Patent Description:
According to statistics from the World Health Organization, about <NUM>% of couples in the world are troubled by infertility, wherein a proportion of male factors is <NUM>% to <NUM>%, and the male factors are reflected on defects of the number or quality of sperm cells. Human pluripotent stem cells have the potential of differentiation into various cell types of human bodies. In-vitro differentiating stem cells into functional sperm cells provide a feasible approach for treatment of infertility.

Sertoli cells are one type of testis somatic cells, exist in seminiferous tubules together with germ cells, and have an important effect on triggering and regulation of spermatogenesis. For construction of a sperm in-vitro differentiation system, the Sertoli cells will be an indispensable component. In addition, the Sertoli cells in testes also evolve a special immune privilege attribute, so that the germ cells can be protected from being attacked by immune system. Such characteristic of the Sertoli cells has been applied to the allogeneic transplantation treatment of cells, tissues and organ, to relieve immune rejection from the host and improve the survival rate. In addition, the Sertoli cells per se also can be used as genetically engineered cells for carrying and expressing a particular protein or drug for treatment. Thus it can be seen that the Sertoli cells are one type of cells with great application potential; but due to restrictions of a source, ethics and the like, it is not realistic to acquire the Sertoli cells from human bodies in a large scale.

Fibroblasts are common cells in animal connective tissues, and can be obtained from animal individuals. In the cell lineage reprogramming research, the fibroblasts are often used as initiating cells; however, currently, there is no report related to in-vitro reprogramming of the fibroblasts into the Sertoli cells. <NPL> deals with embryonic Sertoli-like cells. <NPL> deals with steroidogenic cells and <NPL> discloses the use of a reporter system containing AMH promoter and CAT in labelling Sertoli cells.

The present disclosure aims to prepare Sertoli cells.

The present disclosure firstly provides a method for preparing Sertoli cells, which can include a step a2): increasing the expression level and/or activity of an NR5A1 protein in fibroblasts. The invention provides a method for preparing Sertoli cells according to claims <NUM> to <NUM> and a use of a protein combination for preparing Sertoli cells according to claims 7and <NUM>.

In the method, the "increasing the expression level and/or activity of the NR5A1 protein in the fibroblasts" can achieve an effect of increasing the expression level and/or activity of the NR5A1 protein in the fibroblasts by methods well-known in the art, such as multiple copying, change of a promoter, factor control, transgenosis and the like.

In the method, the "increasing the expression level and/or activity of the NR5A1 protein in the fibroblasts" can be introduction of a substance that increases the expression level and/or activity of the NR5A1 protein into the fibroblasts.

In the method, the "introduction of the substance that increases the expression level and/or activity of the NR5A1 protein into the fibroblasts" can be implemented by introducing nucleic acid molecules (i.e., NR5A1 genes) for encoding the NR5A1 protein into the fibroblasts. The "introducing the nucleic acid molecules for encoding the NR5A1 protein into the fibroblasts" can be implemented by infecting the fibroblasts with a lentivirus; and the lentivirus can express the NR5A1 protein.

In one embodiment of the present disclosure, the lentivirus may be a recombinant lentivirus a (expressing the NR5A1 protein). The recombinant lentivirus a can be formed by packaging of a lentivirus vector a. The lentivirus vector a, a helper plasmid vsvg and a lentivirus vector packaging plasmid pCMV ΔR8. <NUM> can be co-transfected with 293FT cells to obtain the recombinant lentivirus a. A preparation method of the lentivirus vector a can include: inserting the NR5A1 gene to a multiple cloning site (e.g., restriction endonuclease EcoRI) of a pENTR/1A plasmid so as to obtain a vector pENTR/1A-NR5A1; inserting double-stranded DNA molecules (a nucleotide sequence of an EF1α promoter) as shown SEQ ID No. <NUM> in the sequence list to a multiple cloning site (e.g., the restriction endonuclease EcoRI) of a pENTR/<NUM>' topo plasmid so as to obtain a vector pENTR/<NUM>' topo-EF1α; and preparing the lentivirus vector a from the vector pENTR/1A-NR5A1, the vector pENTR/<NUM>' topo-EF1α and a lentivirus plasmid p2K7-NEO by homologous recombination.

In the method, the step a2) may include: increasing "the expression level and/or activity of the NR5A1 protein" and "the expression level and/or activity of a protein combination" in the fibroblasts; and the protein combination is at least one of a GATA4 protein, a WT1 protein, a SOX9 protein and a DMRT1 protein.

