Method for isolating menstrual stem cells

Abstract

The invention is directed to a method for isolating menstrual stem cells (MenSC) comprising the steps of : a. collecting menstrual blood comprising a heterogenous suspension comprising a mixture of cells including menstrual stem cell s in a non-altered state; b. processing said heterogenous suspension through density gradient centrifugation in order to isolate a homogenous suspension comprising a mixture of mononuclear cell s including said menstrual stem cells; c. adding fluorescent markers to said homogenous suspension in order to specifically tag menstrual stem cell s, and subsequently d. running the homogenous suspension with the tagged menstrual stem cell s obtained in step c. through flow cytometry in order to isolate menstrual stem cells from the homogenous suspension, wherein the isolated menstrual stem cell s are in the non-altered state.

Description

Title of the invention: Method for isolating Menstrual Stem Cells The present invention is relative to a method for isolating Menstrual Stem Cells. Menstrual blood-derived stem cells, or more commonly "Menstrual Stem Cells" (MenSCs) are a novel and promising source of stem cells used in many fields, including regenerative medicine. MenSCs are Mesenchymal Stem Cells (MSCs) that have a multipotent differentiation capability. MenSCs were first identified and characterized in 2007 (Meng et al., 2007) and have since then attracted significant attention due to their unique properties. MenSCs are found in the menstrual blood discharge and are referred to as endometrial Mesenchymal Stem Cells (eMSCs) when they reside in the perivascular region of both the basalis and functionalis of the endometrium. These cells may play an important role in the cyclic regeneration and repair of the endometrium. Compared to stem cells from bone marrow and adipose tissues, MenSCs have several advantages. One major advantage is that MenSCs are probably the easiest stem cells to collect from the human body. Further, when compared to other cell types (i.e. non-stem cells) advantages of MenSCs reside in the high proliferation rate, the colony forming unit frequency, the migration capacity and the multilineage differentiation capacities. Aside from these advantages, the potential applications of MenSCs in regenerative medicine are vast. MenSCs have been studied for their potential use in treating a variety of diseases including liver disease, diabetes, stroke, Duchenne muscular dystrophy, ovarian-related disease, myocardial infarction, Asherman syndrome, Alzheimer's disease, acute lung injury, cutaneous wound, endometriosis, and neurodegenerative diseases (Chen et al., 2019). In order to study MenSCs, or more generally MSCs, cell culture is commonly used. Cell culture is a process by which cells are grown under controlled conditions, generally outside of their natural environment, i.e. in vitro. Thus, during cell culture of MSCs, the number of cells is increased by expanding the MSCs in vitro. This in vitro expansion of MSCs is standard practice in cell biology, regenerative medicine and other research areas. However, a cell culture is generally a long and costly process that is accompanied by risks. A major risk for instance is the contamination of the cell culture. This has a negative, often fatal, effect on the study results or the cells themselves. Further, long-term in vitro expansion of MSCs, lead to significant changes in their properties, including alterations in gene expression, in genomic stability and / or in immunomodulatory properties. Also, epigenetic changes, and other morphological and / or functional alterations in MSCs have been observed. The fact that original MSCs (e.g. MenSCs) are altered during cell culture has negative implications for the therapeutic potential of the cells. The changes during cell culture make it impossible to provide accurate results with regard to MenSCs in their original nonaltered state. The present invention improves the situation. To this end, the invention is directed to a method for isolating menstrual stem cells (MenSC) comprising the steps of: a. collecting menstrual blood comprising a heterogenous suspension comprising a mixture of cells including menstrual stem cells in a nonaltered state; b. processing said heterogenous suspension through density gradient centrifugation in order to isolate a homogenous suspension comprising a mixture of mononuclear cells including said menstrual stem cells; c. adding fluorescent markers to said homogenous suspension in order to specifically tag menstrual stem cells, and subsequently d. running the homogenous suspension with the tagged menstrual stem cells obtained in step c. through flow cytometry in order to isolate menstrual stem cells from the homogenous suspension, wherein the isolated menstrual stem cells are in the non-altered state. Thus, the invention is deprived of any cell culturing process. The method of the invention (from the first step to the last step) does not comprise any cell culturing step or process. Step d. is carried out directly subsequently of step c. Step a. can be carried out by means of a menstrual cup. The density gradient centrifugation is preferentially a Ficoll-Paque method. In a preferred embodiment, the fluorescent markers used in step c. comprise a set of positive markers selected from the group consisting of CD90, CD73, CD105, CD44 and a combination thereof. In another preferred embodiment, the fluorescent markers comprise a set of negative