METHOD FOR IN VITRO REPROGRAMMING OF FIBROBLASTS INTO SERTOLI CELLS AND APPLICATION THEREOF

Abstract

Disclosed is a method for in vitro reprogramming of fibroblasts into Sertoli cells and an application thereof. The method includes the following steps: introducing a fluorescent protein reporter system specifically expressed in the Sertoli cells into the fibroblasts; followed by introducing a substance that increases the expression level and/or activity of the NR5A1 protein; adding G418 and culturing for 3-7 d; discarding the liquid phase, adding a medium for culturing fibroblasts and continuing culturing for 3-7 d; and isolating the Sertoli cells from the culture system. Experiments showed that cells prepared by the method fully acquire the characteristics of Sertoli cells, such as attracting endothelial cells, forming lipid droplets, interacting with germ cells, inhibiting lymphocyte growth and interleukin production and having immune privilege. The disclosure has application value in the fields of infertility treatment, allogeneic transplantation, and cell therapy.

Claims

1-24. (canceled)

25. A method for preparing Sertoli cells, comprising a step a2): increasing the expression level and/or activity of an NR5A1 protein in fibroblasts.

26. The method according to claim 25, wherein the step a2) comprises: 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.

27. The method according to claim 26, wherein 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” is 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.

28. The method according to claim 25, further comprising a step a1): introducing a fluorescent protein reporter system specifically expressed in the Sertoli cells into the fibroblasts; wherein 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.

29. The method according to claim 28, wherein the promoter specifically expressed in the Sertoli cells is a promoter of an AMH gene.

30. The method according to claim 29, wherein a nucleotide sequence of the promoter of the AMH gene is as shown SEQ ID No. 1 in the sequence list.

31. The method according to claim 28, wherein the fluorescent protein is a green fluorescent protein, a yellow fluorescent protein or a red fluorescent protein.

32. The method according to claim 25, further comprising steps a3) to a6): a3): after completing the step a2), adding G418 into a culture system of the fibroblasts to obtain a medicinal sieve system; a4): after completing the step a3), taking the medicinal sieve system and culturing for 3-7 d; a5): after completing the step a4), discarding the liquid phase, adding a medium for culturing fibroblasts and continuing culturing for 3-7 d; and a6): after completing the step a5), isolating the Sertoli cells from the culture system.

33. The method according to claim 32, wherein in the step a6), isolating the Sertoli cells is implemented by isolating cells capable of emitting fluorescence.

34. The fluorescent protein reporter system specifically expressed in the Sertoli cells, wherein a promoter specifically expressed in the Sertoli cells promotes expression of a fluorescent protein in the fluorescent protein reporter system.

35. The fluorescent protein reporter system according to claim 34, wherein the fluorescent protein is a green fluorescent protein, a yellow fluorescent protein or a red fluorescent protein.

36. Application of the fluorescent protein reporter system, being at least one of c1) to c4): c1) application as an indication mark of the Sertoli cells; c2) application as a separation mark of the Sertoli cells; c3) application as an enrichment mark of the Sertoli cells; and c4) application to preparation of the Sertoli cells, wherein 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.

37. Application of a product for preparing Sertoli cells, wherein the product containing an NR5A1 protein, nucleic acid molecules for encoding the NR5A1 protein or fibroblasts.

38. The application according to claim 37, the product further containing a protein combination or nucleic acid molecules for encoding the protein combination, the protein combination being at least one of a GATA4 protein, a WT1 protein, a SOX9 protein and a DMRT1 protein.

39. A product for preparing Sertoli cells, containing an NR5A1 protein or nucleic acid molecules for encoding the NR5A1 protein.

40. The product according to claim 39, further containing a protein combination or nucleic acid molecules for encoding the protein combination, the protein combination being at least one of a GATA4 protein, a WT1 protein, a SOX9 protein and a DMRT1 protein.

41. The product according to claim 39, further containing fibroblasts.

42. The product according to claim 39, further containing the fluorescent protein reporter system specifically expressed in the Sertoli cells, wherein a promoter specifically expressed in the Sertoli cells promotes expression of a fluorescent protein in the fluorescent protein reporter system.

43. A method for expressing a target gene, comprising the following step of: inserting a specific DNA molecules to the upstream of any target gene so as to promote target gene expression, wherein the specific DNA molecules as shown SEQ ID No. 1 in the sequence list.

44. The method according to claim 43, wherein the target gene expression is expression of the target gene in Sertoli cells.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0068] FIG. 1 is a location diagram of an AMH: GFP reporter system and blasticidin (BSD) in a recombinant lentivirus.

[0069] FIG. 2 is a flow schematic diagram of in vitro reprogramming of human fibroblasts into hiSCs.