In the method, the "increasing 'the expression quantity and/or level of the NR5A1 protein' and 'the expression level and/or activity of the protein combination' in the fibroblasts" can achieve an effect of increasing "the expression level and/or activity of the NR5A1 protein" and "the expression level and/or activity of the protein combination" in the fibroblasts by methods well-known in the art, such as multiple copying, change of the promoter, factor control, transgenosis and the like.

In the method, the "increasing 'the expression level and/or activity of the NR5A1 protein' and 'the expression level and/or activity of the protein combination' in the fibroblasts" can be introduction of "the substance that increases the expression level and/or activity of the NR5A1 protein" and "a substance that increases the expression level and/or activity of the protein combination" into the fibroblasts.

The "introduction of 'the substance that increases the expression level and/or activity of the NR5A1 protein' and 'the substance that increases the expression level and/or activity of the protein combination' into the fibroblasts" can be implemented by introducing the nucleic acid molecules (i.e., the NR5A1 genes) for encoding the NR5A1 protein and nucleic acid molecules for encoding the protein combination into the fibroblasts. Nucleic acid molecules for encoding the GATA4 protein namely are GATA4 genes. Nucleic acid molecules for encoding the WT1 protein namely are WT1 genes. Nucleic acid molecules for encoding the SOX9 protein namely are SOX9 genes. Nucleic acid molecules for encoding the DMRT1 protein namely are DMRT1 genes. The "introducing the nucleic acid molecules for encoding the NR5A1 protein and the nucleic acid molecules for encoding the protein combination into the fibroblasts" can be implemented by infecting the fibroblasts with a lentivirus; and the lentivirus can express the NR5A1 protein and the protein combination.

In one embodiment of the present disclosure, the lentivirus expressing the NR5A1 protein may be the recombinant lentivirus a.

In one embodiment of the present disclosure, the lentivirus expressing the GATA4 protein may be a recombinant lentivirus b. The recombinant lentivirus b can be formed by packaging of a lentivirus vector b. The lentivirus vector b, the helper plasmid sag and the lentivirus vector packaging plasmid pCMV ΔR8. <NUM> can be co-transfected with the 293FT cells to obtain the recombinant lentivirus b. A preparation method of the lentivirus vector b can include: inserting the GATA4 gene to the multiple cloning site (e.g., the restriction endonuclease EcoRI) of the pENTR/1A plasmid so as to obtain a vector pENTR/1A-GATA4; and preparing the lentivirus vector b from the vector pENTR/1A-GATA1, the vector pENTR/<NUM>' topo-EF1α and the lentivirus plasmid p2K7-NEO by homologous recombination.

In one embodiment of the present disclosure, the lentivirus expressing the WT1 protein may be a recombinant lentivirus c. The recombinant lentivirus c can be formed by packaging of a lentivirus vector c. The lentivirus vector c, the helper plasmid vsvg and the lentivirus vector packaging plasmid pCMV ΔR8. <NUM> can be co-transfected with the 293FT cells to obtain the recombinant lentivirus c. A preparation method of the lentivirus vector c can include: inserting the WT1 gene to the multiple cloning site (e.g., between restriction endonucleases NotI and AscI) of the pENTR/D-topo plasmid so as to obtain a vector pENTR/D-toppo-WT1; and preparing the lentivirus vector c from the vector pENTR/D-topo-WT1, the vector pENTR/<NUM>' topo-EF1α and a lentivirus plasmid 2K7-BSD by homologous recombination.

In one embodiment of the present disclosure, the lentivirus expressing the SOX9 protein may be a recombinant lentivirus d. The recombinant lentivirus d can be formed by packaging of a lentivirus vector d. The lentivirus vector d, the helper plasmid vsvg and the lentivirus vector packaging plasmid pCMV ΔR8. <NUM> can be co-transfected with the 293FT cells to obtain the recombinant lentivirus d. A preparation method of the lentivirus vector d can include: inserting the SOX9 gene to the multiple cloning site (e.g., between the restriction endonucleases NotI and AscI) of the pENTR/D-topo plasmid so as to obtain a vector pENTR/D-topo-SOX9; and preparing the lentivirus vector d from the vector pENTR/D-topo-SOX9, the vector pENTR/<NUM>' topo-EF1α and the lentivirus plasmid p2K7-NEO by homologous recombination.

