CN111484971A - Preparation method, kit and application of blood-derived female autologous reproductive stem cells - Google Patents

Preparation method, kit and application of blood-derived female autologous reproductive stem cells Download PDF

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CN111484971A
CN111484971A CN202010379524.1A CN202010379524A CN111484971A CN 111484971 A CN111484971 A CN 111484971A CN 202010379524 A CN202010379524 A CN 202010379524A CN 111484971 A CN111484971 A CN 111484971A
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stem cells
autologous
cells
female
blood
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林雄斌
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Lin Liuyin
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Lin Liuyin
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Abstract

The invention relates to a preparation method, a kit and application of blood-derived female autologous germ stem cells, and relates to a technology and a method for producing the blood-derived female autologous germ stem cells by non-transgenosis by using female autologous blood cells as raw material cells. The preparation method of the autologous reproductive stem cells of the blood-borne female comprises the following steps: culturing the female autologous blood cells in a cell culture solution O1 for 3 days; continuously culturing in cell culture solution O2 for 6-9 days; then continuously culturing for 6-9 days in a cell culture solution O3; collecting autologous reproductive stem cells of the blood-borne female; the invention also provides a kit for preparing the blood-borne female autologous reproductive stem cells. The method and the kit have remarkable advantages in the aspects of material taking, production speed, yield and purity of the autologous reproductive stem cells of the blood-borne female.

Description

Preparation method, kit and application of blood-derived female autologous reproductive stem cells
Technical Field
The invention relates to the field of biomedical technology, in particular to a cell culture method for generating blood-derived autologous germ stem cells by reversely differentiating female blood cells, the stem cells and application.
Background
Nowadays, with the advent of aging society, aging problems are gradually being emphasized by people. In women, the rate of ovarian aging is significantly faster than other organs of the body. Ovarian aging affects female health, causing the arrival of menopause, cancer and the development of a variety of chronic diseases. However, with the increasingly worsening of living environment, the increasing of living pressure, the damage of the side effects of drug therapy and other factors to the female ovarian function, the ovogenic infertility and the pathological ovarian function decline become great social health problems which plague modern women. Especially with the implementation of the two-birth policy, the demand for quality of life and fertility longevity in elderly women is becoming increasingly strong. Therefore, elucidating the mechanism of the physiological and pathological decline of ovarian function and establishing effective intervention means have become an irresistible research topic in the medical field! The discovery of female germ stem cells in recent years has brought unprecedented hopes for effectively increasing primordial follicle pool, improving ovarian function, delaying ovarian senescence and treating ovarian-derived diseases!
The ovary is an extremely important reproductive organ and life-span determining organ of female mammals and human females, and the physiological processes of generation, development, maturation, ovulation and depletion of reproductive stem cells in the ovary deduct various physiological functions of the life of the female, including fertility, vital capacity, life content and life length. As an important life energy source cell, the consumption and supply of the ovary-derived autologous reproductive stem cells play an important role in the maintenance of female reproductive health and life activities. However, the germ stem cells in the ovaries of women are extremely rare and insufficient to counteract the normal consumption of the body, so that women near the age of 50 years or so essentially lose fertility and suffer from various symptoms caused by the decline of ovarian function in the next third of their lives. Therefore, searching for additional sources of autologous reproductive stem cells except for the ovary, supplementing the number of reproductive stem cells of the aged ovary, maintaining the ovarian function for a long time without decline, and prolonging the ovarian life, so that the reproductive life and the natural life of the female individual are prolonged, and the quality of life is improved, which is an important subject of life science.
A female germ stem cell is a germ cell that has the ability to self-renew and has the potential to differentiate into daughter cells (eggs). In vivo, germ stem cells are usually present in a specific stem cell microenvironment, and maintain stem cell "stem" properties through self-replication while differentiating into oocytes step by step. In women, the germ stem cells in the ovarian gonads are derived from a rare number of primordial germ cells that are transmitted from the embryo. During embryogenesis and ovarian formation, germ stem cells colonize the ovaries, dominate ovarian differentiation, development and maturation. The germ line stem cell develops to form an egg germ cell ► primary follicle ► secondary follicle ► mature follicle ► ovum. The germ stem cell has Mvh+And Dazl+Positive specific cell phenotype. The germ stem cells of the ovary are a slow process of consumption in the process of producing follicles and ova. In the life of a woman, after about 400 to 500 follicular developmental maturity, ovulation and menstruation, reproductive stem cells in ovaries are gradually consumed and exhausted, the ovaries do not produce ovaries and follicles any more, the main sources of estrogen are exhausted at the same time, the ovaries shrink and diminish, the ovaries are aged to lose reproductive functions, and the woman enters the old.
The emergence of the stem cell in vitro differentiation technology is a milestone progress in the research field of stem cell technology. Johnson et al in 2005 found a reserve of "germ stem cells" in the ovaries of female mice. In recent years, research shows that some adult non-germ stem cells can be induced to differentiate into germ stem cells and oocyte-like cells in vitro, but whether the formed germ cells can perform meiosis and further develop into gametes with fertilization capability and development potential is not clear.
Embryonic stem cells have developmental totipotency and can be differentiated into any cell type of the body, including germ stem cells and germ cells. Hubner, et al 2003, first reported the success in inducing mouse embryonic stem cells to differentiate into oocytes in vitro, expressing germline-specific RNA and protein markers. Although embryonic stem cells are an ideal source of female germ stem cells and germ cells, the specific mechanism is still clear, and the potential problems of teratoma formation risk, immunological rejection risk, ethical dispute and the like which cannot be avoided exist, and the research of the embryonic stem cells in human and animals is also limited.
An adult stem cell is a stem cell having repair and regeneration abilities, and is present in various tissues and organs of adults. Experiments show that under certain conditions, adults can self-renew to form new stem cells or differentiate to form functional cells. Bone marrow mesenchymal stem cells (BMSCs) can differentiate into various types of cells, including skeletal muscle cells, nerve cells, cardiac muscle cells, and liver cells, among others. Recent studies have shown that BMSCs also have the potential to differentiate into germ cells. Johnson et al found that germ cell marker-expressing cells were present in rodents, women's peripheral blood and bone marrow and that Mvh expression levels in bone marrow cells of adult females were positively correlated with the estrous cycle and oocyte number, suggesting that germ line stem cells could be supplied from the bone marrow via the circulatory system. However, the content of MSCs in bone marrow is very small, and the number of cells gradually decreases with age, so that such a trace amount of MSCs is difficult to satisfy the needs of research and treatment.
