Methods for restoring and enhancing spermatogenesis for infertility male

Abstract

The present invention relates to a method for obtaining autologous cells enriched with mesenchymal stem cells (MSCs) for restoring and enhancing spermatogenesis for infertility male. The method involves the collection of blood mononuclear cells and skin tissue from a patient, enrichment of the cells with MSCs, and culturing them under specific conditions to induce differentiation towards spermatogenesis. The resulting cell population can be used in reproductive technology and other areas of cell therapy. The invention provides a novel approach to address reproductive problems in adult patients using their own cells.

Description

[0001] METHODS FOR RESTORING AND ENHANCING SPERMATOGENESIS FOR INFERTILITY MALE

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a method for obtaining autologous cells enriched with mesenchymal stem cells (MSCs) and restoring and enhancing spermatogenesis for infertility male. The method involves the collection of blood mononuclear cells and skin tissue from a patient, enrichment of the cells with MSCs, and culturing them under specific conditions to induce differentiation towards spermatogenesis. The resulting cell population can be used in reproductive technology and other areas of cell therapy. The invention provides a novel approach to address reproductive problems in adult patients using their own cells.

[0004] BACKGROUND OF THE INVENTION

[0005] In the field of gametogenesis research, cells derived from the early stages of ontogenesis, such as embryonic tissues or amniotic fluid, have been studied extensively. However, clinical applications require the use of a patient's own genetic material. Various approaches have been explored, including the induction of pluripotency and the use of adult-induced pluripotent stem cells (iPSCs). However, these methods involve complex manipulations and non-natural substances, which may lead to unpredictable consequences, including mutations. Therefore, the search for autologous cell populations containing a high number of stem cells has gained significant attention.

[0006] Prior Art US20140308253A1 disclosed an isolated cell, wherein the cell is mitotically competent and expresses Vasa, Oct-4, Dazl, Stella and optionally, an SSEA. In a specific embodiment, the isolated cell is a female germline stem cell and in another specific embodiment, the isolated cell is a female germline stem cell progenitor that expresses SSEA. Preferably, the female germline stem cells, or their progenitor cells, are non-embryonic, mammalian, and even more preferably, human. A method for the isolation of compositions comprising female germline stem cells and / or female germline stem cell progenitors, said method comprising the steps of: a) homogenizing ovarian tissue; b) contacting the tissue with an agent that binds to an SSEA; and c) isolating female germline stem cells and / or female germline stem cell progenitors. The immunomagnetic method was used in biomedical and diagnostic applications to isolate specific target cells or molecules from a complex sample. It involves the use of magnetic beads coated with antibodies or other molecules that can bind to the target of interest.

[0007] Another prior art disclosed by S Saha et. al., Cells. 2021 Jul; 10(7): 1613. (Application of Stem cell Therapy for Infertility) disclosed Mesenchymal Stem Cells (MSCs) are categorized as bone marrow stromal cells, adipose-derived stem cells, menstrual-blood-derived MSCs and umbilical-cord-derived MSCs, amniotic-fluid-derived MSCs, placental-tissue-derived MSCs, salivary-gland-derived MSCs, and dental-pulp-derived MSCs. Various preclinical and clinical studies have reported the potential therapeutic application of MSCs for the treatment of infertility due to ovarian and endometrial dysfunction. MSCs travel to the damaged ovary and settle there to restore ovarian function through the release of various cytokines by paracrine mechanisms. Cytokines such as vascular endothelial growth factor, insulin-like growth factor, and hepatocyte growth factor induce the formation of new vessels, prevent apoptosis as well as fibrosis and thus ameliorate ovarian dysfunction. MSCs also help in the revival of fertility by endometrial regeneration through the release of various bioactive molecules that modulate inflammation and other immune reactions and activate tissue-specific progenitor cells.

[0008] Meanwhile, prior art by YH Cui, et al. Asian J Androl. 2023 Jan-Feb; 25(1): 13-20. (Generation of male germ cells in vitro from the stem cells) provides methods of obtaining functional male gametes for restoring male fertility. Stem cells, including embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), spermatogonial stem cells (SSCs), and mesenchymal stem cells (MSCs), possess the abilities of both self-renewal and differentiation into germ cells. Significantly, much progress has recently been achieved in the generation of male germ cells in vitro from various kinds of stem cells under the specified conditions, e.g., the coculturing with Sertoli cells, three-dimensional culture system, the addition of growth factors and cytokines, and / or the overexpression of germ cell-related genes.

[0009] However, limitations exist in terms of the quantity and regenerative potential of cells obtained from these sources. Therefore, there is a need for alternative restoring and enhancing spermatogenesis for infertility male. The method involves the collection of blood mononuclear cells and skin tissue from a patient, enrichment of the cells with MSCs, and culturing them under specific conditions to induce differentiation towards spermatogenesis. The resulting cell population can be used in reproductive technology and other areas of cell therapy. The invention provides a novel approach to address reproductive problems in adult patients using their own cells. SUMMARY OF THE INVENTION

[0010] The objective of the present invention is to analyze the possibilities of obtaining an autologous population of viable cells from adult patients with reproductive problems for further influence on gametogenesis in-vitro. The invention involves the collection of blood mononuclear cells and skin tissue from a patient. In the case of blood mononuclear cells, the cells are enriched with MSCs and cultured under specific conditions. In the case of skin tissue, the obtained material is trypsinized and cultured under specific conditions. The resulting cell populations are then subjected to density gradient separation to obtain a viable cell suspension enriched with MSCs. The enriched cell population can be used for further manipulations to induce spermatogenesis.

