Method for the isolation of menstrual stem cells

Abstract

The invention relates to a method for the isolation of menstrual stem cells (MenSC) comprising the steps of: a. collecting menstrual blood comprising a heterogeneous suspension comprising a mixture of cells comprising menstrual stem cells in an unaltered state; b. treating said heterogeneous suspension by density gradient centrifugation to isolate a homogeneous suspension comprising a mixture of mononuclear cells comprising said menstrual stem cells; c. adding fluorescent markers to said homogeneous suspension to specifically label the menstrual stem cells, followed by d. passing the homogeneous suspension having the labeled menstrual stem cells obtained in step c. through flow cytometry to isolate the menstrual stem cells from the homogeneous suspension, wherein the isolated menstrual stem cells are in the unaltered state. Abstract Figure: Figure 2

Description

Title of the invention: Method for the isolation of menstrual stem cells

[0001] The present invention relates to a method for the isolation of menstrual stem cells.

[0002] Stem cells from menstrual blood, or more commonly "menstrual stem cells" (MenSC), are a new and promising source of stem cells used in many fields, including regenerative medicine.

[0003] MenSCs are mesenchymal stem cells (MSCs) that possess multipotent differentiation capacity. MenSCs were first identified and characterized in 2007 (Meng et al., 2007) and have since attracted significant attention due to their unique properties.

[0004] MenSCs are found in menstrual blood loss and are called endometrial mesenchymal stem cells (eMSCs) when they reside in the perivascular region of both the basal and functional layers of the endometrium. These cells may play an important role in the cyclical regeneration and repair of the endometrium.

[0005] Compared to stem cells from bone marrow and adipose tissue, MenSCs have several advantages. A major advantage is that MenSCs are probably the easiest stem cells to collect from the human body.

[0006] Furthermore, when compared to other cell types (i.e., non-stem cells), the advantages of MenSCs lie in the high proliferation rate, colony-forming unit frequency, migration capacity, and multi-lineage differentiation capabilities.

[0007] Besides these advantages, the potential applications of MenSCs in regenerative medicine are vast. MenSCs have been studied for their potential use in the treatment of a variety of diseases including liver disease, diabetes, stroke, Duchenne muscular dystrophy, ovarian-related diseases, myocardial infarction, Asherman's syndrome, Alzheimer's disease, acute lung injury, skin wounds, endometriosis, and neurodegenerative diseases (Chen et al., 2019).

[0008] 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 their natural environment, i.e. in vitro. Thus, during cell culture of MSC, cell quantity is increased by the development of MSCs in vitro. This in vitro development of MSCs is a common practice in cell biology, regenerative medicine and other research areas.

[0009] However, cell culture is generally a long and expensive process that is accompanied by risks. A major risk, for example, is contamination of the cell culture. This has a negative, often fatal, effect on the results of the study or the cells themselves.

[0010] Furthermore, long-term in vitro development of MSCs results in significant changes in their properties, including alterations in gene expression, genomic stability and / or immunomodulatory properties. Also, epigenetic changes, and other morphological and / or functional alterations in MSCs have been observed.

[0011] The fact that native 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 regarding MenSCs in their original, unaltered state.

[0012] The present invention improves the situation.

[0013] To this end, the invention relates to a method for the isolation of menstrual stem cells (MenSC) comprising the steps of:

[0014] a. collecting menstrual blood comprising a heterogeneous suspension comprising a mixture of cells comprising menstrual stem cells in an unaltered state;

[0015] b. treating said heterogeneous suspension by density gradient centrifugation in order to isolate a homogeneous suspension comprising a mixture of mononuclear cells including said menstrual stem cells;

[0016] c. adding fluorescent markers to said homogeneous suspension in order to specifically mark menstrual stem cells, followed by

[0017] d. passing the homogeneous suspension having the labeled menstrual stem cells obtained in step c. through flow cytometry to isolate the menstrual stem cells from the homogeneous suspension, wherein the isolated menstrual stem cells are in the unaltered state.

[0018] Thus, the invention is devoid of any cell culture process. The method of the invention (from the first step to the last step) does not include any cell culture step or process. Step d. is carried out directly following step c.

[0019] Step a. can be carried out using a menstrual cup.

[0020] The density gradient centrifugation is preferably a Ficoll-Paque process.

[0021] 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, CD 105, CD44 and a combination thereof.

