Induced trophoblast stem cells and preparation method and application thereof

Abstract

The present invention discloses an induced trophoblast stem cell, a preparation method and an application thereof, and relates to the field of biomedical technology. The preparation method comprises using human pluripotent stem cells as donor cells, and culturing them in an induction medium to derive induced trophoblast stem cells with the ability to proliferate indefinitely and differentiate into subtypes of trophoblast cells. The induction medium contains vitamin C, epidermal growth factor, a WNT pathway activator, a TGF-β pathway inhibitor, a ROCK pathway inhibitor and a histone deacetylase inhibitor. The present invention can expand the source of trophoblast stem cells and avoid ethical issues, and is helpful to study the mechanism of placental development and the occurrence and development of related diseases, and explore the process of in vitro embryo synthesis and embryo self-assembly.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an induced trophoblast stem cell and a preparation method and application thereof. Background Art

[0002] Trophoblast stem cells (CTs) are specialized cells with the ability to self-renew and differentiate into various trophoblast cell types (syncytiotrophoblasts, extravillous trophoblasts, etc.). Primary mouse trophoblast stem cells were first successfully isolated in 1998. In 2015, it was discovered that mouse fibroblasts could be induced into trophoblast stem cells. In 2018, human primary trophoblast stem cells were first isolated from blastocysts and early pregnancy placentas.

[0003] Compared with various cell models currently used to study trophoblast cell development, human trophoblast stem cells have the following advantages:

[0004] 1) Immortalized trophoblast cell lines, such as HTR8 and JEG-3, are mutant trophoblast cell lines or cancerous trophoblast cells. They cannot truly reflect the normal developmental pathways of human trophoblast cells and the causes of disease. However, the differentiation of human trophoblast stem cells into various trophoblast cell subtypes is consistent with the natural developmental process in vivo.

[0005] 2) There are species differences between mouse and other animal models, their trophoblast stem cells, and induced trophoblast stem cells, and human trophoblast stem cells. For example, the cell states before and after implantation are different, and the morphology, gene expression profiles, and functions of the trophoblast subtypes obtained after differentiation are also significantly different. Diseases caused by human trophoblast cell developmental disorders, such as preeclampsia, do not occur in animals such as mice in nature. Therefore, using animal models can reveal some causal relationships, but it is difficult to reflect research models related to the occurrence and development of human diseases;

[0006] 3) Primary trophoblast cells isolated from villi or placenta cannot self-renew and have limited proliferation capacity during in vitro culture. They do not have the in vitro self-renewal capacity of trophoblast stem cells, making it difficult to perform gene editing and other operations, limiting the possibility of further research on cell functions.

[0007] At present, primary human trophoblast stem cells can only be obtained through blastocysts or early pregnancy placentas. Constructing primary human trophoblast stem cells also destroys the embryo or developing placenta, so it does not meet ethical requirements. The use of discarded embryos or chorionic villi after termination of pregnancy has extremely limited resources.

[0008] In view of this, the present invention is proposed. Summary of the Invention

[0009] The purpose of the present invention is to provide the use of a cell culture medium in preparing induced trophoblast stem cells, induced trophoblast stem cells and a preparation method thereof.

[0010] The present invention is achieved in that:

[0011] In a first aspect, an embodiment provides a method for preparing induced trophoblast stem cells, comprising culturing human pluripotent stem cells into induced trophoblast stem cells capable of unlimited proliferation and differentiation into subtypes of trophoblast cells using a cell culture medium;

[0012] The human pluripotent stem cells include human embryonic stem cells and / or induced pluripotent stem cells;

[0013] The cell culture medium includes a basal culture fluid, and the cell culture medium also contains at least the following components: 0.5-2.5 μg / mL vitamin C, 20-70 ng / mL epidermal growth factor, 1-5 μM WNT pathway activator, 0.3-1.9 μM TGF-β pathway inhibitor, 3-7 μM ROCK pathway inhibitor and 0.6-1.1 mM histone deacetylase inhibitor.

[0014] In a second aspect, the embodiment provides an induced trophoblast stem cell, which is prepared using the preparation method of the induced trophoblast stem cell described in the above embodiment.

[0015] In a third aspect, the embodiments provide the use of the induced trophoblast stem cells described in the aforementioned embodiments in the preparation of a drug for a disease associated with placental developmental disorders.

[0016] In a fourth aspect, the embodiments provide the use of the induced trophoblast stem cells described in the aforementioned embodiments in studying the regulatory network during placental development.

[0017] In a fifth aspect, the embodiments provide the use of the induced trophoblast stem cells described in the aforementioned embodiments in simulating early embryo synthesis or embryonic stem cell self-assembly.