[0070] FIG. 3 is an analysis result of flow cytometry of a step I in an embodiment 2.

[0071] FIG. 4 is a transcriptome map of part of to-be-detected cells.

[0072] FIG. 5 is GO analysis of the transcriptome map.

[0073] FIG. 6 is detection of an expression level of KRT18 in 2F-hiSCs (dH1) by cell immunofluorescence staining.

[0074] FIG. 7 is detection on an ability that the 2F-hiSCs (dH1) attract human umbilical vein endothelial cells (HUVECs).

[0075] FIG. 8 is detection on an ability that the 2F-hiSCs (dH1) form lipid droplets.

[0076] FIG. 9 is detection on an ability that the 2F-hiSCs (dH1) interact with germ cells.

[0077] FIG. 10 shows that the 2F-hiSCs (dH1) can inhibit lymphocyte growth and interleukin production.

[0078] FIG. 11 is detection on immune privilege of the 2F-hiSCs (dH1).

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0079] 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.

[0080] Experimental methods in the under-mentioned embodiments are all conventional methods, unless otherwise specified.

[0081] Materials, reagents and the like used in the under-mentioned embodiments all can be obtained commercially, unless otherwise specified.

[0082] For quantitative tests of the following embodiments, repeated experiments are set for three times, and results are averaged.

[0083] A lentivirus plasmid p2K7-NEO, a pENTR/1A plasmid and a pENTR/5′ 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 ΔR8.9 are products of Invitrogen Corporation. Both WST-1 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 8 μm) 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.

[0084] An ES medium is a Knockout DMEM medium containing 20% (v/v) of KSR, 1% (m/v) of glutamic acid, 1% (m/v) of non-essential amino acid and 4ng/mL of bFGF.

[0085] A differentiation medium is a Knockout DMEM medium containing 10% (v/v) of FBS, 1% (m/v) of glutamic acid and 1% (m/v) of non-essential amino acid.

[0086] An MEF medium is a DMEM medium containing 10% (v/v) of FBS, 1% (m/v) of glutamic acid and 1% (m/v) of non-essential amino acid. The MEF medium is used for culturing MEF.

[0087] Preparation of a dH1 fibroblast strain includes the steps as follows:

[0088] (1) Transferring human embryonic stem cells to a culture plate coated by a 1% Matrigel stock solution, adding the ES medium, and culturing in a 5% CO.sub.2 thermostatic incubator with a temperature of 37° C. for 5 d; and

[0089] (2) After completing the step (1), discarding the medium, adding the differentiation medium, and continuing culturing in the 5% CO.sub.2 thermostatic incubator with the temperature of 37° C. for 7 to 10 d until the embryonic stem cells are differentiated into the fibroblasts, so as to obtain the dH1 fibroblast strain.

[0090] The Gene ID of an NR5A1 protein is 2516. The Refseq number of an NR5A1 gene is NC_000009.12.

[0091] The Gene ID of a GATA4 protein is 2626. The Refseq number of a GATA4 gene is NC_000008.11.

[0092] The Gene ID of a WT1 protein is 7409. The Refseq number of a WT1 gene is NC_000011.10.

[0093] The Gene ID of an SOX9 protein is 6662. The Refseq number of an SOX9 gene is NC_000017.11.

[0094] The Gene ID of a DMRT1 protein is 1761. The Refseq number of a DMRT1 gene is NC_000009.12.

[0095] The Gene ID of a fluorescent protein eGFP is 20473140. The Refseq number of an eGFP gene is NC_025025.1.

Embodiment 1: Construction of Fibroblast Strain Containing AMH:GFP Reporter System

[0096] A fibroblast strain is a dH1 fibroblast strain or a human lung fibroblast strain.

Step I: Construction of the AMH:GFP Reporter System

[0097] 1. An inventor of the present disclosure clones a first promoter of an AMR gene from human genome DNA. A length of the first promoter of the AMR gene is about 1.6 kb, and a nucleotide sequence of the first promoter is as shown in a SEQ ID No. 1 in the sequence list.

[0098] 2. After the step 1 is completed, an encoding gene (i.e., an eGFP gene) of a fluorescent protein eGFP is connected to a 3′ terminal of the first promoter of the AMR gene so as to obtain the AMH: GFP reporter system. In the AMH: GFP reporter system, the first promoter of the AMH gene drives expression of the fluorescent protein eGFP.

[0099] AMH (Gene ID: 268) 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.

Step II: Construction of a Recombinant Lentivirus Containing the AMH: GFP Reporter System

[0100] 1. The eGFP gene is inserted to a recognition site of restriction endonuclease EcoRI of a pENTR/1A plasmid so as to obtain a vector pENTR/1A-eGFP.

