesion, where the endometrium is partially replaced by fibrotic scar tissue. Intravascular injection of MenSCs rapidly repaired the injury by increasing microvessel density, resulting in increased endometrial thickness. Fertility was restored with a higher conception rate and larger numbers of implanted embryos compared to untreated controls. MenSC-conditioned medium promoted angiogenesis in vitro by activating Akt and Erk pathways and overexpression of genes involved in angiogenesis. Autologous cultured MenSCs administered to 7 women with severe Asherman’s syndrome, where intrauterine adhesions have replaced the endometrium, increased endometrial thickness to 7 mm in five of seven patients. Of the four patients who had undergone embryo transfer, two conceived and one had a spontaneous pregnancy, suggesting that MenSC reduced fibrosis and promoted functional endometrial repair of the injured endometrium.

58The application of MenSC, which can differentiate toward glia and neural cells in vitro (Table 3), for neuroprotection is an emerging research area. In an adult rat stroke model, intracerebral or intravenous MenSC transplantation ameliorated motor and behavioral symptoms and diminished neuronal cell death. Implantation of MenSC-seeded gelatin-based scaffolds in rats with sciatic nerve defect improved sciatic nerve function and reduced and gastrocnemius muscle loss to a similar level as bridging the nerve defect with autologous resected nerve segment. MenSC also improved cognition function and memory defects in a mouse APP/PS1 model of Alzheimer’s disease. In the hippocampus, more activated microglia were observed which had altered function as less TNF and IL-1β were produced. These activated microglia had higher expression of insulin degrading enzyme and neprilysin, proteases responsible for Aβ plaque degradation. These results highlight the potential of MenSC in improvement of the pathological and cognitive defects in pre-clinical models of Alzheimer disease.

59The regenerative ability of MenSC to promote muscle regeneration was demonstrated in a murine Mdx model of Duchenne muscular dystrophy, where skeletal muscles degenerate from lack of dystrophin. GFP-labeled MenSC were injected intramuscularly, where they fused with murine myoctes and restored dystrophin in the sarcolemma of muscle fibers. Similarly, MenSC fuse with myocytes in co-culture and subsequently express dystrophin.

60MenSC can stimulate the regeneration of pancreatic islet β cells in a murine model. MenSC transplantation enhanced differentiation of endogenous endocrine progenitor cells into β-cells resulting in more normal islet morphology and structure, with improved hyperglycemia, glucose tolerance, insulin levels, body weight and survival rate. Although not investigated inflammation reduced by MenSC may have also contributed to the observed results.

61In summary, MenSC show potential in regenerative medicine, with early studies suggesting capacity to influence tissue regeneration by paracrine mechanism, fusion and enhancement of endogenous tissue stem cell function.

62Increasingly, the effects of MSC are attributed to their ability to secret extracellular vesicles (EVs), including exosomes. MenSCs also produce functionally active EVs that are homogeneous in size (30–170 nM) and express CD81, CD63, and TSG101. MenSC-EVs contain 895 proteins involved in complement activation, antigen processing and presentation, regulation of adaptive and innate immune responses, apoptosis control and signaling pathways. Licensing MenSCs with pro-inflammatory cytokines such as IFN-γ modulates EV protein cargo, increasing proteins involved in antigen processing and presentation, and miRNA content. MenSC-derived EVs also express high levels of ICAM-1, angiopoetin-2, angiogenin, osteoprotegerin, IL-6, and IL-8. In a mouse model of fulminant hepatic failure, pre-treatment with MenSC-exosomes showed higher survival rates, with reduced serum liver enzymes and pro-inflammatory cytokines, and well-organized hepatic structure. Hepatocyte apoptosis and proliferation of liver macrophages were reduced. In a rat model of diabetes, MenSC-derived EVs were tracked in, and enhanced the number of pancreatic β cell islets and raised serum insulin, without impact on non-fasting blood glucose. MenSC-derived exosomes impacted wound healing in a mouse model of diabetes through M2 macrophage polarization, induction of neoangiogenesis and re-epithelialization.

63In summary, the beneficial impacts of MenSC treatment are mostly attributed to the anti-inflammatory properties of MenSC-derived EVs. In some settings the modulatory effects of EVs were superior to the use of intact MenSC. Therapeutic utilization of MenSC-EVs might be advantageous over whole cells by improving the therapeutic index, by introduction of less protein, avoidance of allo- and xenogeneic reactions and their off-the-shelf potential. Nonetheless, their nano-scale size results in rapid clearance from the body necessitating repeated administration in large quantities, although therapy targeted to damaged tissue may overcome this potential challenge. Taken together, MenSC may qualify as a promising therapeutic cell type for future clinical applications. Nevertheless, as with any other treatment modality, the safety of MenSC in the clinical settings needs addressing.

