26 results about "Spermatogonial stem cells" patented technology

The invention discloses a zebra fish spermatogonial stem cell separation and transplantation method, and belongs to the technical field of fish germline stem cell separation. The method comprises the following steps: washing complete sexual glands of zebra fish testis by adopting penicillin / streptomycin double-antibody-containing PBS (Phosphate Buffer Solution), physically shearing, digesting and dissociating, and filtering by a screen, so as to obtain zebra fish testis cell suspension; performing density gradient centrifugation to obtain zebra fish spermatogonial stem cells; dyeing, marking, centrifuging, washing, re-suspending, and carrying out micro-injection transplantation to enter the back of the abdominal cavity of the acceptor zebrafish juvenile fish. According to different physical properties of different cells, spermatogonial stem cells are separated, purified and transplanted from zebra fish testis cell suspension by adopting a density gradient centrifugation method, and migration and proliferation processes of donor germ cells can be observed in receptor fish after fluorescence labeling. And a new technical path is provided for fish germplasm resource preservation, endangered fish protection, economic cultured fish yield increase and new variety cultivation.

The invention discloses a method for separating, purifying and transplanting spermatogonial stem cells of lateolabrax japonicus, the spermatogonial stem cells of lateolabrax japonicus are separated and purified, marine fish larvae or freshwater fish larvae are used as receptor larvae, and then the spermatogonial stem cells of lateolabrax japonicus are transplanted to abdominal cavities of the receptor larvae. The spermatogonial stem cells obtained through separation are complete in number, structure and function and low in cell clustering rate. After the transplanted spermatogonial stem cells are subjected to fluorescence labeling, the migration and proliferation processes of donor germ cells can be observed in receptor fishes of marine fish fries or freshwater fish fries, so that the receptor fishes with the chimeric germline are obtained. According to the method, through xenotransplantation of the spermatogonial stem cells, a'belly borrowing reproduction 'breeding mode is provided for the marine fishes, a new way for gamete preservation and rapid propagation is provided for the marine fishes with the long reproductive cycle, and a new method and a new thought are provided for genetic improvement and germplasm resource preservation.

The invention discloses a method for separating and transplanting spermatogonial stem cells of Chinese sturgeons, and belongs to the technical field of separation of germline stem cells of fishes. The separation method comprises the following steps: washing Chinese sturgeon testis tissues with an L-15 culture medium containing penicillin / streptomycin double antibodies, and carrying out physical shearing, digestive enzyme enzymolysis and screen filtration to obtain a Chinese sturgeon testis cell suspension; performing percoll density gradient centrifugation to obtain high-purity spermatogonial stem cells of the Chinese sturgeons; and dyeing, washing, resuspending and carrying out micro-injection transplantation to enter the gonad primordium reproductive ridge part of the juvenile acceptor fish. According to the physical characteristics of the spermatogonial stem cells of the Chinese sturgeons, the spermatogonial stem cells with high purity are obtained by adopting percoll density gradient centrifugation, and the purpose of abdomen borrowing reproduction is achieved by adopting a microinjection mode, so that the preservation of germ cells of endangered fishes and the reconstruction of artificial populations are realized; and a new technical approach is provided for protecting genetic resources of the Chinese sturgeons and breaking through artificial propagation bottlenecks.

A sequential cell culture system and methods for the in vitro generation of human spermatozoa from differentiating spermatogonia (dSPGs) and spermatogonial stem cells (SSCs) is provided. The instant disclosure encompasses the discovery and optimization of distinct, stage-specific culture media and conditions that enable, for the first time, the complete progression of human spermatogenesis outside the body. The process includes: (1) inducing entry of dSPGs and SSCs into meiosis; (2) supporting meiotic progression and completion to round spermatids; and (3) promoting spermiogenesis and the formation of elongated, spermatozoa. The invention further provides compositions, systems, and protocols for each stage, as well as markers and methods for identifying successful progression through each developmental transition. This system enables, for the first time, the complete in vitro recapitulation of human spermatogenesis, providing a platform for research, infertility treatment, and assisted reproductive technologies.