[0101] 2. Double-stranded DNA molecules shown in the SEQ ID No. 1 in the sequence list are inserted to a recognition site of restriction endonuclease EcoRI of a pENTR/5′ topo plasmid so as to obtain a vector pENTR/5′ topo-AMH.

[0102] 3. The vector pENTR/1A-eGFP, the vector pENTR/5′ topo-AMH and a lentivirus plasmid p2K7-NEO are taken to prepare a lentivirus vector by homologous recombination.

[0103] 4. The lentivirus vector prepared in the step 3, a helper plasmid vsvg and a lentivirus vector packaging plasmid pCMV ΔR8.9 are co-transfected with 293FT cells to obtain the recombinant lentivirus containing the AMH: GFP reporter system.

[0104] 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. 1.

Step III: Construction of a Fibroblast Strain Containing the AMH:GFP Reporter System

[0105] 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.

[0106] 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.

Embodiment 2: In Vitro Reprogramming of Human Fibroblasts into AMH:GFP+Sertoli Cells (Hereinafter Referred to as hiSCs for Short)

[0107] A flow schematic diagram of in vitro reprogramming of the human fibroblasts into the hiSCs refers to FIG. 2.

Step I: In Vitro Reprogramming of a dH1 Fibroblast Strain Into the hiSCs (dH1)

[0108] 1. A recombinant lentivirus is prepared.

[0109] (1) The NR5A1 gene is inserted to a recognition site of the restriction endonuclease EcoRI of the pENTR/1A plasmid so as to obtain a vector pENTR/1A-NR5A1. The GATA4 gene is inserted to the recognition site of the restriction endonuclease EcoRI of the pENTR/1A plasmid so as to obtain a vector pENTR/1A-GATA4. The WT1 gene is inserted between recognition sites of restriction endonucleases NotI and AscI of a pENTR/D-topo plasmid so as to obtain a vector pENTR/D-topo-WT1. The SOX9 gene is inserted between recognition sites of the restriction endonucleases NotI and AscI of the pENTR/D-topo plasmid so as to obtain a vector pENTR/D-topo-SOX9. The DMRT1 gene is inserted to a recognition site of the restriction endonuclease EcoRI of the pENTR/1A plasmid so as to obtain a vector pENTR/1A-DMRT1.

[0110] (2) Double-stranded DNA molecules (a nucleotide sequence of an EF1α promoter) shown in a SEQ ID No. 2 in the sequence list are inserted to a recognition site of restriction endonuclease EcoRI of a pENTR/5′ topo plasmid so as to obtain a vector pENTR/5′ topo-EF1α.

[0111] (3) The vector pENTR/1A-NR5A1, the vector pENTR/5′ topo-EF1α and the lentivirus plasmid p2K7-NEO are taken to prepare a lentivirus vector a by homologous recombination. The lentivirus vector a, the helper plasmid vsvg and the lentivirus vector packaging plasmid pCMV ΔR8.9 are co-transfected with 293FT cells to obtain the recombinant lentivirus a.

[0112] According to the method in the step (3), 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.

[0113] 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.

[0114] 2. A T175 culture flask is inoculated with the dH1AMH: GFP (an inoculation density is 30% to 60%), and then 1mL of virus fluid (protein combinations expressed by the recombinant lentiviruses in the virus fluid respectively refer to No. 1 to No. 31 in Table 1, an adding amount of each type of viruses is 200 μL, and if a total volume is less than 1 mL, 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 40 to 70%, and concentration of the Polybrene is 8 ng/mL.

[0115] The T175 culture flask is inoculated with the dH1•AMH: GFP (the inoculation density is 30% to 60%), and then 1 mL of MEF medium containing the Polybrene is added to obtain an infection system (as a control, i.e., No. 32 in Table 1). In the infection system, a density of the dH1•AMH: GFP is 40 to 70%, and concentration of the Polybrene is 8 ng/mL.

TABLE-US-00001 TABLE 1 Abbreviation of Protein Protein Combinations Combination NR5A1 GATA4 SOX9 WT1 DMRT1 Number Type Protein Protein Protein Protein Protein 1 S − − + − − 2 W − − − + − 3 N + − − − − 4 G − + − − − 5 D − − − − + 6 SW − − + + − 7 SN + − + − − 8 SG − + + − − 9 SD − − + − + 10 WN + − − + − 11 WG − + − + − 12 WD − − − + + 13 NG + + − − − 14 ND + − − − + 15 GD − + − − + 16 NGD + + − − + 17 WGD − + − + + 18 WND + − − + + 19 WNG + + − + − 20 SGD − + + − + 21 SND + − + − + 22 SNG + + + − − 23 SWD − − + + + 24 SWG − + + + − 25 SWN + − + + − 26 WNGD + + − + + 27 SNGD + + + − + 28 SWGD − + + + + 29 SWND + − + + + 30 SWNG + + + + − 31 SWNGD + + + + + 0 CTRL − − − − − Notes: + represents that the protein combination contains a certain protein, and − represents that the protein combination does not contain a certain protein.