In one embodiment of the present disclosure, the lentivirus expressing the DMRT1 protein may be a recombinant lentivirus e. The recombinant lentivirus e can be formed by packaging of a lentivirus vector e. The lentivirus vector e, the helper plasmid vsvg and the lentivirus vector packaging plasmid pCMV ΔR8. <NUM> can be co-transfected with the 293FT cells to obtain the recombinant lentivirus e. A preparation method of the lentivirus vector e can include: inserting the DMRT1 gene to the multiple cloning site (e.g., the restriction endonuclease EcoRI) of the pENTR/1A plasmid so as to obtain a vector pENTR/1A-DMRT1; and preparing the lentivirus vector e from the vector pENTR/1A-DMRT1, the vector pENTR/<NUM>' topo-EF1α and the lentivirus plasmid p2K7-NEO by homologous recombination.

Any one of the "introducing the nucleic acid molecules for encoding the NR5A1 protein into the fibroblasts" or any one of the "introducing the nucleic acid molecules for encoding the NR5A1 protein and the nucleic acid molecules for encoding the protein combination into the fibroblasts" can be implemented by adopting the corresponding lentivirus infection. Time of the infection may be <NUM> to <NUM> (e.g., <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, <NUM>, <NUM>, <NUM> or <NUM>). After the infection is finished, a medium for culturing the fibroblasts needs to be added, and culture is carried out for <NUM> to <NUM> (e.g., <NUM> to <NUM>, <NUM> to <NUM>, <NUM>, <NUM> or <NUM>) under the conditions of a temperature of <NUM> to <NUM> (e.g., <NUM> to <NUM>, <NUM> to <NUM>, <NUM>, <NUM> or <NUM>) and <NUM>% CO<NUM>.

Any one of the above-mentioned methods may further include a step a1): introducing a fluorescent protein reporter system specifically expressed in the Sertoli cells into the fibroblasts; in the fluorescent protein reporter system specifically expressed in the Sertoli cells, a promoter specifically expressed in the Sertoli cells promotes expression of a fluorescent protein; and the step a1) is carried out before the step a2) is carried out.

In the fluorescent protein reporter system, the promoter specifically expressed in the Sertoli cells may be a promoter of an AMH gene. A nucleotide sequence of the promoter of the AMH gene can be as shown in SEQ ID No. <NUM> in the sequence list.

In the fluorescent protein reporter system, the fluorescent protein is a green fluorescent protein, a yellow fluorescent protein or a red fluorescent protein.

Any one of the above-mentioned mediums for culturing the fibroblasts may be a DMEM medium containing <NUM> to <NUM>% (e.g., <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM>%, <NUM>% or <NUM>%) (v/v) of FBS, <NUM> to <NUM>% (e.g., <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM>%, <NUM>% or <NUM>%) (m/v) of glutamic acid and <NUM>% (e.g., <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM>%, <NUM>% or <NUM>%) (m/v) of non-essential amino acid.

Any one of the above-mentioned methods can further include steps a3) to a6):.

In the step a3), concentration of the G418 may be <NUM> to <NUM>/mL (e.g., <NUM> to <NUM>/mL, <NUM> to <NUM>/mL, <NUM>/mL, <NUM>/mL or <NUM>/mL).

In the step a5), the medium for culturing the fibroblasts may also be a DMEM/F12 medium containing <NUM> to <NUM>% (e.g., <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM>%, <NUM>% or <NUM>%) (v/v) of FBS.

In the step a6), the isolating the Sertoli cells can be implemented by isolating cells capable of emitting fluorescence.

Any one of the above-mentioned cultures can be carried out at a temperature of <NUM> to <NUM> (e.g., <NUM> to <NUM>, <NUM> to <NUM>, <NUM>, <NUM> or <NUM>).

Any one of the above-mentioned fluorescent protein reporter systems specifically expressed in the Sertoli cells also falls within the protection scope of the present disclosure.

The present disclosure further provides an application of any one of the above-mentioned fluorescent protein reporter systems specifically expressed in the Sertoli cells, which may be at least one of c1) to c4):.

The present disclosure further provides S1) or S2):.

The present disclosure further provides T1) or T2):.

The present disclosure further provides an application of fibroblasts to preparation of Sertoli cells.

The present disclosure further provides a product for preparing Sertoli cells; and the product may contain a NR5A1 protein.

The product may further contain a protein combination; and the protein combination may be at least one of a GATA4 protein, a WT1 protein, a SOX9 protein and a DMRT1 protein.

The product may further contain fibroblasts.

The product may further contain any one of the above-mentioned fluorescent protein reporter systems specifically expressed in the Sertoli cells.

The product may further contain fibroblasts introduced into the fluorescent protein reporter system specifically expressed in the Sertoli cells.

