CN107287154B - Preparation method, kit and application of male blood-derived autologous spermatogonial stem cells - Google Patents

Preparation method, kit and application of male blood-derived autologous spermatogonial stem cells Download PDF

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CN107287154B
CN107287154B CN201710589900.8A CN201710589900A CN107287154B CN 107287154 B CN107287154 B CN 107287154B CN 201710589900 A CN201710589900 A CN 201710589900A CN 107287154 B CN107287154 B CN 107287154B
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protein
cells
culture solution
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growth factor
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CN107287154A (en
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林雄斌
汤世坤
任海英
廖唤昭
徐胜美
黄卫红
吴铭
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Shenzhen Bainian Stem Cells Technology Research Institute Co ltd
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Shenzhen Bainian Stem Cells Technology Research Institute Co ltd
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Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to a preparation method for generating blood-derived autologous spermatogonial stem cells by reversely differentiating male blood cells, the stem cells and application thereof. The preparation method comprises the step of culturing autologous cells serving as raw material cells in a cell culture solution S1, a cell culture solution S2 and a cell culture solution S3 in sequence to obtain the blood-derived autologous spermatogonial stem cells. The invention applies the human autologous blood cells as autologous raw material cells for more convenient and simpler reverse differentiation production of human autologous spermatogonial stem cells. The invention applies the cell culture solution formula consisting of small molecular substances, and rapidly makes autologous blood cells of a human body reversely differentiate to generate human blood-derived spermatogonial stem cells under the conditions of not changing the chromosome DNA sequence of the somatic cells of the human body and not inserting any foreign genes or DNA fragments. The invention is obviously superior to the prior art in the aspects of production speed, yield and purity of the male blood-derived autologous spermatogonial stem cells.

Description

Preparation method, kit and application of male blood-derived autologous spermatogonial stem cells
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a preparation method for generating blood-derived autologous spermatogonial stem cells by reversely differentiating male blood cells, the stem cells and application thereof.
Background
Stem cells (Stem cells) are a type of pluripotent cells with the ability to self-replicate. Under certain conditions, it can differentiate into a variety of functional cells. The Stem cells are classified into Embryonic Stem cells (ES cells) and adult Stem cells (Somatic Stem cells) according to the developmental stage of the Stem cells. The Stem cells are classified into three types, Totipotent Stem Cells (TSC), Pluripotent Stem cells (Pluripotent Stem cells) and Unipotent Stem cells (unipotential Stem cells), according to their developmental potential. Stem cells are insufficiently differentiated and immature cells, have potential functions of regenerating various tissues, organs and human bodies, and are called universal cells in the medical field.
Spermatogonial Stem Cells (SSCs) are the mother cells of male germ cells, are the only cells in the male adult stem cells which are closely related to the genetic background of the next generation, and have extremely strong self-renewal and differentiation capacity. The sperm and the ovum are combined to form a fertilized egg, and the fertilized egg is continuously divided and differentiated to form a life individual. In recent years, fertility is low or infertility affects 10% to 15% of fertile couples, with male factors accounting for 40% to 60%. Mammalian germ cells undergo meiosis during development to form haploid sperm or eggs, spermatogenesis being a complex and highly ordered process in which errors at any stage can lead to poor fertility or sterility. Although the assisted reproduction technology can bring offspring for part of male infertility patients, the problem of male infertility is not fundamentally solved, and especially for patients with azoospermia or teratospermia syndrome caused by lack of endogenous germ cells, the assisted reproduction technology or medicines cannot be adopted for treatment because germ cells or healthy haploid sperm cells cannot be generated. The spermatogonial stem cells have the regenerative repair function on the structure of the male gonad, so the production technology of the autologous spermatogonial stem cells has very important value and significance in the treatment field of diseases and injuries of the male reproductive system, the repair treatment application field of male infertility, the anti-aging health care field of spermary and the field of establishing an autologous spermatogonial stem cell repository.
The research of the embryonic stem cell technology and the technology of directionally differentiating the in vitro induced pluripotent stem cell (iPS cell) into the male germ cell brings hope to partial male sterile patients; mouse, primate and human ESCs (hESCs) can be induced to differentiate into Primordial Germ Cells (PGCs) in vitro. In addition, recent studies have shown that human pluripotent stem cells (hPSCs) are capable of entering meiosis and in some cases can give rise to haploid cells. However, because of the problems of difficult material taking, immunological rejection, ethical morality and the like of the ESCs, the iPS cell becomes a research hotspot. However, iPS cells are induced in vitro into male germ cells, and have application prospects and problems. Firstly, the safety is high, scientists use retrovirus (retrovirus) to induce pluripotent stem cells, and then replace the cells with lentivirus, adenovirus and plasmid, and the retrovirus and lentivirus can randomly change the genetic material of human cells, which is easy to cause gene abnormality and cancerization. Thus, a safe and effective method for producing or producing spermatogonial stem cells is lacking.