[0011] A method of enhancing spermatogenesis in an infertile male, comprising: a) collecting mesenchymal stem cells (MSCs) from peripheral blood of the infertile male; b) isolating MSCs from skin tissue of the infertile male; c) directing the enrichment of the cells population from peripheral blood and skin tissue by the MSCs; d) mixing two differentiated populations of enriched by MSCs from peripheral blood and skin tissue in a predetermined ratio; e) transplanting the differentiated MSCs into a testis of the infertile male; f) engrafting the transplanted MSCs into seminiferous epithelium of the testis; and g) promoting the differentiation of the transplanted MSCs into functional sperm cells for enhancing spermatogenesis in the infertile male.

[0012] In one aspect of the present invention, the peripheral blood and skin tissue in a predetermined ratio is 1 :1.

[0013] A method of restoring fertility in an infertile male, comprising: a) administering a therapeutically effective amount of autologous MSCs obtained from the male subject to the subject; b) engrafting the administered autologous MSCs into seminiferous epithelium of the infertile male; c) inducing the differentiation of the engrafted MSCs into functional sperm cells; and d) restoring fertility in the infertile male.

[0014] A method for restoring and enhancing spermatogenesis in an infertile male, comprising: a) collecting blood mononuclear cells and skin tissue from the infertile male; b) enriching the collected blood mononuclear cells and skin tissue with MSCs derived from the collected cells; c) culturing the enriched cells under specific conditions, including a combination of adhesion time, gradients separation, and culture supplements, to induce differentiation towards spermatogenesis; d) subjecting the cultured cells to density gradient separation using iohexol to obtain a viable cell suspension enriched with MSCs; and e) obtaining a cell population enriched with MSCs suitable for restoring and enhancing spermatogenesis in the infertile male.

[0015] In another aspect of the method of the present invention, the specific conditions for culturing the enriched cells further comprise the use of growth factors and cytokines that promote the expansion and differentiation of MSCs.

[0016] In one aspect of the method of the present invention, the enriched cell population is cryoconserved for multiple attempts of germ cell differentiation without requiring the presence of the infertile male.

[0017] In another aspect of the method of the present invention, the enriched cell population obtained from the infertile male comprises at least 35-40% MSCs (CD105+).

[0018] In yet another aspect of the method of the present invention, the enriched cell population obtained from the infertile male is subjected to further characterization and identification to ensure the presence of functional MSCs suitable for restoring and enhancing spermatogenesis.

[0019] A composition comprising an isolated autologous double-derived cell population enriched with MSCs obtained according to the method of the present invention, wherein the composition is in the form of a cell suspension for administration to an infertile male for restoring and enhancing spermatogenesis.

[0020] A composition comprising an isolated autologous double-derived cell population enriched with MSCs obtained according to the method of present invention, wherein the composition is in the form of a therapeutic formulation for administration to an infertile male for restoring and enhancing spermatogenesis. One of the advantages of the methods of the present invention is that by using two types of autologous stem cells from different tissues and preparing them separately, it can indeed increase the reparative potential. Combining different types of stem cells allows for a synergistic effect, as each type of stem cell has its unique characteristics and abilities. Moreover, by using two different types of autologous stem cells, this will harness the benefits of each type and enhance the overall regenerative capacity.

[0021] To avoid the use of magnetic antibodies in the separation and culturing of autologous stem cells for direct clinical application, multistage methods of the present invention can be employed. This approach involves several steps, including tissue harvesting, initial cell separation, density gradient centrifugation, cell sorting, and characterization and quality control. By utilizing these stages, it is possible to isolate and cultivate the desired MSCs without relying on magnetic antibodies. It is important to have these methods to the specific stem cell types and therapeutic goals involved for optimal implementation of obtaining such a population from the tissues of adult patient with reproductive problems for further influence on gametogenesis.

[0022] Further advantages of the system in accordance with the invention and its application can be derived from the description and the accompanying drawings. The above-mentioned features and those to be further described below can be utilized in accordance with the invention individually or collectively in arbitrary combination. The embodiments mentioned are not to be considered exhaustive enumerations, rather have exemplary character. The invention is shown in the drawings and explained more closely with reference to embodiments.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, explain the principles of the invention.

[0025] Figure 1 illustrates human blood cells (adherent fraction) in living not-stained culture. Little cells with big nucleus and dance cytoplasm are well visualized among polymorph cells in accordance of the method of the present invention.

[0026] Figure 2 illustrates human blood cells (adherent fraction) in culture. CD105+ and CD105- cells in accordance of the method of the present invention. Figure 3 illustrates skin cells in living not-stained culture with little cells with big nucleus and dance cytoplasm are well visualized at the surface of polygonal cells monolayer in accordance of the method of the present invention.

[0027] Figure 4 illustrates skin cells in culture with CD105+ and CD105- cells in accordance of the method of the present invention.

[0028] Figure 5 illustrates skin cells in living not-stained culture after retinoic acid addition with little cells with big nucleus and dance cytoplasm are well visualized at the surface of polygonal cells monolayer and sometimes form clusters in accordance of the method of the present invention.

[0029] Figure 6 illustrates skin cells in culture after retinoic acid addition. CD105+ and CD105- cells in accordance of the method of the present invention.