[0116] 3. After the step 2 is completed, the infection systems are respectively taken and placed in the 5% CO.sub.2 thermostatic incubator with the temperature of 37° C. to stand for 24 h.

[0117] 4. The infection systems subjected to the step 3 are respectively taken, a liquid phase is discarded, the MEF medium is added, and the obtained product is placed into the 5% CO.sub.2 thermostatic incubator with the temperature of 37° C. to stand for 24 h (an intermediate state 1 in FIG. 2).

[0118] 5. After the step 4 is completed, G418 is respectively added to obtain a system 1; and in the system 1, concentration of the G418 is 1 mg/mL.

[0119] 6. After the step 5 is completed, the system 1 is respectively taken and placed into the 5% CO.sub.2 thermostatic incubator with the temperature of 37° C. to be cultured for 5 d (an intermediate state 2 in FIG. 2), wherein when culture is carried out on the third to fourth day, the medium needs to be replaced with an MEF culture medium containing 1 mg/mL of G418.

[0120] 7. The system 1 subjected to the step 6 is respectively taken, the medium is discarded, and a DMEM/F12 medium containing 10% (v/v) of FBS is added to obtain a system 2.

[0121] 8. After the step 7 is completed, the system 2 is respectively taken and placed into the 5% CO.sub.2 thermostatic incubator with the temperature of 37° C. to be subjected to static culture for 3 to 7 d.

[0122] 9. The system 2 subjected to the step 8 is respectively taken, and analysis is carried out by adopting flow cytometry.

[0123] A result of the flow cytometry refers to FIG. 3. The result shows that when the protein combination expressed by the recombinant lentivirus in the virus fluid in the step 2 is No. 3, No. 7, No. 10, No. 13, No. 14, No. 16, No. 18, No. 19, No. 21, No. 22, No. 25, No. 26, No. 27, No. 29, No. 30 or No. 31, the AMH: GFP+Sertoli cells (hereinafter referred to as hiSCs (dH1) for short) can be obtained.

[0124] 10. The system 2 subjected to the step 8 is taken, and cells emitting green fluorescence, i.e., the hiSCs (dH1), are collected.

[0125] AMH: GFP+Sertoli cells obtained by the NR5A1 protein and the GATA4 protein (No. 13 in Table 1) expressed by the recombinant lentivirus in the virus fluid are referred to as 2F-hiSCs (dH1) for short.

[0126] AMH: GFP+Sertoli cells obtained by the NR5A1 protein, the GATA4 protein, the WT1 protein, the SOX9 protein and the DMRT1 protein (No. 31 in Table 1) expressed by the recombinant lentivirus in the virus fluid are referred to as 5F-hiSCs (dH1) for short.

[0127] According to the steps 2 to 9, 1 mL of virus fluid containing the recombinant lentivirus and the Polybrene is replaced with 1 mL 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 pCMV ΔR8.9 with the 293FT cells) and the Polybrene (a volume of the empty vector lentivirus is 200 μL, and a total volume is supplemented to 1 mL with the MEF medium), and other steps are all unchanged. The result of the flow cytometry shows that in the system 2 subjected to the step 8, cells are dH1-2K7 cells (hereinafter referred to as dH1-2K7 for short), and the hiSCs (dH1) are nearly not obtained.

[0128] According to the steps 2 to 9, 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 2 subjected to the step 8, the hiSCs (dH1) are nearly not obtained.

[0129] Step II: in vitro reprogramming of a human lung fibroblast strain into the hiSCs (HPF)

[0130] 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 2 is No. 3, No. 7, No. 10, No. 13, No. 14, No. 16, No. 18, No. 19, No. 21, No. 22, No. 25, No. 26, No. 27, No. 29, No. 30 or No. 31, 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. 13 in Table 1) 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. 31 in Table 1) expressed by the recombinant lentivirus in the virus fluid are referred to as 5F-hiSCs (HPF) for short.

[0131] According to the steps 2 to 9 in the step I, 1 mL of virus fluid containing the recombinant lentivirus and the Polybrene is replaced with 1 mL 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 pCMV ΔR8.9 with the 293FT cells) and the Polybrene (a volume of the empty vector lentivirus is 200 μL, and a total volume is supplemented to 1mL 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 2 subjected to the step 8, cells are HPF-2K7 cells (hereinafter referred to as HPF-2K7 for short), and the hiSCs (HPF) are nearly not obtained.