64There are hundreds of clinical trials using MSC either in progress or completed and thousands of patients have been treated. Apart from several spectacular successes the overall results of these clinical trials have been underwhelming. MSC were rushed to the clinic before many issues were resolved, including their mechanism of action and the heterogeneity of MSC products. Heterogeneity arises from variability in isolation methods and during culture expansion. Thawing cryostored MSC induces cell injury lasting up to 24 h, suggesting a lack of fitness for purpose of culture-expanded, thawed MSC. There is also a need for potency assays based on MSC mechanism of action in vivo. These are important considerations for investigators seeking to translate eMSC and MenSC as cell-based therapies.

65The clinical translation of human MSC depends upon largescale production of a homogeneous cell population that is safe and reproducible. Factors contributing to a heterogeneous population are isolation protocol, culture medium and culture environment. The discovery of a single surface marker for human eMSC, SUSD2, has enabled relatively easy purification of a homogeneous starting population. Choice of culture medium and environment majorly influence the generation of large scale homogeneous eMSC and MSC from other sources utilized for clinical use. Until recently, MSC including eMSC have been cultured in fetal calf serum (FCS) containing medium in 20% O2 environment. FCS provides extracellular matrix, growth factors, hormones and many other nutrients promoting cell grow and proliferation. However, FCS is not a defined source of nutrients with batch to batch variation and presence of unknown components that contribute to heterogeneity. Some FCS components induce differentiation, reducing the population of potent MSC. The use of FCS for in vitro MSC expansion also carries a rare risk of xeno-immunization and zoonotic transmission. Defined medium devoid of FCS is required to produce homogeneous, undifferentiated potent MSC using good manufacturing practices (GMP) for safe use in humans. Replacing FCS with human products including human serum, platelet-poor or -rich plasma and platelet lysate have been used. Although more physiological, these alternatives have similar composition variability as FCS and may also induce differentiation, resulting in inconsistent, non-reproducible cell products. Gene profiling of primary CD140b+CD146+ eMSC showed distinct differences to endometrial stromal fibroblasts (CD140b+CD146 –) and extensive eMSC cultivation in FCS medium in 20% O2 led down-regulation of 81% of eMSC-related genes and up-regulation of 55% of fibroblast-associated genes, verifying culture-induced spontaneous differentiation and reduced functionality. A defined medium and physiological O2 environment that generates homogeneous undifferentiated eMSC which are efficacious, safe and reproducible under cGMP guidelines would be appropriate for clinical applications such as pelvic organ prolapse and Asherman’s syndrome.

66While various serum-free media are available for culturing human MSC, vast research shows that “one size does not fit all,” indicting the need to identify the specific niche environment for eMSC. Indeed, eMSC attachment and growth were best supported by a fibronectin matrix in xeno-free DMEM supplemented with FGF2 and EGF (SFM) in 5% O2, compared to other serum-free commercial media, Lonza-TP-SF and Stem Pro-XF giving similar growth rates to serum-containing medium. Despite this, like all MSC, the expanded SUSD2+ eMSC spontaneously differentiated to non-clonogenic stromal fibroblasts with loss of the eMSC surface markers SUSD2, CD140b, and CD146 indicating cellular heterogeneity and decreased potency.

67Understanding the intrinsic signaling pathways involved in stem-cell fate, self-renewal, proliferation and differentiation, and manipulating them using chemical approaches enables generation of homogeneous potent cells for cell-based therapies. Chemically defined media needs optimization for each cell type using relative cell-growth as a guide. Augmenting SFM with a small molecule (A83-01) targeting the transforming growth factor-β1 receptor (TGFβR) signaling pathway involved in cellular differentiation and enhanced cell growth, mitigated loss of undifferentiated, clonogenic eMSC, overcoming this major bottleneck for clinical translation of eMSC. A83-01 maintained ISCT properties of eMSC, promoted proliferation of homogeneous SUSD2+ eMSC and prevented apoptosis and senescence. Correlation between SUSD2 expression and TGFβ-induced senescence and cell death was also demonstrated in cancer cells using small interfering RNA and TGFβ. The potency of A83-01-treated eMSC was further validated by transcriptome profiling, providing insight into the biological nature of eMSC and the probable therapeutic mode of action. The complexity of TGFβR signaling in eMSC was defined through identification of ∼1200 differentially regulated genes by A83-01 involved in anti-inflammatory responses, angiogenesis, cell migration and proliferation, collagen fibril and extracellular matrix organization, anti-fibrosis and anti-apoptosis. The potency of A83-01-treated eMSC was established by demonstrating increased expression of recently described bmMSC potency genes; TWIST1, TWIST2, JAG1, LIFR, and SLIT2. These and our previous findings also highlighted the need for additional surface markers to identify potent MSC and for specific tissues.

