Background

Mesenchymal stromal cells (MSCs) are nonhematopoietic progenitor cells primarily located in umbilical cord, bone marrow, and adipose tissue. MSCs have the potential to differentiate into multiple lineages, including chondrocytes, adipocytes, as well as osteoblasts [1, 2], and are thus considered a promising tool for cell-based regenerative therapy [3]. Data from preclinical studies demonstrate that an intrinsic immunosuppressive capacity of MSCs constitutes a major part of their therapeutic effects [4, 5]. In addition, their low immunogenicity, due to a modest expression of MHC-I and a complete lack of MHC-II [6, 7] and co-stimulatory molecules, helps them to avoid immune surveillance [8].

While MSCs can be isolated from patients and re-applied as autologous MSCs to avoid immune rejection, certain types of diseases restrict patients from supplying MSCs by themselves. For example, myelofibrosis impairs the quality of bone marrow-derived MSCs (BM-MSCs) [9], and systemic diseases, such as diabetes, RA, and SLE alter the intrinsic properties of MSCs [10,11,12]. Patient age also heavily impacts the availability and functionality of MSCs, as it is generally difficult to procure and extract MSCs from infants, while MSCs from the elderly display decreased biological activity and hence deficits in differentiation and immunoregulation potential [13,14,15,16]. In addition, acute diseases such as stroke and myocardial infarction do not allow enough time to extract and expand autologous MSCs and instead need ready-to-use products. Allo-MSCs from young healthy donors are a plausible approach to overcome these difficulties. Indeed, transplantation of human BM-MSCs has been approved for the management of refractory acute GVHD in children unresponsive to systemic steroid therapies. Currently, they are also tested in clinical trials for Crohn's disease [17], GVHD [18, 19], epidermolysis bullosa [20], COVID-19 [21], and for the repair of heart tissue following myocardial infarction [22]. So far, initial results showed that they are well tolerated.

Menstrual blood, monthly shed by women above twelve to fifteen years of age, contains self-renewing stromal cells. In 2007, Meng et al. first isolated MB-MSCs and confirmed their MSC properties, including MSC surface marker expression, self-renewal, and trilineage differentiation potential [23]. MB-MSCs can be collected regularly and non-invasively, providing important potential for biobanking. With regard to stromal cell biological properties, MB-MSCs are comparable to other MSCs with a high proliferation rate [23]. Recent work from the group of Li and her collaboration partners has essentially proven the safety and efficiency of MB-MSCs as allo-MSCs in treating COVID-19 patients [24]. To date, no study has evaluated the therapeutic effects of MB-MSCs in preclinical models of MS.

MS is a neurodegenerative autoimmune disease affecting the central nervous system [25]. It is the most common disabling neurological disorder in young adults and the third largest cause of significant disability for adults between 20 and 40 years [26, 27]. Despite the use of new immunomodulatory agents such as Natalizumab, most patients eventually enter relapsing–remitting phases, accompanied by vicious immunodeficiency complications due to unspecific immune cell depletion or inhibition [28]. Moreover, these therapeutic agents lack the capacity to promote remyelination and therefore the potential for repairing patients´ neurological function [29, 30].

Aiming to overcome these critical drawbacks of current MS therapies, we evaluated the therapeutic effects of MB-MSCs—which hold, among other benefits of MSCs, the advantage of non-invasive and periodical acquisition—in the murine MS model EAE. We first show the disease-ameliorating function of MB-MSCs when transplanted at various stages of EAE and via both intravenous and intraperitoneal routes. Further, we found that the disease-ameliorating effect of MB-MSCs was associated with suppressed inflammatory immune responses in both peripheral lymphoid organs and the CNS, represented by repressed APC activity, lower frequencies of Th1 and Th17 cells, and fewer lymphocytes infiltrating the CNS. Lastly, we show that MB-MSCs had therapeutic effects similar to UC-MSCs. Thus, MB-MSCs have the potential to be developed as a read-to-use allo-MSC therapeutic agent.

Methods

Mice and EAE induction

We used female C57BL/6 mice, 8–12 weeks of age at the start of experiments. Mice were housed in the animal facilities of ** MS [51, 52]. Therapeutic effects of MSCs are more desirable as transplantation could be used as a curative approach. Our data demonstrate that MB-MSCs can suppress EAE when transplanted on day six or ten after EAE induction. Even when transplanted on day 19, MB-MSCs provided a rapid improvement in the recovery phase in this model. Whether this translates into a potential therapeutic effect remains to be tested in detail and could be assessed in non-remitting chronic EAE disease models. Overall, we show that MB-MSCs achieved a similar protective effect as UC-MSCs and thus should be considered a candidate for ready-to-use allo-MSC products.

The administration route determines the microenvironment that MSCs first encounter in recipients and may thus influence their immunosuppressive mechanisms. Intravenous injection is most common due to its convenience; however, MSCs administered via this route are easily trapped in lung capillaries and thus may fail to enter the peripheral immune system [31]. Further, MSC injection via the i.v. route is accompanied by the risk of instant blood-mediated inflammatory reactions that compromise safety and therapeutic efficacy [3, 53, 54]. As an alternative systemic delivery, MB-MSCs can be transplanted via the i.p. route. In this way, the cells are not trapped in the lungs and do not cause hemocompatibility-related issues, while they still generated a comparable protective effect. The main mechanisms of action of MSCs in supporting tissue regeneration and immunomodulation are cell-contact-dependent or -independent mechanisms, mainly mediated via the secretion of trophic and immunomodulatory factors [3, 53, 55]. Hence, the fact that both i.v.- and i.p.-delivered MB-MSCs are therapeutically beneficial provides broader opportunities for MSC administration.

The exact mechanisms by which MB-MSCs mediate their beneficial outcomes have remained ill defined. Our data demonstrate an association of ameliorated disease with suppressed Th1 and Th17 cell responses in the periphery. Interestingly, MB-MSCs delivered i.v. tended to display a stronger suppression of Th1 responses, while MB-MSCs delivered i.p. featured a stronger suppression of Th17 responses. The MB-MSC-induced suppression of CD4 T cell responses was probably mediated by reduced APC activity: The presence of pDC in the spleen was significantly suppressed by both i.v.- and i.p.-delivered MB-MSCs. pDC plays an important role in initiating EAE via promoting the priming of Th1 and Th17 cells [41], so their numeric reduction could explain limited Th1 and Th17 responses in MB-MSC-transplanted mice. CD40 co-stimulation induces IL-12 production and results in the induction of Th1 responses [56, 57]. APCs, especially those of mice that had received MB-MSCs i.v., showed reduced CD40 expression. Thus, this lack of CD40 may provide a molecular explanation of the reduced Th1 response. In addition, surface expression of the co-stimulators CD80, CD86, and OX40L was also reduced on APCs of MB-MSC-transplanted mice.

Conclusions

Human menstrual blood-derived MSCs, which can be isolated non-invasively and could be stored for acute application, ameliorated the disease severity of EAE upon transplantation, regardless of the route or the time of delivery, by suppressing T cell activation in peripheral lymphoid organs and immune cell infiltration into the CNS.