In 2006, Takahashi et al first transfected Oct4, Sox2, c-Myc and Klf4 four transcription factors into mouse skin fibroblasts to successfully obtain iPS cells, and the iPS cells are similar to ESCs in aspects of morphology, growth characteristics, stem cell marker expression, multi-directional differentiation potential and the like, except that the iPS cells do not relate to ethical issues, and eliminate potential immune rejection, can theoretically replace ESCs for basic and clinical research, and become milestone achievements in the field of stem cell research. In 2009, Park et al co-cultured human iPS and human fetal gonad stromal cells in early pregnancy, and found that iPS can be differentiated towards germ stem cells, but differentiated cells have no meiosis-related gene expression and cannot produce gametes. In 2010, Imamura et al successfully induced the iPS line expressing the germ stem cell marker Mvh-RFP into germ stem cells by using BMP4, expressed early germ cell markers, and induced and differentiated into oocyte-like cells by trans-retinoic acid A, but the cells were all arrested in the early stage and could not develop and mature. Because iPS formed by introducing different genes has great difference in differentiation and development potential, establishing an iPS more favorable for the development of germ line cells is particularly important. In addition, transformation efficiency of iPS is low, and the risk of potential abnormalities in cytogenetics and epigenetics arising from iPS reprogramming or in subsequent cell culture processes is increased, limiting clinical applications of iPS.
At present, how to obtain more and better non-ovarian female reproductive stem cells is a worldwide problem, and a satisfactory technical method is not obtained yet.
Here, we report a technical method for producing hematopoietic female germ-like stem cells by chemical induction of rapid retrograde differentiation of blood mononuclear cells. The blood-derived germ-like stem cell not only shows a cell specific phenotype similar to that of the ovary-derived germ-like stem cell, but also can develop and form all levels of growing follicles and mature follicles under the in vitro culture condition.
The combined formula containing a plurality of small molecular protein cell culture solutions is applied to reversely differentiate female somatic cells, particularly blood mononuclear cells to generate blood-borne female totipotent stem cells, and then the differentiation is continued to generate blood-borne female autologous reproductive stem cells. The method comprises the steps of firstly culturing the female blood mononuclear cells by using a cell culture solution A for 3 days, and then culturing by using a culture solution B for 6-9 days, so that the blood mononuclear cells are reversely differentiated to form multi-colony totipotent stem cells. These totipotent stem cells express major cell-specific markers of totipotent stem cells, such as Oct4, Nanog, Klf4, and SOX 2. And then, placing the totipotent stem cells in a cell culture solution C for further culturing for 6-9 days to form new germ stem cells, wherein the new germ stem cells show similar biological characteristics to natural ovary-derived germ stem cells, such as positive expression of germ stem cell specific markers such as Dazl, Mvh, Fragilis and the like. The germ stem cells can be propagated and passaged once in 10-15 days under the culture of cell passage liquid, and normally, autologous germ stem cells can be passaged for 3-5 times. After each generation of cells after cell culture and passage, the cells express cell specific phenotypes of reproductive stem cells such as Dazl, Mvh, Fragilis and the like.
The technology for producing autologous reproductive stem cells of blood-borne women realizes that each female (especially adult female) always has an opportunity to produce and apply autologous stem cells of the own person at any stage and any period in the life process of the female. The autologous stem cell production series technology based on the huge number of somatic cells as raw material cells can produce hundreds of millions of autologous reproductive stem cells within 15-21 days, has huge application value and social value in aspects of scientificity, advancement, safety, effectiveness and the like, and is a life seed cell with actual significance.
The self-reproduction stem cell is an important seed cell of the ovary and has a regenerative repair function on the structure of the ovary, so the production technology of the self-reproduction stem cell has very important value and significance in the treatment field of diseases and damages of the female reproductive system, the repair treatment application field of female infertility, the anti-aging health care field of the ovary and the field of establishing a self-reproduction stem cell repository.
Disclosure of Invention
The technical problem to be solved by the invention is as follows:
1. the human autologous blood cells are used as autologous raw material cells for more conveniently and simply producing the female autologous germ stem cells through reverse differentiation;
2. developing 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 female blood-borne germ stem cells;
3. by using the cell culture solution formula consisting of small molecular substances, the autologous blood cells of a human are quickly differentiated reversely to generate the female blood-derived germ stem cells under the conditions of not changing the chromosome DNA sequence of the somatic cells of the human and not inserting any foreign genes or DNA fragments, and the novel autologous stem cells are obtained.
In order to solve the above technical problems, an object of the present invention is to:
1. providing 1 group of cell culture solution formulas for generating autologous female reproductive stem cells by reversely differentiating human somatic cells;
2. provides a technical method for generating the blood-borne germ stem cells by human somatic cells by adopting the cell culture solution formula;
3. provides a kit of the germ stem cell obtained by the method.
The purpose of the invention is realized by the following technical scheme:
2 × 10 using female autoblood leucocyte as raw material cell6Is seeded in a cell culture dish. The raw material cells include, but are not limited to, umbilical cord blood cells, placental cells, skin cells, blood nucleated cells, adipocytes, etc. of women.