[0018] In a sixth aspect, an embodiment provides a use of a cell culture medium in preparing induced trophoblast stem cells, wherein the preparation of the induced trophoblast stem cells comprises: culturing human pluripotent stem cells into induced trophoblast stem cells capable of unlimited proliferation and differentiation into subtypes of trophoblast cells using a cell culture medium;

[0019] The human pluripotent stem cells include embryonic stem cells and / or induced pluripotent stem cells;

[0020] The cell culture medium includes a basal culture fluid, and the cell culture medium contains at least the following components: 0.5-2.5 μg / mL vitamin C, 20-70 ng / mL epidermal growth factor, 1-5 μM WNT pathway activator, 0.3-1.9 μM TGF-β pathway inhibitor, 3-7 μM ROCK pathway inhibitor and 0.6-1.1 mM histone deacetylase inhibitor.

[0021] The present invention has the following beneficial effects:

[0022] An embodiment of the present invention provides a method for preparing induced trophoblast stem cells, which comprises culturing human pluripotent stem cells into induced trophoblast stem cells using a specific cell culture medium, wherein the transformed induced trophoblast stem cells have the ability to proliferate indefinitely and differentiate into subtypes of trophoblast cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 This is a flow chart for preparing induced trophoblast stem cells according to an embodiment of the present invention;

[0025] Figure 2 This is a diagram showing the identification results of induced trophoblast stem cells in Verification Example 1 of the present invention;

[0026] Figure 3 This is the immunofluorescence staining result of induced trophoblast stem cells in Verification Example 2 of the present invention;

[0027] Figure 4 The results of the detection of GATA3, KRT7 and TFAP2C expression in induced trophoblast stem cells in Validation Example 3 of the present invention are as follows;

[0028] Figure 5 This is the karyotype identification result of the induced trophoblast stem cells in Verification Example 4 of the present invention;

[0029] Figure 6 This is another feature of the induced trophoblast stem cells in Verification Example 4 of the present invention, wherein: Figure 6 A in the middle is the methylation result of ELF5 promoter region, Figure 6 B in the middle is the result of HLA-ABC marker fluorescence immunoassay. Figure 6 Middle C is the result of real-time quantitative PCR detection of miRNA expression encoded by chromosome 19;

[0030] Figure 7 This is the result of the differentiation of induced trophoblast stem cells into extravillous trophoblast cells in Verification Example 5 of the present invention, wherein: Figure 7 A in the middle is the morphology of the differentiated extravillous cytotrophoblast cells. Figure 7 Middle B is the flow cytometry detection result of the extravillous cytotrophoblast cell-specific markers after induction;

[0031] Figure 8 This is the result of the differentiation of induced trophoblast stem cells into syncytiotrophoblast cells in Verification Example 5 of the present invention, wherein: Figure 8 A in the middle is a cell morphology diagram. Figure 8 Middle B is the result of fluorescent immunoassay of syncytiotrophoblast cell-related markers. DETAILED DESCRIPTION

[0032] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0033] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0034] An embodiment of the present invention provides a method for preparing induced trophoblast stem cells, the method comprising culturing human pluripotent stem cells into induced trophoblast stem cells capable of unlimited proliferation and differentiation into subtypes of trophoblast cells using a cell culture medium;

[0035] The human pluripotent stem cells include human embryonic stem cells and / or induced pluripotent stem cells;

[0036] The cell culture medium includes a basal culture fluid, and the cell culture medium also contains at least the following components: 0.5-2.5 μg / mL vitamin C, 20-70 ng / mL epidermal growth factor, 1-5 μM WNT pathway activator, 0.3-1.9 μM TGF-β pathway inhibitor, 3-7 μM ROCK pathway inhibitor and 0.6-1.1 mM histone deacetylase inhibitor.

[0037] It should be noted that the concentrations of the above components are all final concentrations of the components in the cell culture medium, the same below.

[0038] In an optional embodiment, the cell culture medium further comprises one or more of the following components: 0.5-1.5% insulin-like transferrin additive, 0.05-0.15 mM β-mercaptoethanol, and 0.1%-0.5% BSA.

[0039] β-Mercaptoethanol is used as a reducing agent to protect proteins from being oxidized by free radicals; insulin-like transferrin additives provide insulin and metal elements for cell growth; BSA is used to maintain cell growth and stabilize protein activity in the culture medium.

[0040] In an optional embodiment, the cell culture medium further comprises 0.1% to 0.7% penicillin-streptomycin mixture and / or 0.1% to 0.4% FBS.

[0041] In an optional embodiment, the WNT pathway activator is CHIR99021.

[0042] In an optional embodiment, the ROCK pathway inhibitor is Y-27632.

[0043] In an alternative embodiment, the TGF-β pathway inhibitor comprises A83-01 and / or SB431542.

[0044] In an optional embodiment, the TGF-β pathway inhibitor includes A83-01 and SB431542. In the cell culture medium, the final concentration of A83-01 is 0.3 to 0.7 μM, and the final concentration of SB431542 is 0.8 to 1.2 μM.

[0045] In an alternative embodiment, the histone deacetylase inhibitor is sodium valproate (VPA).

[0046] In an optional embodiment, the basal culture fluid is selected from at least one of the following culture media: DMEM / F12, RPMI1640 and HamsF-12.