[0030] Figure 7 illustrates main sperm indicators before and after cells injection into an infertile male.

[0031] BRIEF DESCRIPTIONS OF THE INVENTION

[0032] The present invention will now be described in detail in connection with specific embodiments with reference to the accompanying drawings. Unless the context requires otherwise, throughout the specification and claims which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to”. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity. It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0033] The term “granulocyte colony-stimulating factor” (G-CSF) such as filgrastim (Neupogen®) or pegfilgrastim (Neulasta®), can be given to be administered to a patient to stimulate the mobilization of cells, particularly MSCs.

[0034] Method for obtaining blood mononuclear cells with enriched MSCs

[0035] A method for obtaining blood mononuclear cells enriched with MSCs, comprising: a) administering granulocyte colony-stimulating factor to a patient; b) collecting venous blood from the patient and adding heparin as an anticoagulant; c) separating cells from the blood using a colloidal plasma substitute solution containing modified liquid gelatin; d) resuspending the separated cells in the remaining supernatant; e) culturing the resuspended cells in a culture medium comprising RPMI medium, autologous plasma, and additives; f) fixing the adherent cell fraction obtained after culturing; g) washing the fixed cells and obtaining a cell pellet; h) providing a gradient separation of small-size cells; and i) identifying MSCs within the enriched cultured cells fraction;

[0036] In one aspect of the present invention, the Granulocyte colony-stimulating factor is administered subcutaneously in a dose dependent on the patient's weight and the colloidal plasma substitute solution contains 4% modified liquid gelatin. The purpose of accelerated "spontaneous" sedimentation of erythrocytes of a colloidal plasma substitute solution containing 4% modified liquid gelatin (and which is approved by the Ministry of Health of Ukraine for intravenous use), as well as a change in the temperature regime i.e. sedimentation of erythrocytes is carried out at a temperature of 20-22° C for 45-60 minutes. For concentration, a careful centrifugation procedure is used to reduce the volume of the sample having the nucleated cells after removing the majority of erythrocytes. A change of the condition of adhesion is provided to the way of minimal using foreign biological material. In the present invention, adhesion of blood MSCs is provided to the plastic surface. In addition, the adhesion time is reduced to 8 hours trying to get the fraction of more active MSCs. Meanwhile, culturing of the resuspended cells includes incubation in a carbon dioxide incubator under standard culture conditions with adhesive fraction cultured in vitro during 3 days. During culturing, retinoic acid, bone morphogenetic protein (BMP-4), and Activin A (Act A) are added to the culture medium. Such duration (cultured in vitro during 3 days) is comparable with cell cycle time of most active stem cells and that is why it should be enough for stabilization of cell population and differentiation way direction which is carried out by addition of retinoic acid, BMP-4 and Activin A. The reduction of duration in vitro without control of immune system and absence of culture passaging of cell population promotes gene stability of autologous cell fraction.

[0037] Another important point of the present invention is that the clinical cell application is provided with animal-free culture medium for cell culturing. In one aspect of the present invention, plasma of an infertile patient is obtained at the time of blood sampling. This is to avoid the contact with non-detectable pathogens and inducer of undesirable changes in personal human cell population. Culture condition contributes to implementation of different cells fate program. MSC cells stay small, ready to proliferation and migration unlike other types of cells in population. For separation the fraction of small size cells (7-9 pm), double density gradient of iohexol solutions is used for blood cells separation. A composition comprising blood mononuclear cells enriched with MSCs obtained according to the method of the present invention.

[0038] One aspect of the invention involves a detailed method for obtaining blood mononuclear cells with enriched MSCs. The method comprises the following steps

[0039] 1. Administration of Granulocyte colony-stimulating factor:

[0040] • Granulocyte colony-stimulating factor, is administered to a patient to stimulate the mobilization of cells, particularly MSCs.

[0041] 2. Collection and Anticoagulation of Venous Blood:

[0042] • Venous blood is collected from the patient using standard techniques, and an anticoagulant, such as heparin, is added to prevent clotting.

[0043] 3. Separation of Cells Using Colloidal Plasma Substitute Solution:

[0044] • The collected blood is subjected to cell separation using a colloidal plasma substitute solution containing modified liquid gelatin. This solution facilitates the isolation of MSCs.

[0045] 4. Resuspension and Culture of Separated Cells:

[0046] • The separated cells are resuspended in the remaining supernatant and cultured in a specific culture medium comprising RPMI, autologous plasma, and additives. Non-adherent fraction of cells was removed. This culture step promotes the adherence and proliferation of MSCs. 5. Enrichment of post-culturing cells suspension by MSC

[0047] • After the end of culturing the adherent cells were removed and undergo gradient separation for small-size cells selection. This step allows to avoid antibody binding and stay suitable for further clinical application.

[0048] 6. Cell Fraction Enriched by MSCs expressing CD105+:

[0049] Small aliquot of cell suspension was taken for immunohistochemistry visualization and counting of CD105+.

[0050] • The isolated cell fraction enriched by MSCs expressing CD105+ can be administered to a target tissue for therapeutic purposes, such as transplantation to restore or enhance cellular function.

