Optimized protocol for naive human pluripotent stem cell-derived trophoblast induction

Summary Human trophoblasts arise from the morula as trophectoderm, which differentiates into cytotrophoblast, syncytiotrophoblast, and extravillous trophoblast after implantation. Here, we present a robust step-by-step protocol to induce trophectoderm (TE) from naive human pluripotent stem cells (PSCs) corresponding to pre-implantation epiblast. Our culture system (TE induction and ACE condition) mimics the entire trophoblast development including the molecular events. For complete details on the use and execution of this protocol, please refer to Io et al. (2021).


SUMMARY
Human trophoblasts arise from the morula as trophectoderm, which differentiates into cytotrophoblast, syncytiotrophoblast, and extravillous trophoblast after implantation. Here, we present a robust step-by-step protocol to induce trophectoderm (TE) from naive human pluripotent stem cells (PSCs) corresponding to pre-implantation epiblast. Our culture system (TE induction and ACE condition) mimics the entire trophoblast development including the molecular events. For complete details on the use and execution of this protocol, please refer to Io et al. (2021).

BEFORE YOU BEGIN
The protocol below describes the steps for using a specific human embryonic stem cell line (H9). We have also used this protocol on human induced pluripotent stem cell lines (409B2 and AdiPS1). For simplicity, 6-well plates are usually used. All cells are cultured in 5% O 2 , 5% CO 2 unless noted otherwise. Passages and medium change are performed in normoxia.

Preparation of stock solutions for trophoblast derivation and culture
Timing: 3 h 1. Prepare aliquots of stock solutions to avoid freeze-thaw cycles.

5.
Resuspend 100 mg of bFGF in 10 mL of PBS with 0.01% BSA to a final concentration of 10 mg/mL. Aliquot in a volume of 100 mL. Store the aliquots at À80 C for %6 months. After thawing an aliquot, store it at 4 C for %2 weeks. 6. Resuspend 10 mg of BMP4 in 100 mL of 4 mM HCl with 0.1% BSA to a final concentration of 100 mg/mL. Aliquot in a volume of 10 mL. Store the aliquots at À80 C for %6 months. After thawing an aliquot, store it at 4 C for %2 weeks. 7. Resuspend 200 mg of EGF liquid in 1.8 mL of PBS with 0.1% BSA to a final concentration of 100 mg/mL. Aliquot in a volume of 100 mL. Store the aliquots at À80 C for %6 months. After thawing an aliquot, store it at À20 C for %2 weeks. 8. Resuspend 1 mg of LIF in 100 mL of PBS with 0.1% BSA to a final concentration of 1 mg/mL and aliquot in a volume of 100 mL. Store the aliquots at À80 C for %6 months. The LIF solution should not be stored at 4 C for more than one week. 9. Resuspend 25 mg of CHIR99021 in 5,373 mL of DMSO to a final concentration of 10 mM.
Aliquot in a volume of 20 mL. Store the solution at À80 C for %6 months and avoid repeated freeze-thaw cycles. The CHIR99021 solution should not be stored at 4 C for more than one week.
Note: Toxic. Avoid contact and inhalation, Wear a mask and gloves.
10. Resuspend 50 mg of PD0325901 in 10,359 mL of DMSO to a final concentration of 10 mM. Aliquot in a volume of 20 mL. Store the solution at À80 C for %6 months. The PD0325901 solution should not be stored at 4 C for more than one week. 11. Resuspend 10 mg of Gö 6983 in 4,520 mL of DMSO to a final concentration of 5 mM. Aliquot in a volume of 50 mL. Store the solution at À80 C for %6 months. The Gö 6983 solution should not be stored at 4 C for more than one week. 12. Resuspend 50 mg of Y-27632 in 14.7 mL of distilled water (DW) to a final concentration of 10 mM. Aliquot in a volume of 100 mL. Store the aliquots at À80 C for %6 months. After thawing an aliquot, store it at 4 C for %2 weeks. 13. Resuspend 50 mg of A83-01 in 4,740 mL of DMSO to a final concentration of 2.5 mM. Aliquot in a volume of 50 mL. Store the aliquots at À80 C for %6 months. The A83-01 solution should not be stored at 4 C for more than one week. 14. Resuspend 5 mg of JAK inhibitor I in 323 mL of DMSO to a final concentration of 10 mM. Aliquot in a volume of 30 mL. Store the solution below À80 C for %6 months and avoid repeated freezethaw cycles. 15. Resuspend 10 mg of XAV939 in 1.6 mL of DMSO to a final concentration of 20 mM. Aliquot in a volume of 20 mL. Store the aliquots at À80 C for %6 months. The XAV-939 solution should not be stored at 4 C for more than one week. 16. Resuspend and filter 25 g of valproic acid sodium salt (VPA) in 501 mL of DW to a final concentration of 0.3 M. Aliquot in a volume of 30 mL. 17. Resuspend and filter 50 mg of NRG1 in 500 mL of 20 mM citrate (pH3.0) to a final concentration of 100 mg/mL. Aliquot in a volume of 20 mL. 18. Resuspend 10 mg of Forskolin in 244 mL of DMSO to a final concentration of 100 mM. Aliquot in a volume of 20 mL.

