CN114276984A

CN114276984A - Method for transdifferentiation of female reproductive stem cells into functional sperms and application

Info

Publication number: CN114276984A
Application number: CN202111665381.1A
Authority: CN
Inventors: 吴际; 胡晓鹏
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2021-12-31
Filing date: 2021-12-31
Publication date: 2022-04-05
Anticipated expiration: 2041-12-31
Also published as: CN114276984B

Abstract

The invention provides a method for transdifferentiating female reproductive stem cells into functional sperms and application thereof. The invention provides a method for inducing female reproductive stem cells to differentiate into haploid spermatids for the first time. The method can provide a new strategy for breeding animals such as high-quality cows.

Description

Method for transdifferentiation of female reproductive stem cells into functional sperms and application

Technical Field

The invention belongs to the technical field of assisted reproduction, and particularly relates to a method for transdifferentiation of female reproductive stem cells into functional sperms and application of the method.

Background

The international health organization stipulates that the sperms of the male are not less than 2 million per milliliter, and if the sperms are less than 2 million, the male is classified as oligospermia. The causes of oligospermia are many, including endogenous factors such as varicocele, cryptorchidism, genital tract infection, and autoimmune production of antisperm antibodies. Some exogenous factors also cause oligospermia. There are also some idiopathic oligozoospermia of unknown etiology. Oligospermia causes male sterility, and more people suffer from the problem and have great influence on fertility. Therefore, artificial induction of sperm production in vitro is a feasible approach for the treatment of oligospermia.

Stem cells are a generic term for a group of cells with self-renewal and multipotentiality. The stem cells can be classified into totipotent stem cells, pluripotent stem cells and unipotent stem cells according to the differentiation potential of the stem cells. An Embryonic Stem cell (ES/ESC cell) is a cell isolated from an early embryo or primitive gonad, and can be differentiated into various tissues while having the ability to proliferate indefinitely in vitro. Embryonic stem cells, as an undifferentiated cell, have totipotency, i.e., the property of developing into a variety of different cells. Wherein the human embryonic stem cells can differentiate into various cell tissues, including trophoblast cells, nerve cells, glial cells, hematopoietic cells, cardiac muscle cells, and the like. Morphologically, embryonic stem cells have large nuclei and small cytoplasm, and are relatively simple in structure, with the nuclei of embryonic stem cells being mostly euchromatin. An embryonic stem cell is a highly undifferentiated cell. It has developmental totipotency and can differentiate all tissues and organs of adult animals, including germ cells. However, the differentiation of embryonic stem cells into long spermatids has technical difficulties, and it is difficult to obtain high-activity long spermatids with intact tail structures.

Female germ stem cells are a class of stem cells isolated from the ovaries of animals. Scientists have previously determined that oocytes cease to be produced as soon as a female mammal is born, and have therefore only been reduced; in the previous work of the present inventors, the female germ stem cells were found in the ovaries of adult mammals, and oocytes were continuously differentiated.

However, the ability of female germ stem cells to develop in vitro into functional sperm cells and their in vitro developmental characteristics have not been reported.

Disclosure of Invention

The invention aims to provide a method for transdifferentiating female reproductive stem cells into functional sperms and application

In a first aspect of the invention, there is provided a method of producing functional sperm in vitro comprising: taking female reproductive stem cells as starting cells, mixing the starting cells with testicular somatic cells, and performing pre-culture and induced differentiation; functional sperm are produced from the induced differentiation product.

In one or more embodiments, the pre-culturing comprises: mixing the female germ stem cells and testicular somatic cells in a ratio of 1: 10-10: 1, preferably 1: 5-5: 1, more preferably 1: 2-2: 1, and culturing in a cell culture medium; preferably the cell culture medium is a serum-containing medium; preferably the cell culture medium comprises DMEM medium.

In one or more embodiments, the medium used for the mixed culture is a liquid medium (culture solution).

In one or more embodiments, the medium used for mixed culture contains 5% to 15% fetal bovine serum.

In one or more embodiments, inducing differentiation of the female germ stem cell and testicular somatic cell comprises: induction in stage 1: inducing by Knockout serum substitute, BMP-2, BMP-4, BMP-7, all-trans retinoic acid and activin A; induction in the 2 nd stage: induction with Knockout serum replacement, testosterone, FSH and pituitary extract.

In one or more embodiments, in stage 1, the amounts of the components include: knockout serum replacement 20 + -4% (v/v), BMP-220 + -5 ng/mL, BMP-420 + -5 ng/mL, BMP-720 + -5 ng/mL, all-trans retinoic acid 1 + -0.4 μ M, and activin A100 + -40 ng/mL.

In one or more embodiments, in stage 2, the amounts of the components include: knockout serum replacement 20. + -.4%, testosterone 10. + -.3 mM, FSH 200. + -.30 ng/mL and pituitary extract 50. + -.15 mg/mL.

In one or more embodiments, the individual components are added to the cell culture medium; more preferably, the cell culture medium is MEM α medium.

In one or more embodiments, the medium used for induction is a liquid medium (broth).

In one or more embodiments, in stage 1, the fresh medium is replaced at the appropriate time; preferably, the culture medium is replaced once in 1-2 days; more preferably, the fresh medium is replaced once a day.

In one or more embodiments, in stage 2, the fresh medium is replaced at the appropriate time; preferably, the culture medium is replaced once in 1-2 days; more preferably, the fresh medium is replaced once a day.

In one or more embodiments, the isolated testicular somatic cells are free of germ cells that do not produce sperm cells.