68The reparative and regenerative capacity of MSC is mainly driven by the secretome or EVs that promote angiogenesis, ECM regulation, immunoregulation and antimicrobial activity. Transcriptional profiling of A83-01-treated eMSC revealed important cellular secretome-regulating genes involved in angiogenesis, anti-fibrosis and immunomodulation; HGF, VCAM1, PGF, HPSE, SOD3, SOD2, and SOD1. Secretome analysis of the conditioned medium supported transcriptional findings with increased HGF, PTX3, CCL2, IGFBP-2, CCL2, GM-CSF, IGFBP-2, DKK-1, and EGF and decreased THBS1, EMMPRIN, and OPN. HLA-G, a nonclassical MHC-I molecule, and immunomodulatory cytokine functioning at the maternal-fetal interface and some immune privileged adult tissues, was ubiquitously expressed in A83-01-treated eMSC, together with HLA-A-C and HLA-E and F, but not HLA-II, an indicator of good tolerance for allogeneic transplantation. HLA-G has been identified in several MSC types, and together with other MHC-I molecules correlates directly to their immunosuppressive effect. These molecules are surrogate prognostic factors used to monitor disease progression or efficacy of transplantation. Likewise, RNA-sequencing revealed the first evidence of the ability of A83-01-treated eMSC to secrete exosomes, that may function in their paracrine action. Further transcriptome analysis showed evidence of the antimicrobial potential of A83-01-treated eMSC partially through the secretion of antimicrobial peptides (AMPs), chromogranin B, liver enriched AMP-2 and secretogranin II. Taken together, these findings signify the clinical potential of eMSC and open avenues to examine their use in regenerative medicine as an “off the shelf” as well as cell-derived therapy.

69Spontaneous transformation of MSC is a potential concern for therapeutics, although thorough investigations of earlier studies showed cellular-cross contamination. To date, no cancer due to primary-culture expanded MSC, has been diagnosed in human clinical trials. Nonetheless, the risk of such potential events should be investigated by utilizing surrogate assays, such as telomere length, pluripotency genes, tumor formation, genomic instability, karyotyping and DNA damage response through transcriptomics and in vitro and in vivo assays. A83-01-expanded eMSC promote telomere stability through TERC (Telomerase RNA Component), TERF1 and 2 (Telomeric Repeat Binding Factors 1 and 2), TINF2 (TERF1 Interacting Nuclear Factor 2), TERF2IP (TERF2 Interacting Protein), TNKS (Tankyrase) and POT1 (Protection of Telomere 1) which collaborate to regulate telomere length and protect cells from chromosomal damage. Using our isolation and expansion protocol, SUSD2+ eMSC do not express pluripotency-associated genes, unlike hESC and iPSCs with unlimited growth and tumorigenic potential. Although SFM/5%O2/A83-01 culture-expanded eMSC survived longer in vivo than culture in FCS/20%O2 medium, their lifespan was limited and tumors were not detected. Considerable progress has been made in developing expansion protocols for perivascular eMSC showing promise in their safety profile.

70Age of donor affects many of MSC functions; cytokine secretion, paracrine activity, anti-apoptotic mechanisms, hematopoietic stem cell supporting capacity and proliferation rate, all of which reduce with aging. However, the proliferative capacity of MenSC does not appear donor-dependent in individuals aged up to 40 years, but MenSC from older donors had a weaker potential for long-term passaging.