Any one of the above-mentioned products may further contain G418 and/or the medium for culturing the fibroblasts and/or the DMEM/F12 medium containing <NUM> to <NUM>% (e.g., <NUM> to <NUM>%, <NUM> to <NUM>%, <NUM>%, <NUM>% or <NUM>%) (v/v) of FBS.

The present disclosure further provides specific DNA molecules as shown in the SEQ ID No. <NUM> in the sequence list.

Application of the specific DNA molecules to promotion of target gene expression also falls within the protection scope of the present disclosure.

In the application, the target gene expression may be expression of a target gene in Sertoli cells.

The present disclosure further provides a method for expressing a target gene, including the following step of: inserting the specific DNA molecules to the upstream of any target gene so as to promote target gene expression.

In the method, the target gene expression may be expression of the target gene in Sertoli cells.

Any one of the above-mentioned fibroblasts may be human fibroblasts. The human fibroblasts may be dH1 fibroblast strains or human lung fibroblast strains.

A preparation method of the dH1 fibroblast strains may be as follows:.

Any one of the above-mentioned lentivirus plasmids p2K7-NEO, any one of the above-mentioned pENTR/1A plasmids and any one of the above-mentioned pENTR/<NUM>' topo plasmids all can be products of Invitrogen Corporation. Any one of the above-mentioned DMEM/F12 mediums can be a product of Corning Inc.

Any one of the above-mentioned helper plasmids vsvg and any one of the above-mentioned lentivirus vector packaging plasmids pCMV ΔR8. <NUM> both can be products of Invitrogen Corporation. Any one of the above-mentioned non-essential amino acids can be a product of Gibco Corporation.

The Gene ID of any one of the above-mentioned NR5A1 proteins is <NUM>. The Refseq number of any one of the above-mentioned NR5A1 genes is NC_000009. The Gene ID of any one of the above-mentioned GATA4 proteins is <NUM>. The Refseq number of any one of the above-mentioned GATA4 genes is NC_000008. The Gene ID of any one of the above-mentioned WT1 proteins is <NUM>. The Refseq number of any one of the above-mentioned WT1 genes is NC_000011. The Gene ID of any one of the above-mentioned SOX9 proteins is <NUM>. The Refseq number of any one of the above-mentioned SOX9 genes is NC_000017. The Gene ID of any one of the above-mentioned DMRT1 proteins is <NUM>. The Refseq number of any one of the above-mentioned DMRT1 genes is NC_000009.

Any one of the above-mentioned green fluorescent proteins is a fluorescent protein eGFP (the Gene ID is <NUM>). The Refseq number of a gene (an eGFP gene) for encoding the fluorescent protein eGFP is NC_025025.

Experiments prove that the hiSC prepared by adopting the method provided by the present disclosure highly expresses KRT18 and presents an epithelial appearance, its gene is enriched in the important biological process of the Sertoli cells, and the hiSC has the characteristics of attracting endothelial cells, forming lipid droplets, interacting with germ cells, inhibiting lymphocyte growth and interleukin production, having immune privilege and the like, and fully acquires the characteristics of the Sertoli cells. The present disclosure provides a rich cell source for constructing a complete in-vitro sperm differentiation system and researching an interaction mechanism between the Sertoli cells and male germ cells. The present disclosure has important application value in the fields of infertility treatment, allogeneic transplantation, cell therapy and the like.

The present disclosure will be further described in detail below in combination of particular embodiments, and given embodiments are merely used for illustrating the present disclosure, but not intended to limit the scope of the present disclosure.

Experimental methods in the under-mentioned embodiments are all conventional methods, unless otherwise specified.

Materials, reagents and the like used in the under-mentioned embodiments all can be obtained commercially, unless otherwise specified.

For quantitative tests of the following embodiments, repeated experiments are set for three times, and results are averaged.

A lentivirus plasmid p2K7-NEO, a pENTR/1A plasmid and a pENTR/<NUM>' topo plasmid are all products of Invitrogen Corporation. A human lung fibroblast strain is a product of Concord Cell Bank. Both a helper plasmid vsvg and a lentivirus vector packaging plasmid pCMV ΔR8. <NUM> are products of Invitrogen Corporation. Both WST-<NUM> solution and RIPA solution are products of Beyotime Corporation. Both Matrigel stock solution and a DMEM/F12 medium are products of Corning Inc. Both bFGF and Polybrene are products of R&D Corporation. Non-essential amino acid is a product of Gibco Corporation. HUVECs are products of ATCC Corporation. A Transwell (a pore diameter is <NUM>) is a product of Corning Inc. an ECM medium (i.e., a migration medium) is a product of ScienCell Corporation. A BODIPY dye is a product of Thermofishe Corporation. An ELASA kit is a product of Cusabio Corporation.