In one or more embodiments, in animalsIn the experiment, the testicular somatic cells were isolated from KIT^w/wvA mouse.

In one or more embodiments, the Knockout serum replacement is preferably 20. + -. 3%, more preferably 20. + -. 2%, more preferably 20. + -. 1% in stage 1.

In one or more embodiments, BMP-2 is preferably 20 + -4 ng/mL, more preferably 20 + -3 ng/mL, preferably 20 + -2 ng/mL, preferably 20 + -1 ng/mL in stage 1.

In one or more embodiments, BMP-4 is preferably 20 + -4 ng/mL, more preferably 20 + -3 ng/mL, preferably 20 + -2 ng/mL, preferably 20 + -1 ng/mL in stage 1.

In one or more embodiments, BMP-7 is preferably 20 + -4 ng/mL, more preferably 20 + -3 ng/mL, preferably 20 + -2 ng/mL, preferably 20 + -1 ng/mL in stage 1.

In one or more embodiments, the Knockout serum replacement is preferably 20. + -. 3%, more preferably 20. + -. 2%, more preferably 20. + -. 1% in stage 2.

In one or more embodiments, testosterone is preferably 10 ± 2mM, more preferably 10 ± 1mM, during stage 2.

In one or more embodiments, the FSH is preferably 200 ± 20ng/mL, more preferably 200 ± 15ng/mL, more preferably 200 ± 10ng/mL, more preferably 200 ± 5g/mL in phase 2.

In one or more embodiments, the pituitary extract during stage 2 is preferably 50. + -.12 mg/mL, more preferably 50. + -.10 mg/mL, more preferably 50. + -.5 mg/mL, more preferably 50. + -.3 mg/mL.

In one or more embodiments, the generation of functional sperm from the induced differentiation product comprises: observing the induced differentiation products, and separating functional sperms with tail structures at the tail ends.

In one or more embodiments, the female germ stem cells are cultured (expanded) by the following method: placing female reproductive stem cells in an embryonic fibroblast (STO cell) feeder layer, and culturing in a cell culture medium containing sodium pyruvate, L-glutamine, beta-mercaptoethanol, non-essential amino acids, epidermal growth factor, human basic fibroblast growth factor, glial cell growth factor, and leukemia inhibitory factor; preferably, the cell culture medium comprises MEM alpha medium; preferably the cell culture medium is a serum-containing medium.

In one or more embodiments, the amounts of the individual components include: 1 plus or minus 0.3mM sodium pyruvate, 2 plus or minus 0.6mM L-glutamine, 50 plus or minus 15 mu M beta-mercaptoethanol, 1 plus or minus 0.3mM non-essential amino acid, 20 plus or minus 5ng/mL epidermal growth factor, 10 plus or minus 3ng/mL human basic fibroblast growth factor, 10 plus or minus 3ng/mL glial cell growth factor, 10 plus or minus 3ng/mL leukemia inhibitory factor;

preferably, the individual components are added to the cell culture medium; more preferably, the cell culture medium is MEM α medium; more preferably, the cell culture medium is a serum-containing medium.

In one or more embodiments, the cell culture medium used for the culture of the female germ stem cells is a liquid medium (culture broth).

In one or more embodiments, the cell culture medium for culturing the female germ stem cells contains 5% to 15% fetal bovine serum.

In one or more embodiments, the sodium pyruvate is 1 ± 0.2mM sodium pyruvate, more preferably 1 ± 0.1 mM.

In one or more embodiments, L-glutamine is 2. + -. 0.4mM, more preferably 2. + -. 0.2mM, more preferably 2. + -. 0.1 mM.

In one or more embodiments, the β -mercaptoethanol is 50 ± 12 μ Μ, preferably 50 ± 10, more preferably 50 ± 5; more preferably 50 ± 2.

In one or more embodiments, the non-essential amino acids are 1. + -. 0.2mM, preferably 1. + -. 0.1 mM.

In one or more embodiments, epidermal growth factor 20 + -3 ng/mL, preferably 20 + -1 ng/mL.

In one or more embodiments, the human basic fibroblast growth factor is 10 + 2ng/mL, preferably 10 + 1 ng/mL.

In one or more embodiments, the glial growth factor is 10 + -2 ng/mL, preferably 10 + -1 ng/mL.

In one or more embodiments, leukemia inhibitory factor is 10 ± 2ng/mL, preferably 10 ± 1 ng/mL.

In another aspect of the present invention, there is provided a product of induction culture of female germ stem cells mixed with testicular somatic cells or functional sperm produced therefrom, which is prepared by the method as described above; preferably, it has characteristics selected from the group consisting of: the tail end of the functional sperm carries a tail structure; the male germ cell meiosis marker in the induction system is positive: STRA8, SCP1, SCP2, SCP 3; positive for a spermatogenesis marker; preferably the marker comprises: PRM1, ACROSIN, TNP1, HAPRIN and/or ACROSIN; the functional sperm is haploid karyotype; and/or, upon injection into the cytosol, the functional sperm initiates development and transplantation into the uterus of a living organism produces progeny.

In one or more embodiments, the product of the induction culture of female germ stem cells mixed with testicular somatic cells or functional sperm produced therefrom cannot itself be independently propagated into an animal.

In another aspect of the invention, there is provided the use of female germ stem cells and testicular somatic cells for the preparation of functional sperm in vitro.