Embodiment 3: Characteristics of hiSCs Obtained by In Vitro Reprogramming of Human Fibroblasts in Embodiment 2

Step I: Analysis of a Transcriptome Map

[0132] 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.

[0133] Part of the transcriptome map refers to FIG. 4 (both dH1-2K7-1 and dH1-2K7-2 are dH1-2K7 and used as controls; both hiSCs-1 and hiSCs-2 are 5F-hiSCs (dH1), and both aSCs-1 and aSCs-2 are the adult Sertoli cells).

[0134] Part of the GO analysis result refers to FIG. 5 (both dH1-2K7-1 and dH1-2K7-2 are dH1-2K7 and used as controls; both 2F-hiSCs (dH1)-1 and 2F-hiSCs (dH1)-2 are 2F-hiSCs (dH1), 5F-hiSCs (HPF)-1 is 5F-hiSCs (HPF), both 5F-hiSCs (dH1)-land 5F-hiSCs (dH1)-2 are 5F-hiSCs (dH1), and both aSCs-1 and aSCs-2 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 689, and the entire up-regulated gene numbers all are 512.

[0135] 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.

Step II: Up-Regulation of an Expression Level of KRT18 in the 2F-hiSCs (dH1)

[0136] The KRT18 (Gene ID: 3875) is a marker protein of the Sertoli cells, and represents the epithelization characteristic of the Sertoli cells.

[0137] 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.

[0138] An experimental result refers to FIG. 6 (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 100 μm). The result shows that compared to the dH1-2K7, the expression level of KRT18 in the 2F-hiSCs (dH1) is obviously improved.

Step III: Detection on an Ability that the 2F-hiSCs (dH1) Attract HUVECs

[0139] 1. A six-hole plate is taken, the ECM medium is added into each hole, then the six-hole plate is inoculated with the 2F-hiSCs (dH1), and an inoculation density is 50%.

[0140] 2. After the step 1 is completed, the six-hole plate is taken and placed into the 5% CO.sub.2 thermostatic incubator with the temperature of 37° C. to be subjected to standing culture for 24 h.

[0141] 3. After the step 2 is completed, the six-hole plate is taken, the medium is discarded, then the ECM medium is added, the obtained product is placed into the 5% CO.sub.2 thermostatic incubator with the temperature of 37° C. to be subjected to standing culture for 48 h, and a culture solution is collected. The culture solution is a conditioned medium for the 2F-hiSCs (dH1).

[0142] According to the steps, the 2F-hiSCs (dH1) are replaced with the dH1-2K7, other steps are all unchanged, and a conditioned medium of the dH1-2K7 is obtained and used as a control.

[0143] 4. Another six-hole plate is taken, the ECM medium is added into each hole, then the six-hole plate is inoculated with the HUVCECs, and an inoculation density is 30%.

[0144] 5. After the step 4 is completed, the six-hole plate is taken and placed into the 5% CO.sub.2 thermostatic incubator with the temperature of 37° C. to be subjected to standing culture for 24 h.

[0145] 6. After the step 5 is completed, the six-hole plate is taken, and calcein is added to obtain a first system. In the first system, concentration of the calcein is 2.5 μM.

[0146] 7. After the step 6 is completed, the six-hole plate is taken and placed into the 5% CO.sub.2 thermostatic incubator with the temperature of 37° C. to be subjected to static culture for 1 h, then sufficient digestion is carried out, and cells are collected.

[0147] 8. After the step 7 is completed, the collected cells are taken and resuspended by the ECM medium to obtain a cell suspension with a concentration of 10.sup.6/mL.

[0148] 9. 600 μL of conditioned medium (the conditioned medium of the 2F-hiSCs (dH1) or the conditioned medium of the dH1-2K7) prepared in the step 3 is added into a substrate chamber, and then the obtained product is placed into the Transwell and infiltrated for 5 min.

[0149] 10. After the step 9 is completed, 100 μL of cell suspension prepared in the step 8 is added into the Transwell, the obtained product is placed into the 5% CO.sub.2 thermostatic incubator with the temperature of 37° C. to be subjected to static culture for 20 h, and then observation and counting are carried out under a fluorescence microscope.

[0150] An experimental result refers to FIG. 7 (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.

Step IV: Detection on an Ability that the 2F-hiSCs (dH1) Form Lipid Droplets

[0151] BODIPY storage solution: a BODIPY dye is taken, DMSO is added to dilute to concentration of 1 mg/mL, and the obtained product is preserved in a dark place at a temperature of −20° C. .

[0152] APBS buffer solution is a 0.01 mM PBS buffer solution with pH of 7.2.