[0047] In an alternative embodiment, the subtype of trophoblast cells includes extravillous cytotrophoblast cells and / or syncytiotrophoblast cells.

[0048] In alternative embodiments, the human embryonic stem cells include H1 hESCs and / or HN10 hESCs.

[0049] In an alternative embodiment, the induced pluripotent stem cells are selected from at least one of the following cells: urine cell-derived iPS cells UE005-iPS, extravillous mesenchymal cell-derived iPS cells hAMC-iPS, hAMC002-iPS, and hAMC004-iPS.

[0050] It should be noted that the human embryonic stem cell lines and human induced pluripotent stem cell lines used in the examples of the present invention are not older than 14 days after embryo fertilization.

[0051] In an optional embodiment, when cultured in a cell culture medium, the seeding density of the human embryonic stem cells and / or induced pluripotent stem cells is (1-9)×10 5 cells / mL.

[0052] In an optional embodiment, the preparation method includes subculturing the prepared induced trophoblast stem cells.

[0053] In an optional embodiment, the cell confluence of the subculture is 90-100%.

[0054] In an alternative embodiment, when the cell confluency reaches 90-100%, the subculture includes incubating the cells with a digestive enzyme to obtain a cell solution. The digestive enzyme is 0.3-0.7 mM EDTA. Cells in the early stages of transdifferentiation are close to pluripotent stem cells, and a digestive enzyme suitable for pluripotent stem cells is selected. After digestion with EDTA, cells are mostly present in clusters, which is beneficial for cell survival.

[0055] In an optional embodiment, the subculture ratio is 1:(2-4).

[0056] In an optional embodiment, the subculture medium is the cell culture medium.

[0057] In an optional embodiment, before culturing human embryonic stem cells and / or induced pluripotent stem cells in the cell culture medium, the preparation method includes: pre-culturing human embryonic stem cells and / or induced pluripotent stem cells in the stem cell culture medium at 36-38° C. for 22-26 hours.

[0058] In an optional embodiment, the stem cell culture medium is mTeSR1 medium.

[0059] In an alternative embodiment, the pre-culture is performed in a culture dish coated with Matrigel.

[0060] An embodiment of the present invention provides an induced trophoblast stem cell, which is prepared using the method for preparing induced trophoblast stem cells described in any of the aforementioned embodiments.

[0061] The embodiments of the present invention provide the use of the induced trophoblast stem cells provided in the aforementioned embodiments in the preparation of a medicament for diseases associated with placental development disorders.

[0062] In an optional embodiment, the disease associated with placental developmental disorder includes at least one of the following diseases: preeclampsia, eclampsia, placenta accreta, and fetal growth restriction. It should be noted that the use of induced trophoblast stem cells prepared using the induced trophoblast stem cells or the preparation methods of induced trophoblast stem cells provided in the embodiments of the present invention in the study of other diseases associated with placental developmental disorder falls within the scope of protection of this application.

[0063] The embodiments of the present invention provide the use of the induced trophoblast stem cells provided in the aforementioned embodiments in studying the regulatory network during placental development.

[0064] In an optional embodiment, the regulatory network includes at least one of transcription factors, extracellular signals, epigenetic modifications, metabolism and cell cycle.

[0065] The embodiments of the present invention provide the use of the induced trophoblast stem cells provided in the aforementioned embodiments for simulating early embryo synthesis or embryonic stem cell self-assembly.

[0066] It should be noted that the early embryo synthesis or embryonic stem cell self-assembly refers to the spontaneous assembly of induced pluripotent stem cells and induced trophoblast stem cells derived from the same individual under specific culture conditions into embryos similar to those in the early development process, which is used to study the early embryonic development process.

[0067] In addition, an embodiment of the present invention further provides a use of a cell culture medium in preparing induced trophoblast stem cells, wherein the preparation of the induced trophoblast stem cells comprises: using the cell culture medium to culture human pluripotent stem cells into induced trophoblast stem cells capable of unlimited proliferation and differentiation into subtypes of trophoblast cells;

[0068] The human pluripotent stem cells include embryonic stem cells and / or induced pluripotent stem cells;

[0069] The cell culture medium includes a basal culture fluid, and the cell culture medium contains at least the following components: 0.5-2.5 μg / mL vitamin C, 20-70 ng / mL epidermal growth factor, 1-5 μM WNT pathway activator, 0.3-1.9 μM TGF-β pathway inhibitor, 3-7 μM ROCK pathway inhibitor and 0.6-1.1 mM histone deacetylase inhibitor.

[0070] It should be noted that, in this application, the cell culture medium is the same as the cell culture medium in the preparation method described in the above embodiment, and will not be described in detail here.

[0071] Example

[0072] Experimental materials used in the examples.