[0022] In another preferred embodiment, the fluorescent markers comprise a set of negative markers selected from the group consisting of HLA-DR, CD45, CD 14, CD34, CD235a, CD 19 and a combination thereof.

[0023] In yet another preferred embodiment, the fluorescent markers include both the above positive and negative markers.

[0024] In another embodiment, the method of the invention consists of steps a., b., c. and d.

[0025] Other characteristics and advantages of the invention will emerge and / or become clear from reading the following description, which includes specific examples given in an illustrative and non-limiting manner, as well as from the drawings in which:

[0026] [Fig-1] represents photographs of isolation of blood mononuclear cells menstrual using Ficoll-Paque density gradient centrifugation;

[0027] [Fig.2] depicts FACS tracings of the isolation of menstrual stem cells (MenSC) from uncultured menstrual blood mononuclear cells; and

[0028] [Fig.3] represents a diagram comparing the number of MenSC per milliliter of menstrual blood from a sample with and without hormonal treatment.

[0029] The drawings and description herein contain, as a whole, elements of a defined nature. Accordingly, the description and drawings are used not only to better understand the present invention, but also to contribute to the definition thereof, where appropriate.

[0030] MenSCs are obtained from menstrual blood. The common procedure for menstrual blood collection is using what is called a menstrual cup. The menstrual cup is a device designed to be inserted into the vagina. Collection is primarily planned during the heaviest flows of the menstrual cycle. A cup will typically remain in place for approximately 4 to 12 hours. Once menstrual effluent is acquired, the menstrual cup is removed and the blood is transferred to sterile medium / phosphate buffered saline, PBS, with antibiotics.

[0031] Isolation of stem cells from blood can be performed by density gradient centrifugation. More generally, density gradient centrifugation techniques, such as the so-called Ficoll-Paque technique, are successful in isolating all mononuclear cells from collected menstrual blood. During centrifugation, erythrocytes and granulocytes sediment to the bottom layer, leaving the mononuclear cells easily isolated.

[0032] In the state of the art, mononuclear cells, and in particular MenSCs, exclusively undergo cell culture. Frequently, cells are recovered from menstrual fluid in the form of plastic-adherent cells, similar to bone marrow MSCs (bmMSCs). However, MenSCs are still prepared and placed in 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; by 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 Asl 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 ; Skliuté 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 ; Gonçalves 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-Kazeroni et al., 2019; Feng et al., 2019; Wang et al., 2019. The treatment of MenSCs in art is thus inevitably linked to cell culture procedures.

[0033] Applicant uses a radically different approach to isolate MenSCs for further studies. More particularly, Applicant bypasses any cell culture in order to study MenSCs in an unaltered state (sometimes referred to as native MenSCs).

[0034] Indeed, cell culture is a milestone in modern biology. Moreover, cell culture has revolutionized our understanding of diseases and their treatment. However, as mentioned above, the in vitro development of cells, and in particular mesenchymal stem cells (MSCs), can lead to significant changes in their gene expression, their genomic stability and their immunomodulatory properties. These changes have profound implications for the therapeutic potential of MSCs. Furthermore, all studies that use cell culture can be biased by the fact that the cells studied are altered. In other words, the cells are not in their original state after cell culture. This can lead to significant misinterpretation and / or misunderstanding of study or research results.

[0035] Alteration of cells during cell culture may result from (i) epigenetic changes, (ii) genetic changes, (iii) proteomic changes and / or (iv) morphological changes. All of these changes, taken alone or together, result in MSCs before cell culture (in their original unaltered state) being very different from MSCs after cell culture (altered state).

[0036] (i) Epigenetic changes

[0037] 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 DNA methylation 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 DNA methylation (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 MSC cell culture is associated with a tightly regulated epigenetic program (Wagner, 2012).

[0038] The mechanisms underlying these changes are not fully understood. Some papers (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 lack of selective pressure.