Autologous raw material cells were cultured in cell culture medium 01 (RPMI 1640 medium) at 37 ℃ and 5% CO2 for 3 days. The culture solution 01 contains:
2-10% serum replacement (KSR), preferably 5%;
EPO1-1000 ng/ml, preferably 10ng/ml;
M-GSF 1-1000ng/ml, preferably 10ng/ml;
G-GSF 1-1000ng/ml, preferably 100 ng/ml;
i L-31-1000 ng/ml, preferably 10ng/ml;
i L-61-1000 ng/ml, preferably 10ng/ml;
SCF 1-1000ng/ml, preferably 10ng/ml;
bFGF 1-1000ng/ml, preferably 100 ng/ml;
nanog is 1-1000ng/ml, and the preferred value is 30 ng/ml;
oct41-1000 ng/ml, with the preferred value of 30 ng/ml;
sox 21-1000 ng/ml, preferably 20 ng/ml;
c-myc 1-1000 ng/ml; the preferred value is 20 ng/ml;
the raw material cells were cultured in cell culture broth 02 (M2 broth) at 37 ℃ and 5% CO2 for 6-9 days. The culture solution 02 contains:
2-10% serum replacement (KSR), preferably 5%;
nanog is 1-1000ng/ml, and the preferred value is 30 ng/ml;
oct41-1000 ng/ml, with the preferred value of 30 ng/ml;
sox 21-1000 ng/ml, preferably 20 ng/ml;
c-myc is 1-1000ng/ml, and the preferred value is 20 ng/ml;
DDX4(Mvh) 1-1000ng/ml, preferably 50 ng/ml;
dazl1-1000 ng/ml, preferably 50 ng/ml;
bFGF 1-1000ng/ml, preferably 100 ng/ml;
EGF 1-1000ng/ml, preferably 10ng/ml;
i L-61-1000 ng/ml, preferably 10ng/ml;
sox171-1000 ng/ml, preferably 50 ng/ml;
VASA 1-1000ng/ml, preferably 50 ng/ml;
SCF 1-1000ng/ml, preferably 10ng/ml;
G-GSF 1-1000ng/ml, preferably 10ng/ml;
somatic cells or mononuclear cells were cultured in cell culture broth 03 (M16 broth) at 37 ℃ and 5% CO2 for 6-9 days. The culture solution 03 contains:
2-10% serum replacement (KSR), preferably 5%;
RA is 1-1000 uM, preferably 200 uM;
DMC 11-1000 ng/ml, with 30ng/ml being preferred;
PRDM 11-1000 ng/ml, preferably 30 ng/ml;
PGK 21-1000 ng/ml, preferably 30 ng/ml;
hCG 1-200 iu/ml, preferably 50 iu/ml;
progesterone 1-200 iu/ml, preferably 50 iu/ml;
estradiol 1-200 iu/ml, preferably 50 iu/ml;
FSH1-1000 ng/ml, preferably 100 ng/ml;
DDX4(Mvh) 1-1000ng/ml, preferably 30 ng/ml;
dazl1-1000 ng/ml, preferably 30 ng/ml;
oct41-1000 ng/ml, with the preferred value of 10ng/ml;
sox171-1000 ng/ml, preferably 30 ng/ml;
VASA 1-1000ng/ml, preferably 50 ng/ml;
bFGF 1-1000ng/ml, preferably 100 ng/ml;
VASA 1-1000ng/ml, preferably 50 ng/ml;
FGF 21-1000 ng/ml, preferably 10ng/ml;
FGF 71-1000 ng/ml, preferably 10ng/ml;
BMP 21-1000 ng/ml, preferably 10ng/ml;
BMP 41-1000 ng/ml, preferably 10ng/ml;
i L-61-1000 ng/ml, preferably 10ng/ml;
NT 41-1000 ng/ml, preferably 10ng/ml;
GDNF 1-1000ng/ml, preferably 10ng/ml;
activin A1-1000 ng/ml, with the preferred value of 10ng/ml;
UB 1-1000ng/ml, preferably 100 ng/ml;
17 α,20 β -DHP 1-1000ng/ml, preferably 10ng/ml;
AY 9944-A-71-1000 ng/ml, preferably 10ng/ml;
EGF 1-1000ng/ml, preferably 10ng/ml;
VEGF 1-1000ng/ml, preferably 10ng/ml;
SCF 1-1000ng/ml, preferably 10ng/ml;
IGF 1-1000ng/ml, preferably 10ng/ml;
IGF I1-1000 ng/ml, preferably 10ng/ml;
IGF II1-1000 ng/ml, preferably 10ng/ml;
GRF 1-1000ng/ml, preferably 10ng/ml;
ifitm 31-1000 ng/ml, preferably 10ng/ml;
GH 1-1000ng/ml, preferably 10ng/ml;
dexamethasone 1-1000ng/ml, preferably 10ng/ml;
G-GSF 1-1000ng/ml, preferably 10ng/ml;
and lightly blowing and beating the cells cultured in the cell culture solution 01, the cell culture solution 02 and the cell culture solution 03 respectively to completely suspend the cells, collecting the cells in a centrifuge tube, performing centrifugal operation, then using physiological saline to perform cleaning operation on the suspended cells, repeating the operation for 3 times, and suspending the cells in the physiological saline to obtain the autologous hematopoietic stem cells of the hematogenous female.
The obtained blood-derived autologous germ stem cells are treated with 1 × 105From ml to 1 × 106Mixing the cell density of per ml with the mixed solution of dimethyl sulfoxide and 10% low molecular dextran of the same volume, cooling the mixture to-80 ℃, and then transferring the mixture to liquid nitrogen of-196 ℃ for cryopreservation.
In the present method, the preferred culture medium used is:
the culture solution 01 is RPMI1640 culture solution, which contains:
5% serum replacement (KSR);
EPO 10 ng/ml;
M-GSF 10 ng/ml;
G-GSF 150 ng/ml;
IL-3 10 ng/ml;
IL-6 10 ng/ml;
SCF 10 ng/ml;
bFGF 100 ng/ml;
Nanog 30 ng/ml;
Oct4 30 ng/ml;
Sox2 20 ng/ml;
c-myc 20 ng/ml。
the culture solution 02 is M2 culture solution, which contains:
5% serum replacement (KSR);
Nanog 30 ng/ml;
Oct4 30 ng/ml;
Sox2 10 ng/ml;
c-myc 20 ng/ml;
DDX4(Mvh) 30 ng/ml;
Dazl 30 ng/ml;
bFGF 100 ng/ml;
EGF 10 ng/ml;
IL-6 10 ng/ml;
Sox17 100 ng/ml;
VASA 50 ng/ml；
SCF 10 ng/ml;
G-GSF 10 ng/ml;
culture solution 03 is M16 culture solution, which contains:
RA 200 uM;
DMC1 50 ng/ml;
PRDM1 50 ng/ml;
PGK2 50 ng/ml;
hCG 50iu/ml；
progesterone 50 iu/ml;
estradiol 50 iu/ml;
FSH 100 ng/ml；
DDX4(Mvh) 50 ng/ml;
Dazl 50 ng/ml,
Oct4 10 ng/ml;
Sox17 50 ng/ml;
VASA 50 ng/ml；
bFGF 100 ng/ml;
FGF 2 100 ng/ml;
FGF 7 100 ng/ml;
BMP2 100 ng/ml;
BMP4 100 ng/ml;
IL-6 10 ng/ml;
NT 4 10 ng/ml;
GDNF 10 ng/ml;
Activin A 10 ng/ml;
UB 100 ng/ml;
17α,20β-DHP 10 ng/ml;
AY9944-A-7 10 ng/ml;
EGF 10 ng/ml;
VEGF 10 ng/ml;
SCF 10 ng/ml;
IGF10 ng/ml;
IGF I 10 ng/ml;