[0073] Embryonic stem cells and / or induced pluripotent stem cells: Embryonic stem cell lines were H1 cell line (H1hESC) and HN10 cell line (HN10hESC). Induced pluripotent stem cells were derived from amniotic mesenchymal cells isolated from the placenta after delivery at Nanfang Hospital, Southern Medical University, and reprogrammed using a non-integrating, non-viral approach (for details, see Wei et al. Generation of trophoblast-like cells from the amnion in vitro: A novel cellular model for trophoblast development. Placenta 51:28-37. doi:10.1016 / j.placenta.2017.01.12). This experiment was approved by the Ethics Committee of Nanfang Hospital, Southern Medical University.

[0074] Preparation of mTeSR1 medium: The iPSC (induced pluripotent stem cell) medium mTeSR1 (StemCell) used in subsequent examples is a commercially produced medium, including mTeSR1 basal solution and mTeSR1 supplemental solution. The preparation method is as follows: mTeSR1 supplemental (100 mL) is thawed at 4°C overnight. The next day, in a biological safety cabinet, the thawed mTeSR1 supplemental is added to mTeSR1 basal medium (400 mL). The mixture is thoroughly mixed and dispensed into 50 ml centrifuge tubes (40 mL / tube). The tubes are stored at -20°C and thawed at 4°C overnight before use. The thawed medium is stored at 4°C when not in use and is valid for 2 weeks.

[0075] Cell culture medium (trophoblast stem cell medium): In a biosafety cabinet, add DMEM / F12 basal medium to 1% insulin-like transferrin supplement (ITS-X supplement), 0.1 mM β-mercaptoethanol, 0.3% BSA, 0.5% penicillin-streptomycin, 1.5 g / mL vitamin C, 50 ng / mL epidermal growth factor, 0.2% FBS, 2 μM CHIR99021, 0.5 μM A83-01, 1 μM SB431542, and 5 μM Y-27632. Protect from light, then add 0.8 mM VPA. Mix thoroughly and aliquot into 50 mL centrifuge tubes (40 mL / tube). Store at -20°C. Thaw at 4°C overnight before use. Thawed medium is stored at 4°C when not in use and has a shelf life of 2 weeks.

[0076] MatrigelTM Preparation of working solution: In the subsequent examples, the feeder-free culture system used to culture induced pluripotent stem cells and induced trophoblast stem cells was Corning Matrigel Matrix (Corning). Corning Matrigel Matrix (5 mL) was thawed at 4 ° C overnight according to the instructions in the product manual. The next day, in a biological safety cabinet, the thawed Matrigel was placed on ice and mixed thoroughly. It was then dispensed into 0.5 mL EP tubes (280 μL / tube) to prepare a mother solution and stored at -20 ° C. Thaw at 4 ° C before use, and add 280 ml of Matrigel mother solution to each 50 mL DMEM / F12 and mix well to prepare the working solution.

[0077] Preparation of culture plates for induced trophoblast stem cells and induced pluripotent stem cells: Cell culture plates for pluripotent stem cells and induced trophoblast stem cells should be prepared before cell inoculation. The prepared culture plates should be stored in an incubator and are valid for 3 days. The steps for preparing the culture plates are as follows:

[0078] (1) Matrigel-coated culture dishes: Add 1 mL of Matrigel working solution to each well of a six-well plate and incubate at 37°C for at least 1.5 h to allow the matrix to evenly coat the bottom of the culture plate.

[0079] (2) Medium exchange: Before inoculating pluripotent stem cells and human induced trophoblast stem cells, discard the Matrigel in the culture plate and add 2 mL of mTeSR1 medium or the above cell culture medium. Equilibrate in the incubator for at least one hour before use.

[0080] Preparation of human induced trophoblast stem cell digestion enzyme: The digestion enzyme TrypLE Express (Gibco) used for human induced trophoblast stem cell passage in subsequent examples is a commercially produced cell passage digestion enzyme and is stored at room temperature.

[0081] Preparation of cryopreservation medium: The human induced trophoblast stem cell cryopreservation medium Cell Banker 2 (ZENOAQ) used in the subsequent examples is a commercially produced cell cryopreservation medium and is stored in a refrigerator at 4°C.

[0082] Preparation of induced trophoblast stem cells:

[0083] The methods for inducing trophoblast stem cell transdifferentiation from embryonic stem cells and induced pluripotent stem cells are the same. The following specific steps are described using the embryonic stem cell line H1 (H1 hESC) as an example.

[0084] Human embryonic stem cells H1 were plated at 4 × 10 5Cells were seeded into six-well plates pre-coated with Matrigel; the culture medium was mTeSR1 and cultured in a 37°C, 5% CO2 incubator. After 24 hours, the original mTeSR1 culture medium was removed, the cells were washed once with DMEM / F12, and fresh cell culture medium was added to induce transdifferentiation (the time point was set to D0. Please refer to the attached flowchart for the operation). Figure 1 ) When the cell confluence reached 90%, they were passaged at a ratio of 1:3.

[0085] The method of subculturing is as follows: prepare a six-well plate with Matrigel, remove the Matrigel, add fresh cell culture medium and preheat and equilibrate in a 37°C, 5% CO2 incubator. Discard the old culture medium and add Ca-free 2+ and Mg 2+ Wash twice with PBS. Add 1 mL of 0.5 mM EDTA to each well and place in a 37°C incubator for digestion for 10 min.