[0039] (ii) Genetic changes

[0040] The paper Jeske et al., 2021 discloses that human adipose-derived mesenchymal stem cells (hASCs) undergo a transition from an anti-inflammatory to a pro-inflammatory phenotype during in vitro development (i.e., cell culture). In addition, some hASCs exhibit reduced RNA methylation levels for genes responsible for osteogenic and adipogenic differentiation, as well as genes involved in cell signaling pathways that correlate with the pro-inflammatory phenotype. Other studies have discovered similar changes in gene expression during in vitro development of MSCs from different sources. For example, the paper Gu et al., 2016 discloses that RNA methylation levels of P21, P53, and APE1 / Ref-1 are significantly increased in the mid- and late-phase in vitro development of human umbilical cord mesenchymal stem cells (hucMSCs). Xiang et al., 2019 discloses that BMSCs from passage 6 and passage 10 (note that the word “passage” refers to a transfer of cells to a 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. Redaelli et al., 2012, examined the genomic stability of human bone marrow mesenchymal stem cells (hBM-MSCs) during in vitro development.They found that hBM-MSCs generally possess a normal karyotype, but there is a variable tendency toward random chromosomal losses and, in rare cases, clonal aneuploidies. Telomere length decreases with increasing passage number. Other papers disclose evidence of genomic instability during in vitro MSC development. For example, Jiang et al., 2017 disclose that passage 3 BMSCs possess downregulated cell cycle regulation, DNA replication, and mismatch repair pathways. Moreover, several genes associated with telomerase activity and chromosomal stability are also notably suppressed in passage 3 BMSCs.

[0041] Furthermore, MSC cell culture exhibited an accumulation of yH2AX foci, a well-known marker of genomic instability. Therefore, selection of the appropriate passage is a critical procedure prior to transplantation of allogeneic MSCs into recipient patients, since in vitro propagations may cause MSCs to acquire genetic changes that may result in malignant transformation (Pustovalova et al., 2016; Al-Azab et al., 2022).

[0042] (iii) Proteomic changes

[0043] Protein expression changes in human adipose-derived mesenchymal stem cells (hASCs) and bone marrow mesenchymal stem cells (BMSCs) during in vitro development. Jeske et al. 2021 discloses that in vitro development of hASCs results in global alterations in protein expression, affecting key functions such as cellular morphology, assembly, and organization; carbohydrate metabolism; small molecule biochemistry; and post-translational modification. Xiang et al. 2019 disclosed that passage 6 and passage 10 BMSCs exhibit differences in the protein levels of conserved genes involved in the cell cycle, DNA replication, and oocyte meiosis. Weighted gene co-expression network analysis confirmed these findings and also revealed how cell passage affects the response to irradiation in BMSCs.

[0044] (iv) Morphological changes

[0045] Human adipose-derived mesenchymal stem cells (hASCs) undergo morphological transformations and exhibit changes in their functional properties during cell culture (i.e., in vitro development). They transition from a spindle-like shape to a flattened and elongated appearance. Moreover, the colony-forming unit (CFU) capacity significantly decreases in higher passages (passage 12) compared to earlier passages (passage 5). Furthermore, increased SA-[3-Gal] activity indicates enhanced senescence in passage 12 hASCs. Notably, energy metabolism and NAD-Sirt pathways associated with cellular senescence remain stable throughout hASC in vitro development (Jeske et al., 2021). In another paper, hASCs initially retain low levels of aneuploidy in early passages (passage 0 to passage 4).However, prolonged culture development (5–16 passages) results in a significant increase in aneuploidy percentages, without triggering malignancy (Roemeling-van Rhijn et al., 2013).

[0046] In the case of human umbilical cord mesenchymal stem cells (hucMSCs), some documents disclose 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. The proliferation rates of hucMSCs also decline over time. In addition, there is an increase in senescence, G0 / G1 cell cycle arrest, and reduced differentiation capacity. One document discloses that hucMSCs undergo replicative senescence during prolonged in vitro culture (Gu et al., 2016). Moreover, human embryonic stem cells exhibit unstable chromosomal alterations in differentiated MSCs, resulting in replicative senescence after prolonged culture (Karagiannidou et al., 2014).

[0047] 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 passage and irradiation in two stages, passage 6 and passage 10. Comparing BMSCs from passage 6 and passage 10, differences in the percentage of apoptotic cells and cell cycle distribution were observed. BMSCs from passage 6 exhibited lower apoptosis rates, a reduced proportion of cells in the S phase, and a higher proportion in the G1 phase than BMSCs of passage 10. 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. BMSCs of passage 3 also demonstrated reduced telomerase activity and alterations in chromosomal structure, indicative of cellular senescence (Jiang et al., 2017). After the first passage, BMSCs display a notable reduction in their proliferation rate and gradually lose their multi-differentiation potential. Their in vivo bone formation efficiency significantly decreases compared to fresh bone marrow (Banfi et al., 2000).