IGF II 10 ng/ml;
GRF 10 ng/ml;
Ifitm3 10 ng/ml;
GH 10 ng/ml;
dexamethasone 100 ng/ml;
G-GSF 10 ng/ml。
the invention also provides a kit for preparing the blood-borne female autologous reproductive stem cells. The kit comprises cell culture solution 01, cell culture solution 02 and cell culture solution 03. Wherein:
the culture solution 01 is RPMI1640 culture solution, which contains:
2-10% serum replacement (KSR), preferably 5%;
EPO1-1000 ng/ml, preferably 10ng/ml;
M-GSF 1-1000ng/ml, preferably 10ng/ml;
G-GSF 1-1000ng/ml, preferably 100 ng/ml;
i L-31-1000 ng/ml, preferably 10ng/ml;
i L-61-1000 ng/ml, preferably 10ng/ml;
SCF 1-1000ng/ml, preferably 10ng/ml;
bFGF 1-1000ng/ml, preferably 100 ng/ml;
nanog is 1-1000ng/ml, and the preferred value is 30 ng/ml;
oct41-1000 ng/ml, with the preferred value of 30 ng/ml;
sox 21-1000 ng/ml, preferably 20 ng/ml;
c-myc 1-1000 ng/ml; the preferred value is 20 ng/ml;
the culture solution 02 is M2 culture solution, which contains:
2-10% serum replacement (KSR), preferably 5%;
nanog is 1-1000ng/ml, and the preferred value is 30 ng/ml;
oct41-1000 ng/ml, with the preferred value of 30 ng/ml;
sox 21-1000 ng/ml, preferably 20 ng/ml;
c-myc is 1-1000ng/ml, and the preferred value is 20 ng/ml;
DDX4(Mvh) 1-1000ng/ml, preferably 50 ng/ml;
dazl1-1000 ng/ml, preferably 50 ng/ml;
bFGF 1-1000ng/ml, preferably 100 ng/ml;
EGF 1-1000ng/ml, preferably 10ng/ml;
i L-61-1000 ng/ml, preferably 10ng/ml;
sox171-1000 ng/ml, preferably 50 ng/ml;
VASA 1-1000ng/ml, preferably 50 ng/ml;
SCF 1-1000ng/ml, preferably 10ng/ml;
G-GSF 1-1000ng/ml, preferably 10ng/ml;
culture solution 03 is M16 culture solution, which contains:
2-10% serum replacement (KSR), preferably 5%;
RA is 1-1000 uM, preferably 200 uM;
DMC 11-1000 ng/ml, with 30ng/ml being preferred;
PRDM 11-1000 ng/ml, preferably 30 ng/ml;
PGK 21-1000 ng/ml, preferably 30 ng/ml;
hCG 1-200 iu/ml, preferably 50 iu/ml;
progesterone 1-200 iu/ml, preferably 50 iu/ml;
estradiol 1-200 iu/ml, preferably 50 iu/ml;
FSH1-1000 ng/ml, preferably 100 ng/ml;
DDX4(Mvh) 1-1000ng/ml, preferably 30 ng/ml;
dazl1-1000 ng/ml, preferably 30 ng/ml;
oct41-1000 ng/ml, with the preferred value of 10ng/ml;
sox171-1000 ng/ml, preferably 30 ng/ml;
VASA 1-1000ng/ml, preferably 50 ng/ml;
bFGF 1-1000ng/ml, preferably 100 ng/ml;
VASA 1-1000ng/ml, preferably 50 ng/ml;
FGF 21-1000 ng/ml, preferably 10ng/ml;
FGF 71-1000 ng/ml, preferably 10ng/ml;
BMP 21-1000 ng/ml, preferably 10ng/ml;
BMP 41-1000 ng/ml, preferably 10ng/ml;
i L-61-1000 ng/ml, preferably 10ng/ml;
NT 41-1000 ng/ml, preferably 10ng/ml;
GDNF 1-1000ng/ml, preferably 10ng/ml;
activin A1-1000 ng/ml, with the preferred value of 10ng/ml;
UB 1-1000ng/ml, preferably 100 ng/ml;
17 α,20 β -DHP 1-1000ng/ml, preferably 10ng/ml;
AY 9944-A-71-1000 ng/ml, preferably 10ng/ml;
EGF 1-1000ng/ml, preferably 10ng/ml;
VEGF 1-1000ng/ml, preferably 10ng/ml;
SCF 1-1000ng/ml, preferably 10ng/ml;
IGF 1-1000ng/ml, preferably 10ng/ml;
IGF I1-1000 ng/ml, preferably 10ng/ml;
IGF II1-1000 ng/ml, preferably 10ng/ml;
GRF 1-1000ng/ml, preferably 10ng/ml;
ifitm 31-1000 ng/ml, preferably 10ng/ml;
GH 1-1000ng/ml, preferably 10ng/ml;
dexamethasone 1-1000ng/ml, preferably 10ng/ml;
G-GSF 1-1000ng/ml, preferably 10ng/ml;
preferably, the composition of the kit comprises:
the culture solution 01 is RPMI1640 culture solution, which contains:
5% serum replacement (KSR);
EPO 10 ng/ml;
M-GSF 10 ng/ml;
G-GSF 150 ng/ml;
IL-3 10 ng/ml;
IL-6 10 ng/ml;
SCF 10 ng/ml;
bFGF 100 ng/ml;
Nanog 30 ng/ml;
Oct4 30 ng/ml;
Sox2 20 ng/ml;
c-myc 20 ng/ml。
the culture solution 02 is M2 culture solution, which contains:
5% serum replacement (KSR);
Nanog 30 ng/ml;
Oct4 30 ng/ml;
Sox2 10 ng/ml;
c-myc 20 ng/ml;
DDX4(Mvh) 30 ng/ml;
Dazl 30 ng/ml;
bFGF 100 ng/ml;
EGF 10 ng/ml;
IL-6 10 ng/ml;
Sox17 100 ng/ml;
VASA 50 ng/ml；
SCF 10 ng/ml;
G-GSF 10 ng/ml;
culture solution 03 is M16 culture solution, which contains:
RA 200 uM;
DMC1 50 ng/ml;
PRDM1 50 ng/ml;
PGK2 50 ng/ml;
hCG 50iu/ml；
progesterone 50 iu/ml;
estradiol 50 iu/ml;
FSH 100 ng/ml；
DDX4(Mvh) 50 ng/ml;
Dazl 50 ng/ml,
Oct4 10 ng/ml;
Sox17 50 ng/ml;
VASA 50 ng/ml；
bFGF 100 ng/ml;
FGF 2 100 ng/ml;
FGF 7 100 ng/ml;
BMP2 100 ng/ml;
BMP4 100 ng/ml;
IL-6 10 ng/ml;
NT 4 10 ng/ml;
GDNF 10 ng/ml;
Activin A 10 ng/ml;
UB 100 ng/ml;
17α,20β-DHP 10 ng/ml;
AY9944-A-7 10 ng/ml;
EGF 10 ng/ml;
VEGF 10 ng/ml;
SCF 10 ng/ml;
IGF 10 ng/ml;
IGF I 10 ng/ml;
IGF II 10 ng/ml;
GRF 10 ng/ml;
Ifitm3 10 ng/ml;
GH 10 ng/ml;
dexamethasone 100 ng/ml;
G-GSF 10 ng/ml。
the kit can be applied to the preparation of autologous reproductive stem cells of blood-borne females; the autologous female reproductive stem cells prepared by the kit relate to the application in the fields of disease treatment of ovary regeneration and repair, ovary health care, ovary anti-aging and the like;
the method and the kit create a new production technology for the autologous female reproductive stem cells which use the autologous blood cells as raw material cells and are derived from blood through non-transgenic genes. The invention has obvious advantages in the aspects of production speed, yield and purity of the autologous reproductive stem cells of the blood-borne female.