[0051] Examples

[0052] Prior to blood sampling, the patient is prescribed a Granulocyte colony-stimulating factor (30- 60 Mill-dose depends of patient weight) was injected subcutaneously. After 24 hours taking the Granulocyte colony-stimulating factor, venous blood (200 ml) was collected from the patient. Heparin (2500 III per 100 ml of blood) was added as anticoagulant. About 20 ml of blood was used for autological plasma was obtained. The preparation of the cell suspension according to a prototype [Harris D. T., Schumacher M. J., Rychlik S. et al. Collection, separation, and cryopreservation of umbilical cord blood for use in transplantation / / Bone Marrow Transplant. - 1994. - B13. - P. 135-143] and according to the method was carried out in parallel on the same samples of biomaterial. Biological material for research was taken in the same quantity. When carrying out the separation of cells according to the prototype, a solution was used, which contained gelatin in a concentration of 3% (the composition of the solution also included glucose, acidic sodium citrate, bi-distilled water). After preparation of the separation of the cells, the solution was poured into vials with a capacity of 50 ml, which were sealed and sterilized in the autoclave mode (1.2 atmospheres for 30 minutes). Mixing was carried out gradually by layering gelatin solution on native blood in a 1 :1 ratio. After careful mixing of the gelatin solution by shaking and ensuring thorough distribution of the gelatin solution within the blood, the gelatin solution on native blood was left for free accelerated sedimentation of erythrocytes at a temperature of +4°C on a tripod in a refrigerator chamber for 45, 55, 60 minutes. Then, the supernatant was transferred to a special container using a plasma extractor and centrifuged at 1200 rpm for 8 minutes. After that, the cell-free plasma was carefully removed, and the cell sediment was resuspended in the remaining plasma. Thereafter, the cells in the suspension were counted, their viability was studied by the method of supravital staining, and the cell composition of the suspension in smears was also studied. When performing the examples according to the above method, a colloidal plasma substitutes solution containing 4% of modified liquid gelatin was carefully added to the blood container in a ratio of 1 : 1 , after which exposures were made for 45, 55, 60 minutes (the duration of the settling procedure depended on the characteristics of the specific sample) at a temperature of 20-22 °C. Blood leukoconcentrate was concentrated by gentle centrifugation at 1200 rpm for 8 minutes at room temperature. The cell-free supernatant was carefully removed, and the cells were resuspended in the remaining supernatant.

[0053] It was found that the percentage of nucleated cells isolated by the prototype is lower than when isolated by the method of the present invention. In addition, when using the prototype method, the loss of nucleated cells in general is much greater and this can be observed on the quality of the samples. The results indicate that the sedimentation of erythrocytes according to the prototype is much slower, and thus the sedimentation procedure is prolonged. At the same time, there are conditions that prevent the free sedimentation of erythrocytes i.e. the gelatin solution forms a gel-like structure when the temperature drops, which contributes both to the retention of erythrocytes and to the "mixing" of nucleated cells, which have a high density with erythrocytes. In addition, at a temperature close to zero, nucleated cells are sensitized to low temperature conditions, which additionally causes a decrease in their preservation during further freezing and thawing. The results of bacteriological control show that the samples prepared according to the prototype have a significantly higher percentage of microflora contamination than the samples prepared according to the method of the present invention. This indicates an increase in the probability of a violation of sterility, which may be related to the need to prepare a 3% gelatin solution, as well as the need to bring containers with the material into a refrigerator for precipitation at a temperature of +4°C. The method of the present invention has better results in the preservation of nucleated cells, which includes stem cells. At the same time, the use of a 4% solution of succinylated gelatin significantly simplifies and shortens the procedure for the selection of nucleated cells. All stages of the procedure are such as to protect the cells from severe mechanical damage.

[0054] Due to single-use materials that have permission for clinical use, as sterile, non-toxic, the method of the present invention meets the conditions of sterility in working with blood and other human biological material. The resulting cell pellet was examined with a light microscope and the total number of cells and the percentage of living were counted using a Makler’s chamber after staining with trypan blue. Thereafter, the cells were resuspended in culture medium, which consist of RPMI, 20% autological plasma of patient, then transferred to culture flasks and kept for 8 hours in a carbon dioxide (CO2) incubator under standard culture conditions (t - 370C, 5% CO2) to fix the adherent cell fraction. At the end of the experiment, the non-adherent fraction was removed, and the fixed layer of cells was washed 3 times with physiological saline.

[0055] After that cell pellet was culturing in the medium, which consists of RPMI (growth medium used in cell culture), 20% autological plasma of patient, 1% GlutaMax Supplement, 1% Antifungal Antibiotic, in a CO2 incubator under standard culture conditions (t - 370C, 5% CO2) during 48 hours. After that 1 pM retinoic acid, 20 ng / ml BMP-4, Activin A 100 ng / ml were added to the culture medium and culturing 24 hours. Visual control and photography were carried out with using of inverted microscope. During the 12-24 hours of cultivation, the adherent fraction of blood mononuclear cells becomes polymorphic. It was observed that cells of different shapes and sizes - round, spindle-shaped, triangular, with processes and without. Some of these cells acquire a very large size and form a vacuolated cytoplasm and a small asymmetric nucleus as illustrated in Figure 1. MSC CD105 positive cells was stained using monoclonal antibody and secondary antibody according with manual protocol. Visualization of positive cells was made by Multivision Polymer Detection. Cell nuclei were stained by hematoxylin. After that Percentage of CD105+ were calculated in the tan fields of view in each sample. Among these cells, there are rounded small cells with dense cytoplasm, a large nucleus and the absence of processes. According to immunohistochemistry, they are CD105+ MSCs as shown in Figure 1 and Figure 2. The number of such cells in the experiment was 16 ± 1 .7% of all nuclear cells in the population. After gradient separation the cell population enriched by MSC was obtained. The number of CD105+ consisted 35 ± 2.1 %.