markers selected from the group consisting of HLA-DR, CD45, CD14, CD34, CD235a, CD19 and a combination thereof. In yet another preferred embodiment, fluorescent markers comprise both the above positive and negative markers. In another embodiment, the method of the invention consists of steps a . , b., c., and d. Other features and advantages of the invention will stand out and / or become clear upon reading the following description, which comprises specific examples given in an illustrative and non-limiting manner, as well as from the drawings in which: [Fig. 1] shows photographs from Menstrual Blood Mononuclear Cells isolation using Ficoll-Paque Density Gradient Centrifugation; [Fig. 2] shows FACS plots from the isolation of Menstrual Stem Cells (MenSC) from non cultured Menstrual Blood Mononuclear cells; and [Fig. 3] shows a diagramm comparing the number of MenSCs per mililiter of menstrual blood of a sample with and without hormone treatment The drawings and the description herein contain, for the most part, elements of definite nature. Therefore, description and drawings not only are being used to better understand the present invention, but also to contribute to the definition therefor, when appropriate. MenSCs are obtained from menstrual blood. The common procedure for collecting menstrual blood is to use a so called menstrual cup. The menstrual cup is a device designed to be inserted into the vagina. The collection is mostly scheduled during the heaviest flow of the menstrual cycle. A cup generally will remain in place for about 4 to 12 hours. Once the menstrual effluents are procured, the menstrual cup is removed and the blood is transferred to sterile media / PBS with antibiotics . The isolation of stem cells from the blood can be done by density gradient centrifugation. More generally, density gradient centrifugation techniques, such as the so called Ficoll-Paque technique, manage to isolate all mononuclear cells from the collected menstrual blood. During centrifugation, erythrocytes and granulocytes sediment to the bottom layer, leaving the mononuclear cells easily isolated. In the state of the art, the mononuclear cells, and in particular MenSCs, exclusively undergo cell culture. Often, the cells are retrieved from menstrual fluid as plastic adherent cells, similar to bone marrow MSC (bmMSC). Yet, MenSCs are always prepared and placed into cell culture, cf. Meng et al., 2007; Borlogan et al., 2010; Patel et al., 2008; Allickson et al., 2011; Cui et al., 2007; Khanjani et al., 2014; Chen et al., 2017; Chen et al., 2017; Zhao et al., 2018; Xiang et al., 2017; Khanmohammadi et al., 2014; Nikoo et al., 2014; Alcayaga-Miranda et al., 2015; Khanjani et al., 2015; Lai et al., 2015; Ren et al., 2016; Tan et al., 2016; Moreno et al., 2017; Wang et al., 2017; Cuenca et al., 2018; Liu et al., 2018; Liu et al., 2018; Xu et al., 2023; Du et al., 2016; de Pedro et al., 2023; Faramarzi et al., 2016; Darzi et al., 2012; Li et al., 2023; Li et al., 2019; Yang et al., 2023; Davoodi Asi et al., 2023; Manshori et al., 2023; Chen et al., 2023 ; Mirzadegan et al., 2023; Zhou et al., 2023; Roodbari et al., 2023; Lian et al., 2023; Manshori et al., 2023; Hojjat et al., 2023; de Pedro et al., 2023; Izanlou et al., 2023; Zafardoust et al., 2023; Yang et al., 2022; Hu et al., 2022; Miller et al., 2022; Sun et al., 2022; Fu et al., 2022; Mahdipour, 2022; Manshori et al., 2022; Mirzadegan et al., 2022; Sun et al., 2022; He et al., 2022; Li et al., 2022 ; Qiu and Tan, 2022; Sahraei et al., 2022; Hao et al., 2022; Skliute et al., 2021; Dalirfardouei et al., 2021; Wang et al., 2021; Zhang et al. 2021; Yamchi et al., 2021; Arezoo et al., 2021; Sheikholeslami et al., 2021; Uzieliene et al., 2021; Aleahmad et al., 2021; Ghanavatinejad et al., 2021; de Pedro et al., 2021; Chang et al., 2020; Lopez-Caraballo et al., 2020; Chen et al., 2020; Ma et al., 2020; Gonqalves et al., 2020; Sun et al., 2019; Fathi-Kazerooni and Tavoosidana, 2019; Sun et al., 2019; Rosenberger et al., 2019; Yan et al., 2019; Varas-Godoy et al., 2019; Mahdipour et al., 2019; Dalirfardouei et al., 2019; Cen et al., 2019; Guo et al., 2019; Hu et al., 2019; Shokri et al., 2019; Liu et al., 2019; Li et al., 2019; Manshadi et al., 2019; Zhu et al., 2019; Wu et al., 2019; Fathi-Kazerooni et al., 2019; Feng et al., 2019; Wang et al., 2019. Processing of MenSC in the art is thus inevitably linked to cell culture procedures . The Applicant uses a radically different approach to isolate MenSCs for subsequent studies. More particularly, the Applicant by-passes any cell culture in order to study the MenSCs in a non-altered state (sometimes referred to as MenSCs in native state). In fact, cell culture is a milestone of modern biology. Even more, cell culture has revolutionized our understanding of diseases and their treatment. However, as mentionned above, in vitro expansion of cells, and in particular Mesenchymal Stem Cells (MSCs), can lead to significant changes in their gene expression, genomic stability, and immunomodulatory properties. These changes have profound implications for the therapeutic potential of MSCs. Even more, all studies that use cell culture may be flawed by the fact that the studied cells are altered. Said otherwise, the cells are not in an original state after cell culture. This may lead to signficant misinterpretation and / or misunderstanding of study or research results. Alteration of the cells during cell culture can come from, (i) epigenetic changes, (ii) genetic