Drawings
FIG. 1 is a scheme of differentiation of blood mononuclear cells into autologous germ stem cells by reverse induction and in vitro and in vivo differentiation,
FIG. 2 is a pictorial view under a microscope of venous blood mononuclear cells (PBMC),
FIG. 3 is a microscopic view of hematopoietic female autologous pluripotent stem cells obtained after PBMC retrodifferentiation,
FIG. 4 is a phenotypic test chart showing the identification result of autologous pluripotent stem cells of a blood-derived female,
FIG. 5 is a microscopic view of hematopoietic female autologous germ stem cells obtained after PBMC retrodifferentiation,
FIG. 6 is a phenotype test chart showing the result of the identification of autologous germline stem cells of a blood-borne female,
figure 7 is a passage chart showing autologous reproductive stem cells of a female,
figure 8 is a graph showing the detection of first generation female autologous germline stem cell markers,
figure 9 is a graph showing the detection of a fifth generation female autologous germline stem cell marker,
FIG. 10 is a diagram showing in vitro differentiation and identification of mature follicles from autologous germline stem cells of a blood-borne female.
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments that can be derived by one of ordinary skill in the art from the embodiments given herein are intended to be within the scope of the present invention.
EXAMPLE 1 preparation of the kit
The preparation is carried out in a safe operating platform with grade cleanliness of 10-100 and is prepared under the low-temperature condition of 4-10 ℃.
To 500ml of RPMI 640 medium, were added:
5% serum replacement (KSR);
EPO 10 ng/ml;
M-GSF 10 ng/ml;
G-GSF 150 ng/ml;
IL-3 10 ng/ml;
IL-6 10 ng/ml;
SCF 10 ng/ml;
bFGF 100 ng/ml;
Nanog 30 ng/ml;
Oct4 30 ng/ml;
Sox2 20 ng/ml;
c-myc 20 ng/ml。
fully dissolving, filtering by a filter with the pore size of 0.22 micron, and sterilizing to prepare the cell culture solution 01.
To 500ml of M2 culture solution, were added:
5% serum replacement (KSR);
Nanog 30 ng/ml;
Oct4 30 ng/ml;
Sox2 10 ng/ml;
c-myc 20 ng/ml;
DDX4(Mvh) 30 ng/ml;
Dazl 30 ng/ml;
bFGF 100 ng/ml;
EGF 10 ng/ml;
IL-6 10 ng/ml;
Sox17 100 ng/ml;
VASA 50 ng/ml；
SCF 10 ng/ml;
G-GSF 10 ng/ml;
fully dissolved, filtered and sterilized by a filter with the pore size of 0.22 micron, and prepared into a cell culture solution 02.
To 500ml of M16 culture solution, were added:
RA 200 uM;
DMC1 50 ng/ml;
PRDM1 50 ng/ml;
PGK2 50 ng/ml;
hCG 50iu/ml；
progesterone 50 iu/ml;
estradiol 50 iu/ml;
FSH 100 ng/ml；
DDX4(Mvh) 50 ng/ml;
Dazl 50 ng/ml,
Oct4 10 ng/ml;
Sox17 50 ng/ml;
VASA 50 ng/ml；
bFGF 100 ng/ml;
FGF 2 100 ng/ml;
FGF 7 100 ng/ml;
BMP2 100 ng/ml;
BMP4 100 ng/ml;
IL-6 10 ng/ml;
NT 4 10 ng/ml;
GDNF 10 ng/ml;
Activin A 10 ng/ml;
UB 100 ng/ml;
17α,20β-DHP 10 ng/ml;
AY9944-A-7 10 ng/ml;
EGF 10 ng/ml;
VEGF 10 ng/ml;
SCF 10 ng/ml;
IGF 10 ng/ml;
IGF I 10 ng/ml;
IGF II 10 ng/ml;
GRF 10 ng/ml;
Ifitm3 10 ng/ml;
GH 10 ng/ml;
dexamethasone 100 ng/ml;
G-GSF 10 ng/ml。
fully dissolving, filtering by a filter with the pore size of 0.22 micron, and sterilizing to prepare the cell culture solution 03.
And respectively placing the cell culture solution 01, the cell culture solution 02 and the cell culture solution 03 into three mutually isolated containers of the kit, thereby preparing the kit for preparing the blood-derived female autologous reproductive stem cells.
Example 2 preparation of autologous hematopoietic Stem cells for blood-derived women
50ml of female venous blood (from volunteers) was drawn, and the venous blood was centrifuged using a conventional Ficoll centrifugation technique, thereby obtaining blood mononuclear cells. Subjecting the obtained mononuclear cells to5x106The cell culture solution 01 prepared in example 1 was cultured at 37 ℃ in a 5% CO2 incubator for 3 days; the cell culture broth 02 prepared in example 1 was then used instead, and mononuclear cells were continued at 5X106Culturing for 6-9 days to obtain the autologous totipotent stem cells of the blood-borne female; the cell culture solution 03 prepared in example 1 was then used instead, and the mononuclear cells were continued at 5X106The cell culture density is 6-9 days, the cells cultured in the cell culture solution 01, the cell culture solution 02 and the cell culture solution 03 are lightly blown and beaten to completely suspend the cells, the cells are collected in a centrifuge tube to be centrifuged, then the suspended cells are cleaned by physiological saline, after 3 times of repetition, the cells are suspended in the physiological saline to obtain the blood-derived female autologous reproductive stem cells, and the obtained blood-derived female autologous reproductive stem cells are mixed with 1 × 105From ml to 1 × 106Mixing the cell density of per ml with the mixed solution of dimethyl sulfoxide and 10% low molecular dextran of the same volume, cooling the mixture to-80 ℃, and then transferring the mixture to liquid nitrogen of-196 ℃ for cryopreservation.
Immunofluorescence assay for cellular phenotype
The optimum cell suspension concentration of the autologous hematopoietic stem cells and autologous reproductive stem cells of the female with blood origin prepared in example 2 was selected by using a pre-prepared slide glass of poly-D-lysine, and the slide glass was prepared by rotating and polishing the slide glass at 1800RCF for 2 minutes by using a cell slide centrifuge and labeled, and the cell density (uniform cells and appropriate density) on the slide glass was obtained. And (5) airing the polished section, and storing the polished section in a refrigerator with the temperature of-20 ℃ for freezing storage.