Preparation of mouse embryonic fibroblast (MEF) feeder plates
Timing: 1-2 weeks 19. Thawing MEF feeder cells a. Prepare 10 cm dishes and the MEF culture medium. b. Add 4 mL of 0.1% gelatin solution to each dish and incubate for 10 min at room temperature. c. Remove the cryovial containing MEF from liquid nitrogen and thaw for 1-2 min in a 37 C water bath. d. Transfer the MEF in the cryovial to a 15-mL tube and add 5 mL of MEF culture medium. e. Centrifuge at 1,300 rpm (330 g) for 3 min, aspirate the supernatant, and resuspend the pellet with MEF culture medium at a density of 5.0 3 10 4 cells/mL. f. Aspirate the gelatin solution from the well and add 10 mL of MEF suspension to each dish. 20. Passage of MEF feeder cells a. Incubate the MEF feeder dishes at 37 C until the culture cells reach subconfluence. b. Wash the dishes with PBS 2 times. c. Add 1 mL of trypsin and incubate at 37 C for 5-10 min. d. Transfer the dissociated cells into a 50-mL tube and add MEF culture medium at 5 times the amount. e. Centrifuge at 1,300 rpm for 3 min, aspirate the supernatant, and resuspend the pellet with MEF culture medium. f. Seed the cells on newly prepared gelatin-coated 10 cm dishes at a 1:4-1:6 split ratio. 21. Inactivate the MEF feeder cells.
a. Harvest the MEF feeder cells with trypsin after the cells reach subconfluence. b. Resuspend the dissociated cells with MEF culture medium at a density of 1.0-5.0 3 10 6 cells/mL. c. Irradiate the cells at 3,285 cGy/min for 40 min with a GAMMACELL 40 EXACTOR. d. Centrifuge at 1,300 rpm for 3 min, aspirate the supernatant, and resuspend the pellet with CELLBANKER 1 (ZENOGEN PHAMA) at a density of 2.0 3 10 6 cells/mL. e. Aliquot in a volume of 500 mL into cryovials. f. Cryopreserve the cells in liquid nitrogen. 22. Thawing the inactivated MEF feeder cells for use in PSC culture.
a. Thaw MEF in the cryovial for 1-2 min in a 37 C water bath. b. Transfer the content of the cryovial to a 15-mL tube and then add 5 mL of MEF culture medium. c. Centrifuge at 1,300 rpm for 3 min, aspirate the supernatant, and resuspend the pellet with MEF culture medium. d. Seed the cells to a gelatin-coated 6-well plate (1.0 3 10 6 cells/plate). e. Incubate the plate overnight at 37 C.
Note: We usually establish MEF feeder cells from embryonic day 13.5 Jcl:ICR mouse embryos.
Note: MEF feeder cells can be expanded more in 5% oxygen than in 20% oxygen. The expanding MEF feeder cells should be used within 10 passages.
Note: The MEF feeder cells can be also inactivated with Mitomycin C (MMC) treatment. When you use MMC, add the MMC-containing medium at a density of 10 mg/mL and incubate for 2-3 hours before harvesting the cells.
Note: Prepared dishes with the inactivated MEF layer should be used within 1 week.
Note: Store the solution at 4 C for up to 8 weeks.