In another aspect of the present invention, there is provided a method of preparing an animal progeny comprising: (1) preparing functional sperm by the method described above; (2) introducing the functional sperm of (1) into the cytosol of an egg cell (ICSI), fertilizing the egg, and developing in vitro (e.g., to the 2-cell stage, 4-cell stage, or blastocyst stage); preferably, the cells are transplanted to uterus, implanted and developed into healthy offspring at a suitable stage of in vitro development.

In one or more embodiments, the animal comprises a human.

In one or more embodiments, the animal comprises a non-human mammal, preferably including (but not limited to): rodents (including mice, rats, hamsters, etc.), non-human primates (e.g., monkeys, chimpanzees, etc.), and domestic animals (e.g., cows, sheep, dogs, pigs, rabbits, etc.).

In one or more embodiments, the animal comprises an endangered animal.

In another aspect of the present invention, there is provided a kit for in vitro preparation of functional sperm comprising: female reproductive stem cells; testicular somatic cells; a pre-culture agent comprising a cell culture medium; preferably the cell culture medium is a serum-containing medium; preferably the cell culture medium comprises DMEM medium; stage 1 inducers including Knockout serum replacement, BMP-2, BMP-4, BMP-7, retinoic acid, and activin A; the preferred amounts of each component include: knockout serum replacement 20 + -4% (v/v) BMP-220 + -5 ng/mL, BMP-420 + -5 ng/mL, BMP-720 + -5 ng/mL, retinoic acid 1 + -0.4 μ M and activin A100 + -40 ng/mL; phase 2 inducers including Knockout Serum Replacement (KSR), testosterone (T), Follicle Stimulating Hormone (FSH) and pituitary extract (BPE); the preferred amounts of each component include: knockout serum replacement 20. + -. 4%, testosterone 10mM, FSH200ng/mL and pituitary extract 50 mg/mL.

In one or more embodiments, the components of the inducer are added to the cell culture medium.

In one or more embodiments, the kit further comprises a medium/agent for culturing and propagating female reproductive stem cells, and/or a medium/agent for isolating and maintaining testicular somatic cells.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1 is a schematic diagram of the differentiation system of the female germ stem cell into a sperm cell according to the present invention.

FIG. 2, female germ stem cell in vitro spermatogenesis;

A) a characteristic morphological diagram of the development of female reproductive stem cells in an in vitro transdifferentiation system;

B) immunofluorescence observation of the formation of synaptonemal complexes (SCP1/SCP3) and meiotic processes (thin line, even line, thick line, double line);

C) RT-PCR examined the expression of male meiosis (STRA8, SCP1, SCP2, SCP3) and spermatogenesis markers (PRM1, ACROSIN, TNP1, HAPRIN) by the culture products.

D) Analyzing the haploid by a flow cytometer;

E) analyzing a haploid by karyotype;

F) immunofluorescent spermatogenesis marker ACROSIN.

FIG. 3 functional characterization of sperm cells (differentiated in vitro from female germ stem cells).

A) B), C), D), E), F), G), H) an in vitro developmental process (2 cells, 4 cells, blastocyst) injected from a sperm-like cell into the oocyst plasma;

I)2, the cells are transplanted to the uterus of the mouse to give birth to offspring, and the born mouse gives birth naturally after transplantation;

J) identifying the GFP sequence in the born offspring by a Sourther blot method;

K) bisulfite methylation sequencing identifies that the methylation state of the genes of the birth offspring blots is not changed.

Detailed Description

The present inventors have developed a method system for inducing differentiation of female germ stem cells in vitro based on intensive studies, thereby obtaining spermatids (preferably, haploid spermatids). The invention provides in vitro induced female reproductive stem cells for the first time, which are capable of differentiating into haploid spermatids. The method can provide a sperm cell source for clinical and scientific research, and provides a new approach for the technical field of assisted reproduction.

Term(s) for

As used herein, the term "cell culture medium" is in the present invention a "basal medium", which is a medium that is universally suitable for the culture of cells (stem cells), and that provides sufficient nutrients to the cells (stem cells) for the cells to grow.

As used herein, the term "female germ stem cell" or "ovarian female germ stem cell" are used interchangeably.

As used herein, a "mammal" is an animal of the Mammalia class (Mammalia) of the phylum Chordata (Chordata) vertebrate subphyla (Vertebrata). Such mammals include, but are not limited to, primates (including humans), artiodactyla, carnivora, rodentia, and the like. The mammals of the present invention include humans, and also non-human mammals. Such non-human mammals include, for example and without limitation, rodents (e.g., mice, rats), primates (e.g., apes, monkeys, orangutans), domestic animals (e.g., rabbits, dogs, rabbits, pigs, cows, sheep, horses), and the like.

In some embodiments of the invention, the model organism is a mouse, which is in close proximity to humans in terms of genomic composition, individual development, metabolic patterns, organ anatomy, disease pathogenesis, etc., as compared to humans; therefore, some of the cases listed in the present invention applicable to mice can be applied to other mammals such as humans without any doubt.

As used herein, the terms "comprising," having, "or" including "include" comprising, "" consisting essentially of … …, "" consisting essentially of … …, "and" consisting of … …; "consisting essentially of … …", "consisting essentially of … …", and "consisting of … …" are subordinate concepts of "comprising", "having", or "including".

As used herein, "functional" means that the sperm obtained according to the described method has the same or similar function as intended.