[0048] Typically, mesenchymal stem cells (MSCs) exhibit a spindle-shaped morphology with a small cell body, few thin 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, senescence induced by prolonged in vitro development of adipose tissue MSCs results in morphological alterations, including increased size and complexity, giving them a “fried egg-like” appearance (Truong et al., 2019). The presence of reactive oxygen species (ROS) negatively impacts the ability of MSCs to suppress immune cells, such as T cells.Oxidative stress plays a central role in the replicative senescence of MSCs, limiting their passage number and 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 compared to passage 1. This is indicative of decreased proliferation and decreased capacity of MSCs with aging (Chen et al., 2005).

[0049] Some of the references described above do not directly concern studies with MenSCs. However, they at least concern MSCs of other types. 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 directly affect MenSCs.

[0050] More generally, in vitro development (or cell culture) of MSCs is a common practice in cell biology and regenerative medicine. However, the above describes that cell culture can result in significant changes in cells, including alterations in gene expression and genomic stability, epigenetic changes, and other morphological and functional alterations.

[0051] The present invention supports the isolation of fresh MenSC. Fresh MenSC means cells that have not been altered in any way by cell culture. The MenSC isolated with the present invention are in their original state. The MenSC obtained with the present invention possess the physiological properties as 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 example with respect to methylation sites.

[0052] Freshly isolated MenSCs, i.e., unaltered MenSCs, are at least close to their natural state. They are isolated prior to any cell culture, and therefore are spared the potentially transformative effects of in vitro development. This means that they retain their original gene expression profiles and genomic stability, making them more effective for therapeutic and / or diagnostic applications. In addition, 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 accompanies cell culture. Another advantage is that freshly isolated cells can provide a more accurate picture of the cell population in the body.On the other hand, cultured cells may overrepresent certain subpopulations due to differential growth rates.

[0053] The invention uses fluorescence activated cell sorting (FACS).

[0054] 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 on the scattering of specific light and fluorescence characteristics of each cell. It is a known scientific operation because it provides a rapid, objective, and quantitative recording of fluorescence signals from individual cells as well as physical separation of cells of particular interest.

[0055] The use of FACS in the present invention for the isolation of MenSCs involves four main steps: (1) Sample preparation, (2) Cell labeling, (3) Cell sorting, and (4) Cell collection.

[0056] (1) Sample Preparation: Menstrual blood is collected, typically at using a menstrual cup. The collected sample is then processed to isolate mononuclear cells by density gradient centrifugation, such as Ficoll-Paque.

[0057] (2) Labeling of cells with fluorescent markers: Following isolation cells, the invention uses specific fluorescent markers for the identification and isolation of MenSCs. These markers target MenSC-specific surface antigens, such as CD90, CD73, CD 105 and CD44, while also excluding unwanted cell populations in the sample by targeting markers such as HLA-DR, CD45, CD 14, CD34, CD235a and CD 19.

[0058] (3) Cell sorting with FACS: The labeled cell suspension is loaded in the FACS machine. When cells pass through the machine's laser beam, they will produce a fluorescence signal. The FACS machine detects this fluorescence and separates the cells based on their signal.

[0059] (4) Cell Collection: The sorted MenSCs are then collected for a further use.

[0060] The present invention is devoid of any cell culture. The invention provides a simple and reliable source for collecting freshly isolated MenSC for subsequent use. The invention allows cells that come directly from a patient to be processed for analysis, thereby avoiding the addition of intermediate cell culture processes that alter the cells of interest.

[0061] Although in vitro development has its place in cell biology and regenerative medicine, the use of freshly isolated and unaltered cells offers valuable reinforcement in these technical fields. The invention provides a more accurate picture of in vivo conditions, and thus improves the development of safer and more effective therapeutic strategies.

[0062] The state of the art discloses that MenSCs can be identified and / or isolated by specific cell markers. However, the specific markers disclosed in the art are used solely and exclusively after cell culture. The present invention utilizes the knowledge derived from the teachings of the opposing prior art regarding cell markers. In fact, MenSCs possess the classical markers of the International Society for Cellular Therapy (ISCT). They are positive for MSC markers including CD29, CD44, CD73, CD90, CD 105 and STRO-1. They do not express hematopoietic lineage markers such as CD34, CD45, CD133, CD14, CD38 and the human leukocyte antigen DR isotype (HLA-DR).