Maintenance of human primed PSCs
Timing: 1-2 weeks 23. Prepare an inactivated MEF plate the day before step 24. 24. Thawing human primed PSCs a. Prepare an inactivated MEF-feeder plate and primed PSC medium. b. Warm the primed PSC medium in a 37 C water bath. c. Remove the cryovial of primed PSCs from liquid nitrogen and thaw 1-2 min in the 37 C water bath. d. Transfer the contents of the cryovial to a 15-mL tube and then add 5 mL of primed PSC medium. e. Centrifuge at 1,300 rpm for 3 min, aspirate the supernatant, and resuspend the pellet with 2 mL of primed PSC medium. f. Aspirate the MEF culture medium from the inactivated MEF-feeder plate and wash the wells with 1 mL of PBS 2 times. g. Add 2 mL of primed PSCs suspension to the well. 25. Replenish the medium every day until the culture cells reach 70-80% confluence. 26. The primed PSCs are passaged on the newly prepared MEF plates as clumps after treatment with CTK solution for 15-30 min.
Note: If you use a cryopreservation reagent that has a strong cytotoxic effect, such as DAP213, you should add prewarmed medium to the cryovials and thaw quickly. Note: Aliquot and freeze the solution at À20 C or less.

Chemical conversion to naive PSCs
The chemical conversion to naive PSCs is performed as previously described (Guo et al., 2017). The 5i/L/A and NK2 transgene methods can also be used for primed-to-naive resetting (Theunissen et al., 2014;Takashima et al., 2014).
28. Single-cell collection of primed PSCs. a. Incubate the primed PSCs on MEF feeder cells in primed PSC medium with 10 mM Y-27632 for more than one hour. b. Aspirate the medium of the well and wash the well with 1 mL of PBS 2 times. c. Add 500 mL TrypLE Express and incubate in a humidified incubator at 37 C for 5 min. d. Transfer the dissociated cells to a 15-mL tube and add 5 mL of Wash medium. e. Centrifuge at 1,300 rpm for 3 min, aspirate the supernatant, and resuspend the pellet with 1 mL primed PSC medium with 10 mM Y-27632. f. Prepare a 6-well plate and add 1 mL of 0.1% gelatin solution to a well. g. Aspirate the gelatin solution after 10 min of incubation at room temperature and transfer the cell suspension in 1 mL primed PSC medium with 10 mM Y-27632 on the gelatin-coated well. h. Incubate at 37 C for 2 h to make the MEF feeder cells attach onto the well. i. Collect the medium with floating PSCs and count the number of cells. 29. Seed the PSCs at a density of 1 3 10 4 cells/cm 2 on inactivated MEF feeder cells in primed PSC medium with 10 mM Y-27632. 30. On the next day, switch the medium to cRM-1 medium. 31. On day 3, replace the medium with cRM-2 medium. 32. Dome-shaped naive PSC colonies are observed around two weeks after plating. 33. Cells are split every 5-7 days after dissociation with Accutase. Fully reset naive PSCs are passaged and maintained on MEF feeders in t2iLGö medium ( Figure 1A). 34. After the medium is changed to t2iLGö medium, the cells can be dissociated with Accutase and passaged on newly prepared MEF-coated plates at a 1:4 split ratio every 3-5 days. Note: Naive PSCs should be passaged every three to five days. Over-confluency affects cell division and may reduce the efficiency of subsequent naive PSC-derived trophectoderm (nTE) and naive PSC-derived cytotrophoblasts (nCTs) differentiation.
Note: Mycoplasma contamination is also possible should the cells not grow. Mycoplasma contamination can be checked using a MycoAlert Mycoplasma Detection Kit.
Note: Store the solution at 4 C for up to 8 weeks.
Note: Store the solution at 4 C for up to one week.  Protocol Note: Store the solution at 4 C for up to one week.
Note: Store the solution at 4 C for up to one week.
Note: Store the solution at 4 C for up to one week.
Note: Store the solution at 4 C for up to one week.
Note: Store the solution at 4 C for up to one week. Note: Store the solution at 4 C for up to one week.
Note: Store the solution at 4 C for up to one week.