An ES medium is a Knockout DMEM medium containing <NUM>% (v/v) of KSR, <NUM>% (m/v) of glutamic acid, <NUM>% (m/v) of non-essential amino acid and 4ng/mL of bFGF.

A differentiation medium is a Knockout DMEM medium containing <NUM>% (v/v) of FBS, <NUM>% (m/v) of glutamic acid and <NUM>% (m/v) of non-essential amino acid.

An MEF medium is a DMEM medium containing <NUM>% (v/v) of FBS, <NUM>% (m/v) of glutamic acid and <NUM>% (m/v) of non-essential amino acid. The MEF medium is used for culturing MEF.

Preparation of a dH1 fibroblast strain includes the steps as follows:.

The Gene ID of an NR5A1 protein is <NUM>. The Refseq number of an NR5A1 gene is NC_000009.

The Gene ID of a GATA4 protein is <NUM>. The Refseq number of a GATA4 gene is NC_000008.

The Gene ID of a WT1 protein is <NUM>. The Refseq number of a WT1 gene is NC_000011.

The Gene ID of an SOX9 protein is <NUM>. The Refseq number of an SOX9 gene is NC_000017.

The Gene ID of a DMRT1 protein is <NUM>. The Refseq number of a DMRT1 gene is NC_000009.

The Gene ID of a fluorescent protein eGFP is <NUM>. The Refseq number of an eGFP gene is NC_025025.

A fibroblast strain is a dH1 fibroblast strain or a human lung fibroblast strain.

AMH (Gene ID: <NUM>) is a marker protein specifically expressed in the early period of development of Sertoli cells. If the Sertoli cells are formed in cells introduced into the AMH: GFP reporter system, green fluorescence will be emitted.

In the recombinant lentivirus containing the AMH: GFP reporter system, a location diagram of the AMH: GFP reporter system and blasticidin (BSD) refers to <FIG>.

By a lentivirus infection manner, the recombinant lentivirus containing the AMH: GFP reporter system obtained in the step II is integrated to a fibroblast strain (the BSD carried by the recombinant lentivirus containing the AMH: GFP reporter system is used as a selection marker), so as to obtain the fibroblast strain containing the AMH: GFP reporter system.

Hereinafter, the dH1 fibroblast strain containing the AMH: GFP reporter system is referred to as dH1•AMH: GFP for short. An HPF fibroblast strain containing the AMH: GFP reporter system is referred to as HPF•AMH: GFP for short.

A flow schematic diagram of in vitro reprogramming of the human fibroblasts into the hiSCs refers to <FIG>.

According to the method in the step (<NUM>), the vector pENTR/1A-NR5A1 is respectively replaced with the vector pENTR/1A-GATA4, the vector pENTR/1A-WT1, the vector pENTR/1A-SOX9 and the vector pENTR/1A-DMRT1, and other steps are unchanged, so as to sequentially obtain a recombinant lentivirus b, a recombinant lentivirus c, a recombinant lentivirus d and a recombinant lentivirus e.

The recombinant lentivirus an express the NR5A1 protein. The recombinant lentivirus b expresses the GATA4 protein. The recombinant lentivirus c expresses the WT1 protein. The recombinant lentivirus d expresses the SOX9 protein. The recombinant lentivirus e expresses the DMRT1 protein.

A T175 culture flask is inoculated with the dH1•AMH: GFP (an inoculation density is <NUM>% to <NUM>%), and then <NUM> of virus fluid (protein combinations expressed by the recombinant lentiviruses in the virus fluid respectively refer to No.<NUM> to No.<NUM> in Table <NUM>, an adding amount of each type of viruses is 200µL, and if a total volume is less than <NUM>, supplementation is carried out by the MEF medium) containing the recombinant lentiviruses and Polybrene is added to obtain an infection system. In the infection system, a density of the dH1•AMH: GFP is <NUM> to <NUM>%, and concentration of the Polybrene is 8ng/mL.

The T175 culture flask is inoculated with the dH1•AMH: GFP (the inoculation density is <NUM>% to <NUM>%), and then <NUM> of MEF medium containing the Polybrene is added to obtain an infection system (as a control, i.e., No.<NUM> in Table <NUM>). In the infection system, a density of the dH1•AMH: GFP is <NUM> to <NUM>%, and concentration of the Polybrene is 8ng/mL. Combinations NG, NGD, WNG, SNG, WNGD, SNGD and SWNG are according to the invention. The other combinations are not according to the invention.