[0153] 1. A six-hole plate is taken, the MEF medium is added into each hole, then the six-hole plate is inoculated with the 2F-hiSCs (dH1), and an inoculation density is 50%.

[0154] 2. After the step 1 is completed, the six-hole plate is taken and placed into the 5% CO.sub.2 thermostatic incubator with the temperature of 37° C. to be static cultured for 24 h.

[0155] 3. After the step 2 is completed, the six-hole plate is taken, the medium is discarded, and then the obtained product is washed once with the PBS buffer solution for 5 min each time; and then a 4% (v/v) PFA aqueous solution is added, the obtained product is allowed to be static at 37° C. for 20 min (for fixation).

[0156] 4. After the step 3 is completed, the six-hole plate is taken, the liquid phase is discarded, a BODIPY working solution (formed by mixing 1 part by volume of BODIPY storage solution and 500 parts by volume of PBS buffer solution) is added, and staining is carried out in a dark place for 60 min.

[0157] 5. After the step 4 is completed, the six-hole plate is taken, the liquid phase is discarded, and the obtained product is washed for three times with the PBS buffer solution for 5 min each time; and then the PBS buffer solution containing 1 μg/mL of DAPI is added, and is static for 10 min (for carrying out cell nucleus staining).

[0158] 6. After the step 4 is completed, the six-hole plate is taken, the liquid phase is discarded, and the obtained product is washed for three times with the PBS buffer solution for 5 min each time; and then the number of BODIPY positive cells is observed and counted under the fluorescence microscope.

[0159] 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.

[0160] An experimental result refers to FIG. 8 (DAPI is a DAPI staining result, BODIPY-488 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.

Step V: Detection on an Ability that the 2F-hiSCs (dH1) Interact with Germ Cells

[0161] Mouse spermatogonia are derived from a C57BL/6 male mouse on the age of 6 days, and a separation method refers to the following literature: Kanatsu Shinohara M, et al. Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biology of Reproduction 69.2(2003):612.

[0162] 1. A 48-hole plate is taken, and each hole is inoculated with 5×10.sup.4 2F-hiSCs (dH1).

[0163] 2. After the step 1 is completed, the 48-hole plate is taken, a liquid phase is discarded, the obtained product is washed for three times with the PBS buffer solution (for washing away dead falling cells).

[0164] 3. After the step 2 is completed, the 48-hole plate is taken, 500 μL of mouse spermatogonium suspension (which is formed by resuspending the mouse spermatogonia by the DMEMF/12 medium and has concentration of 2×10.sup.5/mL) is added into each hole, and the obtained product is placed into the 5% CO.sub.2 thermostatic incubator with the temperature of 37° C. to be static cultured for 48 h.

[0165] 4. After the step 3 is completed, the 48-hole plate is taken, and immunofluorescence identification of KRT18 (indicating the Sertoli cells) and DAZL (indicating the spermatogonia) marker proteins is carried out.

[0166] 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.

[0167] An experimental result refers to FIG. 9 (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)

[0168] 1. Inhibition of JurKat E6-1 cell proliferation by the 2F-hiSCs (dH1)

[0169] (1) A six-hole plate is taken, the MEF medium is added into each hole, then the six-hole plate is inoculated with the 2F-hiSCs (dH1), and an inoculation density is 50%.

[0170] (2) After the step (1) is completed, the six-hole plate is taken and placed into the 5% CO.sub.2 thermostatic incubator with the temperature of 37° C. to be subjected to static culture for 24h.

[0171] (3) After the step (2) is completed, the six-hole plate is taken, the medium is discarded, then a 1640 medium containing 10% (v/v) of FBS, 1% (m/v) of glutamic acid, 1% (m/v) of non-essential amino acid and 1% (m/v) of mercaptoethanol is added, the obtained product is placed into the 5% CO.sub.2 thermostatic incubator with the temperature of 37° C. to be subjected to static culture for 48 h, and a culture solution is collected. The culture solution is a conditioned medium.

[0172] (4) The conditioned medium is taken, and the 1640 medium is added to carry out dilution (a volume ratio of the conditioned medium to the 1640 medium is 4:1, 2:3, 3:2 or 1:4) so as to obtain a conditioned medium diluent.

[0173] (5) JurKat E6-1 cells are resuspended by the conditioned medium diluent to obtain a cell suspension. The JurKat E6-1 cells are resuspended by the conditioned medium to obtain a cell suspension (used as a blank control).

[0174] (6) After the step (5) is completed, a 96-hole plate is taken, the cell suspension obtained in the step (5) is added into each hole, each hole is inoculated with 2×10.sup.4 JurKat E6-1 cells (about 120 μL), and the obtained product is placed into the 5% CO.sub.2 thermostatic incubator with the temperature of 37° C. to be subjected to static culture for 72 h.