[0086] Then, add 1 mL of the above-mentioned cell culture medium to each well, gently pipette the cells off the well dish, transfer them to a 14 mL centrifuge tube, and centrifuge at 380 × g for 1 min. After centrifugation, aspirate the supernatant, add the above-mentioned cell culture medium, gently mix thoroughly, and inoculate at a 1:3 ratio into a new six-well plate pre-filled with the above-mentioned cell culture medium. After gently shaking to mix, place in a 37°C, 5% CO2 incubator to complete the subculture.

[0087] On days 7-8, trophoblast stem cell-like clones appeared in the culture plate and expanded over the days. On days 10-12, the induced trophoblast stem cell-like clones were mechanically picked out using a homemade glass needle and a 1 mL pipette and transferred to a new six-well plate pre-coated with Matrigel. The culture medium was the same as above, and the culture conditions were 37°C, 5% CO2.

[0088] After three passages, the cells were purified into uniform induced trophoblast stem cells that grew rapidly and were then identified and cryopreserved.

[0089] Maintenance and passaging of induced trophoblast stem cells

[0090] When the human induced trophoblast cells prepared in the above "Method for Preparing Induced Trophoblast Stem Cells" grow to 60%-90%, they can be passaged. The specific steps are as follows (taking a six-well plate as an example):

[0091] Prepare a six-well plate with Matrigel, remove the Matrigel, add fresh cell culture medium and preheat and balance in a 37℃, 5% CO2 incubator; discard the old culture medium and add Ca-free 2+ and Mg 2+Wash twice with PBS; add 1 mL of TrypLE digestion enzyme to each well and digest in a 37°C incubator for 8-12 minutes; add 1 mL of the above-mentioned cell culture medium to each well, use a pipette to blow the cells off the well dish, and transfer them to a 14 mL centrifuge tube; centrifuge at 380×g for 1 minute; after centrifugation, aspirate the supernatant, add the above-mentioned cell culture medium, mix thoroughly, and then inoculate into a new six-well plate pre-filled with the above-mentioned cell culture medium at a ratio of 1:3. After gently shaking to mix, place in a 37°C, 5% CO2 incubator to complete the subculture.

[0092] In this embodiment, the induced trophoblast stem cells were cultured once every 24 hours and passaged once every 48 to 72 hours.

[0093] Cryopreservation of induced trophoblast stem cells

[0094] When the cell confluence of the induced trophoblast stem cells prepared in the above maintenance and passaging steps reaches about 80%, cryopreservation can be performed, specifically including:

[0095] Digest the induced trophoblast stem cells from the culture plate, remove the centrifugal supernatant, add 1 mL of freezing solution to the cell pellet in the centrifuge tube, and gently pipette to evenly distribute the cells. Then, aliquot the cells into cryopreservation tubes, 1 mL per tube, and label each tube with the name, generation number, and time. Place the cryopreservation tubes containing the cells in a program freezing box and place them in a -80°C freezer overnight. After 24 hours, remove the tubes from the program freezing box and place them in a liquid nitrogen container.

[0096] Thawing and recovery of induced trophoblast stem cells

[0097] In this embodiment, the specific steps for resuscitating induced trophoblast stem cells from the liquid nitrogen container are as follows.

[0098] Remove the cryovial from the liquid nitrogen container and immerse it in a 37°C water bath while gently shaking to accelerate melting. When most of the cells have melted and a few ice crystals are visible, open the lid in the biosafety cabinet, aspirate the cell suspension, add it to a centrifuge tube, add 10x mTeSR1 medium dropwise, and mix thoroughly. Centrifuge at 380×g for 1 min, aspirate the supernatant, add mTeSR1 medium, count the cells, and plate them into a six-well plate, inoculating 100,000 cells per well.

[0099] Verification Example 1

[0100] The induced trophoblast stem cells provided in the examples were identified.

[0101] 1. Using the method of "preparation of induced trophoblast stem cells" provided in the example, 6 groups of experimental examples were set up. Experimental examples 1 to 6 used cell culture medium to culture H1 hESCs, HN10 hESCs, hTS UE005-iPS 、hTShAMC-iPS 、hTS hAMC002-iPS and hTS hAMC004-iPS Transformed into induced trophoblast stem cells.

[0102] After preparation, the induced trophoblast stem cells were passaged three times and identified. The identification results of induced trophoblast stem cells are shown in the attached figure. Figure 2 .

[0103] Verification Example 2

[0104] Identification by immunofluorescence staining.

[0105] The cell culture medium and preparation method provided in the example were used to transform H1 hESCs and HN10 hESCs into induced trophoblast stem cells. The induced trophoblast stem cell slices were then placed in a twelve-well plate and plated with Matrigel. TM Coated cell slides.