[0056] Obtaining of autological cells suspension derived from blood mononuclear enriched by MSCs and committed for spermatogenesis

[0057] The research study group included five male patients with an average age of 27- 39 years old. Exclusion criteria of the patients were chronic disease and a BMI higher than 25. Prior to 24 hours before blood sampling patient underwent the Neupogen stimulation (60MILI). His venous blood (200 ml) collected, and heparin (2500 III per 100 ml of blood) was added as an anticoagulant. The fraction of nucleated cells isolated using gelofusine, a succinylated gelatin solution. It is a colloid solution that is commonly used as a volume expander or plasma substitute in medical settings. Then washed twice in the Hank's balanced salt solution (HBSS). The resulting cell pellet examined with a light microscope, the total number of cells and the percentage of living counted. The living mononuclear accounted 97% of mononuclear suspension. Thereafter, the cells resuspended in culture medium, which consist of RPMI, 20% autological plasma of patient, 1 % GlutaMax Supplement, 1 % Antibiotic-Antimycotic, in a carbon dioxide (CO2) incubator under standard culture conditions (temperature - 370C, 5% CO2) during 48 hours. Non-adherent cell fraction was removed after first 8 hours during this period. After that 1 M RA, 20 ng / ml BMP-4, Activin A 100 ng / ml were added to the culture medium and culturing next 24 hours. Then cells were removed mechanically and separated in the density gradient of iohexol solutions. Mononuclear cells suspension diluted with HBSS is layered on double density gradient of iohexol solutions (upper layer is 1080 kg / m3, and lower - 1090 kg / m3). After centrifugation at 700g, a ring containing cells 7-9 pm appears on the boundary between these gradients. These cells were collected and washed twice by physiological saline. Living cells accounted 91%, CD105+ cells - 35% of this suspension.

[0058] Method for Obtaining Skin Cells Enriched with MSCs a) collecting a piece of skin tissue from a patient; b) trypsinizing the skin tissue in trypsin-EDTA diluted with saline; c) incubating the resulting solution and biomaterial at 37°C; d) washing the incubated mixture in Hepes-solution; e) placing the washed mixture in culture flasks; f) culturing the skin cells in a culture medium comprising RPMI medium, autologous plasma, and additives; g) providing enrichment of post-culturing cell suspension by MSCs using gradient separation of small-size cells; and h) identifying MSCs within the enriched cultured skin cells fraction.

[0059] In one aspect of the present invention, the culturing of the skin cells includes incubation in a carbon dioxide incubator under standard culture conditions. During culturing, retinoic acid, bone morphogenetic protein (BMP-4), and Activin A (Act A) are added to the culture medium. Suspension human skin cells without feeder layer is grown, using autologous patient plasma and avoiding long-term culture or passaging in 3-day duration in vitro is comparable with cell cycle time of most active stem cells. Other trophic factors such as retinoic acid, Brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are provided to directly stimulate cultured skin cells. At a final day (the 3rdday) the BMP-4 and Activin A are added in culture medium for germ way differentiation direction. MSC cells stay small, ready to proliferation and migration unlike other types of cells in population. For separation the fraction of small size cells (7-9 pm), double density gradient of iohexol solutions is used for blood cells separation. A composition comprising skin cells enriched with MSCs obtained according to the method of the present invention. Another aspect of the invention involves a detailed method for obtaining skin cells enriched with MSCs. The method comprises the following steps:

[0060] 1. Collection of Skin Tissue:

[0061] • A piece of skin tissue is collected from the patient using standard surgical techniques.

[0062] 2. Trypsinization and Incubation:

[0063] • The collected skin tissue is treated with trypsin-EDTA diluted with saline to obtain a cell solution. The cell solution is then incubated at 37°C for a specified period of time.

[0064] 3. Washing and Plating of Cell Solution:

[0065] • The incubated cell solution is washed in Hepes-solution and placed in culture flasks containing a culture medium comprising autologous plasma. This allows for the adherence and growth of MSCs.

[0066] 4. Culture and Identification of MSCs:

[0067] • The skin cells are cultured under specific conditions, including the use of a culture medium comprising RPMI 1640, autologous plasma, and specific additives. MSCs within the cultured skin cells are identified in small aliquot of cell suspension using immunocytochemistry.

[0068] 5. Enrichment of post-culturing cell suspension by MSCs

[0069] • The removed cells undergo gradient separation for small-size cells selection. This step allows to avoid antibody binding and stay suitable for further clinical application.