The invention provides a recombinant lentivirus capable of indicating bovine spermatogonial stem cell differentiation, a spermatogonial stem cell line and application, and belongs to the technical field of cell biology. The invention provides a method for constructing a spermatogonial stem cell line for indicating bovine sperm differentiation on the basis of a lentivirus transduction technology, and a visual detection system for differentiating spermatogonial stem cells into spermatoblasts is constructed by regulating expression of mCherry protein by utilizing a PRM1 promoter. According to the method, the expression of PRM1 is indicated by fluorescent protein mCherry, so that the differentiation process of the bovine spermatogonial stem cells is indicated, and a good tool is provided for the induction of differentiation of the bovine spermatogonial stem cells to sperms, the research of a development mechanism and the optimization of a differentiation system of the bovine spermatogonial stem cells. The method is a new way for solving the neck clamping problem that good-variety bovine sperms are difficult to obtain in scientific research and production, can also be used for optimization of a spermatogonial stem cell differentiation system, sorting and purification of sperms, even downstream research of drug screening and the like, and has a good application prospect.

The invention discloses construction of a Guangdong small-ear spotted pig spermatogonial stem cell in-vitro culture system and screening of surface markers, and belongs to the technical field of cell culture.The technical scheme includes that testis are broken firstly, then enzyme digestion, density gradient centrifugal purification and differential adherent purification are conducted, and purified spermatogonial stem cells are obtained; inoculating the obtained spermatogonial stem cells to feeder layer cells and culturing in a culture medium; the culture medium is a DMEM culture medium added with FBS and growth factors; the mass fraction of the FBS in the DMEM culture medium is 8-12%, an enzyme digestion method, a density gradient centrifugation method and a differential adhesion method are combined for application, the Guangdong small-ear spotted pig spermatogonia stem cells with certain purity are obtained, cells enriched and purified in vitro can proliferate massively, and an in-vitro culture system of the Guangdong small-ear spotted pig spermatogonia stem cells is established.

A sequential cell culture system and methods for the in vitro generation of human spermatozoa from differentiating spermatogonia (dSPGs) and spermatogonial stem cells (SSCs) is provided. The instant disclosure encompasses the discovery and optimization of distinct, stage-specific culture media and conditions that enable, for the first time, the complete progression of human spermatogenesis outside the body. The process includes: (1) inducing entry of dSPGs and SSCs into meiosis; (2) supporting meiotic progression and completion to round spermatids; and (3) promoting spermiogenesis and the formation of elongated, spermatozoa. The invention further provides compositions, systems, and protocols for each stage, as well as markers and methods for identifying successful progression through each developmental transition. This system enables, for the first time, the complete in vitro recapitulation of human spermatogenesis, providing a platform for research, infertility treatment, and assisted reproductive technologies.

The present disclosure provides bovine animals and methods to create recipient bovine animals for spermatogenic stem cell transplantation / complementation through modulation of the NANOS2 gene. In one embodiment genome editing was used to create bovine animals with insertions or deletions (indels) that inactivate or otherwise modulate NANOS2 gene activity so that resulting male bovines lack functional germ cells yet retain functional testicular somatic cells, and bovine females are fertile. These bovine males can then be transplanted or complemented with donor cells capable of giving rise to cells of the spermatogenic lineage, or spermatogenic stem cells and used for breeding.

The invention discloses an oryzias latipe spermatogonial stem cell strain capable of stably expressing Cas9 protein as well as a construction method and application of the oryzias latipe spermatogonial stem cell strain. The oryzias latipe spermatogonial stem cell strain is designed and constructed based on a Tol2 transposon system, wherein the transposon vector comprises a coding sequence of a Cas9 gene, a hygromycin B resistance gene and left and right arm sequences of a Tol2 transposon; constructing a Tol2 transposase expression vector; co-transfecting the transposon vector and the expression vector to the spermatogonial stem cells SG3 of the oryzias latipe; screening in an ESM4 culture medium containing hygromycin B to obtain a drug-resistant cell population capable of stably expressing Cas9; selecting monoclonal colonies under a microscope, and carrying out continuous subculture to obtain an oryzias latipe spermatogonial stem cell strain which stably expresses Cas9 protein and is named as oryzias latipe gonad cells SG3-Cas9. The method has the advantages of short construction period, high efficiency, good expression stability and the like, and provides a reliable cell platform for fish cell level gene editing research.

The invention discloses a rapid breeding method for generating larimichthys crocea sperms based on spermatogonial stem cell transplantation. The rapid breeding method comprises the following steps: step S1, performing whole cryopreservation on larimichthys crocea testis; s2, separating the spermatogonial stem cells of the pseudosciaena crocea; s3, dyeing the spermatogonial stem cells of the pseudosciaena crocea; step S4, treatment of nibea albiflora receptors; and S5, transplanting the dyed donor larimichthys crocea spermatogonial stem cells into the nibea albiflora receptor testis. According to the method, when the nibea albiflora acceptor is treated, the traditional busulfan concentration of 40 mg / kg, twice injection and 35 DEG C high-temperature assistance is optimized into 60 mg / kg, single injection and 22 DEG C normal-temperature culture, optimal parameters induced by busulfan are screened out through experiments, the operation process is simplified under the action of ensuring that endogenous germ cells of the acceptor are reduced and immunological rejection is reduced, and the method is suitable for large-scale popularization and application. The harsh requirement of a high-temperature environment is avoided, and meanwhile the field planting efficiency of donor cells in receptors and the survival rate of the receptors are improved.