[0175] (7) After the step (6) is completed, the 96-hole plate is taken, 12 μL of WST-1 solution is added into each hole, and the obtained product is placed into the 5% CO.sub.2 thermostatic incubator with the temperature of 37° C. to be subjected to static culture (in a dark place) for 3 h.

[0176] (8) After the step (7) is completed, the 96-hole plate is taken, absorbance of the solution in each hole at the position of OD.sub.450 nm is read by a microplate reader, and then the relative number of the JurKat E6-1 cells in each hole is calculated. The relative number of the JurKat E6-1 cells in a certain hole=the absorbance of the solution in the hole at the position of OD.sub.450 nm/the absorbance of the blank control at the position of OD.sub.450 nm.

[0177] The relative number of the JurKat E6-1 cells in the hole where the blank control is positioned is set as 1.

[0178] 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.

[0179] An experimental result refers to A (2F-hiSCs CM are the 2F-hiSCs (dH1), and dH1-2K7 CM are the dH1-2K7) in FIG. 10. The result shows that secreta of the 2F-hiSCs (dH1) can inhibit proliferation of the JurKat E6-1 cells.

[0180] 2. Inhibition from production of interleukin IL-2 of JurKat cells

[0181] (1) A six-hole plate is taken, the MEF medium is added into each hole, then the six-hole plate is inoculated with 2F-hiSCs (dH1), and an inoculation density is 50%.

[0182] (2) After the step (1) is completed, the six-hole plate is taken and placed into the 5% CO.sub.2 thermostatic incubator with the temperature of 37° C. to be subjected to static culture for 24h.

[0183] (3) After the step (2) is completed, the six-hole plate is taken, the medium is discarded, then the 1640 medium containing 10% (v/v) of FBS, 1% (m/v) of glutamic acid, 1% (m/v) of non-essential amino acid and 1% (m/v) of mercaptoethanol is added, the obtained product is placed into the 5% CO.sub.2 thermostatic incubator with the temperature of 37° C. to be subjected to static culture for 48 h, and a culture solution is collected. The culture solution is a conditioned medium.

[0184] (4) One part by volume of conditioned medium and one part by volume of 1640 medium are mixed to obtain a diluent. The JurKat E6-1 cells are resuspended by the diluent to obtain a cell suspension.

[0185] (5) After the step (4) is completed, the six-hole plate is taken, the cell suspension obtained in the step (4) is added into each hole, each hole is inoculated with 2×10.sup.5 JurKat E6-1 cells (about 2 mL), and the obtained product is placed into the 5% CO.sub.2 thermostatic incubator with the temperature of 37° C. to be subjected to static culture for 72 h.

[0186] (6) After the step (5) is completed, centrifugation is carried out, precipitates are collected, then 200 μL of RIPA solution is added to carry out pyrolysis so as to obtain a lysate. IL-2 concentration in the lysate is detected by adopting the ELASA kit.

[0187] 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.

[0188] An experimental result refers to B (2F-hiSCs CM are the 2F-hiSCs (dH1), and dH1-2K7 CM are the dH1-2K7) in FIG. 10. The result shows that the secreta of the 2F-hiSCs (dH1) can inhibit production of the interleukin IL-2.

[0189] 3. In-vivo cotransplantation experiment of human 293FT cells in mice

[0190] (1) The 293FT cells containing a luciferase reporter system is constructed.

[0191] a. A luciferase gene (the genebank number is: MF693179.1) is inserted to a recognition site of the restriction endonuclease EcoRI of the pENTR/1A plasmid so as to obtain a vector pENTR/1A-luc.

[0192] b. Double-stranded DNA molecules (a nucleotide sequence of an EF 1 a promoter) as shown in the SEQ ID No. 2 in the sequence list are inserted to the recognition site of the restriction endonuclease EcoRI of the pENTR/5′ topo plasmid so as to obtain a vector pENTR/5′ topo-EF1α.

[0193] c. The vector pENTR/1A-luc, the vector pENTR/5′ topo-EF1α and the lentivirus plasmid p2K7-NEO are taken to prepare a lentivirus vector by homologous recombination.

[0194] d. The lentivirus vector prepared in the step c, the helper plasmid vsvg and the lentivirus vector packaging plasmid pCMV ΔR8.9 are co-transfected with 293FT cells to obtain the 293FT cells containing the luciferase reporter system.