changes, (iii) proteomic changes and / or (iv) morphological changes. All thoses changes, taken alone or together, have the result that MSCs before cell culture (in their original non-altered state) are very different from MSCs after cell culture (altered state). (i) Epigenetic changes Epigenetic modifications, such as DNA methylation (DNAm) and histone modification, play a role in the development, differentiation, and aging of cells and organisms. In the context of MSCs, cell culture is associated with specific DNAm changes, particularly in developmental genes and homeobox genes (Redaelli et al., 2012; Schellenberg et al., 2011; Koch et al., 2013; Bork et al., 2010) . These senescence-associated DNAm (SA-DNAm) changes are enriched in intergenic regions and appear to be associated with repressive histone marks (Schellenberg et al., 2011; Koch et al., 2012) . These findings suggest that cell culture of MSCs is associated with a tightly regulated epigenetic program (Wagner, 2012). The mechanisms underlying these changes are not fully understood. Some documents (Franzen et al. 2021) suggest that the changes are random and undirected, and that they may accumulate over time during cell culture due to the absence of selective pressure. (ii) Genetic changes The document Jeske et al., 2021 discloses that human adipose-derived mesenchymal stem cells (hASCs) undergo a shift from an antiinflammatory to a pro-inflammatory phenotype during in vitro expansion (i.e. cell culture) . Additionally, some hASCs show decreased mRNA levels for genes responsible for osteogenic and adipogenic differentiation, as well as genes involved in cell signaling pathways that are correlated with the pro-inflammatory phenotype. Other studies have found similar changes in gene expression during in vitro expansion of MSCs from different sources. For example, the document Gu et al., 2016 discloses that P21, P53, and APEl / Ref-1 mRNA levels are significantly increased in the middle and late phases of in vitro expansion of human umbilical cord mesenchymal stem cells (hucMSCs). The document Xiang et al., 2019 discloses that passage 6 and passage 10 BMSCs (note that the word "passage" refers to transferring cells to new recipient with fresh medium to maintain optimal density for growth) consistently express a distinct set of genes, including genes involved in the cell cycle, DNA replication, and oocyte meiosis. The document Redaelli et al., 2012, examined the genomic stability of human bone marrow mesenchymal stem cells (hBM-MSCs) during in vitro expansion. They found that hBM-MSCs generally have a normal karyotype, but there is a variable trend towards random chromosome losses and, in rare cases, clonal aneuploidies. Telomere length decreases with increasing passage number. Other documents disclose evidence of genomic instability during in vitro expansion of MSCs. For example, Jiang et al., 2017 discloses that passage 3 BMSCs have downregulated cell cycle regulation, DNA replication, and mismatch repair pathways. Additionally, several genes associated with telomerase activity and chromosomal stability are also markedly suppressed in passage 3 BMSCs. Further, cell culture of MSCs showed an accumulation of yH2AX foci, a well-known marker of genomic instability. As a result, selecting the appropriate passage is a critical procedure before transplanting allogeneic MSCs into recipient patients, since in vitro propagations can cause MSCs to acquire genetic changes that can lead to malignant transformation (Pustovalova et al., 2016; Al-Azab et al., 2022) . (iii) Proteomic changes Protein expression changes in human adipose-derived mesenchymal stem cells (hASCs) and bone marrow mesenchymal stem cells (BMSCs) during in vitro expansion. The document Jeske et al. 2021 discloses that in vitro expansion of hASCs leads to global alterations in protein expression, affecting key functions such as cell morphology, assembly, and organization; carbohydrate metabolism; small molecule biochemistry; and post-translational modification. The document Xiang et al. 2019 disclosed that passage 6 and passage 10 BMSCs show differences in protein levels of conserved genes involved in the cell cycle, DNA replication, and oocyte meiosis. Weighted gene coexpression network analysis confirmed these findings and also revealed how cell passaging affects the irradiation response in BMSCs. (iv) Morphological changes Human adipose-derived mesenchymal stem cells (hASCs) undergo morphological transformations and exhibit changes in their functional properties during cell culture (i.e. in vitro expansion). They transition from a spindle shape to a flattened and elongated appearance. Additionally, the colony-forming units ability (CFU) decreases significantly in higher passages (passage 12) compared to earlier passages (passage 5) . Moreover, the increase in SA-[3-Gal activity indicates enhanced senescence in passage 12 hASCs. Notably, the energy metabolism and NAD-Sirt pathways associated with cellular senescence remain stable throughout hASC in vitro expansion (Jeske et al., 2021). In another document, hASCs initially maintain low aneuploidy levels in early passages (passage 0 - passage 4). However, extended culture expansion (5-16 passages) results in a significant increase in aneuploidy percentages, without triggering malignancy (Roemeling-van Rhijn et al., 2013). In the case of human umbilical cord mesenchymal stem cells (hucMSCs), some documents disclose a replicative senescence during prolonged in vitro culture. These cells