Disclosure of Invention
Aiming at the problems of difficult technical source, complex process, long time consumption, small quantity and doubtful safety of the male autologous spermatogonial stem cells obtained in the prior art, the invention provides a preparation method of male blood-derived autologous spermatogonial stem cells (blood-derived autologous germ stem cells), which is simple, convenient, rapid, safe and high in yield.
In one aspect, the present invention provides a method for preparing male blood-derived autologous spermatogonial stem cells, comprising:
(1) 1 × 10 using autologous cells as raw material cells6～5×106Is inoculated in a cell culture dish;
(2) at 37 ℃ and 5% CO2Culturing the raw material cells in the cell culture solution S1 for 3 days to obtain cells 1; the cell culture solution S1 is RPMI 1640 culture solution containing the following components:
2-10% of Knockout Serum Replacement (KSR), 1-1000 ng/mL of Erythropoietin (EPO), 1-1000 ng/mL of macrophage colony stimulating factor (M-CSF), 1-1000 ng/mL of granulocyte colony stimulating factor (G-CSF), 1-1000 ng/mL of interleukin-3 (IL-3), 1-1000 ng/mL of interleukin-6 (IL-6), 1-1000 ng/mL of Stem Cell Factor (SCF), 1-1000 ng/mL of basic fibroblast growth factor (bFGF), 1-1000 ng/mL of Nanog protein, 1-1000 ng/mL of Oct4 protein, 1-1000 ng/mL of Sox2 protein and 1-1000 ng/mL of C-myc protein;
(3) at 37 ℃ and 5% CO2Culturing the cell 1 obtained in the step (2) in a cell culture solution S2 for 3-6 days to obtain a cell 2; the cell culture solution S2 is an M2 culture solution containing the following components:
2-10% of Knockout Serum Replacement (KSR), 1-1000 ng/mL of Nanog protein, 1-1000 ng/mL of Oct4 protein, 1-1000 ng/mL of Sox2 protein, 1-1000 ng/mL of C-myc protein, 1-1000 ng/mL of germ cell specific protein DDX4/MVH, 1-1000 ng/mL of Dazl protein, 1-1000 ng/mL of basic fibroblast growth factor (bFGF), 1-1000 ng/mL of Epidermal Growth Factor (EGF), 1-1000 ng/mL of interleukin-6 (IL-6), 1-1000 ng/mL of Sox17 protein, 1-1000 ng/mL of VASA protein, 1-1000 ng/mL of Stem Cell Factor (SCF) and 1-1000 ng/mL of granulocyte colony stimulating factor (G-CSF);
(4) at 37 ℃ and 5% CO2Culturing the cell 2 obtained in the step (3) in a cell culture solution S3 for 3-6 days to obtain a cell 3, wherein the cell culture solution S3 is an M16 culture solution containing the following components:
2-10% Knockout Serum Replacement (KSR), 1-1000 μ M nicotinic acid (RA), 1-1000 ng/mL DMC1 protein, 1-1000 ng/mL PRDM1 protein, 1-1000 ng/mL phosphoglycerate kinase 2(PGK2), 1-1000 ng/mL testosterone, 1-1000 ng/mL Follicle Stimulating Hormone (FSH), 1-1000 ng/mL germ cell specific protein DDX4/MVH, 1-1000 ng/mL Dazl protein, 1-1000 ng/mL Oct4 protein, 1-1000 ng/mL Sox17 protein, 1-1000 ng/mL VASA protein, 1-1000 ng/mL basic fibroblast growth factor (bCSF), 1-1000 ng/mL fibroblast growth factor 2 (bFGF), 1-1000 ng/mL fibroblast growth factor 7 (7), 1-1000 ng/mL fibroblast growth factor 7 (FGF), 1000ng/mL osteogenesis factor 2 (FGF), 1000-1000 ng/mL VEGF-1000A-1000 ng/mL VEGF-1 (EGF), 1000-1000 ng/VEGF-1000 ng/mL VEGF-1000 ng/mL VEGF-1 (VEGF-1000-1-1000 ng/mL), 1000ng VEGF-1000-1-1000 ng-1-;
(5) gently blowing and beating the cells 3 obtained after the cells are cultured in the cell culture solution S1, the cell culture solution S2 and the cell culture solution S3 in sequence, collecting the cells 3 in a centrifuge tube after the cells 3 are completely suspended, carrying out centrifugal operation, then using physiological saline to carry out cleaning operation on the suspended cells 3, repeating the cleaning operation for 3 times, and suspending the cells 3 in the physiological saline to obtain the male blood-derived autologous spermatogonial stem cells (blood-derived autologous germ stem cells);
(6) the obtained blood-derived autologous spermatogonial stem cells are expressed as 1 × 105from/mL to 1 × 106Mixing the cell density of/mL with a mixed solution of dimethyl sulfoxide and 10% low molecular dextran with the same volume, cooling the mixture to-80 ℃, and then transferring the mixture to liquid nitrogen with the temperature of-186 ℃ for cryopreservation.