[0106] The induced trophoblast stem cells were passaged into the twelve-well plate according to the passage method of the embodiment, and were used for immunofluorescence staining after growing for 3 days. The specific steps of immunofluorescence staining are as follows.

[0107] Before staining, discard the old medium, wash three times with 1 ml of PBS, and add 0.5 ml of 4% paraformaldehyde for 30 minutes at room temperature. Then, discard the fixative and wash three times with 1 ml of PBS, shaking on a shaker for 5 minutes each time. After shaking, add 0.5 ml of permeabilization solution (0.2% Triton-X100 in PBS) and permeabilize at room temperature for 30 minutes. After permeabilization, discard the permeabilization solution and wash three times with 1 ml of PBST, shaking on a shaker for 5 minutes each time. Then, add 0.5 ml of blocking solution (10% goat serum, 5% BSA in PBST) and block at room temperature for 1 hour.

[0108] After blocking, discard the blocking solution and add 0.5 ml of primary antibody (rabbit anti-GATA3 IgG, Abcam, ab19857; mouse anti-KRT7 IgG, Abcam, ab16287; mouse anti-TP63 IgG, Millipore, MAB4360; and mouse anti-TEAD4 IgG, Millipore, MAB4381) to a 12-well plate. Cover the liquid surface with sealing film slightly smaller than the 12-well plate and incubate in a humidified chamber at 4°C in the dark for 1 hour. Discard the primary antibody and wash three times with 1 ml of PBST, shaking on a shaker for 5 minutes each wash.

[0109] Then, 0.5 ml of secondary antibodies (including goat anti-rabbit IgM Alexa Flour 488, Invitrogen, A11008; goat anti-mouse IgM Dylight 488, GeneTex, GTX76752; and goat anti-mouse IgG Alexa Flour® 488, Abcam, ab150117) were added to a 12-well plate and incubated in a humidified chamber for 1 hour at room temperature in the dark. The secondary antibody was discarded, and the plates were washed three times with 1 ml of PBST, rocking on a shaker for 5 minutes each wash. 0.5 ml of DAPI was added and the plates were incubated in a humidified chamber for 5 minutes in the dark.

[0110] Discard the DAPI stain and wash twice with 0.5ml of PBS in the dark, shaking on a shaker for 5 minutes each time. Add 0.5ml of fresh PBS and observe and photograph under an Olympus inverted microscope, or mount the slides with mounting medium for observation and storage. This allows for the detection of pluripotency marker expression in thawed and subcultured iPSC clones.

[0111] Depend on Figure 3 It can be seen that the induced trophoblast stem cells obtained according to the embodiment of the present invention were subjected to immunofluorescence staining of GATA3, KRT7, TP63 and TEAD4, and the detection results were all positive.

[0112] Verification Example 3

[0113] Flow cytometry assay

[0114] Take the induced trophoblast stem cells provided in the example, remove the original culture medium from the cells to be treated, wash once with PBS, and then digest with 0.5 mM EDTA in a 37°C incubator for several minutes until the cells are dispersed. Then, add the above cell culture medium to terminate the digestion. Pipet the cells to ensure that they are dispersed and in a single cell state, and centrifuge at 400 × g for 1 minute.

[0115] After centrifugation, discard the supernatant, resuspend the cells in flow cytometry buffer (PBS containing 2% FBS or BSA), transfer to a 1.5 ml EP tube, and centrifuge at 400 × g for 1 min. After centrifugation, discard the supernatant, add 200 μl of 1% paraformaldehyde, and fix at 37°C for 10 min. Centrifuge at 400 × g for 1 min, discard the supernatant, wash once with flow cytometry buffer, centrifuge again at 400 × g for 1 min, discard the supernatant, add 200 μl of ice-cold 90% methanol, and permeabilize on ice for 30 min. After permeabilization, centrifuge at 400 × g for 1 min, rinse twice with flow cytometry buffer, and discard the supernatant.

[0116] Dilute the primary antibody in flow cytometry buffer at an appropriate ratio. Resuspend the cells in 50 μl of diluted primary antibody and incubate at 37°C for 30 minutes. During incubation, flick the cells several times to minimize aggregation at the bottom of the EP tube, which could affect antibody binding. After incubation, centrifuge at 200 × g for 5 minutes and wash twice with flow cytometry buffer.

[0117] The following operations should be performed under light-proof conditions.

[0118] First, dilute the secondary antibody in flow cytometry buffer at an appropriate ratio. Resuspend the cells in 200 μl of the diluted secondary antibody and incubate at 37°C for 30 minutes, flicking the cells several times during incubation. After incubation, centrifuge at 400 × g for 1 minute and wash once with flow cytometry buffer. Remove the supernatant, resuspend in 200–300 μl of flow cytometry buffer, filter, and analyze using a flow cytometer at an appropriate wavelength.

[0119] Depend on Figure 4 It can be seen that the induced trophoblast stem cells obtained according to the embodiment of the present invention were tested by flow cytometry for the expression of GATA3, KRT7 and TFAP2C, and the test results were all positive and high expression.