A result of the flow cytometry refers to <FIG>. The result shows that when the protein combination expressed by the recombinant lentivirus in the virus fluid in the step <NUM> is No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM> or No.<NUM>, the AMH: GFP+Sertoli cells (hereinafter referred to as hiSCs (dH1) for short) can be obtained.

The system <NUM> subjected to the step <NUM> is taken, and cells emitting green fluorescence, i.e., the hiSCs (dH1), are collected.

AMH: GFP+Sertoli cells obtained by the NR5A1 protein and the GATA4 protein (No.13in Table <NUM>) expressed by the recombinant lentivirus in the virus fluid are referred to as 2F-hiSCs (dH1) for short.

AMH: GFP+Sertoli cells obtained by the NR5A1 protein, the GATA4 protein, the WT1 protein, the SOX9 protein and the DMRT1 protein (No.<NUM> in Table <NUM>) expressed by the recombinant lentivirus in the virus fluid are referred to as 5F-hiSCs (dH1) for short.

According to the steps <NUM> to <NUM>, <NUM> of virus fluid containing the recombinant lentivirus and the Polybrene is replaced with <NUM> of virus fluid containing an empty vector lentivirus (obtained by co-transfecting the lentivirus plasmid p2K7-NEO, the helper plasmid vsvg and the lentivirus vector packaging plasmid pCMVΔR8. <NUM> with the 293FT cells) and the Polybrene (a volume of the empty vector lentivirus is 200µL, and a total volume is supplemented to <NUM> with the MEF medium), and other steps are all unchanged. The result of the flow cytometry shows that in the system <NUM> subjected to the step <NUM>, cells are dH1-2K7 cells (hereinafter referred to as dH1-2K7 for short), and the hiSCs (dH1) are nearly not obtained.

According to the steps <NUM> to <NUM>, the dH1•AMH: GFP is replaced with the dH1 fibroblast strain, and other steps are all unchanged. The result of the flow cytometry shows that in the system <NUM> subjected to the step <NUM>, the hiSCs (dH1) are nearly not obtained.

According to the step I, the dH1 fibroblast strain is replaced with the human lung fibroblast strain, and other steps are all unchanged. A result shows that when the protein combination expressed by the recombinant lentivirus in the virus fluid in the step <NUM> is No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM> or No.<NUM>, the AMH: GFP+Sertoli cells (hereinafter referred to as hiSCs (HPF) for short) can be obtained. AMH: GFP+Sertoli cells obtained by the NR5A1 protein and the GATA4 protein (No.<NUM> in Table <NUM>) expressed by the recombinant lentivirus in the virus fluid are referred to as 2F-hiSCs (HPF) for short. AMH: GFP+Sertoli cells obtained by the NR5A1 protein, the GATA4 protein, the WT1 protein, the SOX9 protein and the DMRT1 protein (No.<NUM> in Table <NUM>) expressed by the recombinant lentivirus in the virus fluid are referred to as 5F-hiSCs (HPF) for short.

According to the steps <NUM> to <NUM> in the step I, <NUM> of virus fluid containing the recombinant lentivirus and the Polybrene is replaced with <NUM> of virus fluid containing an empty vector lentivirus (obtained by co-transfecting the lentivirus plasmid p2K7-NEO, the helper plasmid vsvg and the lentivirus vector packaging plasmid pCMVΔR8. <NUM> with the 293FT cells) and the Polybrene (a volume of the empty vector lentivirus is 200µL, and a total volume is supplemented to <NUM> with the MEF medium), and dH1•AMH:GFP is replaced with HPF•AMH:GFP, and other steps are all unchanged. The result of the flow cytometry shows that in the system <NUM> subjected to the step <NUM>, cells are HPF-2K7 cells (hereinafter referred to as HPF-2K7 for short), and the hiSCs (HPF) are nearly not obtained.

The transcriptome map of to-be-detected cells (2F-hiSCs (dH1), 5F-hiSCs (dH1), 2F-hiSCs (HPF), 5F-hiSCs (HPF), adult Sertoli cells or dH1-2K7) is compared by adopting an RNA sequencing method, and then GO analysis is carried out.