[0070] Examples

[0071] The piece of skin tissue (size 10 x 5 x 8 mm) was taken with special scissors and syringe needles. The obtained skin tissue as biomaterial was trypsinized in trypsin-EDTA (0.25%), which was diluted with saline concentration of 1 : 1. Thereafter, the resulting solution together with the biomaterial was put on display for 1 hour at 37°C. Next, the resulting mixture was washed in Hepes-solution and placed into culture flacons. Skin cells were incubated in carbon dioxide (CO2) incubator under standard culture conditions (temperature - 370C, 5% carbon dioxide (CO2)) during 48 hours. Culture medium consists of RPMI 1640, 20% autological plasma of patient, 1% GlutaMax Supplement, 1% Antibiotic-Antimycotic, 40 ng / ml BDNF, 80 ng / ml NGF, 1 pM retinoic acid. After that 1 pM retinoic acid, 20 ng / ml BMP-4, Activin A 100 ng / ml were added to the culture medium and culturing 24 hours. Visual control and photography were carried out with using of inverted microscope. After period of culturing, cell material was removed using trypsin. At the early period of culturing (12- 24hours), it was observed adhesion of skin cells of two main phenotypic appearance (some cells had big elongated nucleus with several nucleoli, some cells had one large round nucleus) at the stages of settling and fixation to the plastic surface as shown in Figure 3. The adhesion of skin cells of different phenotypic begins to form a monolayer, which consists of polygonal cells of the same type that are closely adjacent to each other. MSCs CD105 positive cells was stained in small aliquot of cell suspension using monoclonal antibody and secondary antibody according with manual protocol. Visualization of positive cells was made by Multivision Polymer Detection. Cell nuclei were stained by hematoxylin. After that Percentage of CD105+ were calculated in the tan fields of view in each sample. Among them there are single small rounded cells with dense cytoplasm and a large nucleus, which are CD 105 positive according to histochemical staining as illustrated in Figure 3 and Figure 4. The number of such cells 18.0 ± 2.5% of all living cells in the population. In this case, it was observed the formation of cell clusters, which consisted of 5-20 small rounded cells of the same type as depicted in Figure 5 and Figure 6. After gradient separation, the cell population enriched by MSCs was obtained. The number of CD105+ cells consisted 41 ± 3.2%. Mononuclear cells suspension was diluted with Hank’s solution and was layered on double density gradient of iohexol solutions. Density gradient of the upper layer is 1080 kg / m3, and lower - 1090 kg / m3. After centrifugation at 700 g force, a ring containing cells 7-9 pm appears on the boundary between these gradients. This method provides reduced contamination of the released cells 7-9 pm size with other cell types while maintaining a sufficient portion of viable cells.

[0072] Obtaining of autoloqical cells suspension derived from skin enriched by MSCs and for restoring and enhancing spermatogenesis for infertility male

[0073] Few pieces of skin tissues were taken with special scissors and syringe needles. The obtained material trypsinized in trypsin-EDTA (0.25%), which diluted with saline 1 : 1 , after of which the resulting solution together with the biomaterial put on displayed for 1 hour at 37° C. Then, the resulting mixture was washed in Hepes-solution and placed into culture flacons. Culture medium consisted of RPMI, 20% autological plasma of patient, 1% GlutaMax Supplement, retinoic acid 1 pM, 80 ng / ml NGF (nerve growth factor) and 40 ng / ml BDNF (brain-derived neurotrophic factor), 1% Antibiotic-Antimycotic, in a CO2 incubator under standard culture conditions (temperature - 370C, 5% CO2) during 48 hours. Non-adherent cell fraction was removed after first 8 hours during this period. After that 20 ng / ml BMP-4, Activin A 100 ng / ml were added to the culture medium and culturing next 24 hours. Then cells were removed using trypsin (0.25%), which was diluted with culture medium 1 : 1 , after which the resulting mixture together with the cell complexes were placed for 10 minutes of exposure to the incubator (temperature = 37 C) followed by pipetting. After that cell suspension was rinsed by Hank's balanced salt solution (HBSS) 3 times by centrifugation and separated in the density gradient of iohexol solutions. Mononuclear cells suspension diluted with Hank's balanced salt solution (HBSS) was layered on double density gradient of iohexol solutions (upper layer is 1080 kg / m3, and lower - 1090 kg / m3). After centrifugation at centrifugation at 700 g force, a ring containing cells 7-9 m appears on the boundary between these gradients. These cells were collected and washed twice by physiological saline. Living cells accounted 85%, CD105+ cells - 40% of this suspension.

[0074] The present invention further encompasses methods for restoring and enhancing spermatogenesis for infertility male. These methods involve the utilization of the isolated autological double-derived cell population MSCs. The steps involved include collecting MSCs from peripheral blood, isolating MSCs from skin tissue, directing their differentiation in a shortterm animal-free culture, and transplanting them into the testes of male subjects. The engraftment and differentiation of MSCs into sperm cells in the seminiferous epithelium help restore or enhance spermatogenesis and fertility in male subjects.

[0075] An isolated autologous double-derived cell population enriched by MSCs obtained from both skin and blood sources for transplantation to a patient. The isolated cell population enriched by MSCs are mammalian cells and non-embryonic cells. In addition, these cells are human cells and are derived from the patient's own body.

[0076] A method of enhancing spermatogenesis in an infertile male, comprising: a) collecting mesenchymal stem cells (MSCs) from peripheral blood of the infertile male; b) isolating MSCs from skin tissue of the infertile male; c) directing the enrichment of the cells population from peripheral blood and skin tissue by the MSCs; d) mixing two differentiated populations of enriched by MSCs from peripheral blood and skin tissue in a predetermined ratio; e) transplanting the differentiated MSCs into a testis of the infertile male; f) engrafting the transplanted MSCs into seminiferous epithelium of the testis; and g) promoting the differentiation of the transplanted MSCs into functional sperm cells for enhancing spermatogenesis in the infertile male.