The invention provides a trachinotus ovatus spermatogonial stem cell line as well as establishment, cryopreservation and recovery methods and application thereof, and relates to the technical field of biologication.The method comprises the steps that tissue cells of trachinotus ovatus testis are placed in a complete culture medium to be cultured, and a fresh culture medium is replaced once every 2-4 days within 15 days before culture; culturing until the bottom of the bottle is full of cells, adding a trypsin solution to digest the cells, sucking out the digested cells every 30 seconds, respectively adding a complete culture medium for culturing for three times, screening out high-proportion spermatogonial stem cells for subsequent continuous culture, subculturing to a new culture bottle according to the proportion of 1: (1-3), and culturing to obtain the trachinotus ovatus spermatogonial stem cell line. The trachinotus ovatus spermatogonial stem cells with relatively high purity are obtained by a differential digestion method, and the trachinotus ovatus spermatogonial stem cell line is obtained by setting a passage proportion for culture and can be stably passed. And gene transfection research is carried out, so that technical support is provided for extensive research on spermatogonial stem cells of trachinotus ovatus.

The present invention relates to in vitro methods for production of Sertoli cells and related organoids. The Sertoli cells and testis like organoids may be used for therapeutic purposes including facilitation of the production of spermatogonia from pro-spermatogonia stem cells.

The invention provides a construction method of a viral reproductive dysfunction model based on spermatogonial stem cells and application of the model, and belongs to the cross technical field of biotechnology and veterinary medicine. The construction method comprises the following steps: placing spermatogonial stem cells in a spermatogonial stem cell culture medium for culture, and infecting the spermatogonial stem cells with bovine viral diarrhea viruses to obtain the viral reproductive function disorder model based on the spermatogonial stem cells. In order to solve the problems that in the prior art, a BVDV reproductive injury mechanism is unknown, and an effective research model is lacked, the reproductive stem cells are used for simulating direct injury of BVDV to a male reproductive system for the first time, and the defect that a traditional model (such as MDBK cells) cannot reflect specific biological reaction of reproductive cells is overcome; meanwhile, experiments show that the model constructed by the invention can support standardized processes of virus strain typing and drug screening, the research and development efficiency is remarkably improved, and the experiment cost is reduced.

The invention relates to the crossing field of a gene editing technology and a veterinary biotechnology, in particular to a goat spermatogonial stem cell anti-BVDV functional target based on CD46 gene editing and application of the goat spermatogonial stem cell anti-BVDV functional target. An anti-virus cell strain is obtained by designing sgRNA of a targeted CD46 CCP1 structural domain and knocking out the gene through a CRISPR-Cas9 system. An infection experiment shows that the virus load of the delta CD46-SSCs is reduced by 90%, and the survival rate is recovered to 85%. The invention reveals that CD46 is a key receptor of goat BVDV infection for the first time, and provides a new strategy for disease-resistant breeding and targeted drug development.

The present invention discloses the application of uridine in promoting the injury regeneration or proliferation of spermatogonial stem cells. The present invention discovers for the first time this new use of uridine in promoting the injury regeneration or proliferation of spermatogonial stem cells. Uridine not only has an obvious promoting effect on the proliferation of spermatogonial stem cells in vivo, but also has the same effect on promoting the proliferation of spermatogonial stem cells in vitro cultured spermatogonial stem cells, spermatogonial stem cells in testicular organoids cultured in vitro, and spermatogonial stem cells in senescent testes, and can repair problems such as the reduction in the number of spermatogonial stem cells caused by testicular injury induced by chemotherapeutic drugs, thereby providing a new solution for the clinical treatment of male infertility and the protection of male fertility.