undergo morphological changes that include a reduction in the number and length of microvilli and swelling of the nuclei. Proliferation rates of hucMSCs also decline over time. Additionally, there's an increase in senescence, G0 / G1 cell cycle arrest, and reduced differentiation capacity. A document discloses that hucMSCs experience replicative senescence during extended in vitro culture (Gu et al., 2016) . Additionally, human embryonic stem cells exhibit unstable chromosomal alterations in differentiated MSCs, leading to replicative senescence after prolonged culture (Karagiannidou et al., 2014) . Another document discloses differences in the percentage of apoptotic cells and cell cycle distribution (Xiang et al., 2019). Bone marrow stromal cells (BMSCs) were subjected to in vitro passaging and irradiation at two stages, passage 6 and passage 10. Comparing passage 6 and passage 10 BMSCs, differences in the percentage of apoptotic cells and cell cycle distribution were observed. Passage 6 BMSCs exhibited lower apoptosis rates, a reduced proportion of cells in the S phase, and a higher proportion in the G1 phase than passage 10 BMSCs. Also, BMSCs at passage 3 exhibited lower chondrogenic potential and higher genomic instability than freshly isolated bone marrow mononuclear cells (BMMNCs). This trend was consistent both in the context of cartilage repair and in vitro settings. Passage 3 BMSCs also demonstrated decreased telomerase activity and alterations in chromosomal structure, indicative of cellular senescence (Jiang et al., 2017) . After the first passage, BMSCs display a marked decrease in their proliferation rate and gradually lose their multidifferentiation potential. Their in vivo bone-forming efficiency decreases significantly compared to fresh bone marrow (Banfi et al., 2000) . Typically, mesenchymal stem cells (MSCs) exhibit a spindle-shaped morphology with a small cell body, few slender processes, and a large nucleus with a differentiated nucleolus during in vitro culture. However, with prolonged culture, these MSCs undergo morphological changes, becoming larger and losing their spindle-like characteristics, often appearing flattened. Notably, extended in vitro expansion-induced senescence of adipose tissue MSCs results in morphological alterations, including increased size and complexity, giving them a "fried-egg-like" appearance (Truong et al., 2019). Reactive Oxygen Species (ROS) presence negatively impacts the ability of MSCs to suppress immune cells, such as T cells. Oxidative stress plays a pivotal role in the replicative senescence of MSCs, limiting their number of passages and their cellular potency (Denu and Hematti, 2016; Al-Azab et al., 2022) . Furthermore, a decline in both ALP activity and calcium deposition is observed in MSCs at passage 2 in comparison to passage 1. This is indicative of a decrease in proliferation and a decrease in the capability of MSCs with aging (Chen et al., 2005). Some of the above described references do not directly concern studies with MenSCs. However, they do at least concern other types of MSCs. Given the fact that MenSCs are particular MSCs, it is highly likely that said (i) epigenetic, (ii) genetic, (iii) proteomic and / or (iv) morphological changes direcly affect MenSCs. More generally, in vitro expansion (or cell culture) MSCs is a common practice in cell biology and regenerative medicine. However, the above describes that cell culture can lead to significant changes in the cells, including alterations in gene expression and genomic stability, epigenetic changes and other morphological and functional alterations. The present invention manages the isolation of fresh MenSCs. By fresh MenSCs it is meant cells that have non been altered in any way by cell culture. The MenSCs isolated with the present invention are in its original state. The MenSCs obtained with the present invention have the physiological properties as they are found in the human body. They have not been altered by (i) epigenetic, (ii) genetic, (iii) proteomic and / or (iv) morphological changes. The invention thus provides cells that are highly relevant for research in any field, and in particular in the fields of therapeutics and / or regenerative medicine. The cells of the invention have not been modified by any cell culture process, for exemple with regard to methylation sites. Freshly isolated MenSCs, i.e. non altered MenSCs, are at least close to their natural state. They are isolated before any cell culture, and consequently are spared the potentially transformative effects of in vitro expansion. This means that they retain their original gene expression patterns and genomic stability, making them more effective for therapeutic and / or diagnosis applications. Moreover, freshly isolated cells have not been exposed to the artificial environment of a culture medium, which can induce stress responses and other changes in cells. They are also free from the risk of contamination that comes with cell culture. Another advantage is that freshly isolated cells can provide a more accurate reflection of the cell population in the body. In contrast, cultured cells may over-represent certain subpopulations due to differential growth rates. The invention uses Fluorescence-Activated Cell Sorting (FACS). FACS is a specialized type of flow cytometry that provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. It is a known scientific operation as it provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest. The use of FACS