[0063] In a preferred embodiment of the invention, the fluorescent markers are selected from the group consisting of CD90, CD73, CD 105, CD44, HLA-DR, CD45, CD 14, CD34, CD235a, CD 19 or any combination thereof.

[0064] CD90 is a marker found on the surface of T lymphocytes, hematopoietic cells and MSCs, including MenSCs. Its presence is key to confirming the identity of these cells. CD73 is an enzyme known as ecto-5'-nucleotidase and plays a role in MSC migration. It is a marker important for the identification of 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 the identification of MenSCs. HLA-DR serves as a marker for dendritic cells and macrophages, both of which are antigen-presenting cells. The presence of HLA-DR helps to identify and exclude these cell types. CD45 is a pan-leukocyte marker, meaning it is expressed on the surface of most white blood cells. In the context of FACS analysis, it is essential for the exclusion of non-target leukocyte populations. CD14 is a monocyte-specific marker. Monocytes are a type of white blood cell, and their presence can be excluded using this marker.CD34 is a marker for hematopoietic stem cells. It is used to exclude hematopoietic stem cells from 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 analysis. CD19 is a marker that identifies B lymphocytes, a type of lymphocyte. This marker is used to exclude B lymphocytes from analysis.

[0065] In another embodiment of the present invention, the positive markers that can be used in the present invention are for example: CD9 (Marker of MSCs, associated with angiogenesis - Also marker of eosinophils, megakaryocytes and platelets); CD29 (Adhesion molecule on mesenchymal and hepatic stem cells); CD73 (Ecto-5'-nucleotidase, involved in the migration of MSCs); CD41a (Marker of platelets and megakaryocytes); CD44 (Hyaluronic acid receptor found on tissue stem cells and MSCs); CD90 (Marker of T lymphocytes, hematopoietic cells and MSCs); CD 105 (Tissue and MSC marker); Oct-4 (Marker of embryonic stem cells); CXCR4 (Marker of hematopoietic and endothelial cells, neurons and stem cells (embryonic and adult)); CD 166 (Marker of human mesenchymal stromal cells);CD49f (Marker of long-term hematopoietic stem cells (LT-HSC)); MHC I (HLA-ABC) (present in all nucleated cells); CD 13 (Marker of fibroblasts, pericytes, epithelial cells, tumor-initiating cells and stem cells); CD54 (Marker of antigen-presenting cells); CD55 (Marker of leukocytes, erythrocytes, platelets and NK cells); Vimentin (Marker of endothelial and mesenchymal cells); CD59 (Marker of hematopoietic stem cells); CD 10 (Marker of lymphocytes and stromal cells); CD 140b (PDGFR[3) (Marker of MSCs and fibroblasts); SUSD2 (Marker of naive human pluripotent stem cells).

[0066] In one embodiment of the present invention, negative markers that can be used in the present invention are for example: CD 14 (Marker of monocytes); CD34 (Marker of hematopoietic stem cells); CD38 (Marker of differentiated hematopoietic stem cells); CD45 (Pan-leukocyte marker); CD133 (Marker of hematopoietic cells / angioblasts); MHC II (HLA-DR) (Marker of dendritic cells and macrophages (antigen presenting cells)); LIN (Undifferentiated human embryonic stem cells); CD31 (Marker of monocytes, megakaryocytes and platelets, also endothelial marker); CD50 (Expressed almost exclusively on hematopoietic cells); CD271 (Multipotent mesenchymal stem cell marker); EpCAM (Epithelial marker); SSEA-3 (Stem cell marker); TRA-1-60 (Human pluripotent stem cell marker);CD40 (B-cell marker); CD83 (Mature dendritic cell marker); CD86 (Macrophage and B-cell marker); CD 19 (B-cell marker); CD79a (B-cell marker); CD80 (Dendritic cells, activated B cells, T cells and macrophages); CD5 (T-cell marker); CD8a (Cytotoxic and suppressor T-cell marker); CD 15 (Myeloid cell marker); CD20 (Immune cell marker); CD 144 (Endothelial cell marker); CD 11b (Monocyte and macrophage marker).

[0067] The state of the art has characterized these cells with the aforementioned markers only after culturing the MenSCs.