Culture and induction method

In the invention, the female germ stem cells are used as starting cells for the first time and are mixed with testicular somatic cells to successfully induce and generate spermatids. Based on the characteristics of the female reproductive stem cells, the inventors have conducted extensive research experiments, culture for induction and induction protocols. The culture and induction method comprises the steps of separating, mixing and pre-culturing female reproductive stem cells and testicular somatic cells, and performing stage 1 induction and stage 2 induction. Under the action of in vitro culture conditions of a microenvironment consisting of testicular body cells, cytokines (activin, BMP2, BMP4, BMP7, FSH, BPE, testosterone), all-trans Retinoic Acid (RA) and the like, the epigenetic state and gene expression of the Y-chromosome-free female germ stem cells are remarkably changed, male meiosis can be completed, and functional sperm-like cells can be developed. The differentiation system of the present invention for differentiating female reproductive stem cells into sperm cells is schematically shown in FIG. 1.

Accordingly, the present invention provides a method for the in vitro production of functional sperm comprising: taking female reproductive stem cells as starting cells, mixing the starting cells with testicular somatic cells, and performing pre-culture and induced differentiation; functional sperm are produced from the induced differentiation product. During the culture, fresh medium may be replaced according to the culture conditions.

In the invention, the female reproductive stem cell can be a female reproductive stem cell from an animal body, and can also be a female reproductive stem cell which is expanded and cultured/passaged by the prior art in the field. For example, the present inventors have expanded female reproductive stem cells in previous studies, and these in vitro expanded female reproductive stem cells can be used. Preferably, female germ stem cells from animals and cells passaged therefrom are used.

As a preferred mode of the present invention, the female reproductive stem cells include those obtained by culturing (expanding) the following method: placing female reproductive stem cells in an embryonic fibroblast (STO cell) feeder layer, and culturing in a cell culture medium containing sodium pyruvate, L-glutamine, beta-mercaptoethanol, non-essential amino acids, epidermal growth factor, human basic fibroblast growth factor, glial cell growth factor, and leukemia inhibitory factor.

In the present invention, the testicular somatic cells are preferably animals derived from germ cells that remain stable after isolation. The testicular somatic cells are isolated from KIT^w/wvA mouse.

In a preferred mode of the present invention, the pre-culturing comprises: the female germ stem cells and testicular somatic cells are mixed at a ratio of 1:10 to 10:1, preferably 1:5 to 5:1, more preferably 1:2 to 2:1, and even more preferably 1: 1. Culturing in a cell culture medium; preferably the cell culture medium is a serum-containing medium. Preferably, the medium used for the mixed culture is a liquid medium (culture solution). Preferably, the medium used for mixed culture contains fetal bovine serum. The inventors found that, according to the characteristics of the female reproductive stem cells, the mixed culture at this stage greatly promotes the efficiency of the subsequent induction culture stage; if the induction culture is performed directly, the transformation efficiency is relatively low and the induction phase takes longer.

In a preferred embodiment of the present invention, the inducing differentiation of the female germ stem cell and the testicular somatic cell comprises: induction in stage 1: inducing with Knockout Serum Replacement (KSR), BMP-2, BMP-4, BMP-7, all-trans retinoic acid and activin A; induction in the 2 nd stage: induction with Knockout serum replacement, testosterone, FSH and pituitary extract. The inventors have optimized the amount of components, e.g. the Knockout serum replacement preferably 20 ± 4%, based on the characteristics of the female germ stem cells, and have found that the expression of the spermatogenesis marker, the cell product, after the induction phase, is barely detectable when the Knockout serum replacement is 10% or less. As another example, the pituitary extract is preferably 50. + -.15 mg/mL, which, if at a lower level, results in a low induction efficiency.

Unless otherwise specified, the medium used for the culture or for the induction in the present invention is a liquid medium (culture solution).

The generation of the functional sperm from the differentiation inducing product comprises the following steps: observing the induced differentiation products, and separating functional sperms with tail structures at the tail ends. It is understood that the sperm with tail structure is more similar in shape and vitality to the natural sperm.

Preferably, the amounts of the components include: 1mM sodium pyruvate, 2mM L-glutamine, 50 μ M β -mercaptoethanol, 1mM non-essential amino acid, 20ng/mL mouse epidermal growth factor, 10ng/mL human basic fibroblast growth factor, 10ng/mL glial cell growth factor, 10ng/mL mouse leukemia inhibitory factor;

The culture method and the culture medium of the present invention can be used for two-dimensional or three-dimensional culture systems.

In a specific embodiment of the present invention, the female germ stem cells and testicular somatic cells are first obtained, mixed, and then transdifferentiation into sperm cells is achieved by pre-culturing and adjusting cell growth factors and other components in the culture medium in stages. The first stage (stage 1) of the staged culture is a component that promotes growth and induces differentiation mainly, and the later stage is a component that matures and differentiates mainly. The invention induces and differentiates the female reproductive stem cell into the spermatid within about 16 to 18 days (preferably 17 days) of in vitro culture, and verifies the characteristics and functions of the obtained spermatid through a series of in vitro experiments (cell staining, RT-PCR, flow identification of haploid cells, immunofluorescence method observation of formation of synaptophytic complex and meiosis process, and the like) and in vivo experiments of animals.

The present invention also provides a method of preparing an animal comprising: (1) functional sperm cells were prepared as described previously; (2) introducing the functional sperm of (1) into the cytosol of an egg cell, fertilizing the egg, and developing in vitro (e.g., to the 2-cell stage, 4-cell stage, or blastocyst stage); preferably, the cells are transplanted to uterus, implanted and developed into animals at a proper stage of in vitro development.