The cells of autologous origin include, but are not limited to, umbilical cord blood cells, placental cells, skin cells, blood nucleated cells, adipocytes, etc. of men.
In a preferred embodiment, the preparation method is that the cell culture solution S1 is RPMI 1640 culture solution containing the following components: 5% Knockout Serum Replacement (KSR), 10ng/mL Erythropoietin (EPO), 10ng/mL macrophage colony stimulating factor (M-CSF), 150ng/mL granulocyte colony stimulating factor (G-CSF), 10ng/mL interleukin-3 (IL-3), 10ng/mL interleukin-6 (IL-6), 10ng/mL Stem Cell Factor (SCF), 100ng/mL basic fibroblast growth factor (bFGF), 30ng/mL Nanog protein, 30ng/mL Oct4 protein, 20ng/mL Sox2 protein and 20ng/mL C-myc protein.
In a preferred embodiment, the preparation method is that the cell culture solution S2 is M2 culture solution containing the following components: 5% Knockout Serum Replacement (KSR), 30ng/mL Nanog protein, 30ng/mL Oct4 protein, 10ng/mL Sox2 protein, 20ng/mL C-myc protein, 30ng/mL germ cell specific protein DDX4/MVH, 30ng/mL Dazl protein, 100ng/mL basic fibroblast growth factor (bFGF), 10ng/mL Epidermal Growth Factor (EGF), 10ng/mL interleukin-6 (IL-6), 100ng/mL Sox17 protein, 50ng/mL VASA protein, 10ng/mL Stem Cell Factor (SCF) and 10ng/mL granulocyte colony stimulating factor (G-CSF).
In a preferred embodiment, the cell culture medium S3 is M16 medium containing 5% Knockout Serum Replacement (KSR), 200. mu.M nicotinic acid (RA), 50ng/mL DMC1 protein, 50ng/mL PRDM1 protein, 50ng/mL phosphoglycerate kinase 2(PGK2), 10ng/mL testosterone, 100ng/mL Follicle Stimulating Hormone (FSH), 50ng/mL germ-specific protein DDX4/MVH, 50ng/mL Dazl protein, 10ng/mL Oct4 protein, 50ng/mL Sox17 protein, 50ng/mL VASA protein, 100ng/mL basic fibroblast growth factor (bFGF), 100ng/mL fibroblast growth factor 2(FGF2), 100ng/mL fibroblast growth factor 7(FGF7), 100ng/mL osteogenesis factor 2 (FGF), 10ng/mL VEGF-derived growth factor 10-VEGF), 10ng/mL VEGF-derived growth factor (VEGF), 10-activating hormone (VEGF), 10-activating factor I), 10 mL VEGF-activating factor (VEGF), 10 mL), 10-activating hormone (VEGF-activating factor I-80, 10 mL), 10 mL, 10-EGF-activating factor (VEGF), 10-activating factor (VEGF-activating factor-7, 5-mL).
In another aspect, the present invention provides a set of culture solutions for retrodifferentiating human somatic cells into autologous spermatogonial stem cells, characterized in that: the group of culture solutions comprises the following components: cell culture fluid S1, cell culture fluid S2 and cell culture fluid S3.
In another aspect, the invention also provides a kit for preparing male blood-derived autologous spermatogonial stem cells, which comprises the cell culture solution S1, the cell culture solution S2 and the cell culture solution S3.