[0068] Some documents disclose an enrichment of MenSCs during cell culture. For this, specific markers can be used to sort the cells. This was done with the marker CD117 (Patel et al., 2008; Borlogan et al., 2010; Allickson et al., 2011). However, other papers show that MenSCs are 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; Skliuté et al., 2021; Dalirfardouei et al., 2021; Yang et al., 2022; Zhou et al., 2023). The enrichment of MenSCs with CD117 remains controversial. CD 146 has also been used to select MenSCs for enrichment (Yamchi et al., 2021), but again, another paper shows that MenSCs are low to negative for CD 146 (Hu et al., 2019). This marker is also controversial. Besides the use of controversial markers to distinguish MenSCs, the state of the art uses cell sorting as a tool to enrich and / or . select their population. Regardless of the enrichment or cell sorting technique that is used, after this step, the cells continue to undergo cell culture.

[0069] In other embodiments of the present invention, additional markers may be used in the present invention. However, some markers show controversial results and are classified as positive markers in some studies, and as negative markers in others. Examples of controversial markers are: SSEA-4 (Embryonic stem cell marker); Nanog (Stem cell marker); CD 117 (c-kit) (Hematopoietic stem cell multipotent progenitor marker); CD49a (NK cell marker); Sox2 (Multipotent progenitor marker); CD 106 (Macrophage marker); STRO-1 (MSC marker); CD 146 (Endothelial and pericyte marker); (c-myc Multipotent marker). EXAMPLES

[0070] Menstrual blood is collected using a menstrual cup (any commercially available 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). Optionally, a rapid HIV test can be performed (e.g. Hexagon HIV 1&2 57002P - Servibio) in order to proceed with a sample that is HIV negative.

[0071] Mononuclear cells are isolated using density gradient centrifugation with Ficoll-Paque (Dutscher, 17-5446-02), using the following specific protocol.

[0072] A. WASHING THE SAMPLE

[0073] AL Transfer the sample into a 50 ml Falcon tube.

[0074] A2. Add PBS to a volume of 50 ml.

[0075] A3. Centrifuge the tube at 400g for 10 minutes.

[0076] A4. Carefully remove the supernatant, leaving only the cell pellet.

[0077] A5. Repeat a washing step.

[0078] A6. Remove the supernatant without disturbing the cell pellet.

[0079] A7. Use a 70 qm filter to filter the blood and remove any endometrial debris.

[0080] B. FICOLL INSULATION

[0081] Bl. Mix the Ficoll (Ficoll-Paque PREMIUM 1.084) by turning the container several times.

[0082] B2. Remove the cap from the container and use a pipette to measure the volume required of Ficoll (3 ml).

[0083] B3. Prepare two centrifuge tubes for each blood sample and add these 3 ml of Ficoll to each tube.

[0084] B4. Carefully place 4 ml of diluted blood on the Ficoll in each tube, avoiding mixing.

[0085] B5. Centrifuge the tubes at 400g for 40 minutes at 20°C, without the brake.

[0086] B6. Carefully collect the cell layer (cell layer ring mononuclear) above the Ficoll, taking care not to collect the Ficoll phase.

[0087] B7. Transfer the mononuclear cell layer into a sterile centrifuge tube. of 15 ml.

[0088] B8. Fill the tube with PBS and mix gently to homogenize the content.

[0089] B9. Centrifuge the tube at 500g for 15 minutes at 20°C.

[0090] B10. Remove the supernatant.

[0091] B11. Fill the tube with PBS and mix gently to homogenize the contents.

[0092] B12. Centrifuge the tube at 500g for 15 minutes at 20°C.

[0093] B13. Remove the supernatant.

[0094] C. FREEZING OF CELLS

[0095] CL Resuspend the cell pellet in StemMACS Cryo-Brew at 5 million cells per ml.

[0096] C2. Rapidly transfer the cell suspension into cryogenic vials.

[0097] C3. Place the vials in an isopropanol freezing tank and store immediately to -80°C.

[0098] [Fig.l] shows photographs of menstrual blood mononuclear cell isolation using Ficoll-Paque density gradient centrifugation. Panel A of [Fig.l] shows a photograph of the mononuclear cell layer obtained following density gradient centrifugation. Panel B of [Fig.l] shows a photograph of isolated menstrual blood mononuclear cells extracted from the mononuclear cell layer and subsequently washed with PBS.

[0099] Cells are loaded into the FACS device and sorted based on their fluorescence intensity. In this example, menstrual blood mononuclear cells are counted using a MACSQuant® Tyto® cell sorter. The following protocol is applied.