In a specific embodiment of the invention, functional sperm (spermatozoa-like cells) are successfully induced to differentiate from female germ stem cells. In vitro transdifferentiation system, female germ stem cell can express male meiosis and spermatogenesis markers STRA8, SCP1, SCP2, SCP3, PRM1, ACROSIN, TNP1, HAPRIN, etc., and immunofluorescence method can observe formation of synaptal complex (SCP1/SCP3) and meiosis process (thin line, even line, thick line and double line). The sperm-like cells injected into the oocyte plasma can develop in vitro to cause blastocysts, and 2 cells are transplanted into the uterus of a mouse to produce offspring. The mice born after transplantation give birth by spontaneous birth. The sequence of the birth offspring containing GFP is identified by a Sourthern blot method. Bisulfite methylation sequencing identifies that the methylation state of the genes of the birth offspring blots is not changed.

Several main features and advantages of the method of the invention are as follows: firstly, the human cell growth factor and the chemical molecule are adopted in the whole culture system, and no exogenous gene related to the reprogramming or transdifferentiation function is introduced (but a reporter gene can be introduced), so that the interference of the exogenous gene on the genome stability of the original stem cell and the transplantation potential safety hazard related to the exogenous cell can be avoided. Secondly, the sperm cell prepared by the female germ stem cell has a tail-shaped structure and good activity.

Culture medium

The present inventors provide a medium for preculture and induction culture at each stage, comprising: a pre-culture medium, which may be a basal medium, to which serum is added; a stage 1 culture medium, which is added with KSR, BMP-2, BMP-4, BMP-7, retinoic acid and activin A on the basis of a cell culture medium; stage 2 medium supplemented with KSR, testosterone, FSH and pituitary extract on a cell culture medium basis.

Cytokines or chemical components known in the art to have the same or similar functions to them may be used in the present invention in addition to the specific cytokines or chemical components listed in the examples of the present invention. Analogues, homofunctional proteins (e.g. of growth factors) or compounds of the specifically listed components, equivalent compounds, analogues, derivatives and/or their salts, hydrates or precursors which induce the same target may also be used in place of the specifically listed components to achieve the same technical effect. Such analogs, homofunctional proteins or compounds are also intended to be encompassed by the present invention. Analogs of the compounds include, but are not limited to: isomers and racemates of the compounds. The compounds have one or more asymmetric centers. Thus, these compounds may exist as racemic mixtures, individual enantiomers, individual diastereomers, mixtures of diastereomers, cis or trans isomers. The term "precursor of a compound" refers to a compound which, when applied or treated by a suitable method, is converted in a culture medium to a compound of any of the above compounds, or a salt or solution of a compound of any of the above compounds.

In a preferred form of the invention, the culture medium is also supplemented with components for preventing bacterial contamination of the cell culture, in particular gram-positive and gram-negative bacterial contamination, such as some antibiotics.

The cell culture medium (basal medium) may be, for example, but not limited to: DMEM/F12, MEM, DMEM, RPMI1640, Neuronal basal or Fischer, and the like. It will be appreciated that those skilled in the art are familiar with the formulation or purchase route of the basal cell culture medium. Preferred cell culture media are provided in the examples of the present invention.

The invention also provides a kit, which contains the pre-culture medium, the 1 st stage culture medium and the 2 nd stage culture medium; it also contains starting cells, including female germ stem cells and testicular somatic cells. Preferably, the kit further contains a culture medium/reagent for culturing and expanding female reproductive stem cells and a culture medium/reagent for separating and maintaining testicular somatic cells, if necessary. Preferably, the kit further comprises instructions for use, thereby facilitating the study or clinical use of the kit by a person skilled in the art.

Based on the new discovery, the invention also provides a culture product of the female reproductive stem cell obtained by the method and the sperm cell produced by the culture product. The inventor finds that the functional sperm end obtained by the method of the invention carries a tail structure and presents a good state. Meanwhile, the sperm cell has typical sperm formation markers including: PRM1, ACROSIN, TNP1, HAPRIN and/or ACROSIN, etc.; and the functional sperm is of a haploid karyotype.

Methods for enriching or isolating purified cells from cell cultures are also well known to those skilled in the art, e.g., enrichment can be based on morphological characteristics of sperm cells; or selection for collection (e.g., using specific antibodies or ligands) based on the particular protein (e.g., Albumin, etc.) or molecular marker expressed by the sperm cell. As an alternative embodiment, the cells may be isolated and purified by molecular labeling of the surface of the sperm cells using flow cytometric sorting techniques.

The sperm cells cultured by the invention can be applied to assisted reproduction. According to the embodiment of the present invention, the in vitro development process can be smoothly continued after the injection into the cytoplasm, and the offspring can be smoothly generated after the uterus of the recipient animal is transplanted at a proper time.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, or according to the manufacturer's recommendations.

Materials and methods

1. Laboratory animal

KIT^w/wvMice were purchased from jackson laboratories (usa).

C57BL/6 mice were purchased from Shanghai Si Laike laboratory animals, Inc.

2、RT-PCR

Extracting total RNA from cells, and using

II Q RT SuperMix (+ gDNA wiper) kit (Vazyme, R223-01) reverse transcription was performed according to the instructions. RT-PCR was performed using Taq polymerase (Takara, R10T1M) with primers as shown in Table 1 for 35 cycles. The PCR-amplified GAPDH gene was used as an internal control. Electrophoresis was performed using a 2% agarose gel, and the DNA bands were stained with Ethidium Bromide (EB). PCR products were obtained and sequencing was used to determine whether the band was the corresponding gene.