In a preferred embodiment, the kit for preparing the male blood-derived autologous spermatogonial stem cells comprises:
the cell culture solution S1 is RPMI 1640 culture solution containing the following components: 5% Knockout Serum Replacement (KSR), 10ng/mL Erythropoietin (EPO), 10ng/mL macrophage colony stimulating factor (M-CSF), 150ng/mL granulocyte colony stimulating factor (G-CSF), 10ng/mL interleukin-3 (IL-3), 10ng/mL interleukin-6 (IL-6), 10ng/mL Stem Cell Factor (SCF), 100ng/mL basic fibroblast growth factor (bFGF), 30ng/mL Nanog protein, 30ng/mL Oct4 protein, 20ng/mL Sox2 protein and 20ng/mL C-myc protein;
the cell culture solution S2 is M2 culture solution containing the following components: 5% Knockout Serum Replacement (KSR), 30ng/mL Nanog protein, 30ng/mL Oct4 protein, 10ng/mL Sox2 protein, 20ng/mL C-myc protein, 30ng/mL germ cell specific protein DDX4/MVH, 30ng/mL Dazl protein, 100ng/mL basic fibroblast growth factor (bFGF), 10ng/mL Epidermal Growth Factor (EGF), 10ng/mL interleukin-6 (IL-6), 100ng/mL Sox17 protein, 50ng/mL VASA protein, 10ng/mL Stem Cell Factor (SCF) and 10ng/mL granulocyte colony stimulating factor (G-CSF);
cell culture fluid S3 is M16 culture fluid containing 5% Knockout Serum Replacement (KSR), 200. mu.M nicotinic acid (RA), 50ng/mL DMC1 protein, 50ng/mL PRDM1 protein, 50ng/mL phosphoglycerate kinase 2(PGK2), 10ng/mL testosterone, 100ng/mL Follicle Stimulating Hormone (FSH), 50ng/mL germ cell specific protein DDX4/MVH, 50ng/mL Dazl protein, 10ng/mL Oct4 protein, 50ng/mL Sox17 protein, 50ng/mL VASA protein, 100ng/mL basic fibroblast growth factor (bFGF), 100ng/mL fibroblast growth factor 2(FGF2), 100ng/mL fibroblast growth factor 7(FGF7), 100ng/mL osteogenesis morphogenetic protein 2(FGF 3), 100ng/mL osteogenesis factor 2 (FGF), 10ng/mL VEGF-derived growth factor (VEGF), 10-EGF-activating factor 7-VEGF-10-VEGF-activating factor (VEGF-activating factor), 10-VEGF-activating factor (VEGF-activating factor-VEGF-activating factor (VEGF-activating factor-80-activating factor.
In another aspect, the invention provides application of the kit for preparing the male blood-derived autologous spermatogonial stem cells in preparation of medicaments for male testis regeneration and repair, testis health care and anti-aging and medicaments for treating male reproductive diseases.
The invention also provides a preparation method or a kit for preparing the male blood-derived autologous spermatogonial stem cells and application of the blood-derived autologous spermatogonial stem cells prepared by a group of culture solutions in preparation of medicaments for male testis regeneration and repair, testis health care and anti-aging.
The invention also provides a preparation method or a kit for preparing the male blood-derived autologous spermatogonial stem cells and application of the blood-derived autologous spermatogonial stem cells prepared by a group of culture solutions in preparation of medicaments for treating male reproductive diseases.
The male reproductive diseases include but are not limited to male oligospermia and azoospermia, sterility, reproductive/sexual hypofunction, senilism, aging and the like.
The invention also provides a preparation method or a kit for preparing the male blood-derived autologous spermatogonial stem cells and application of the blood-derived autologous spermatogonial stem cells prepared by a group of culture solutions in preparation of production storage banks, in-vitro autologous tissue organ engineering and production.
All the components involved in the cell culture solution S1, the cell culture solution S2 and the cell culture solution S3 of the present invention can be purchased directly from commercial sources or extracted from organisms; the protein component can also be a corresponding recombinant protein obtained by gene recombination technology.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention applies the human autologous blood cells as autologous raw material cells for more convenient and simpler reverse differentiation production of human autologous spermatogonial stem cells.
2. The invention develops a group of cell culture solution formulas which are composed of various small molecular substances and can lead human somatic cells to be reversely differentiated to generate human blood-derived spermatogonial stem cells.
3. The invention applies the cell culture solution formula consisting of small molecular substances, and rapidly makes autologous blood cells of a human body reversely differentiate to generate human blood-derived spermatogonial stem cells under the conditions of not changing the chromosome DNA sequence of the somatic cells of the human body and not inserting any foreign genes or DNA fragments.
4. The method and the kit create a novel technology for producing the male blood-derived autologous spermatogonial stem cells by non-transgenic mode by using the autologous blood cells as raw material cells. The invention is obviously superior to the prior art in the aspects of production speed, yield and purity of the male blood-derived autologous spermatogonial stem cells.
Drawings
Fig. 1 is a view under a microscope of venous blood mononuclear cells (PBMCs) in example 2.
FIG. 2 is a microscopic image of male hematopoietic autologous spermatogonial stem cells obtained after the retrograde differentiation of PBMCs in example 2.
FIG. 3 is a fluorescent photograph of the male blood-derived autologous spermatogonial stem cells reacted with the primary antibody and the secondary antibody in Experimental example 1.
FIG. 4 is a graph showing the results of the measurement of the conversion rate of male blood-derived autologous spermatogonial stem cells in Experimental example 1.