[0100] Menstrual blood mononuclear cells are centrifuged for 5 min, at 400g, at 20°C and the resulting pellet is resuspended in 100 μL of PBS / 10% BSA / 2 mM EDTA. Menstrual blood mononuclear cells are stained by Viobility (1 μL per 1.107 cells) and antibodies targeting surface proteins at a dilution of 1 / 50 which is equivalent to 2 μL / Ab per 106 cells. Mononuclear cells are stained with the following antibodies: CD105 VioBright V423 (Miltenyi 130-129-182), CD44-VioBrightB515 (Miltenyi 130-126-975), CD73-PE (Miltenyi 130-129-182) CD90 APC (Miltenyi 130-114-861), 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 130-110-521), CD235a PE Vio770 (Miltenyi 130-120-474). Incubate for 20 min at 4°C in the dark and then wash with 1 ml of PBS / 10% BSA / 2 mM EDTA. Centrifuge for 5 min at 400g at 20°C. Prime the cartridge (this step is specific to the use of the MACSQuant® Tyto® cell sorter machine used in this example). Add 500 µl of PBS / 10% BSA / 2 mM EDTA to the inlet compartment of the cartridge. Inject air into a syringe, close the vent to prime the negative fraction. Wait 5 seconds and observe the negative fraction fill.Place the cartridge on the magnet and repeat the same process for the positive fraction. Remove any excess buffer from the inlet. For optimal yield, use the Poisson distribution and resuspend the cell pellet in an appropriate volume of PBS / 10% BSA / 2 mM EDTA. Filter the sample. Use a 30 µm filter at a minimum, with 20 µm recommended.

[0101] [Fig.2] shows FACS plots and a triggering strategy from the isolation of menstrual stem cells (MenSC) from uncultured menstrual blood mononuclear cells. MenSC are defined by Lineage- CD90+ CD73+ CD 105+ CD44+. Lineage is defined by CD45+ CD34+ CD 19+ CD 14+ HLA-DR+.

[0102] The present example thus isolates unaltered MenSCs from a blood sample.

[0103] The sensitivity of the present invention opens new avenues for applications in therapeutic and regenerative medicine. Furthermore, the invention provides a new tool for diagnosis.

[0104] In this regard, one application is related to the cell counting of MenSCs in menstrual blood.

[0105] Hormonal contraceptives have been shown to impact stem cell numbers by altering the perivascular microenvironment where a key type of endometrial stem cell is found (Schwab and Gargett., 2007; Spitzer et al., 2012). Other studies have shown that hormonal treatment with a progestin resulted in endometrial stromal cell differentiation (Deligdisch-Schor and Mareg Miceli, 2020), which is ultimately associated with a reduction in stem cells present in the endometrial tissue. Hormonal contraceptive treatments have also been linked to so-called endometrial thinning (Meresman et al., 2002). A thinner endometrium has a reduced number of stem cells.

[0106] Overall, it appears that hormonal contraceptive treatments have an impact on the number of stem cells in menstrual blood, i.e., on the MenSC level. The present invention provides a powerful tool for rapidly identifying abnormal cell counts.

[0107] [Fig.3] represents a diagram comparing the number of MenSC quantity per milliliter of menstrual blood of a sample with and without hormonal treatment.

[0108] In the present description, the invention is described with reference to cell sorting by flow cytometry. 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.

[0109] Magnetically activated cell sorting (MACS): MACS uses antibody-coated magnetic beads 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. In addition, with MACS techniques, it is generally difficult to achieve a result with high purity.

[0110] Cell plating technique: Cell plating uses antibodies to capture specific cell types on a dish or other surface. The cells are incubated with an antibody-coated surface that captures the target cell types. Unbound cells are removed through washing steps. The bound cells are then collected for further analysis or experimentation. Cell plating 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. Again, it is generally difficult to achieve a result with high purity.

[0111] 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 on 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.

[0112] 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 fields electrical devices to separate cells based on their size, shape, and other properties. Microfluidic devices are becoming increasingly popular for cell sorting, as they offer several advantages over conventional methods, such as high throughput, small sample volumes, and minimal cell damage. However, the devices remain very expensive and are not suitable for the detection of secreted materials. There is also a risk of cell damage, and scaling up is problematic.