[0077] Meanwhile, a method of restoring fertility to an infertile male (who has undergone chemotherapy or radiotherapy, or both, and desires restored fertility), comprising: a) administering a therapeutically effective amount of autologous MSCs to obtain from the infertile male; b) engrafting the MSCs into the seminiferous epithelium of the infertile male; c) inducing the differentiation of the engrafted MSCs into functional sperm cells; and d) restoring fertility in the infertile male. In another aspect of the present invention, a method for restoring and enhancing spermatogenesis in an infertile male, comprising: a) collecting blood mononuclear cells and skin tissue from the infertile male; b) enriching the collected blood mononuclear cells and skin tissue with MSCs derived from the collected cells; c) culturing the enriched cells under specific conditions, including a combination of adhesion time, gradients separation, and culture supplements, to induce differentiation towards spermatogenesis; d) subjecting the cultured cells to density gradient separation using iohexol to obtain a viable cell suspension enriched with MSCs; and e) obtaining a cell population enriched with MSCs suitable for restoring and enhancing spermatogenesis in the infertile male.

[0078] In another aspect of the present invention, the step b) enriching the blood mononuclear cells or skin tissue with MSCs comprises administering a granulocyte colony-stimulating factor (G- CSF) to the patient to stimulate the production of MSCs in the bone marrow. In addition, the step b) enriching the blood mononuclear cells or skin tissue with MSCs comprises culturing the cells in a suitable medium with growth factors and cytokines that promote the expansion and differentiation of MSCs.

[0079] In one aspect of the present invention, the specific conditions for culturing the enriched cells comprise a combination of adhesion time, gradients separation and culture supplements. The enriched cell population is cryoconserved for multiple attempts of germ cell differentiation without requiring the presence of the infertile male. The enriched cell population obtained from the infertile male comprises at least 35-40% MSCs (CD105+). The enriched cell population obtained from the infertile male is subjected to further characterization and identification to ensure the presence of functional MSCs suitable for restoring and enhancing spermatogenesis.

[0080] In one aspect of the present invention, the step e) obtaining a cell population enriched with MSCs for restoring and enhancing spermatogenesis for infertility male comprises subjecting the cultured cells to density gradient separation to obtain a viable cell suspension enriched with MSCs. The density gradient separation of the present invention is performed using iohexol. The method of the present invention further comprising injecting the autologous cell population enriched with MSCs back into the patient for therapeutic purposes. The therapeutic purposes of the method include treating reproductive problems in the infertile patient. Compositions

[0081] The present invention also claims compositions comprising the isolated autological doublederived cell population enriched by MSCs obtained according to the described methods. These compositions can be in the form of cell suspensions, preparations, or therapeutic formulations suitable for administration and use in various therapeutic applications.

[0082] The present invention provides an in-depth description of the isolated autological doublederived cell population enriched with MSCs and detailed methods for obtaining and utilizing these cells for therapeutic purposes. The methods encompass multiple steps, including the collection of blood and skin tissues, cell separation, culturing, identification, and administration of MSCs. The invention also extends to methods for restoring or enhancing spermatogenesis and fertility, particularly in male subjects. The disclosed invention offers a novel approach to stem cell therapy and regenerative medicine, enhancing the potential for successful treatments and cellular restoration in patients.

[0083] A composition comprising an isolated autologous double-derived cell population enriched with MSCs obtained according to the method for restoring and enhancing spermatogenesis in an infertile male, wherein the composition is in the form of a cell suspension for administration to an infertile male for restoring and enhancing spermatogenesis.

[0084] A composition comprising an isolated autologous double-derived cell population enriched with MSCs obtained according to the method for restoring and enhancing spermatogenesis in an infertile male, wherein the composition is in the form of a therapeutic formulation for administration to an infertile male for restoring and enhancing spermatogenesis.

[0085] Intratesticular injections were done by a direct injection of the MSCs into the testicle tissue once a week for three months. The results of cell treatment were controlled by semen analysis before therapy and one week after last injection. Using the new combination of methods of the present invention, the autological cell populations from adult patient, containing 35-40% of MSCs (CD105+), which effectively stimulated spermatogenesis after injection. It’s can be cryoconserved for many attempts of germ way differentiation without patient’s presence any time. The combined methods of the present invention are provided to obtain the cell population enriched by MSC from the tissue of adult patient with reproductive problem and direct the differentiation of this cells to germ way. The results of confirmed that it was possible to obtain an autologous cell population containing 35-40% MSCs (CD105+) from the tissues (blood and skin) of an adult person and improve the spermogram indicators after injection. It should be noted that this is a primary culture that was not passaged in vitro that contribute to the accumulation of mutations and other undesirable changes in cells.

[0086] Injection of double-derived autologous cell population enriched of MSCs CD105+ to an infertile patient

[0087] Two populations of MSCs (from skin and blood) prepared as described previously were mixed in a predetermined ratio of 1 :1. About 65 million cells, and 89% of those cells are living (65 x 106 cells) (living - 89%) were injected to an infertile patient.

[0088] The wav of cells transplantation (injection)

[0089] Under deep sedation the testicles are sterilised first with a sterilising solution. 0.9% Sodium Chloride solution is added into a previously prepared solution of stem cells of room temperature. Later, the testicle is divided into 4 equal squares by drowning two imaginary perpendicular lines. A retrograde injection approximately 10mm in depth is made with a sterile 30 G needle into all 4 quadrants of the testicle.