in the present invention for isolating MenSCs involves four main steps: (1) Sample Preparation, (2) Cell Labelling, (3) Cell Sorting, and (4) Cell Collection. (1) Sample Preparation: Menstrual blood is collected, typically using a menstrual cup. The collected sample is then processed to isolate mononuclear cells through density gradient centrifugation, such as Ficoll-Paque. (2) Labeling Cells with Fluorescent Markers: Following cell isolation, the invention uses specific fluorescent markers for the identification and isolation of MenSCs. These markers target MenSC-specific surface antigens, such as CD90, CD73, CD105, and CD44, while also excluding unwanted cell populations in the sample by targeting markers like HLA-DR, CD45, CD14, CD34, CD235a, and CD19. (3) Cell Sorting with FACS: The labeled cell suspension is loaded into the FACS machine. As the cells pass through the laser beam of the machine, they will produce a fluorescent signal. The FACS machine detects this fluorescence and separates the cells depending on their signal. (4) Cell Collection: The sorted MenSCs are then collected for further use . The present invention is deprived of any cell culture. The invention provides a simple and reliable collection source of freshly isolated MenSCs for later use. The invention enables cells that come directly from a patient to be processed for analysis, thus avoiding the addition of intermediate cell culture processes that alter the cells of interrest. Altough in vitro expansion has its place in cell biology and regenerative medicine, the use of freshly isolated, non-altered, cells offers a valuable enhancement in these technical fields. The invention provides a more accurate reflection of in vivo conditions, and thus improves the developpement of safer and more effective therapeutic strategies . The state of the art discloses that MenSCs may be identified and / or isolated by specific cell markers. However, the specific markers disclosed in the art are used only and exclusively after cell culture. The present invention uses the knowledge from the opposed prior art teachings regarding the cell markers. In fact, MenSCs possess the classic International Society for Cellular Therapy (ISCT) markers. They are positive for MSC markers including CD29, CD44, CD73, CD90, CD105 and STRO-1. They do not express hematopoietic lineage markers such as CD34, CD45, CD133, CD14, CD38, and human leukocyte antigen-DR isotype (HLA-DR). In a preferred embodiment of the invention, the fluorescent markers are selected from the group consisting of CD90, CD73, CD105, CD44, HLA-DR, CD45, CD14, CD34, CD235a, CD19 or any combination thereof. CD90 is a marker found on the surface of T cells, hematopoietic cells, and MSCs, including MenSCs. Its presence is key in confirming the identity of these cells. CD73 is an enzyme known as ecto-5'-nucleotidase and plays a role in the migration of MSCs. It is an important marker for identifying MSCs, including MenSCs. CD105 is a marker associated with tissue and Mesenchymal Stem Cells (MSCs). Its presence serves to identify MenSCs and other MSCs. CD44 is a receptor for hyaluronic acid and is commonly found on tissue stem cells and MSCs, making it a crucial marker for MenSC identification. HLA-DR serves as a marker for dendritic cells and macrophages, both of which are antigen-presenting cells. The presence of HLA-DR helps identify and exclude these cell types. CD45 is a pan-leukocyte marker, which means it's expressed on the surface of most white blood cells. In the context of FACS analysis, it's essential for excluding non-targeted leukocyte populations. CD14 is a specific marker for monocytes. Monocytes are a type of white blood cell, and their presence can be excluded using this marker. CD34 is a hematopoietic stem cell marker. It is employed to exclude hematopoietic stem cells from the analysis, ensuring that only MenSCs and other relevant cell types are considered. CD235a is a marker specific to erythrocytes, or red blood cells. Its use ensures the exclusion of red blood cells from the analysis. CD19 is a marker that identifies B cells, a type of lymphocyte. This marker is used to exclude B cells from the analysis. In another embodiment of the present invention, positive markers that may be used in the present invention are for instance: CD9 (MSC marker, associated with angiogenesis - Also, eosinophil, megakaryocye and platelet marker); CD29 (Adhesion molecule on mesenchymal and hepatic stem cells); CD73 (Ecto-5'-nucleotidase, involved in migration of MSC) ; CD41a (Platelets and megakariocites marker); CD44 (Hyaluronic acid receptor found on tissue stem cells and MSC); CD90 (Marker of T cells, hematopoietic and MSC); CD105 (Marker of tissue and MSC); Oct-4 (Embryonic stem cell marker); CXCR4 (Hematopoietic and endothelial cells, neurons and stem cells (embryonic and adult) marker); CD166 (Human mesenchymal stromal cell marker); CD49f (Long Term Hematopoietic Stem Cell (LT-HSC) marker); MHC I (HLA-ABC) (Present in all nucleated cells); CD13 (fibroblasts, pericytes, epithelial cells, tumor-initiating cells, and stem cells marker); CD54 (Antigen presenting cell marker); CD55 (Leucocyte, eritrocyte, platelet and NK cell marker); Vimentin (Endothelial and mesenchymal cell marker); CD59 (Hematopoietic Stem Cell Marker); CD10 (Linfocite and stromal cell marker) ; CD140b (PDGFR[3) (MSC and fibroblast marker) ; SUSD2 (Naive human pluripotent stem cells marker). In an embodiment of the present invention, negative markers that may be used