The cell slide was taken out of a-20 degree refrigerator, air-dried at room temperature, and area-defined on the reverse side with a marker, and then fixed with 4% paraformaldehyde, and after punching with 2% Triton X-100 for 10 minutes and washing with phosphate buffer (PBS, pH 7.4), the sample was blocked with 10% normal goat serum at room temperature for 1 hour to obtain a sample, a first antibody reagent was added to the sample, the first antibody used was Mvh, Dazl, Fragilis, Oct4, Nanog, K L F4, SOX2, the antigen in the sample was reacted with the first antibody at 4 ℃ for 12 hours (overnight), then after washing the sample with Phosphate Buffer (PBS), a second antibody corresponding to the first antibody was added to the sample, the first antibody in the sample was reacted with the second antibody for 1 hour, a nuclear staining procedure was performed with pi (1:1000) at room temperature, after washing with phosphate buffer, the sample was photographed with PBS, the sample was subjected to a photograph, and the blocking procedure was performed with glycerol at room temperature, and the blocking was performed with PBS = PBS, and finally, and the fluorescence was observed in a microscope, the sample was observed for 0.8: 8.
Fig. 4 shows that the cells obtained in the preparation process of example 2 can observe a fluorescent moiety in a fluorescence microscope after reacting with primary antibodies (Oct 4, Nanog, K L F4, SOX 2) and secondary antibodies, thereby illustrating that the cells prepared in example 2 are autologous hematopoietic female stem cells.
FIG. 6 shows that the cells prepared in example 2 can be observed in a fluorescence microscope to obtain a fluorescence portion after reacting with primary antibodies (Mvh, Dazl, Fragilis) and secondary antibodies, thereby illustrating that the cells prepared in example 2 are autologous hematopoietic stem cells of female.
Flow cytometry characterization of cell phenotypes
Adherent female autologous pluripotent stem cells were collected, centrifuged, resuspended in PBS and counted on ice, then cells were labeled with primary anti-Oct 4, Nanog, K L F4, SOX2 monoclonal antibodies, washed with PBS at 4 ℃ for 30min, and FITC or PE labeled secondary antibodies were incubated at room temperature for 30min after PBS washing.
Fig. 4 also shows that the cells obtained in the preparation process of example 2 can observe antibody positive parts in a flow cytometer after reacting with primary antibodies (Oct 4, Nanog, K L F4, SOX 2) and secondary antibodies, thereby illustrating that the cells obtained in the preparation process of example 2 are autologous hematopoietic female stem cells.
FIG. 6 also shows that the cells prepared in example 2 can observe antibody positive parts in a flow cytometer after reacting with primary antibodies (Mvh, Dazl, Fragilis) and secondary antibodies, thereby illustrating that the cells prepared in example 2 are autologous hematopoietic stem cells of female.
Example 3 passage of autologous germline stem cells from blood-borne females
When 80% -90% of the bottle bottom is fully paved with the blood-derived female autologous germ stem cells obtained in example 2, the ratio of 1: passage 3. Washing with appropriate amount of PBS for 2 times, digesting with collanase type IV 2ml for about l-2min, observing under inverted microscope, if cytoplasm retraction and intercellular space increase, immediately adding serum-containing culture medium to terminate digestion, collecting liquid, centrifuging at 1000rpm for 8min, removing supernatant, adding complete culture medium for resuspension, and inoculating for culture.
Fig. 7 shows a passage map of female autologous germ stem cells.
FIGS. 8 and 9 show detection charts of autologous germline stem cell markers for first and fifth generations of blood-derived females, respectively
Example 4 in vitro differentiation of autologous female germline stem cells of blood origin
The autologous hematopoietic stem cells obtained in example 2 were transformed into single cells by collagenic type IV, and then resuspended to 5 × 10 using a germ cell culture solution 016Cell suspensions of/m L, according to 5 × 106The cell amount of each hole is inoculated in 6 holes, the cells are fully and uniformly shaken and then put into an incubator at 37 ℃, and the culture solution is changed every three days after culture by 5 percent CO 2. The germ cell culture solution is induced for 10-20 days, and cells similar to primary oocyte-like cells, secondary oocyte-like cells and mature oocyte-like cells appear in the culture plate. The change of cell morphology was observed under the microscope every day and photographed in time. Detection of haploid, meiotic markers and levels of oocyte markers was performed at 10, 12, 14, 16, 18, 20d, respectively.
FIG. 10 shows graphs of in vitro differentiation and identification of mature follicles from autologous female reproductive stem cells of blood origin, with markers at various stages of oocytes identified by Ovary-Specific acid, ZP2, ER α, GDF9, Stra8, respectively, in the set of graphs, the graphs show respectively (a) autologous reproductive stem cells of blood origin, (b) primary-like follicles, (c) secondary-like follicles, (d) mature-like follicles, (e) detection of anti-ovum antibodies, (f) detection of anti-zona pellucida antibodies, (g) detection of the meiotic marker Stra8, (h) detection of the meiotic marker GDF9, (i) detection of the estrogen receptor ER α, (j) detection of single cells during germ cell division.
Example 5 application of blood-borne female autologous germline stem cells for the treatment of diseases of regeneration and repair of the female reproductive system, ovarian health, ovarian anti-aging, and the like
After the autologous hematopoietic stem cells obtained in example 2 were transformed into single cells by collagenic type IV, they were resuspended in cell suspensions of different concentrations using physiological saline, and then transplanted by reinfusion according to different reinfusion methods.
Venous return: the intravenous infusion path was established and maintained aseptically, and was slowly instilled/injected over a 30 minute period at 1-3 × 10 of the above-described 30ml specification7Autologous germ stem cells; to prevent possible allergic reactions, an antiallergic agent, for example, dexamethasone at 1-2mg is administered as an intravenous bolus injection, 10-30 minutes before reinfusion if necessary.
The abdominal cavity micro-incision ovary direct injection back transfusion method comprises the following steps: usually, the lower abdomen of local anesthesia is subjected to a 2 cm micro-incision, the ovary is hooked out and exposed, and 3-5ml of the produced autologous stem cells subjected to quality and safety detection are directly injected back into each side of the ovary. The ovary was then returned to the pelvic cavity and the abdomen closed with two needles. The operation can be quickly, simply and conveniently carried out in an operating room of a hospital. The micro-operation type ovarian autologous reproductive stem cell reinfusion method has the advantages of high operation reinfusion success rate, high reinfusion accuracy and no more than 30 minutes of operation process. The operation incision and the wound are small; is suitable for women of various ages, especially women with obviously atrophied ovary, and has high success rate of operation. To prevent possible allergic reactions, an antiallergic agent, for example, dexamethasone at 1-2mg is administered as an intravenous bolus injection, 10-30 minutes before reinfusion if necessary.
B ultrasonic positioning ovary direct puncture reinfusion method: 3-dimensional or 4-dimensional B ultrasound is applied to position the ovary, the ovary is punctured from the fornix behind the vagina to directly reinfuse the produced various blood-borne female autologous stem cells, and 3-5ml of ovary at each side is reinfused. The advantages of this method of implantation are minimal invasive and painless rapidity. The disadvantage is that the success rate of the surgery is not high if the ovary atrophy seriously at the age of the client. To prevent possible allergic reactions, an antiallergic agent, for example, dexamethasone at 1-2mg is administered as an intravenous bolus injection, 10-30 minutes before reinfusion if necessary.