Part of the transcriptome map refers to <FIG> (both dH1-2K7-<NUM> and dH1-2K7-<NUM> are dH1-2K7 and used as controls; both hiSCs-<NUM> and hiSCs-<NUM> are 5F-hiSCs (dH1), and both aSCs-<NUM> and aSCs-<NUM> are the adult Sertoli cells).

Part of the GO analysis result refers to <FIG> (both dH1-2K7-<NUM> and dH1-2K7-<NUM> are dH1-2K7 and used as controls; both 2F-hiSCs (dH1)-<NUM> and 2F-hiSCs (dH1)-<NUM> are 2F-hiSCs (dH1), 5F-hiSCs (HPF)-<NUM> is 5F-hiSCs (HPF), both 5F-hiSCs (dH1)-1and 5F-hiSCs (dH1)-<NUM> are 5F-hiSCs (dH1), and both aSCs-<NUM> and aSCs-<NUM> are the adult Sertoli cells). A result shows that compared to the dH1-2K7 or the HPF-2K7, the entire down-regulated gene numbers of the 2F-hiSCs (HPF), the 5F-hiSCs (HPF), the 2F-hiSCs (dH1), the 5F-hiSCs (dH1) and the adult Sertoli cells all are <NUM>, and the entire up-regulated gene numbers all are <NUM>.

The result shows that the 2F-hiSCs (HPF), the 5F-hiSCs (HPF), the 2F-hiSCs (dH1), the 5F-hiSCs (dH1) and the adult Sertoli cells express similar genes, are enriched in the important biological process of the Sertoli cells, and are obviously different from the dH1-2K7 or the HPF-2K7 in expression situation.

The KRT18 (Gene ID: <NUM>) is a marker protein of the Sertoli cells, and represents the epithelization characteristic of the Sertoli cells.

The to-be-detected cells (2F-hiSCs (dH1) or dH1-2K7) are taken, and cell immunofluorescence staining is carried out. The dH1-2K7 is used as a control.

An experimental result refers to <FIG> (2F-hiSCs are 2F-hiSCs (dH1), cell nucleuses are marked with blue (DAPI), Anti-KRT18 is KRT18 protein immunofluorescence, AMH: GFP is fluorescence of the intracellular AMH: GFP reporter system, Merged is a staining overlay diagram, and a proportional scale is <NUM>). The result shows that compared to the dH1-2K7, the expression level of KRT18 in the 2F-hiSCs (dH1) is obviously improved.

An experimental result refers to <FIG> (the upper left is a schematic diagram of a detection device, the upper right shows migration numbers of the HUVECs of five different regions, the lower left shows an observation result under the fluorescence microscope, the lower right is a statistic result of the number of the HUVECs emitting fluorescence, 2F-hiSCs CM are the 2F-hiSCs (dH1), and dH1-2K7 CM are the dH1-2K7). The result shows that the conditioned medium for the 2F-hiSCs (dH1) have the ability of attracting more HUVECs.

BODIPY storage solution: a BODIPY dye is taken, DMSO is added to dilute to concentration of <NUM>/mL, and the obtained product is preserved in a dark place at a temperature of -<NUM>.

APBS buffer solution is a <NUM> PBS buffer solution with pH of <NUM>.

According to the steps, the 2F-hiSCs (dH1) are replaced with the dH1-2K7, and other steps are all unchanged, and the obtained result is used as a control.

An experimental result refers to <FIG> (DAPI is a DAPI staining result, BODIPY-<NUM> is a BODIPY dye staining result, Merged is staining overlay, and the 2F-hiSCs are the 2F-hiSCs (dH1)). The result shows that the 2F-hiSCs (dH1) have the ability of forming the lipid droplets.

Mouse spermatogonia are derived from a C57BL/<NUM> male mouse on the age of <NUM> days, and a separation method refers to the following literature: <NPL>.

According to the steps, the 2F-hiSCs (dH1) are replaced with the dH1-2K7, other steps are all unchanged, and the obtained result is used as a control.

An experimental result refers to <FIG> (cell nucleuses are marked with blue (DAPI), Merged is staining overlay, with 2F-hiSCs are the 2F-hiSCs (dH1), and with dH1 is the dH1-2K7). The result shows that the 2F-hiSCs (dH1) can support attachment of the mouse germ cells better, i.e., have the ability of interacting with the germ cells.

Step VI: detection on immune privilege of the 2F-hiSCs (dH1).

The relative number of the JurKat E6-<NUM> cells in the hole where the blank control is positioned is set as <NUM>.

An experimental result refers to A (2F-hiSCs CM are the 2F-hiSCs (dH1), and dH1-2K7 CM are the dH1-2K7) in <FIG>. The result shows that secreta of the 2F-hiSCs (dH1) can inhibit proliferation of the JurKat E6-<NUM> cells.