in the present invention are for instance: CD14 (Monocyte marker); CD34 (Hematopoietic stem cell marker); CD38 (Differentiating hematopoietic stem cell marker); CD45 (Pan-leukocyte marker); CD133 (Hematopoietic / angioblast marker); MHC II (HLA-DR) (Dendritic cells and macrophage marker (antigen presenting cells)); LIN (Undifferentiated human embryonic stem cells); CD31 (Monocyte, megakaryocite and platelet marker, also endothelial marker); CD50 (Expressed almost exclusively on hematopoietic cells); CD271 (Multipotent mesenchymal stem cells marker) ; EpCAM (Epithelial marker); SSEA-3 (Stem cell marker); TRA-1-60 (Human pluripotent stem cell markers); CD40 (B-cell marker); CD83 (Mature dendritic cells marker); CD86 (Macrophage and B-cell marker); CD19 (B-cell marker); CD79a (B-cell marker); CD80 (Dendritic cells, activated B-cells, T-cells and macrophages) ; CD5 (T cell marker) ; CD8a (Cytotoxic & Suppressor T-Cell Marker); CD15 (Myeloid cell marker); CD20 (Inmune cell marker); CD144 (Endothelial cell marker); CDllb (Monocyte and macrophage marker). The state of the art has only characterized these cells with the aforementioned markers after culturing the MenSCs. Some documents diclose enrichment of MenSC during cell culture. For this, specific markers may be used to sort out the cells. This has been done with the marker CD117 (Patel et al., 2008; Borlogan et al., 2010; Allickson et al., 2011). Yet, other documents show that MenSCs is negative for CD117 (Cui et al., 2007; Chen et al., 2017; Chen et al., 2017; Xiang et al., 2017; Wang et al., 2017; Zhao et al., 2018; Mahdipour et al., 2019; Dalirfardouei et al., 2019; Cen et al., 2019; Guo et al., 2019; Zhu et al., 2019; Wu et al., 2019; Chen et al., 2020; Skliute et al., 2021; Dalirfardouei et al., 2021; Yang et al., 2022; Zhou et al., 2023) . The enrichment of MenSCs with CD117 remains controversial. CD146 has also been used to select MenSCs for enrichment (Yamchi et al., 2021), but here again, another document shows that MenSCs are low-to-negative for CD146 (Hu et al., 2019) . This marker is also controversial. Apart from using controversial markers to distinguish MenSCs, the state of the art uses cell sorting as a tool to enrich and / or select their population. Whatever enrichment or cell sorting technique is used, after this step, the cells continue to undergo cell culture. In other embodiments of the present invention, further markers may be used in the present invention. However, some markers show controversial results and are classified as positive markers in some studies, and negative markers in others. Examples of controversial markers are: SSEA-4 (Embryonic stem cell marker); Nanog (Stemm cell marker); CD117 (c-kit) (Hematopoietic stem cells multipotent progenitors marker); CD49a (NK cell marker); Sox2 (Multipotent progenitor marker); CD106 (Macrophage marker); STRO-1 (MSC marker); CD146 (Endothelial and pericite marker); (c-myc Multipotent marker). EXAMPLES Menstrual blood is collected using a menstrual cup (any commercial cup is suitable) and poured into a tube containing collection medium (DMEM, Pen / Strep 100 U / mL, Amphotericin B, L-Glutamine 2 mM, EDTA 2 mM) . Optionnally, a HIV rapid test may be carried out (e.g. Hexagon HIV 1&2 57002P - Servibio) in order to proceed with a sample that is HIV negative. Mononuclear cells are isolated using density gradient centrifugation with Ficoll-Paque (Dutscher, 17-5446-02), using the following specific protocol. A. SAMPLE WASHING Al. Transfer the sample to a 50 mL Falcon tube. A2 . Add PBS up to volume of 50 mL. A3. Centrifuge the tube at 400g for 10 minutes. A4. Carefully remove the supernatant, leaving only the cell pellet. A5. Repeat a washing step. A6. Remove the supernatant without disturbing the cell pellet. A7. Use a 70 pm filter to filter the blood and remove any endometrial debris . B. FICOLL ISOLATION Bl. Mix the Ficoll (Ficoll-Paque PREMIUM 1.084) by inverting the container several times. B2. Remove the cap of the container and use a pipette to measure out the required volume of Ficoll (3 mL). B3. Prepare two centrifuge tubes for each blood sample and add this 3 mL of Ficoll to each tube. B4. Carefully layer 4 mL of diluted blood onto the Ficoll in each tube, avoiding mixing. B5. Centrifuge the tubes at 400g for 40 minutes at 20°C, without the brake . B6. Carefully collect the cell layer (ring of mononuclear cell layer) above the Ficoll, taking care not to collect the Ficoll phase. B7. Transfer the mononuclear cell layer to a sterile 15 mL centrifuge tube . B8. Fill the tube with PBS and gently mix to homogenize the contents. B9. Centrifuge the tube at 500g for 15 minutes at 20°C. B10. Remove the supernatant. Bll. Fill the tube with PBS and gently mix to homogenize the contents. B12. Centrifuge the tube at 500g for 15 minutes at 20°C. B13. Remove the supernatant. C. FREEZING OF THE CELLS Cl. Resuspend the cell pellet in StemMACS Cryo-Brew to 5 million cells per mL. C2. Quickly transfer the cell suspension into cryogenic vials. C3. Place the vials into an isopropanol freezing container and immediately store at -80 °C. Figure 1 shows photographs from Menstrual Blood Mononuclear Cells isolation using Ficoll-Paque Density Gradient Centrifugation. Panel A of figure 1 shows a photograph of the mononuclear cell layer obtained following density gradient centrifugation. Panel B of figure 1 shows a photograph of isolated menstrual blood mononuclear cells, extracted from the mononuclear cell layer and subsequently washed with PBS. The