Detailed Description
The following description of the embodiments is only intended to aid in the understanding of the method of the invention and its core ideas. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention. The following description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The molecular biological tests, which are not specifically described in the following examples, were carried out according to the methods specified in molecular cloning, a laboratory manual (third edition) J. SammBruke, or according to the kit and product instructions; the kit biomaterials, if not specifically indicated, are commercially available.
Example 1A kit for preparing male blood-derived autologous spermatogonial stem cells
The kit is operated in a safe operation table with 10-100 grade cleanliness and prepared at a low temperature of 4-10 ℃.
The kit comprises a cell culture solution S1, a cell culture solution S2 and a cell culture solution S3.
Preparation of cell culture solution S1: to 500mL of RPMI 1640 culture solution were added: 5% Knockout serum replacement, 10ng/mL erythropoietin, 10ng/mL macrophage colony stimulating factor, 150ng/mL granulocyte colony stimulating factor, 10ng/mL interleukin-3, 10ng/mL interleukin-6, 10ng/mL stem cell factor, 100ng/mL basic fibroblast growth factor, 30ng/mL Nanog protein, 30ng/mL Oct4 protein, 20ng/mL Sox2 protein, 20ng/mL C-myc protein; fully dissolved, filtered by a filter with a pore size of 0.22 micron, and sterilized to prepare a cell culture solution S1.
Preparation of cell culture solution S2: to 500mL of M2 culture solution were added: 5% Knockout serum replacement, 30ng/mL Nanog protein, 30ng/mL Oct4 protein, 10ng/mL Sox2 protein, 20ng/mL C-myc protein, 30ng/mL germ cell specific protein DDX4/MVH, 30ng/mL Dazl protein, 100ng/mL basic fibroblast growth factor, 10ng/mL epidermal growth factor, 10ng/mL interleukin-6, 100ng/mL Sox17 protein, 50ng/mL VASA protein, 10ng/mL stem cell factor, 10ng/mL granulocyte colony stimulating factor; fully dissolved, filtered by a filter with a pore size of 0.22 micron, and sterilized to prepare a cell culture solution S2.
Preparation of cell culture solution S3: 5% Knockout serum replacement, 200. mu.M nicotinic acid, 50ng/mL DMC1 protein, 50ng/mL PRDM1 protein, 50ng/mL phosphoglycerate kinase 2, 100ng/mL testosterone, 100ng/mL follicle stimulating hormone, 50ng/mL germ cell specific protein DDX4/MVH, 50ng/mL Dazl protein, 10ng/mL Oct4 protein, 50ng/mL Sox17 protein, 50ng/mL VASA protein, 100ng/mL basic fibroblast growth factor, 100ng/mL fibroblast growth factor 2, 100ng/mL fibroblast growth factor 7, 100ng/mL bone morphogenetic protein 2, 100ng/mL osteogenic morphogenetic protein 4, 10ng/mL leukocyte growth factor, 10ng/mL neurocyte growth factor, 10ng/mL fibroblast growth factor, 10ng/mL fibroblast growth factor, 10 ng/10 ng, 10 ng-g, 10ng, and the like growth factor, 10 micro-stimulating factor, and the like growth factor.
And respectively placing the cell culture solution S1, the cell culture solution S2 and the cell culture solution S3 into three mutually isolated containers of the kit, thereby preparing the kit for preparing the male blood-derived autologous spermatogonial stem cells.
Example 2 preparation of Male blood-derived autologous spermatogonial Stem cells
(1) Drawing 50mL of male venous blood (from volunteers) and centrifuging the venous blood by using a conventional Ficoll centrifugal separation technology to obtain blood mononuclear cells, wherein a view under a microscope is shown in figure 1;
(2) the obtained mononuclear cells were washed at 5 × 106The density of (1) was determined using the cell culture solution S1 prepared in example 1 at 37 ℃ and 5% CO2Culturing in an incubator for 3 days to obtain cells 1; then, the cell culture solution S2 prepared in example 1 was used in place of the culture solution, and the amount of the culture solution was 5X10 for cell 16At 37 ℃ and 5% CO2Culturing in an incubator for 3-6 days to obtain cells 2; then, the cell culture solution S3 prepared in example 1 was used in place of the culture solution, and the amount of cells 2 was 5X106At 37 ℃ and 5% CO2Culturing in an incubator for 3-6 days to obtain cells 3;
(3) gently blowing and beating the cells 3 obtained after the cells are cultured in the cell culture solution S1, the cell culture solution S2 and the cell culture solution S3 in sequence, collecting the cells 3 in a centrifuge tube after the cells 3 are completely suspended, carrying out centrifugal operation, then using physiological saline to carry out cleaning operation on the suspended cells 3, repeating the cleaning operation for 3 times, and suspending the cells 3 in the physiological saline to obtain the male blood-derived autologous spermatogonial stem cells, wherein the view of the cells under a microscope is shown in figure 2;
(4) the obtained male blood-derived autologous spermatogonial stem cells are expressed as 1 × 105from/mL to 1 × 106Mixing the cell density of/mL with a mixed solution of dimethyl sulfoxide and 10% of low molecular dextran with the same volume, cooling the mixture to-80 ℃, and then transferring the mixture to liquid nitrogen with the temperature of-186 ℃ for cryopreservation.