A laparoscope/pelioscope direct ovary reinfusion method: the produced blood-derived autologous stem cells are respectively punctured and reinfused to the ovaries at both sides by a laparoscopic/pelvic endoscope operation mode, and 3-5ml of blood-derived autologous stem cells are reinfused to each ovary. To prevent possible allergic reactions, an antiallergic agent, for example, dexamethasone at 1-2mg is administered as an intravenous bolus injection, 10-30 minutes before reinfusion if necessary.
Example 6 production and storage of blood-derived female autologous reproductive stem cells and establishment of autologous reproductive stem cell banks
The autologous hematopoietic stem cells obtained in example 2 were unicellular transformed by collaenase type IV, suspended in 7.2 m L culture medium 03, and the collected cells were counted, and 1.8 m L frozen stock solution was slowly added dropwise to 7.2 m L cell suspension, and the tube was shaken to prepare a cell frozen suspension having a cell concentration of 5 × 107cell/m L, mixing uniformly, taking 1 m L cell suspension for pollution detection, subpackaging the rest 8m L into 6 tubes in a cryopreservation tube marked completely, wherein 5 tubes are 1.5 m L/tube for cryopreservation of stem cells, the last tube is 0.5 m L for detecting recovery of stem cells, after tight sealing, noting the name, number and date of the cells, then reducing the temperature from room temperature to-120 ℃ at the speed of-1 to-3 ℃/min by using a programmed cooling instrument, and finally placing in a liquid nitrogen tank at-196 ℃ for long-term cryopreservation.
The method is characterized in that a blood-derived female autologous germ stem cell production technology is taken as a core, and a stem cell bank project with autologous germ stem cell production/detection/storage/supply capacity is constructed, wherein the bank project comprises five major departments, namely an autologous stem cell production center, an autologous stem cell quality and safety detection center, an autologous stem cell storage center, an autologous stem cell scientific research center, an autologous germ stem cell supply center and the like. The female autologous reproductive stem cell bank advocates the people to produce and store autologous reproductive stem cells in young and healthy states so as to charge own lives in the future, maintain and promote the functions and activities of the lives and prolong the length of the lives. The project plans that the blood-derived female autologous reproductive stem cell bank is constructed as a first female life energy production, storage and energy supply station in China, and the storage popularization rate of female autologous stem cells is taken as an important index for measuring the national and social health level.

Claims (14)

1. A method for preparing autologous reproductive stem cells of a blood-borne female, which comprises the following steps:
human somatic cells are placed inCell culture solution 01Culturing for 3 days in medium culture medium (RPMI 1640) containing 2-10% of serum substitute (KSR), EPO1-1000 ng/ml, M-GSF 1-1000ng/ml, G-GSF 1-1000ng/ml, I L-31-1000 ng/ml, I L-61-1000 ng/ml, SCF 1-1000ng/ml, bFGF 1-1000ng/ml, Nanog1-1000 ng/ml, Oct41-1000 ng/ml, Sox 21-1000 ng/ml, c-myc 1-1000ng/ml, and replacing with other medium (RPMI 1640) and/or serum substitute (KSR)Cell culture solution 02Culturing for 6-9 days, wherein the cell culture solution 02 is M2 culture solution and contains 2-10% of serum substitute (KSR), Nanog1-1000 ng/ml, Oct41-1000 ng/ml, Sox 21-1000 ng/ml, myc 1-1000ng/ml, DDX4(Mvh) 1-1000ng/ml, Dazl1-1000 ng/ml, bFGF 1-1000ng/ml, EGF 1-1000ng/ml, I L-61-1000 ng/ml, Sox171-1000 ng/ml, VASA 1-1000ng/ml, SCF 1-1000ng/ml, G-GSF 1-1000ng/ml to obtain female blood-derived totipotent stem cells, and replacing withCell culture solution 03Continuing to culture for 6-9 days, wherein the cell culture solution 03 is M16Culture solutionAnd contains 2-10% serum substitute (KSR), RA 1-1000 uM, DMC 11-1000 ng/ml, PRDM 11-1000 ng/ml, PGK 21-1000 ng/ml, hCG 1-300iu/ml, progesterone 1-300iu/ml, estradiol 1-300iu/ml, FSH1-1000 ng/ml, DDX4(Mvh) 1-1000ng/ml, Dazl1-1000 ng/ml, Oct 68-1000 ng/ml, Sox171-1000 ng/ml, VASA 1-1000ng/ml, bFGF 1-1000ng/ml, VASA 1-1000ng/ml, GF 21-1000 ng/ml, 71-1000 ng/ml, 21-1000 ng/ml, BMP 41-1000 ng/ml, I2-61-1000 ng/ml, NT 41-1000 ng/ml, GDNF 1-1000ng/ml, DHNF 1-1000ng/ml, UBA 1-1000ng/ml, UB 1-l; EGF 1-1000 ng/ml; VEGF 1-1000 ng/ml; 1-1000ng/ml of SCF; IGF 1-1000 ng/ml; 1-1000ng/ml of IGF I; IGF II1-1000 ng/ml; GRF 1-1000 ng/ml; ifitm 31-1000 ng/ml; GH 1-1000 ng/ml; dexamethasone 1-1000 ng/ml; G-GSF 1-1000 ng/ml; obtaining autologous reproductive stem cells of a blood-borne female;
culture conditions at 5% CO2And cell culture was performed at 37 ℃.