cells are loaded into the FACS device and sorted based on their fluorescence intensity. In the present example, menstrual blood mononuclear cells are counted using a MACSQuant® Tyto® Cell sorter device. Following protocol is applied. Menstrual blood mononuclear cells are centrifuged 5 min, 400 g, 20 °C and the resulting pellet is resuspended in 100 pL de PBS / BSA 10% / EDTA 2mM. Menstrual blood mononuclear cells are stained with Viobility (1 pL for 1.107 cells) and antibodies targetting surface proteins at a dilution of 1 / 50 which is equivalent to soit 2 pL / Ac par 106 cellule. The mononuclear cells are stained with following antibodies: CD105 VioBright V423 (Miltenyi 130-129-182), CD44-VioBright B515 (Miltenyi 130-126-975), CD73-PE (Miltenyi 130-129-182) CD90 APC (Miltenyi 130114-861), CD19 PE Vio770 (Miltenyi 130-113-647), CD34 PE Vio770 (Miltenyi 130-124-456), CD45 PE Vio770 (Miltenyi 130-110-634), HLA-DR PE Vio770 (Miltenyi 130-111-791), CD14 PE Vio770 (Miltenyi 130-110521), CD235a PE Vio770 (Miltenyi 130-120-474). Incubate 20 min at 4°C in the dark and then wash with 1 mL de PBS / BSA 10% / EDTA 2mM. Centrifuge 5 min, 400 g, 20°C. Prime the cartridge (this step is specific for the use of the MACSQuant® Tyto® Cell Sorter machine used in the present example). Add 500 pL of PBS / BSA 10% / EDTA 2mM to the input compartment of the cartridge. Inject air into a syringe, seal the vent to prime the negative fraction. Wait for 5 seconds and observe the negative fraction filling. Place the cartridge on the magnet and repeat the same process for the positive fraction. Remove any excess buffer from the input. For optimal yield, use the Poisson distribution and resuspend the cell pellet in an appropriate volume of PBS / BSA 10% / EDTA 2mM. Filter the sample. Use a minimum 30 pm filter, with 20 pm recommended. Figure 2 shows FACS plots and gating strategy from the isolation of Menstrual Stem Cells (MenSC) from non cultured Menstrual Blood Mononuclear cells. MenSCs are defined as Linenage- CD90+ CD73+ CD105+ CD44+. Lineage is defined as CD45+ CD34+ CD19+ CD14+ HLA-DR+. The present example thus isolates non-altered MenSCs from a blood sample . The sensitivity of the present invention opens new ways for therapeutic and regenerative medicine applications. Further, the invention provides a new tool for diagnostics. In this regard, one application is linked to the cell count of MenScs in menstrual blood. It has been shown that hormonal contraceptives impact stem cell numbers by altering the perivascular microenvironment where a key type of endometrial stem cells is found (Schwab and Gargett., 2007; Spitzer et al., 2012). Other studies showed that hormone treatment with progestin lead to differentiation of the endometrial stromal cells (Deligdisch-Schor and Mare§ Miceli, 2020), which ultimately is associated with a decrease of stem cells present in endometrial tissue. Contraceptive hormone treatments have also been linked to so called endometrial thinning (Meresman et al., 2002) . A thinner endometrium has a decreased number of stem cells. Taken together, it appears that hormonal contraceptive treatments impact stem cell numbers in menstrual blood, i.e. MenSC rate. The present invention provides a strong tool to rapidly identify anormal cell numbers. Figure 3 shows a diagramm comparing the number of MenSC quantity per mililiter of menstrual blood of a sample with and without hormone treatment. In the present description, the invention is described with reference to flow cytometry cell sorting. However, there are a number of alternatives to cell sorting, each with its own advantages and disadvantages. Some of the most common alternatives are briefly described below. Magnetic-activated cell sorting (MACS): MACS uses magnetic beads coated with antibodies to target specific cell types. The beads are incubated with the cell suspension, and a magnetic field is used to isolate the targeted cells from the rest of the sample. MACS is a relatively simple and inexpensive technique, but it is not as versatile as other cell sorting techniques. Further, with MACS techniques it is generally difficult to achieve a result having high purity. Cell panning technique: cell panning uses antibodies to capture specific cell types onto a dish or other surface. The cells are incubated with an antibody-coated surface that captures the target cell types. Unbound cells are eliminated by means of washing steps. The bound cells are then collected for further analysis or experimentation. Cell panning is a gentle technique that can be used to isolate cells without damaging them. However, it is not as efficient as other cell sorting techniques. Here again, it is generally difficult to achieve a result having high purity. Advanced density gradient centrifugation: advanced density gradient centrifugation is a technique that uses a gradient of different densities to separate cells based on their size and weight. The cell suspension is layered onto the gradient, and the cells are then centrifuged. The cells will migrate to different levels of the gradient depending on their size and weight. The desired cell population can then be collected from the appropriate layer of the gradient. Density gradient centrifugation is a versatile and efficient technique. However, this technique is generally difficult to optimize for specific cell types. Microfluidic devices: microfluidic devices are small-scale devices that are used to manipulate and sort cells. These devices typically use a combination