An experimental result refers to B (2F-hiSCs CM are the 2F-hiSCs (dH1), and dH1-2K7 CM are the dH1-2K7) in <FIG>. The result shows that the secreta of the 2F-hiSCs (dH1) can inhibit production of the interleukin IL-<NUM>.

An experimental result refers to <FIG> (from left to right, they sequentially are the mice on the first day, the fourth day and the sixth day of injection). The result shows that the 2F-hiSCs (dH1) can prolong the allogeneic survival ability of the 293FT cells, and has an immune privilege function.

According to the steps II to VI, the 2F-hiSCs (dH1) are replaced with the 5F-hiSCs (dH1), the 2F-hiSCs (HPF) or the 5F-hiSCs (HPF), and other steps are all unchanged. A result shows that compared to the 2F-hiSCs (dH1), the 5F-hiSCs (dH1), the 2F-hiSCs (HPF) and the 5F-hiSCs (HPF) have no obvious difference in result.

According to the steps, the 2F-hiSCs (dH1) are replaced with other hiSCs (dH1) (i.e., the protein combination expressed by the recombinant lentivirus in the virus fluid in the step <NUM> of the step I in Embodiment <NUM> is No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM> or No.<NUM> in Table <NUM>) and other hiSCs (HPF) (i.e., the protein combination expressed by the recombinant lentivirus in the virus fluid in the step II in Embodiment <NUM> is No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM>, No.<NUM> or No.<NUM>), and other steps are all unchanged. A result shows that compared to the 2F-hiSCs (dH1), other hiSCs (dH1) and other hiSCs (HPF) have no obvious difference in result.

From the above, the hiSCs (dH1) and the hiSCs (HPF) prepared in Embodiment <NUM> highly express the KRT18 and present an epithelial appearance, their genes are enriched in the important biological process of the Sertoli cells, and the hiSCs (dH1) and the hiSCs (HPF) have the characteristics of attracting the endothelial cells, forming the lipid droplets, interacting with the germ cells, inhibiting lymphocyte growth and interleukin production, having immune privilege and the like, and fully acquire the characteristics of the Sertoli cells. Thus it can be seen that both the hiSCs (dH1) and the hiSCs (HPF) prepared in Embodiment <NUM> are the Sertoli cells.

Step III: according to the step III in Embodiment <NUM> and the steps in Embodiment <NUM> and the steps in Embodiment <NUM>, replacement of the AMH:GFP reporter system in the step III in Embodiment <NUM> with the AMH-D:GFP reporter system without changing other steps.

Part of results are as follows: when the AMH-D:GFP reporter system is adopted, in the system <NUM> (the NR5A1 protein and the GATA4 protein expressed by the recombinant lentivirus in the virus fluid) subjected to the step <NUM> in the step I in Embodiment <NUM>, <NUM>% of cells are AMH-D:GFP+Sertoli cells; and in the system <NUM> (the NR5A1 protein, the GATA4 protein, the WT1 protein, the SOX9 protein and the DMRT1 protein expressed by the recombinant lentivirus in the virus fluid) subjected to the step <NUM> in the step II in Embodiment <NUM>, <NUM>% of cells are AMH-D:GFP+Sertoli cells. Those AMH-D:GFP+Sertoli cells highly express the KRT18 and present the epithelial appearance, their genes are enriched in the important biological process of the Sertoli cells, and the AMH-D:GFP+Sertoli cells have the characteristics of attracting the endothelial cells, forming the lipid droplets, interacting with the germ cells, inhibiting lymphocyte growth and interleukin production, having immune privilege and the like, and fully acquire the characteristics of the Sertoli cells. Thus, it can be seen that the Sertoli cells can also be obtained by adopting the AMH-D:GFP reporter system, but a proportion of the obtained Sertoli cells is obviously reduced.

Claim 1:
A method for preparing Sertoli cells, comprising a step a2): increasing the expression level and/or activity of a protein combination in fibroblasts,
wherein the protein combination is selected from one of the following groups:
B2) NR5A1 and GATA4,
B6) NR5A1, GATA4 and WT1,
B7) NR5A1, GATA4 and SOX9,
B8) NR5A1, GATA4 and DMRT1,
B12) NR5A1, GATA4, WT1 and SOX9,
B13) NR5A1, GATA4, WT1 and DMRT1,
B14) NR5A1, GATA4, SOX9 and DMRT1.