[0113] In addition to these conventional alternatives, there are a number of emerging technologies that are currently being developed for cell sorting. These technologies include, for example, the following.

[0114] Acoustic cell sorting: Acoustic cell sorting uses sound waves to separate cells based on their size and compressibility. It is a non-invasive technique that can be used to sort cells without damaging them.

[0115] Optical cell sorting: Optical cell sorting uses light to separate cells based on their size, shape, and refractive index. It is a high-throughput technique that can be used at very high speeds.

[0116] Dielectrophoretic cell sorting: Dielectrophoretic cell sorting uses electric fields to separate cells based on their dielectric properties. It is a versatile technique that can be used to sort cells based on a variety of factors, such as size, shape, and surface charge.

[0117] The above alternative techniques in terms of cell sorting, as well as emerging technologies, are listed in Table 1 below:

[0118] [Tables 1] ALTERNATIVE ADVANTAGE(S) DISADVANTAGE(S) MACS Simple, inexpensive, gentle Not as versatile as cell sorting, difficult to achieve high purity Cells adhere to plastic 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 May be difficult to optimize for specific cell types Microfluidic Devices High throughput, small sample volumes, minimal cell damage Expensive Acoustic Cell Sorting Non-invasive Still in development Optical Cell Sorting High throughput Expensive & still in development Dielectrophoretic Cell Sorting Versatile Expensive & still in development

[0119] Table 1: Alternative techniques and emerging technologies in cell sorting

[0120] Therefore, given the alternative techniques and emerging technologies in cell sorting, the present invention can be broadly defined as a method for the isolation of menstrual stem cells (MenSC) comprising the steps of:

[0121] 1. the collection of menstrual blood comprising a heterogeneous suspension comprising a mixture of cells comprising menstrual stem cells in an unaltered (or native) state;

[0122] 2. passing said heterogeneous suspension through a cell sorting operation in order to to isolate menstrual stem cells in an unaltered (or native) state, wherein said cell sorting technique is devoid of any cell culture process.

[0123] The cell sorting operation may combine different cell sorting techniques / technologies.

[0124] In the preferred embodiment of the invention described herein, step 2. comprises the substeps of:

[0125] i. treating said heterogeneous suspension by density gradient centrifugation in order to isolate a homogeneous suspension comprising a mixture of mononuclear cells;

[0126] ii. adding fluorescent markers to said homogeneous suspension in order to specifically label menstrual stem cells, followed by

[0127] iii. passing the homogeneous suspension having the labeled menstrual stem cells obtained in step c. through flow cytometry to isolate the menstrual stem cells from the homogeneous suspension, wherein the isolated menstrual stem cells are in the unaltered (or native) state.

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[0242]

Claims

Claims

1. A method for the isolation of menstrual stem cells (MenSC) comprising the steps of: a. collecting menstrual blood comprising a heterogeneous suspension comprising a mixture of cells comprising menstrual stem cells in an unaltered state; b. treating said heterogeneous suspension by density gradient centrifugation to isolate a homogeneous suspension comprising a mixture of mononuclear cells comprising said menstrual stem cells; c. adding fluorescent markers to said homogeneous suspension to specifically label the menstrual stem cells, followed by d. passing the homogeneous suspension having the labeled menstrual stem cells obtained in step c. through flow cytometry to isolate the menstrual stem cells from the homogeneous suspension, wherein the isolated menstrual stem cells are in the unaltered state.

2. The method of claim 1, wherein step d. is carried out following step c. and is devoid of any cell culture process.

3. A method according to any preceding claim, wherein said method is devoid of any cell culture process.

4. A method according to any preceding claim, wherein step a. is carried out by means of a menstrual cup.

5. A method according to any preceding claim, wherein said density gradient centrifugation is a Ficoll-Paque process.

6. A method according to any preceding claim, wherein said fluorescent markers comprise a set of positive markers selected from the group consisting of CD90, CD73, CD 105, CD44 and a combination thereof.

7. A method according to any preceding claim, wherein said fluorescent markers comprise a set of negative markers selected from the group consisting of HLA- 33 DR, CD45, CD 14, CD34, CD235a, CD 19 and a combination of these.

8. A method according to any preceding claim, wherein said fluorescent markers consist of CD90, CD73, CD 105, CD44, HLA-DR, CD45, CD 14, CD34, CD235a and CD19.

9. A method according to any preceding claim, consisting of steps a., b., c. and d.

Images