2. The method for preparing autologous female reproductive stem cells according to claim 1, wherein said human somatic cells include, but are not limited to, human umbilical cord blood cells, placental cells, skin cells, blood nucleated cells, adipocytes, blood;
culturing cells in cell culture medium 01 (RPMI 1640) containing 5% serum replacement (KSR), EPO 10ng/ml, M-GSF 10ng/ml, G-GSF 150 ng/ml, I L-310 ng/ml, I L-610 ng/ml, SCF 10ng/ml, bFGF100 ng/ml, Nanog 30ng/ml, Oct430 ng/ml, Sox 220 ng/ml, c-myc 20ng/ml for 72 hr, and continuously culturing in cell culture medium 02 for 5% CO2And culturing at 37 deg.C for 6-9 days, wherein the cell culture solution 02 is M2 culture solution containing 5% serum substitute (KSR), Nanog 30ng/ml, Oct430 ng/ml, Sox 210 ng/ml, c-myc 20ng/ml, DDX4(Mvh) 30ng/ml, Dazl 30ng/ml, bFGF100 ng/ml, EGF 10ng/ml, I L-610 ng/ml, Sox 17100 ng/ml, VASA 50ng/ml, SCF 10ng/ml, G-GSF 10ng/ml to obtain female blood-derived autologous stem cells, and replacing with cell culture solution 03 continuing at 5% CO2And culturing at 37 deg.C for 6-9 days, wherein the cell culture solution 03 is M16 culture solution containing RA 200 uM, DMC 150 ng/ml, PRDM 150 ng/ml, PGK 250 ng/ml, hCG 30iu/ml, progesterone 30iu/ml, estradiol 30iu/ml, FSH 100ng/ml, DDX4(Mvh) 50ng/ml, Dazl 50ng/ml, Oct 410 ng/ml, Sox 1750 ng/ml, VASA 50ng/ml, bFGF100 ng/ml, FGF 2100 ng/ml, FGF 7100 ng/ml, BMP 2100 ng/ml, BMP 0ng/ml, BMP L-610 ng/ml, NT 410 ng/ml, GDNF 10ng/ml, Activin A10 ng/ml, UB 100ng/ml, 17 α,20 β -P10 ng/ml, AY 9944-Ing/ml, IGF 10ng/ml, EGF 10ng/ml, IGF 10ng/ml, EGF/ml, IGF 10ng/ml, EGF/ml, IGF 10ng/ml, VEGF 10ng/mlitm 310 ng/ml, GH 10ng/ml, dexamethasone 100ng/ml and G-GSF 10ng/ml to obtain autologous reproductive stem cells of blood-borne females.
3. The method for preparing autologous hematopoietic stem cells according to any one of claims 1 to 2, wherein the autologous hematopoietic stem cells are prepared.
4. The method for preparing autologous female reproductive stem cells according to claim 3, which relates to the application in the fields of disease treatment for regeneration and repair of female reproductive system, ovarian health, ovarian aging resistance, etc.;
such disorders include, but are not limited to, female infertility, hypoovarianism, premature ovarian failure, ovarian senescence/menopause.
5. The method for preparing autologous female reproductive stem cells according to claim 4, wherein the method is applied to drugs for treating female infertility, hypoovarianism, premature ovarian failure, ovarian senescence/menopause and the like and drugs for treating diseases related to organ regeneration, reconstruction and repair.
6. The method for preparing autologous female reproductive stem cells derived from blood according to claim 4, wherein the method is used for production storage, in vitro autologous tissue organ engineering and production.
7. A group of culture solution for reversely differentiating the somatic cells of female is characterized by that said culture solution is selected from cell culture solution 01, 02 and 03, and the described cell culture solution 01 is RPIM1640 and contains 2-10% of serum substitute (KSR), EPO1-1000 ng/ml, M-GSF 1-1000ng/ml, G-GSF 1-1000ng/ml, I L-31-1000 ng/ml, I L-61-1000 ng/ml, SCF 1-1000ng/ml, bFGF 1-1000ng/ml, Nanog1-1000 ng/ml, Oct41-1000 ng/ml, Sox 21-1000/ml, c-myc 1-1000ng/ml, then using themCell culture solution 02Culturing for 6-9 days, wherein the cell culture solution 02 is M2 culture solution and contains 2-10% of serum substitute (KSR), Nanog1-1000 ng/ml, Oct41-1000 ng/ml, Sox 21-1000 ng/ml, myc 1-1000ng/ml, DDX4(Mvh) 1-1000ng/ml, Dazl1-1000 ng/ml, bFGF 1-1000ng/ml, EGF 1-1000ng/ml, I L-61-1000 ng/ml, Sox171-1000 ng/ml, VASA 1-1000ng/ml, SCF 1-1000ng/ml, G-GSF 1-1000ng/ml, obtaining blood-derived female autologous stem cells, and replacing with new onesCell culture solution 03Continuing to culture for 6-9 days, wherein the cell culture solution 03 is M16Culture solutionThe composition comprises 2-10% of serum replacement (KSR), 1-1000 uM of RA, 1-1000ng/ml of DMC 11-1000 ng/ml of PRDM 11-1000 ng/ml of PGK 21-1000 ng/ml of hCG 1-300iu/ml of progesterone 1-300iu/ml of estradiol 1-300iu/ml of FSH1-1000 ng/ml of FSH, 1-1000ng/ml of DDX4(Mvh), 1-1000ng/ml of Dazl, 41-1000 ng/ml of Oct, 171-1000 ng/ml of Sox, 1-1000ng/ml of VASA, 1-1000ng/ml of bFGF 1-1000ng/ml of FGF, 21-1000 ng/ml of GF, 71-1000 ng/ml of IGF 71-1000 ng/ml of FGF P, 1000 ml of GPF 71-1000 ng/ml of GPF, 1000ng/ml of GPF 71-1000 ng/ml of GPF, 1000ng/ml of GPG 7-1000 ml of autologous mature blood, 1000 ml of GPF 1-1000 ml of GPF, 1000 ml of GPF 1-1000 ml of GPF, 1000 ml of GPF 7-1000 ml of GPF, 1000 ml of GPF, 1000ng/ml of GPF 1-1000, 1000ng/ml of GPF, 1000 ml of GPF 7-1000 ng/ml of GPF, 1000.
8. A set of culture fluids for the retrodifferentiation of somatic cells in women according to claim 7, wherein the use of cell culture fluids 01, 02 and 03 in the culture of human somatic cells for the retrodifferentiation to give autologous germ stem cells.
9. A kit for preparing autologous female reproductive stem cells derived from blood, comprising the cell culture solutions 01, 02 and 03 of claim 8;
the kit further comprises somatic cells of a female;
the somatic cells include, but are not limited to, umbilical cord blood cells, placental cells, skin cells, blood nucleated cells, and fat cells of women.
10. The kit for preparing the autologous hematopoietic stem cells of the blood-derived female according to claim 9, wherein the kit is used for treating female infertility, hypoovarianism, premature ovarian failure, and aging/menopause in preparing the autologous hematopoietic stem cells of the blood-derived female.
11. The use of a kit for preparing autologous female reproductive stem cells derived from blood according to claim 10, wherein the diseases include but are not limited to female infertility, hypoovarianism, premature ovarian failure, ovarian senescence/menopause.
12. The kit for preparing the hematopoietic stem cells of claim 9, wherein the kit is used for establishing a hematopoietic stem cell bank and a hematopoietic stem cell bank.
13. The method for preparing autologous hematopoietic stem cells for female according to claims 1 to 6, wherein the autologous hematopoietic stem cells are used in the fields of treatment of diseases related to ovarian regeneration and repair, female infertility, ovarian hypofunction, premature ovarian failure, ovarian aging/menopause, etc.
14. The method for preparing autologous hematopoietic stem cells of claim 1 to 6, wherein the autologous hematopoietic stem cells are used for establishing a bank of autologous hematopoietic stem cells for storing and storing the autologous hematopoietic stem cells.
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