of fluid flow and electrical fields to separate cells based on their size, shape, and other properties. Microfluidic devices are becoming increasingly popular for cell sorting, as they offer a number of advantages over traditional methods, such as high throughput, low sample volumes, and minimal cell damage. However the devices remain very expensive and are not suitable for detection of secreted material. Also there remains a risk of cell damage and a scale-up is problematic. In addition to these traditional alternatives, there are a number of emerging technologies that are currently being developed for cell sorting. These technologies include for instance the below. Acoustic cell sorting: acoustic cell sorting uses sound waves to separate cells based on their size and compressibility. This is a non-invasive technique that may be used to sort cells without damaging them. Optical cell sorting: optical cell sorting uses light to separate cells based on their size, shape, and refractive index. This is a high-throughput technique that may be used to sort cells at very high rates . Dielectrophoresis cell sorting: dielectrophoresis cell sorting uses electrical fields to separate cells based on their dielectric properties. This is a versatile technique that may be used to sort cells based on a variety of factors, such as size, shape, and surface charge . The above alternative techniques in cell sorting, as well as emerging technologies are listed in table 1 below: [Table 1] ALTERNATIVE ADVANTAGE (S) DISADVANTAGE (S) MACS Simple, inexpensive, gentle Not as versatile as cell sorting, difficult to achieve high purity Cell panning Gentle, can be used to isolate cells without damaging them Not as efficient as cell sorting, difficult to achieve high purity Density gradient centrifugation Versatile, efficient Can be difficult to optimize for specific cell types Microfluidic devices High throughput, low sample volumes, minimal cell damage Expensive Acoustic     cell sorting Non-invasive Still under development Optical      cell sorting High throughput Expensive & still under development Dielectrophoresis cell sorting Versatile Expensive & still under development Table 1 : Alternative techniques and emerging technologies in cell sorting Consequently, given the alternative techniques and emerging technologies in cell sorting, the present invention can be defined in a broad manner as a method for isolating menstrual stem cells (MenSC) comprising the steps of: 1. collecting menstrual blood comprising a heterogenous suspension comprising a mixture of cells including menstrual stem cells in a nonaltered (or native) state; 2. running said heterogenous suspension through a cell sorting operation in order to isolate said menstrual stem cells in a nonaltered (or native) state, wherein said cell sorting technique is deprived of any cell culturing process. The cell sorting operation may combine different cell sorting techniques / technologies . In the preferred embodiment of the invention described herein, step 2. comprises the sub-steps of: i. processing said heterogenous suspension through density gradient centrifugation in order to isolate a homogenous suspension comprising a mixture of mononuclear cells; ii. adding fluorescent markers to said homogenous suspension in order to specifically tag menstrual stem cells, and subsequently iii. running the homogenous suspension with the tagged menstrual stem cells obtained in step ii. through flow cytometry in order to isolate menstrual stem cells from the homogenous suspension, wherein the isolated menstrual stem cells are in the non-altered (or native) state. BIBLIOGRAPHY:

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Claims

1. Method for isolating menstrual stem cells (MenSC)comprising the steps of:a. collecting menstrual blood comprising a heterogenous suspension comprising a mixture of cells including menstrual stem cells in a non-altered state;b. processing said heterogenous suspension through density gradient centrifugation in order to isolate a homogenous suspension comprising a mixture of mononuclear cells including said menstrual stem cells;c. adding fluorescent markers to said homogenous suspension in order to specifically tag menstrual stem cells, and subsequentlyd. running the homogenous suspension with the tagged menstrual stem cells obtained in step c. through flow cytometry in order to isolate menstrual stem cells from the homogenous suspension, wherein the isolated menstrual stem cells are in the non-altered state,wherein said method is deprived of any cell culturing process.

2. Method according to any of the preceding claims, wherein step a. is carried out by means of a menstrual cup.

3. Method according to any of the preceding claims, wherein said density gradient centrifugation is a Ficoll-Paque method.

4. Method according to any of the preceding claims, wherein said fluorescent markers comprise a set of positive markers selected from the group consisting of CD90, CD73, CD105, CD44 and a combination thereof.

5. Method according to any of the preceding claims, wherein said fluorescent markers comprise a set of negative markers selected from the group consisting of HLA-DR, CD45, CD14, CD34, CD235a, CD19 and a combination thereof.

6. Method according to any of the preceding claims, wherein said fluorescent markers consist of CD90, CD73, CD105, CD44, HLA-DR, CD45, CD14, CD34, CD235a and CD19.

7. Method according to any of the preceding claims, consisting of steps a., b., c., and d.

Images