[0120] Verification Example 4

[0121] Karyotype analysis.

[0122] The cell culture medium and preparation method provided in the examples were used to transform H1 hESCs and HN10 hESCs into induced trophoblast stem cells.

[0123] The prepared induced trophoblast stem cells were then passaged onto Matrigel TM When the cell density in two wells of the coated six-well plate reaches 80%, it can be used for karyotype analysis. The specific steps of karyotype analysis are as follows:

[0124] Aspirate and discard the old culture medium and replace with 2 mL of fresh mTeSR1 TM Culture medium. Drop 14 ml (20 μg / ml) of colcemid (to make the final working concentration: 0.14 mg / ml) and treat for 3 hours. Aspirate the culture medium containing colcemid, wash twice with 2 mL PBS, and digest the cells with 1 mL 0.25% trypsin-EDTA for 1 minute. Add 1 mL of culture medium containing 10% FBS to stop digestion, blow down the cells (require as many single cells as possible), transfer to a 15 ml centrifuge tube, and centrifuge at 1500 rpm for 5 minutes. Discard the supernatant and keep the precipitate. Add 8 ml of 0.075 mM KCl pre-warmed at 37°C to the 15 ml centrifuge tube, repeatedly use a pipette to disperse the cells, and place in a 37°C water bath for hypotonic treatment for 12 minutes.

[0125] Slowly add 2 ml of fixative (methanol and acetic acid in a volume ratio of 3:1) to the hypotonic cell suspension, mix gently, and fix at room temperature for 3 minutes, then centrifuge at 1500 rpm for 5 minutes. Discard the supernatant and keep the pellet. Slowly add 8 ml of fixative along the wall of the tube, mix gently, and pre-fix in a 37°C water bath for 30 minutes, then centrifuge at 1500 rpm for 5 minutes. Discard the supernatant, keep the pelleted cells, and add 1 ml of fixative again (the amount of fixative added should be adjusted according to the amount of pellet and the distribution of cells after preparation). After mixing, the liquid will become frosted glass, drop ice chips on it, 2 drops per slide, and dry over an alcohol lamp.

[0126] Bake the prepared slides in a 90°C oven for 25 minutes. Preheat three 50ml vertical staining jars (labeled as staining jars I, II, and III) in a 37°C water bath for 30 minutes. Add 49ml of phosphate buffered saline and 1ml of 1.25% trypsin to staining jar I, mix thoroughly, and use for cytogenetic digestion and band visualization for 1 minute 20 seconds. Add approximately 50ml of normal saline to staining jar II for rinsing. Add 45ml of double-distilled water and two tubes of staining solution to staining jar III, mix thoroughly, and use for staining for 4 minutes. Remove the slides, rinse with tap water, and dry them in an oven or air dry.

[0127] An Olympus upright microscope was used to observe 15-20 karyotypes, which were then scanned and analyzed using an automatic chromosome scanning and analysis system (Zeiss MetaSystems).

[0128] Test results such as Figure 4 and 5 shown.

[0129] Depend on Figure 5 and 6 It can be seen that the karyotype of human induced trophoblast stem cells derived from two embryonic stem cell lines (H1 and HN10) at the 10th generation of culture was normal and had typical trophoblast stem cell characteristics: low expression of HLA-I class molecules, low methylation of ELF5 promoter, and high expression of specific microRNA.

[0130] Verification Example 5

[0131] The differentiation potential of the induced trophoblast stem cells prepared in the examples was detected.

[0132] In the embodiment, human induced trophoblast stem cells are differentiated into extravillous cytotrophoblast cells, and the specific steps are as follows:

[0133] The passaged induced trophoblast stem cells were seeded into six-well plates pre-coated with Matrigel, with 4×10 cells per well. 5Each culture medium was cultured with EVT (extravillous cytotrophoblast) Differentiation Medium #1 in an incubator at 37°C, 5% CO2. After three days, the original medium was aspirated and replaced with EVT Differentiation Medium #2. Around day 6-8, cells were harvested for identification and analysis.

[0134] The specific steps for the induced trophoblast stem cells in the embodiment to differentiate into syncytiotrophoblast cells are as follows:

[0135] The passaged human induced trophoblast stem cells were seeded into six-well plates pre-coated with Matrigel, with 1×10 cells per well. 5 The cells were cultured in STB (syncytiotrophoblast) differentiation medium #1 in an incubator at 37°C, 5% CO2. After three days, the original medium was removed and replaced with fresh STB medium for differentiation. On day 6-7, the cells were harvested for identification and analysis.

[0136] Please refer to the attached Figure 7 and Figure 8 As shown in the accompanying figures, induced trophoblast stem cells derived from human embryonic stem cells differentiated into trophoblast subtypes (including EVT and STB), had typical morphological characteristics, and expressed the corresponding markers HLA-G (EVT) and SDC1 / HCG (STB). The ability of induced trophoblast stem cells obtained according to the embodiments of the present invention to differentiate into trophoblast subtypes (including EVT and STB) is demonstrated.