The invention provides a specific marker gene lhh2a. 2.2 of spermatogonium of larimichthys crocea as well as a screening method and application of the specific marker gene lhh2a. 2.2, and belongs to the field of molecular markers and the field of germline stem cell research. According to the invention, the nucleotide sequence of the lhh2a. 2.2 gene is as shown in SEQ ID NO: 1; through screening and analysis of a single cell transcription sequencing technology, and through in-situ hybridization, Vasa gene and PCNA gene co-localization, it is determined that the lhh2a. 2.2 gene can specifically identify spermatogonium in a proliferation state; a core tool and a prerequisite condition can be provided for establishment of a high-efficiency germ cell transplantation technology, purification of spermatogonial stem cells for in-vitro culture and construction of a transgenic pseudosciaena crocea strain.

The present invention provides a method for culturing spermatogonial stem cells (SSCs) The process involves the development of a bioartificial testis by repopulating a decellularized testicular extracellular matrix (ECM) with isolated SSCs and culturing them in the presence of seminal exosomes (SEs). The invention encompasses a method for the complete in vitro reconstitution of spermatogenesis. This is achieved through the co-culture of SSCs on a biomechanically and biochemically optimized decellularized testicular ECM scaffold, further supplemented with seminal exosomes as a key bioactive component.

The invention discloses sgRNA for accurately editing a PSEN1 p.M146V site of a tree shrew and application of the sgRNA, and belongs to the technical field of gene editing. The nucleotide sequence of the sgRNA designed by the invention is shown as SEQ ID NO.1. The sgRNA can guide a base editor to be precisely positioned to a PSEN1 p.M146V site, a spermatogonial stem cell line for precisely editing the PSEN1 p.M146V site of the tree shrew is obtained, the precisely edited spermatogonial stem cell line is obtained through single cell cloning, screening and enrichment, and the stably inherited PSEN1 p.M146V gene is obtained through reproductive line transmission to edit the tree shrew. The sgRNA provided by the invention is accurate in design and high in editing efficiency, and the editing efficiency of the obtained tree shrew spermatogonial stem cells is as high as 100%. The spermatogonial stem cell line for precisely editing the tree shrew PSEN1 p.M146V site and the stably inherited PSEN1 p.M146V gene editing tree shrew are obtained for the first time, a research basis is provided for researching the degenerative phenotype, pathological characteristics and molecular mechanism of the Alzheimer's disease caused by the mutation, and an important experimental platform is provided for screening related drugs.

The invention belongs to the technical field of in-vitro culture of germline stem cells, and particularly relates to a separation and in-vitro culture method of spermatogonial stem cells of grass carp. The method comprises the following steps: by taking male grass carps as donors, anesthetizing, killing, cleaning, disinfecting, dissecting and separating gonad tissues, crushing into fragments by adopting a mode of combining magnetic bead crushing with shearing, digesting with trypsin, filtering and centrifuging to obtain high-purity spermatogonial stem cells, and performing in-vitro culture on the spermatogonial stem cells by adopting a culture solution. By optimizing the age of the male grass carp, an enzyme digestion process and an in-vitro culture system and adopting a one-step enzyme digestion method, the digestion time is shortened, the operation process is simplified, and the industrial problems that the spermatogonial stem cells of the grass carp are low in separation efficiency and culture is easy to differentiate are solved. According to the invention, a separation, purification and primary culture technical system of the spermatogonial stem cells of the grass carp is systematically established for the first time, and important cell materials and technical supports are provided for breeding technologies such as transplantation of germline stem cells of the grass carp, gene editing and the like.

The application provides a preparation for enhancing the in-vitro proliferation capacity of pig spermatogonial stem cells, and belongs to the technical field of livestock reproduction. The preparation is composed of a basic culture solution, basic maintenance components, supportive growth factors and a core active component polypeptide Japonicin-1Npb. Research shows that under the condition of approaching the physiological temperature (38.5 DEG C) of pigs, the addition of 25-100 mu g / mL of the polypeptide Japonicin-1Npb can significantly improve the EdU positive rate of pig spermatogonial stem cells, promote cell proliferation, specifically up-regulate the expression of stemness genes PLZF, UCHL1 and GFR alpha 1, and inhibit the differentiation gene c-KIT. The application solves the problems of low in-vitro expansion efficiency and easy loss of stemness of pig spermatogonial stem cells, and the cells still retain good meiotic differentiation potential after the removal of the preparation, thereby providing technical support for the development of high-quality genetic resources of pigs and the research of reproductive biology.