TABLE 1 PCR primers for analysis of differentiation of genes from female germ stem cells to sperm

3. Spermatocyte surface spreading technology

Spermatocyte surface spreading techniques are used to identify spermatocytes produced during spermatogenesis in vitro. Cells were digested into single cell suspensions with 0.05% trypsin (Gibco) and then washed with Phosphate Buffered Saline (PBS) at room temperature. The cells were then placed in freshly prepared buffer (30mM tris, pH8.2, 50mM sucrose, 17mM citric acid, 5mM EDTA, 0.5mM DTT, 0.1mM PMSF, PH 8.2-8.4) for 30-60 min. The cells were centrifuged (350g, 5min) and 100mM sucrose (ph8.2) added for 20 min. Finally, the cell suspension was placed on a glass slide containing 1% paraformaldehyde PFA (pH 9.2) and 0.15% triton X-100PBS and the slide was dried overnight in a high humidity enclosure.

4. Immunofluorescence staining

Cells were fixed in culture with 4% PFA for 30min, washed 3 times with 0.1% Tween 20 (PBST), and then incubated in 1% triton X-100PBS for 15min (for cells spread with PMSF, this step was omitted). Cells or slides were blocked with 5% goat serum in PBS for 30min at room temperature, then incubated overnight with PBS diluted primary antibody at 4 ℃. Primary anti-dilution ratio: MVH (1: 200; Abcam), FRAGILIS (1:100, Abcam), OCT4(1:100, Abcam), SCP3(1:200, Santa cruz), SCP1(1:100, Abcam) and ACR (1:200, Santa cruz).

After overnight incubation, the cells were washed 3 times with PBS, then incubated with secondary antibody (1:200) at 37 ℃ and washed 3 times with PBS, followed by staining of the nuclei with DAPI and observation of the fluorescence signal with a fluorescence microscope.

5. Cytosolic sperm microinjection (ICSI)

BDF1 mice were used as oocyte donors. Sorting sperm-like cells (GFP positive cells or haploid cells) by flow cytometric sorting. The sorted sperm-like cells were cytoplasmatically removed and injected into cumulus-free oocytes. The fluorescent microscope is used for observing the change of the fluorescent protein in different development stages. Transferring the embryo reaching 2-cell stage after 24h culture into oviduct of 0.5-day-0.5 CD1 pseudopregnant female mouse at embryo stage or further culturing in KSOMaa culture medium to blastocyst stage (37 deg.C, 5% CO)₂. The mice born after transplantation give birth by spontaneous birth. The sequence of the birth offspring containing GFP is identified by a Sourthern blot method. Bisulfite methylation sequencing identifies that the methylation state of the genes of the birth offspring blots is not changed.

Example 1 Induction of differentiation of female reproductive Stem cells into sperm

1. Mouse female reproductive stem cell isolation

Separating, purifying and culturing female germ stem cells in vitro from the ovary (6 days after birth, C57BL/6) of a newborn mouse; female germ stem cells were isolated and purified from β -ACTIN-GFP transgenic mice (methods ref: Zou, K., Z. yuan, Z. Yang, et al., Production of offset from a germ line Cell line derived from neural cells Biol, 2009.11(5): p.631-636). The female germ stem cell can obtain a wild mouse; where necessary, mice bearing GFP were used for in vitro cell differentiation or progeny mouse tracking.

Female germ stem cells were cultured on mouse embryonic fibroblast (STO cell) feeder layers in MEM alpha medium containing 10% Fetal Bovine Serum (FBS), 1mM sodium pyruvate (Amresco), 2mM L-glutamine (Amresco), 50. mu.M beta-mercaptoethanol (Biotech), 1mM non-essential amino acids (NEAA) (invitrogen), 20ng/mL mouse epidermal growth factor (Peprotech), 10ng/mL human basic fibroblast growth factor (bFGF) (Peprotech), 10ng/mL mouse glial cell growth factor (GDNF) (Peprotech), 10ng/mL mouse Leukemia Inhibitory Factor (LIF) (Santa cruz).

And identifying the female reproductive stem cells by immunofluorescence and RT-PCR.

2、KIT^w/wvMouse testicle somatic cell separation

Collecting and extracting KIT 2-7 days after birth^w/wwMouse testicular somatic cell (KIT)^w/wwThe absence of germ cells in the male testis ensures that the differentiated sperm-like cells are not derived from KIT^w/wwTesticular cells of male mice). Mice were sacrificed by cervical dislocation, their testis removed aseptically, cut into small pieces, digested with collagenase IV (1mg/mL, Sigma) in a water bath at 37 ℃ for 10min, and then digested with 0.05% trypsin (Gibco) for 6 min.

After filtering with a 72 μm cell sieve, a single cell suspension was obtained, and the cells were collected by centrifugation.

3. Transdifferentiation

3-1 transdifferentiation 1

Transdifferentiation was induced from day 0 to day 6 using MEM α medium containing 20% Knockout serum replacement (abbreviated as serum replacement or KSR) (Gibco), BMPs-2/4/7 (20 ng/mL each, R & D), all-trans retinoic acid (1 μ M, Sigma), and activin A (100ng/mL, R & D), with fluid changes once a day.

Immediately thereafter, from day 7 to day 14, differentiation was induced in MEM α medium containing 20% serum replacement (KSR) (Gibco), 10mM testosterone, 200ng/mL FSH, 50mg/mL pituitary extract (BPE, Stem cell), and the medium was changed once a day. Cells were in 5% CO₂Medium culture and differentiation induction at 37 ℃.