[0137] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for preparing induced trophoblast stem cells, characterized in that: The method comprises using a cell culture medium to culture human pluripotent stem cells into induced trophoblast stem cells with the ability to proliferate indefinitely and differentiate into subtype trophoblast cells; The human pluripotent stem cells include human embryonic stem cells and / or induced pluripotent stem cells; The cell culture medium consists of a basal culture medium and the following components: 0.5-2.5 μg / mL vitamin C, 20-70 ng / mL epidermal growth factor, 1-5 μM WNT pathway activator, 0.3-1.9 μM TGF-β pathway inhibitor, 3-7 μM ROCK pathway inhibitor, 0.6-1.1 mM histone deacetylase inhibitor, 0.5-1.5% insulin-like transferrin additive, 0.05-0.15 mM β-mercaptoethanol, 0.1%-0.5% BSA, 0.1-0.7% penicillin-streptomycin mixture, and 0.1-0.4% FBS; The WNT pathway activator is CHIR99021, the ROCK pathway inhibitor is Y-27632, the TGF-β pathway inhibitor consists of A83-01 at a final concentration of 0.3-0.7 μM and SB431542 at a final concentration of 0.8-1.2 μM, and the histone deacetylase inhibitor is sodium valproate; The preparation method comprises subculturing the prepared induced trophoblast stem cells; the culture medium for the subculturing is the cell culture medium; The preparation method further includes pre-culturing the human embryonic stem cells and / or induced pluripotent stem cells in a stem cell culture medium at 36-38° C. for 22-26 hours before culturing the human embryonic stem cells and / or induced pluripotent stem cells in the cell culture medium; the stem cell culture medium is mTeSR1 medium; and the pre-culture is performed in a culture dish coated with Matrigel.

2. The method for preparing induced trophoblast stem cells according to claim 1, characterized in that: The basal culture fluid is selected from at least one of the following culture media: DMEM / F12, RPMI1640 and HamsF-12.

3. The method for preparing induced trophoblast stem cells according to claim 1, characterized in that: The subtypes of trophoblast cells include extravillous cytotrophoblast cells and / or syncytiotrophoblast cells.

4. The method for preparing induced trophoblast stem cells according to claim 1, wherein: The human embryonic stem cells include H1 hESC and / or HN10 hESC.

5. The method for preparing induced trophoblast stem cells according to claim 1, wherein: The induced pluripotent stem cells are selected from at least one of the following cells: urine cell-derived iPS cells UE005-iPS, extravillous mesenchymal cell-derived iPS cells hAMC-iPS, hAMC002-iPS, and hAMC004-iPS.

6. The method for preparing induced trophoblast stem cells according to any one of claims 1 to 5, characterized in that: When cultured in cell culture medium, the seeding density of human embryonic stem cells and / or induced pluripotent stem cells is (1-9)×10 5 cells / mL.

7. The method for preparing induced trophoblast stem cells according to any one of claims 1 to 5, characterized in that: The cell fusion degree of the subculture is 90-100%.

8. The method for preparing induced trophoblast stem cells according to claim 7, wherein: When the cell confluence reaches 90-100%, the subculture includes incubating the cells with a digestive enzyme to obtain a cell solution, wherein the digestive enzyme is 0.3-0.7 mM EDTA.

9. The method for preparing induced trophoblast stem cells according to any one of claims 1 to 5, characterized in that: The ratio of the subculture is 1:(2~4).

10. Use of a cell culture medium in preparing induced trophoblast stem cells, characterized in that: The preparation of induced trophoblast stem cells comprises: using a cell culture medium to culture human pluripotent stem cells into induced trophoblast stem cells capable of unlimited proliferation and differentiation into subtype trophoblast cells; The human pluripotent stem cells include embryonic stem cells and / or induced pluripotent stem cells; The cell culture medium consists of a basal culture medium and the following components: 0.5-2.5 μg / mL vitamin C, 20-70 ng / mL epidermal growth factor, 1-5 μM WNT pathway activator, 0.3-1.9 μM TGF-β pathway inhibitor, 3-7 μM ROCK pathway inhibitor, 0.6-1.1 mM histone deacetylase inhibitor, 0.5-1.5% insulin-like transferrin additive, 0.05-0.15 mM β-mercaptoethanol, 0.1%-0.5% BSA, 0.1-0.7% penicillin-streptomycin mixture, and 0.1-0.4% FBS; The WNT pathway activator is CHIR99021, the ROCK pathway inhibitor is Y-27632, the TGF-β pathway inhibitor consists of A83-01 with a final concentration of 0.3-0.7 μM and SB431542 with a final concentration of 0.8-1.2 μM, and the histone deacetylase inhibitor is sodium valproate.

11. Use of the cell culture medium according to claim 10 in preparing induced trophoblast stem cells, characterized in that: The basal culture fluid is selected from at least one of the following culture media: DMEM / F12, RPMI1640 and HamsF-12.

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