The present invention discloses a method for establishing an APP / PSEN1 double transgenic AD tree shrew model. For the first time, lentivirus carrying pathogenic genes APP and PSEN1 is used to infect the spermatogonial stem cells of tree shrews. The AD transgenic spermatogonial stem cells of tree shrews are injected into the testes of male tree shrews treated with busulfan. Then, the recipient male tree shrews are allowed to mate with wild-type female tree shrews. The obtained offspring AD transgenic tree shrews are identified to obtain a tree shrew model that produces overexpressed mutant APP and PSEN1 double transgenic Alzheimer's disease. The present invention is the first AD transgenic disease model of tree shrews in the world. A stable and heritable new tree shrew strain can be obtained through continuous hybridization of existing transgenic tree shrews; it can simulate the generation and metabolic process of the main pathogenic genes of AD pathology in vivo, reproduce the disease process, and prove through behavioral indicators that this model is significantly different from other AD models, and can be used for research on the pathogenic mechanism of AD, development of drug targets, and drug screening.

The invention belongs to the technical field of biology, and particularly relates to a culture medium for in-vitro induced differentiation of paralichthys olivaceus spermatogonial stem cells and application. According to the method, by optimizing an induced differentiation culture medium formula, combining specific growth factors and hormones and establishing a step-by-step collection and artificial insemination procedure, directional differentiation from long-term passage spermatogonial stem cells to sperms with insemination ability is successfully realized, and the paralichthys olivaceus offspring which normally develops is finally obtained. The method breaks through the key technical bottleneck that the sperm function is incomplete after induced differentiation of long-term passage spermatogonial stem cells, and survival offspring cannot be obtained, and provides a reliable technical platform for bastard halibut germplasm resource preservation, genetic breeding acceleration and reproductive development research.

This invention provides a method for isolating spermatogonial stem cells from frozen testicular tissue, belonging to the field of cell biology. The isolation method provided by this invention includes the following steps: thawing frozen testicular tissue; digesting the thawed tissue with collagenase IV and DNase I until the seminiferous tubules are exposed and their walls become rough; washing with DPBS buffer, centrifuging, and culturing until cells migrate; collecting the migrated cells and purifying them for spermatogonial stem cell enrichment. The method described in this invention can maximally preserve the internal environment of spermatogonial stem cells, maintain cell viability, and reduce cell damage, and can be used for isolating spermatogonial stem cells from frozen testicular tissue.

The present application provides a method for directly transdifferentiating spermatogonial stem cells (SSCs) into neural stem cell-like cells (iNSCs). The iNSCs have proliferative activity, can be stably subcultured in vitro, and have the potential to differentiate into other neural cells such as neurons, astrocytes and oligodendrocytes. The method of the present application has high efficiency, can obtain more iNSCs in a relatively short period in vitro, and can obtain iNSCs with high purity, wherein the double positive rate of Nestin and Pax6 is more than 95%.

The invention discloses a method for efficiently differentiating monkey spermatogonial stem cells in vitro and application. The invention belongs to the technical field of biology, and particularly relates to a method for efficiently differentiating monkey spermatogonial stem cells in vitro and application. The active ingredients of the composition for promoting in-vitro differentiation of the monkey spermatogonial stem cells are retinoic acid, BMP4, testosterone and a basic differentiation medium. The concentration of the retinoic acid in the composition is 1 [mu] mol / L, the concentration of the BMP4 is 50 [mu] g / L, and the concentration of the testosterone is 0.1 mmol / L. According to the method, a culture system for in-vitro differentiation of the monkey spermatogonial stem cells (SSCs) into the functional haploid sperms is successfully established, the haploid differentiation efficiency can be obviously improved (the proportion exceeds 8%) after 96 hours of continuous treatment, and the blank of research on in-vitro differentiation systems and molecular mechanisms of the monkey SSCs is filled.

The invention discloses a method for efficiently editing a sex control gene cyp19a1a in a spermatogonial stem cell (ObSSC) of opsariichthys bidens, which comprises the following steps: aiming at the cyp19a1a gene of opsariichthys bidens, designing four sgRNA (small guide ribonucleic acid) knockout target spots based on a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas9 (clustered regularly interspaced short palindromic repeats) system; the method comprises the following steps: synthesizing sgRNA, and incubating the sgRNA and Cas9 protein at room temperature to form an RNP compound; the RNP compound is further incubated with ObSSC, and is electrically transferred to the ObSSC under the optimal transfection condition. The method disclosed by the invention has the advantages of high efficiency, simplicity, convenience, short period and the like, ensures that exogenous molecules are efficiently introduced into cells while maintaining in-vitro survival and proliferation of the ObSSC, lays a foundation for subsequently obtaining functional gametes carrying edited genes through ObSSC transplantation and analyzing a sex determination mechanism of the functional gametes, solves the fundamental problem of genetic modification cross-generation delivery, and has a wide application prospect. And a new technical path can be provided for fish genetic breeding.