The process directly induces the mouse testicular somatic cells and the female germ stem cells in stages. However, the present inventors found that the transformation efficiency was low and the time was long during the induction process, and the expression of the cell products spermatogenesis markers PRM1, ACROSIN, TNP1 and HAPRIN was low after 14 days.

3-2 transdifferentiation 2

(1) Mixed culture

Will KIT^w/wvThe mouse testicular cell and the female germ stem cell were directly mixed at a ratio of 1:1, and cultured in DMEM medium containing 10% FBS for 3 days.

(2) Induced differentiation

Transdifferentiation was induced from day 0 to day 6 using MEM α medium containing 10% serum replacement (KSR) (Gibco), BMPs-2/4/7 (20 ng/mL each, R & D), all-trans retinoic acid (1. mu.M, Sigma), and activin A (100ng/mL, R & D), with a change of medium every day.

Immediately thereafter, from day 7 to day 14, differentiation was induced in MEM α medium containing 10% serum replacement (KSR) (Gibco), 10mM testosterone, 200ng/mL FSH, 50mg/mL pituitary extract (BPE, Stem cell), and the medium was changed once a day. Cells were in 5% CO₂Medium culture and differentiation induction at 37 ℃.

In the process, mouse testicular somatic cells and female germ stem cells are pre-cultured and then are induced in stages. However, the present inventors found that the expression of the cell products spermatogenesis markers PRM1, ACROSIN, TNP1, HAPRIN was low after 14 days.

3-3, transdifferentiation 3

(1) Mixed culture

(2) Induced differentiation

Transdifferentiation was induced from day 0 to day 6 using MEM α medium containing 20% serum replacement (KSR) (Gibco), BMPs-2/4/7 (20 ng/mL each, R & D), all-trans retinoic acid (1. mu.M, Sigma), and activin A (100ng/mL, R & D), with a change of medium every day.

Immediately thereafter, from day 7 to day 14, differentiation was induced in MEM α medium containing 20% serum replacement (KSR) (Gibco), 10mM testosterone, 200ng/mL FSH, 50ug/mL pituitary extract (BPE, Stem cell), with changing every day. Cells were in 5% CO₂Medium culture and differentiation induction at 37 ℃.

In the process, mouse testicular somatic cells and female germ stem cells are pre-cultured and then are induced in stages. However, the present inventors found that the transformation efficiency during induction was relatively low and cells produced during differentiation did not have a tail.

3-4, transdifferentiation 4

(1) Mixed culture (and culture)

The mouse testicular somatic cells and the female germ stem cells were directly mixed at a ratio of 1:1, and cultured in DMEM medium containing 10% FBS for 3 days.

(2) Induced differentiation

In the process, mouse testicular somatic cells and female germ stem cells are pre-cultured and then are induced in stages. The transformation efficiency of the induction process is high, the sperm formation marker is very obvious, and functional sperm can be efficiently formed, which is specifically shown in the following example 2.

Example 2 and verification of differentiation-inducing products 3 to 4 in example 1

(1) Cell morphology

The development morphology of female germ stem cells in the in vitro transdifferentiation system is shown in figure 2A. It is particularly unexpected that sperm formed after induction by the method of the present invention have a distinct tail structure, which is not possible in the prior art.

(2) Formation of synaptotic complexes and meiotic processes

Cells were obtained during differentiation (D6-D10), and formation of synaptonemal complexes and meiotic processes were observed by immunofluorescence.

As a result, in FIG. 2B, formation of synaptonemal complexes (SCP1/SCP3) and meiotic processes (thin line, even line, thick line, double line) were observed.

(3) Sperm formation marker analysis

Total RNA was extracted from cells on days D1, D3, D6, D8, D10, and D14 after induction culture, and male meiosis (STRA8, SCP1, SCP2, SCP3), a sperm formation marker (PRM1, ACROSIN, TNP1, HAPRIN), and the like were expressed by RT-PCR analysis.

As shown in FIG. 2C, the RT-PCR detection result shows that the spermatogenesis marker is positive successively along with the increase of the induction time, which proves that the method of the invention can well induce the in vitro spermatogenesis of the female germ stem cells.

(4) Haploid analysis

For sperm (sperm-like cells) produced in vitro from female germ stem cells, the inventors analyzed their ploidy using flow cytometry, and STO cells, FGSC cells, fSLC cells, Sertoli cells as controls. The results are shown in FIG. 2D, and it can be seen that the produced sperm was haploid.

For spermatozoa (spermatozoa-like cells) produced in vitro by female germ stem cells, the inventors also performed karyotyping, resulting in the appearance of a haploid karyotype, as shown in fig. 2E.

The karyotype of the female reproductive stem cell is XX, and the sperm can be determined to be X sperm, so that female offspring can be obtained.

(5) Immunofluorescence analysis spermatogenesis marker

In addition to analysis of the spermatogenesis marker at the mRNA level by RT-PCR, the present inventors also used immunofluorescence to detect the presence of the spermatogenesis marker ACROSIN in spermatozoa (spermatozoa-like cells) produced in vitro from female germ stem cells.

The results are shown in FIG. 2F, where ACROSIN was found to be positive.

Example 3, functional characterization of sperm cells obtained after 3-4 induced differentiation in example 1

The present inventors injected sperm cells obtained by in vitro differentiation of female germ stem cells into the cytoplasm and observed the in vitro development process.

As shown in fig. 3A-H, after injection of sperm-like cells into the cytosol, the cells can initiate development, including from 2 cells, 4 cells to blastocysts.