71Menstrual blood can be processed up to 72 h after collection without significant change in properties of the plastic adherent MenSC. As mentioned, culture expansion of MSC is required to obtain enough cells for preclinical or clinical applications. The proliferation rate of MenSC gradually decreased with increasing passage number, with highly passaged cells increasing in size, losing fibroblastic morphology and appearing senescent. Important signaling molecules, including MAPK, molecules involved in carcinogenesis (PPAR and P53) and immune responses changed with MenSC passage number. Passaging induces aging of MenSC and alters genes involved in transcriptional regulation, stress response, cell proliferation, development and apoptosis, and karyotype at high passages (P20). MenSC underwent aging after 45 PD, but even after 68 PDs of a single MenSC sample, maintained a normal karyotype and did not develop tumors. Multiplex ligation-dependent probe amplification of MenSC genomic DNA at passages two and twelve showed that MenSC maintained a diploid phenotype without chromosomal aberrations, consistent with other reports of a normal female karyotype without chromosomal aberrations using standard cytogenetics at passage 12. Pluripotency protein expression reduced during passaging of MenSCs, from 97.5% OCT-4+ cells at the first passage to 19.4% at passage twelve, important for clinical translation. These observations warrant standardization of MenSC isolation and culture protocols for clinical applications.

72The risk of tumorigenicity, toxicity, autoimmunity, and/or endometriosis following MenSCs injection are crucial safety issues to be established before using MenSC in the clinic. Angiogenic activity of MenSC, raises concern of promoting tumor progression. In a C6 model of rat glioma MenSCs not only failed to stimulate, but also inhibited tumor growth. MenSCs showed no tumor formation in a xenograft rat model of brain stroke with no concurrent immunosuppression. Several acute and chronic tumorigenicity/toxicity experiments in immunocompromised mice using MenSC in clinically relevant doses showed no toxic effects on body and organ weight, biochemistry following necropsy or histopathological changes at injection sites. Concerns of ectopic bone formation on clinical application of bmMSC, are mitigated by the weak osteogenic differentiation capacity of MenSC and could be advantageous for clinical translation.

73The next step prior to clinical development of MenSC is to undertake cell tracking of reliably labeled MenSC in vivo and determining their mechanism of action. Also important is the development of relevant large animal pre-clinical models of the targeted disease for evaluating MenSC, to improve the likelihood of translating rodent studies into clinical practice. In 2008, four multiple sclerosis patients were injected both intrathecally and intravenously with in vitro expanded allogeneic MenSC. Functional and radiological evaluations demonstrated no disease status progression and none of the treated patients showed allergic reactions or ectopic tissue formation. No adverse effects were observed in the patients 4 years later in the last reported follow up. Clearly more investigation is required to determine the safety of MenSC for clinical translation.

74Identification of eMSC potency makers and serum-free expansion media have enabled gene sequencing of the primary cells and their expanded progeny. Comprehensive RNA-seq analysis of SFM/5%O2/A83-01-treated eMSC described the gene expression profiles and uncovered vast range of regulatory pathways which not only aided in understating their mechanism of action but also suggested their potential for broader applications. Much progress are made in understanding the in vitro and in vivo roles of eMSC, setting a strong foundation for clinical application. Further research to understand and validate eMSC’s potential through careful titration of assays and selection of animal models relative to their potential use is vital.

75eMSC and MenSC have fulfilled the ISCT criteria for MSC, but thus far, no comparative study has been performed to delineate the potential link between these two MSC types. Nonetheless, considering that eMSC comprise a small cell population residing around endometrial blood vessels while MenSC represent majority of endometrial stromal cells, it seems that the latter is more heterogeneous. Although multi-differentiation capacity of eMSC and MenSC has been documented in vitro, the reparative properties of these cells in vivo seem to stem from their paracrine rather than differentiation capacity and integration into tissue. Akin to MSC from other sources, MenSC and eMSC have profound immunomodulatory effects and attenuate inflammation, although there are distinct differences highlighting their unique tissue of origin. eMSC and MenSC have been evaluated in numerous preclinical animal models of disease showing substantial angiogenic, anti-fibrotic and anti-inflammatory effects although it is too early to draw a firm conclusion on the same impacts in clinical settings. Culture protocols have been developed that maintain perivascular eMSC in the undifferentiated state during culture expansion, which now need testing in animal models to determine if this more homogeneous MSC product further improves in vivo angiogenesis, antifibrosis, anti-inflammatory and prohealing effects. Both eMSC, easily procured without anesthetic, and non-invasively obtained MenSC, imbued with both reparative and immunomodulatory properties are likely promising therapeutic cell types for future clinical applications. Nevertheless, multiple variables need to be standardized particularly heterogeneity of MenSC and the safety and therapeutic potency of eMSC and MenSC in clinical settings needs to be extensively addressed.

76CG and A-HZ contributed to the conception and review design and revised the manuscript. MB, SG, SD, SN, SK, A-HZ, and CG wrote sections of the manuscript. All authors contributed to the article and approved the submitted version.

77The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.