[0090] The results of treatment are shown in a spermogram, (also known as a semen analysis or sperm analysis, is a laboratory test used to evaluate various parameters of semen and sperm quality) below in Table 1.

[0091] Table 1. Preoperative and posttreatment simplified semen analysis data according to WHO 6th Edition Criteria The research study group included five male patients with an average age of 29-45 years old. Exclusion criteria of the patients were chronic disease and a BMI higher than 25.

[0092] Five couples suffered from infertility for more than 3 years and were diagnosed with male factor infertility (oligoteratozoospermia). Four couples undergone In vitro fertilization (IVF) and Intracytoplasmic Sperm Injection (ICSI) procedures twice before. One third of the embryos had stopped developing on the third day, the rest reached to a blastocyst stage. All blastocysts undergone a biopsy of trophectoderm cells for next-generation sequencing (NGS). About 95% of the examined blastocysts were aneuploid and 5% of blastocysts according to NGS results were euploid. It was recommended to proceed for embryo transferred process. However, pregnancy did not occur after embryo transferred. One married couple undergone the IVF and ICSI procedures for five times. However, the embryos had not successfully reached the blastocyst stage.

[0093] Meanwhile, semen analysis was prepared three times during a period of treatment (on a first day of a first injection of stem cells into the testicles, one month after the first injection and one month after a last injection) with an obligatory condition of 3 days of abstinence with complete exclusion of smoking and alcohol consumption by the infertile male or the female prior to the spermogram was made. Intratesticular injection of stem cells was carried out 3 times with an interval of 30 days.

[0094] The results of pretreatment and posttreatment of the blood test results of by the infertile male or the female are provided in Table 2 below.

[0095] Table 2. Pretreatment and posttreatment blood test results Blood sampling was carried out twice - on the day of the first intratesticular injection and 30 days after the last intratesticular injection. Figure 7 illustrates main sperm indicators before and after cells injection into an infertile male. Test results showed no significant changes in the course of treatment. In the posttreatment column, it can be observed that a significant improvement in the quality of sperm according to all evaluation criteria, specifically, sperm volume, sperm concentration, total sperm count, sperm motility. During the course of treatment, one couple had a spontaneous pregnancy and the pregnancy was progressing at 20 weeks.

Claims

AMENDED CLAIMS received by the International Bureau on 15 November 2023 (15.11.2023)1. A method of enhancing spermatogenesis in an infertile male, comprising: a) collecting mesenchymal stem cells (MSCs) from peripheral blood of the infertile male; b) isolating MSCs from skin tissue of the infertile male; c) directing the enrichment of the cells population from peripheral blood and skin tissue by the MSCs; d) mixing two differentiated populations of enriched by MSCs from peripheral blood and skin tissue in a predetermined ratio; e) transplanting the differentiated MSCs into a testis of the infertile male; f) engrafting the transplanted MSCs into seminiferous epithelium of the testis; and g) promoting the differentiation of the transplanted MSCs into functional sperm cells for enhancing spermatogenesis in the infertile male.

2. The method as claimed in Claim 1 wherein the peripheral blood and skin tissue in a predetermined ratio is 1 :1.

3. A method of restoring fertility in an infertile male, comprising: a) administering a therapeutically effective amount of autologous MSCs to obtain from the infertile male; b) engrafting the MSCs into the seminiferous epithelium of the infertile male; c) inducing the differentiation of the engrafted MSCs into functional sperm cells; and d) restoring fertility in the infertile male.

4. A method for restoring and enhancing spermatogenesis in an infertile male, comprising: a) collecting blood mononuclear cells and skin tissue from the infertile male; b) enriching the collected blood mononuclear cells and skin tissue with MSCs derived from the collected cells; c) culturing the enriched cells under specific conditions, including a combination of adhesion time, gradients separation, and culture supplements, to induce differentiation towards spermatogenesis; d) subjecting the cultured cells to density gradient separation using iohexol to obtain a viable cell suspension enriched with MSCs; and e) obtaining a cell population enriched with MSCs suitable for restoring and enhancing spermatogenesis in the infertile male.23AMENDED SHEET (ARTICLE 19)5. The method of claim 4, wherein the specific conditions for culturing the enriched cells further comprise the use of growth factors and cytokines that promote the expansion and differentiation of MSCs.

6. The method of claim 4, wherein the enriched cell population is cryoconserved for multiple attempts of germ cell differentiation without requiring the presence of the infertile male.

7. The method of claim 4, wherein the enriched cell population obtained from the infertile male comprises at least 35-40% MSCs (CD105+).

8. The method of claim 4, wherein the enriched cell population obtained from the infertile male is subjected to further characterization and identification to ensure the presence of functional MSCs suitable for restoring and enhancing spermatogenesis.

9. A composition comprising an isolated autologous double-derived cell population enriched with MSCs obtained according to the method of anyone of Claims 1 - 2 and Claims 4-8 wherein the composition is in the form of a cell suspension for administration to an infertile male for restoring and enhancing spermatogenesis.

10. A composition comprising an isolated autologous double-derived cell population enriched with MSCs obtained according to the method of anyone of claims 1-2 and Claims 4-8, wherein the composition is in the form of a therapeutic formulation for administration to an infertile male for restoring and enhancing spermatogenesis.24AMENDED SHEET (ARTICLE 19)

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