The present inventors transplanted cells at the 2-cell stage into the uterus of mice. As a result, the mice can give rise to offspring, and the mice are naturally delivered after transplantation, and the mice are born as offspring, for example, 3I (TG in the figure refers to TransGene mice).

The Sourther Blot method was used to identify the born progeny, which were seen to contain the GFP sequence, as shown in FIG. 3J.

And (3) identifying the methylation state of the blot gene of the birth offspring by bisulfite methylation sequencing. The results showed no change in methylation status, as shown in FIG. 3K.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims. Also, all references cited herein are incorporated by reference in this application as if each reference were individually incorporated by reference.

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Claims

1. A method of producing functional sperm in vitro, the method comprising: taking female reproductive stem cells as starting cells, mixing the starting cells with testicular somatic cells, and performing pre-culture and induced differentiation; functional sperm are produced from the induced differentiation product.

2. The method of claim 1, wherein the pre-culturing comprises: mixing the female germ stem cells and testicular somatic cells in a ratio of 1: 10-10: 1, preferably 1: 5-5: 1, more preferably 1: 2-2: 1, and culturing in a cell culture medium; preferably the cell culture medium is a serum-containing medium; preferably the cell culture medium comprises DMEM medium.

3. The method of claim 1, wherein inducing differentiation of the female germ stem cells and testicular somatic cells comprises:

induction in stage 1: inducing by Knockout serum substitute, BMP-2, BMP-4, BMP-7, all-trans retinoic acid and activin A;

induction in the 2 nd stage: induction with Knockout serum replacement, testosterone, FSH and pituitary extract.

4. The method of claim 3, wherein in stage 1, the amounts of each component comprise: knockout serum substitute 20 + -4% (v/v), BMP-220 + -5 ng/mL, BMP-420 + -5 ng/mL, BMP-720 + -5 ng/mL, all-trans retinoic acid 1 + -0.4 μ M and activin A100 + -40 ng/mL;

in the 2 nd stage, the dosage of each component comprises: knockout serum replacement 20 + -4%, testosterone 10 + -3 mM, FSH200 + -30 ng/mL and pituitary extract 50 + -15 mg/mL;

preferably, the individual components are added to the cell culture medium; more preferably, the cell culture medium is MEM α medium.

5. The method of claim 1, wherein said producing functional sperm from an induced differentiation product comprises: observing the induced differentiation products, and separating functional sperms with tail structures at the tail ends.

6. The method of claim 1, wherein said female reproductive stem cells are cultured by a method comprising: placing female reproductive stem cells in an embryonic fibroblast feeder layer, and culturing in a cell culture medium containing sodium pyruvate, L-glutamine, beta-mercaptoethanol, non-essential amino acids, epidermal growth factor, human basic fibroblast growth factor, glial cell growth factor, and leukemia inhibitory factor; preferably, the cell culture medium comprises MEM alpha medium; preferably the cell culture medium is a serum-containing medium;

preferably, the amounts of the components include: 1 plus or minus 0.3mM sodium pyruvate, 2 plus or minus 0.6mM L-glutamine, 50 plus or minus 15 mu M beta-mercaptoethanol, 1 plus or minus 0.3mM non-essential amino acid, 20 plus or minus 5ng/mL epidermal growth factor, 10 plus or minus 3ng/mL human basic fibroblast growth factor, 10 plus or minus 3ng/mL glial cell growth factor, 10 plus or minus 3ng/mL leukemia inhibitory factor;

7. A product of induction culture of female germ stem cells and testicular somatic cells or functional sperm produced by the product, which is prepared by the method of any one of claims 1 to 6; preferably, it has characteristics selected from the group consisting of:

the tail end of the functional sperm carries a tail structure;

the male germ cell meiosis marker in the induction system is positive: STRA8, SCP1, SCP2, SCP 3;

positive for a spermatogenesis marker; preferably the marker comprises: PRM1, ACROSIN, TNP1, HAPRIN and/or ACROSIN;

the functional sperm is haploid karyotype; and/or

After injection into the cytosol, the functional sperm initiates development and transplantation into the uterus of a living organism can produce progeny.

8. The application of female germ stem cell and testis cell in preparing functional sperm in vitro.

9. A method of producing an animal progeny comprising:

(1) preparing functional sperm by the method of any one of claims 1 to 6;

(2) introducing the functional sperm of (1) into the cytoplasm of the egg cell, fertilizing the egg and developing in vitro; preferably, the cells are transplanted to uterus, implanted and developed into healthy offspring at a suitable stage of in vitro development.

10. A kit for the in vitro preparation of functional sperm, comprising:

female reproductive stem cells;

testicular somatic cells;

a pre-culture agent comprising a cell culture medium; preferably the cell culture medium is a serum-containing medium; preferably the cell culture medium comprises DMEM medium;

stage 1 inducers including Knockout serum replacement, BMP-2, BMP-4, BMP-7, retinoic acid, and activin A; the preferred amounts of each component include: knockout serum replacement 20 + -4% (v/v) BMP-220 + -5 ng/mL, BMP-420 + -5 ng/mL, BMP-720 + -5 ng/mL, retinoic acid 1 + -0.4 μ M and activin A100 + -40 ng/mL;

phase 2 inducers including Knockout serum replacement, testosterone, FSH and pituitary extract; the preferred amounts of each component include: knockout serum replacement 20 + -4%, testosterone 10mM, FSH200ng/mL and pituitary extract 50 mg/mL;

preferably, the components of the inducer are added to the cell culture medium.