[0195] (2) 100 μL of Matrigel stock solution, about 1.3×10.sup.6 293FT cells containing the luciferase reporter system and about 2.5×10.sup.5 2F-hiSCs (dH1) are mixed to obtain mixed liquid 1. 100 μL of Matrigel stock solution, about 1.3×10.sup.6 293FT cells containing the luciferase reporter system and about 2.5×10.sup.5 dH1-2K7 are mixed to obtain mixed liquid 2. Both the mixed liquid 1 and the mixed liquid 2 are placed on ice to be taken into an animal operation platform.

[0196] (3) Three healthy C57B6/6J mice are taken and are anesthetized by intraperitoneal injection of 1.2% (m/v) of Avertin solution, and an injection dosage is 0.2 mL per 10 g of weight.

[0197] (4) After the step (3) is completed, the mixed liquid 1 is subcutaneously injected to armpits of left fore limbs of the mice, the mixed liquid 2 is subcutaneously injected to armpits of right fore limbs of the mice, after the Matrigel stock solution is solidified, the C57B6/6J mice are place back to a culture cage, and then survival conditions of 293FT at the mouse transplantation positions (i.e., the armpits of the fore limbs of the mice) are monitored by utilizing an in-vivo imager. The operation of in-vivo imaging is as follows: 15min before monitoring, a luciferase substrate aqueous solution with a concentration of 15 mg/mL is intraperitoneally injected, an injection dosage is 10 μL per 1 g of weight; and the mice are placed in the in-vivo imager to detect strength of bioluminescence.

[0198] An experimental result refers to FIG. 11 (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.

[0199] 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.

[0200] 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 2 of the step I in Embodiment 2 is No. 3, No. 7, No. 10, No. 14, No. 16, No. 18, No. 19, No. 21, No. 22, No. 25, No. 26, No. 27, No. 29 or No. 30 in Table 1) and other hiSCs (HPF) (i.e., the protein combination expressed by the recombinant lentivirus in the virus fluid in the step II in Embodiment 2 is No. 3, No. 7, No. 10, No. 14, No. 16, No. 18, No. 19, No. 21, No. 22, No. 25, No. 26, No. 27, No. 29 or No. 30), 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.

[0201] From the above, the hiSCs (dH1) and the hiSCs (HPF) prepared in Embodiment 2 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 2 are the Sertoli cells.

Embodiment 4: Influence of AMH:GFP Reporter System on Acquisition of Sertoli Cells

Step I: Construction of an AMH-D:GFP Reporter System

[0202] 1. The inventor of the present disclosure clones a second promoter of the AMH gene from the human genome DNA. A length of the second promoter of the AMH gene is about 726 bp, and a nucleotide sequence of the second promoter is as shown in a SEQ ID No. 3 in the sequence list.

[0203] 2. After the step 1 is completed, an encoding gene (i.e., an eGFP gene) of a fluorescent protein eGFP is connected to a 3′ terminal of the second promoter of the AMH gene so as to obtain the AMH-D:GFP reporter system. In the AMH-D:GFP reporter system, the second promoter of the AMH gene drives expression of the fluorescent protein eGFP.

Step II: Construction of a Recombinant Lentivirus Containing the AMH-D:GFP reporter System

[0204] 1. The eGFP gene is inserted to the recognition site of the restriction endonuclease EcoRI of the pENTR/1A plasmid so as to obtain the vector pENTR/1A-eGFP.

[0205] 2. Double-stranded DNA molecules as shown in the SEQ ID No. 3 in the sequence list are inserted to the recognition site of the restriction endonuclease EcoRI of the pENTR/5′ topo plasmid so as to obtain a vector pENTR/5′ topo-AMH-D.

[0206] 3. The vector pENTR/1A-eGFP, the vector pENTR/5′ topo-AMH-D and the lentivirus plasmid p2K7-NEO are taken to prepare a lentivirus vector by homologous recombination.

[0207] 4. The lentivirus vector prepared in the step 3, the helper plasmid vsvg and the lentivirus vector packaging plasmid pCMV ΔR8.9 are co-transfected with the 293FT cells to obtain the recombinant lentivirus containing the AMH-D:GFP reporter system.

[0208] Step III: according to the step III in Embodiment 1 and the steps in Embodiment 2 and the steps in Embodiment 3, replacement of the AMH:GFP reporter system in the step III in Embodiment 1 with the AMH-D:GFP reporter system without changing other steps.

[0209] Part of results are as follows: when the AMH-D:GFP reporter system is adopted, in the system 2 (the NR5A1 protein and the GATA4 protein expressed by the recombinant lentivirus in the virus fluid) subjected to the step 8 in the step I in Embodiment 2, 1.23% of cells are AMH-D:GFP+Sertoli cells; and in the system 2 (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 8 in the step II in Embodiment 2, 0.86% 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.

INDUSTRIAL APPLICATION

[0210] The Sertoli cells prepared by adopting the method provided by 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.