Experimental example 1 detection of blood-derived autologous spermatogonial stem cells
(1) Coating the male blood-derived autologous spermatogonial stem cells prepared in the example 2 on a poly-D-lysine glass slide, using a cell slide-making centrifuge to rotate and slide for 2 minutes at 1800RCF to prepare a cell slide, marking, airing the cell slide, and storing the cell slide in a refrigerator at the temperature of-20 ℃ for freezing;
(2) taking out the cell slide from a refrigerator at the temperature of-20 ℃, airing at room temperature, and drawing a region on the reverse side by using a mark; then fixing with 4% paraformaldehyde, perforating with 0.1% Triton X-100 for 5 min, washing with phosphate buffer (PBS, pH 7.4), and sealing with 10% normal goat serum at room temperature for 1 hr to obtain sample;
(3) adding a first antibody reagent to the sample, wherein the first antibody is a rabbit anti-human DAZL polyclonal antibody (1:100 dilution), and reacting the antigen in the sample with the first antibody at 4 ℃ for 12 hours (overnight);
(4) washing the sample reacted with the first antibody using Phosphate Buffered Saline (PBS), and then adding a second antibody corresponding to the first antibody, which is FITC-labeled goat anti-rabbit IgG (1:200 dilution), to the sample to react the first antibody with the second antibody for 1 hour;
(5) performing a nuclear staining step by using DAPI (1:1000) at room temperature, then washing by using Phosphate Buffered Saline (PBS), and performing a mounting agent mounting operation on the sample, wherein the mounting agent is a glycerol/PBS mounting agent (glycerol: PBS is 1: 9, and the pH value is 8.0-8.5);
(6) and observing the sample by using an inverted fluorescence microscope, photographing, and finishing photographing within 6-8 hours.
FIG. 3 shows fluorescence photographs of male blood-derived autologous spermatogonial stem cells prepared in example 2 after sequentially reacting with a primary antibody (rabbit anti-human DAZL polyclonal antibody) and a secondary antibody (FITC-labeled goat anti-rabbit IgG). Fig. 3 shows that the male blood-derived autologous spermatogonial stem cells prepared in example 2 can be observed by a fluorescence microscope after sequentially reacting with a first antibody (rabbit anti-human DAZL polyclonal antibody) and a second antibody (FITC-labeled goat anti-rabbit IgG), which indicates that the cells prepared in example 2 are male blood-derived autologous spermatogonial stem cells.
FIG. 4 is a graph showing the results of the conversion rate measurement of male blood-derived autologous spermatogonial stem cells.
FIG. 3 shows that the cells prepared in example 2 can be observed in a fluorescence microscope as fluorescent parts after reacting with primary antibody (rabbit anti-human DAZL polyclonal antibody) and secondary antibody (FITC-labeled goat anti-rabbit IgG), thereby illustrating that the cells prepared in example 2 are male blood-derived autologous spermatogonial stem cells.

Claims (6)

1. A preparation method of male blood-derived autologous spermatogonial stem cells is characterized by comprising the following steps: the preparation method comprises the following steps:
(1) 1 × 10 using autologous cells as raw material cells6～5×106Is inoculated in a cell culture dish;
(2) at 37 ℃ and 5% CO2Culturing the raw material cells in the cell culture solution S1 for 3 days to obtain cells 1; the cell culture solution S1 is RPMI 1640 culture solution containing the following components:
2-10% of Knockout serum substitute, 1-1000 ng/mL of erythropoietin, 1-1000 ng/mL of macrophage colony stimulating factor, 1-1000 ng/mL of granulocyte colony stimulating factor, 1-1000 ng/mL of interleukin-3, 1-1000 ng/mL of interleukin-6, 1-1000 ng/mL of stem cell factor, 1-1000 ng/mL of basic fibroblast growth factor, 1-1000 ng/mL of Nanog protein, 1-1000 ng/mL of Oct4 protein, 1-1000 ng/mL of Sox2 protein and 1-1000 ng/mL of C-myc protein;
(3) at 37 ℃ and 5% CO2Culturing the cell 1 obtained in the step (2) in a cell culture solution S2 for 3-6 days to obtain a cell 2; the cell culture solution S2 is an M2 culture solution containing the following components:
2-10% of Knockout serum substitute, 1-1000 ng/mL of Nanog protein, 1-1000 ng/mL of Oct4 protein, 1-1000 ng/mL of Sox2 protein, 1-1000 ng/mL of C-myc protein, 1-1000 ng/mL of germ cell specific protein DDX4/MVH, 1-1000 ng/mL of Dazl protein, 1-1000 ng/mL of basic fibroblast growth factor, 1-1000 ng/mL of epidermal cell growth factor, 1-1000 ng/mL of interleukin-6, 1-1000 ng/mL of Sox17 protein, 1-1000 ng/mL of VASA protein, 1-1000 ng/mL of stem cell factor and 1-1000 ng/mL of granulocyte colony stimulating factor;
(4) at 37 ℃ and 5% CO2Culturing the cell 2 obtained in the step (3) in a cell culture solution S3 for 3-6 days to obtain a cell 3, wherein the cell culture solution S3 is an M16 culture solution containing the following components:
2-10% of Knockout serum substitute, 1-1000 μ M of nicotinic acid, 1-1000 ng/mL of DMC1 protein, 1-1000 ng/mL of PRDM1 protein, 1-1000 ng/mL of phosphoglycerate kinase 2, 1-1000 ng/mL of testosterone, 1-1000 ng/mL of follicle stimulating hormone, 1-1000 ng/mL of germ cell specific protein DDX4/MVH, 1-1000 ng/mL of Dazl protein, 1-1000 ng/mL of Oct4 protein, 1-1000 ng/mLSox17 protein, 1-1000 ng/mL of VASA protein, 1-1000 ng/mL of basic fibroblast growth factor, 1-1000 ng/mL of fibroblast growth factor 2, 1-1000 ng/mL of fibroblast growth factor 7, 1-1000 ng/mL of fibroblast growth factor 2, 1-1000 ng/mL of osteogenic protein 2, 1-1000 ng/mL of osteogenic morphogenetic protein, 1-1000 ng/mL of angiogenin growth factor 4, 1-1000 ng/mL of angiopoietic factor 1-1000 ng/mL, 1000ng/mL of angiopoietic factor I-1000 ng/mL of hematopoietic factor, 1000-1000 ng/1-1000 ng/mL of hematophagostimulin stimulating hormone, 1-1000 ng/mL of insulin growth factor I, 1-1000 ng/1-1000 ng of insulin growth factor II, 1-1000 ng-1000 ng-1-1000 ng stimulating hormone, 1-1000 ng-1-1000 ng-1-1000 ng hormone, 1-1000 ng-1-1000 ng hormone, 1-;
(5) and lightly blowing and beating the cells 3 obtained after the cells are cultured in the cell culture solution S1, the cell culture solution S2 and the cell culture solution S3 in sequence, collecting the cells 3 in a centrifuge tube after the cells 3 are completely suspended, carrying out centrifugal operation, then using physiological saline to carry out cleaning operation on the suspended cells 3, repeating the cleaning operation for 3 times, and suspending the cells 3 in the physiological saline to obtain the male blood-derived autologous spermatogonial stem cells.
2. The method of claim 1, wherein: the autologous cells are umbilical cord blood cells, placenta cells, skin cells, blood nucleated cells or fat cells of the male.
3. The method of claim 2, wherein: the autologous cells are blood mononuclear cells of the male.
4. The method according to any one of claims 1 to 3, wherein: the cell culture solution S1 is RPMI 1640 culture solution containing the following components:
5% Knockout serum replacement, 10ng/mL erythropoietin, 10ng/mL macrophage colony stimulating factor, 150ng/mL granulocyte colony stimulating factor, 10ng/mL interleukin-3, 10ng/mL interleukin-6, 10ng/mL stem cell factor, 100ng/mL basic fibroblast growth factor, 30ng/mL Nanog protein, 30ng/mL Oct4 protein, 20ng/mL Sox2 protein, and 20ng/mL C-myc protein;
the cell culture solution S2 is an M2 culture solution containing the following components:
5% Knockout serum replacement, 30ng/mL Nanog protein, 30ng/mL Oct4 protein, 10ng/mL Sox2 protein, 20ng/mL C-myc protein, 30ng/mL germ cell specific protein DDX4/MVH, 30ng/mL Dazl protein, 100ng/mL basic fibroblast growth factor, 10ng/mL epidermal growth factor, 10ng/mL interleukin-6, 100ng/mL Sox17 protein, 50ng/mL VASA protein, 10ng/mL stem cell factor and 10ng/mL granulocyte colony stimulating factor;
the cell culture solution S3 is an M16 culture solution containing the following components:
5% Knockout serum replacement, 200. mu.M niacin, 50ng/mL DMC1 protein, 50ng/mL PRDM1 protein, 50ng/mL phosphoglycerate kinase 2, 10ng/mL testosterone, 100ng/mL follicle stimulating hormone, 50ng/mL germ cell specific protein DDX4/MVH, 50ng/mL Dazl protein, 10ng/mL Oct4 protein, 50ng/mLSox17 protein, 50ng/mL VASA protein, 100ng/mL basic fibroblast growth factor, 100ng/mL fibroblast growth factor 2, 100ng/mL fibroblast growth factor 7, 100ng/mL osteogenic morphogenetic protein 2, 100ng/mL osteogenic morphogenetic protein 4, 10/mL interleukin-6, 10ng/mL neurotrophic factor 4, 10ng/mL glial derived neurotrophic factor, 10ng/mL glial cell derived neurotrophic factor, 10ng/mL activating factor 10ng/mL, 10ng/mL angiopoietin-activating factor, 10ng/mL, 10ng growth factor, 10-activating factor, 10 mL, 10ng/mL endothelial growth factor, 10-releasing hormone, 10-activating factor, 10-10 mL, 10-mL endothelial growth factor, and a-10-g hormone.
5. A group of culture solutions for reversely differentiating human somatic cells into autologous spermatogonial stem cells is characterized in that: the group of culture solutions comprises: cell culture fluid S1, cell culture fluid S2, and cell culture fluid S3;
the cell culture solution S1 is RPMI 1640 culture solution containing the following components:
2-10% of Knockout serum substitute, 1-1000 ng/mL of erythropoietin, 1-1000 ng/mL of macrophage colony stimulating factor, 1-1000 ng/mL of granulocyte colony stimulating factor, 1-1000 ng/mL of interleukin-3, 1-1000 ng/mL of interleukin-6, 1-1000 ng/mL of stem cell factor, 1-1000 ng/mL of basic fibroblast growth factor, 1-1000 ng/mL of Nanog protein, 1-1000 ng/mL of Oct4 protein, 1-1000 ng/mL of Sox2 protein and 1-1000 ng/mL of C-myc protein;
the cell culture solution S2 is an M2 culture solution containing the following components:
2-10% of Knockout serum substitute, 1-1000 ng/mL of Nanog protein, 1-1000 ng/mL of Oct4 protein, 1-1000 ng/mL of Sox2 protein, 1-1000 ng/mL of C-myc protein, 1-1000 ng/mL of germ cell specific protein DDX4/MVH, 1-1000 ng/mL of Dazl protein, 1-1000 ng/mL of basic fibroblast growth factor, 1-1000 ng/mL of epidermal cell growth factor, 1-1000 ng/mL of interleukin-6, 1-1000 ng/mL of Sox17 protein, 1-1000 ng/mL of VASA protein, 1-1000 ng/mL of stem cell factor and 1-1000 ng/mL of granulocyte colony stimulating factor;
the cell culture solution S3 is an M16 culture solution containing the following components:
2-10% Knockout serum substitute, 1-1000 μ M nicotinic acid, 1-1000 ng/mL DMC1 protein, 1-1000 ng/mL PRDM1 protein, 1-1000 ng/mL phosphoglycerate kinase 2, 1-1000 ng/mL testosterone, 1-1000 ng/mL follicle stimulating hormone, 1-1000 ng/mL germ cell specific protein DDX4/MVH, 1-1000 ng/mL Dazl protein, 1-1000 ng/mL Oct4 protein, 1-1000 ng/mLSox17 protein, 1-1000 ng/mL VASA protein, 1-1000 ng/mL basic fibroblast growth factor, 1-1000 ng/mL fibroblast growth factor 2, 1-1000 ng/mL fibroblast growth factor 7, 1-1000 ng/mL osteogenin morphogenetic protein 2, 1-1000 ng/mL osteogenesis factor 4, 1-1000 ng/1 ng/mL, 1000 ng/1-1000 ng/mL VEGF-1000 ng/1-1000 ng/mL, 1000 ng/1-1000 ng/mL insulin-stimulating hormone, 1000 ng-1-1000 ng/mL VEGF-1000 ng stimulating hormone, 1-1000 ng-1-1000-mL fibroblast growth factor, 1-1000-1-1000 ng-1-mL fibroblast growth factor, and-1000-1-.
6. A kit for preparing human blood-derived autologous spermatogonial stem cells, which is characterized in that: the kit comprises the cell culture solution S1, the cell culture solution S2 and the cell culture solution S3 of claim 5.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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