Induction of nTE
Timing: 2-3 days 1. Prepare Laminin-E8-coated wells beforehand. a. Dilute 1.5 mg (3 mL) of Laminin-E8 (iMatrix-511) in 1 mL/well of PBS and pipette the solution into one well of a newly prepared 6-well plate. b. Incubate at 37 C for at least 30 min.
Note: The Laminin-E8 concentration is critical and is 0.15 mg/cm 2 (0.3 mL/cm 2 ). Instead of precoating, Laminin-E8 can be added to the medium and pipetted up and down several times after the naive PSCs are seeded in Step 5.
Note: MEF feeder cells are not used for the nTE induction.

Dissociation of naive PSCs.
a. Aspirate the medium from the wells and rinse with PBS two times. b. Add 500 mL Accutase and incubate at 37 C for 10-15 min. c. Add 1 mL of Wash medium and gently pipette up and down to dislodge the naive PSCs from the well. d. Transfer the cells to a 15-mL tube and add 5 mL of Wash medium. e. Centrifuge at 1,300 rpm for 3 min. f. Aspirate the supernatant and resuspend the pellet with 1 mL of t2iLGö medium with Y-27632. g. Prepare a 6-well plate and add 1 mL of 0.1% gelatin solution to a well. h. Aspirate the gelatin solution after 10 min of incubation at room temperature and incubate the cell suspension in 1 mL t2iLGö medium with 10 mM Y-27632 on the gelatin-coated well. i. Incubate at 37 C for 2 h to make the MEF feeder cells attach to the well. j. Collect the medium with floating PSCs into a 15-mL tube and count the number of cells.
Optional: nTE can be induced without 10 ng/mL recombinant human BMP4 for the first 24 hours of the induction, although at a lower efficiency.
CRITICAL: The step of depriving MEF is critical. The residual MEF inhibits the differentiation of naive PSCs to nTE.
Note: A higher concentration of Laminin-E8 reduces the induction efficiency of nTE. A concentration of 0.10-0.25 mg/cm 2 Laminin-E8 is recommended.
Note: hPSCs should be cultured with 10 mM Y-27632 for 24 h upon/after seeding.
Note: Confirm that cell morphology on day 2 or 3 is polygonal and flat under the microscope ( Figure 1B).
Note: nTE strongly attaches to the dish and takes 20-30 minutes to dissociate using Accutase.
Note: RNA sequencing confirmed that naive PSC-derived TACSTD2 + ENPEPcells on day 2 and naive PSC-derived TACSTD2 + ENPEP + cells on day 3 have a high correlation with preimplantation trophectoderm in vivo.

Induction of nCTs
Timing: 4 h; 3-6 days until next splitting nTE differentiates into nCT under ACE condition (Io et al., 2021). 9. Prepare Laminin-E8-coated wells beforehand. a. Add 1.5 mg (3 mL) of Laminin-E8 to 1 mL of PBS and pipette up and down several times. b. Add Laminin-E8/PBS solution to one well of 6-well plates. c. Incubate at 37 C for at least 30 min. 10. Dissociation of nTE from step 8. a. Aspirate the medium from the wells and rinse with PBS two times. b. Add 500 mL Accutase and incubate at 37 C for 20-30 min. c. Add 1 mL of Wash medium and gently pipette up and down to dislodge the cells from the well. d. Transfer the cells to a 15-mL tube and add 5 mL of Wash medium. e. Centrifuge at 1,300 rpm for 3 min. 11. Aspirate the supernatant and resuspend the pellet with 2 mL HBSS with 1% BSA buffer (1% BSA/ HBSS) and Y-27632. 12. Incubate at 4 C for at least 30 min. 13. Centrifuge at 1,300 rpm for 3 min, aspirate the supernatant, and resuspend the pellet with 1% BSA/HBSS with anti-TACSTD2, biotin-conjugated antibody, and anti-ENPEP, PE-conjugated antibody. 14. Incubate at 4 C for 30 min in the dark. 15. Resuspend with 1 mL of 1% BSA/HBSS. 16. Centrifuge at 1,300 rpm for 3 min and aspirate the supernatant. 17. Resuspend with 1% BSA/HBSS with anti-Streptavidin, APC-conjugated antibody. 18. Incubate at 4 C for 30 min. 19. Resuspend with 1 mL of 1% BSA/HBSS. 20. Centrifuge at 1,300 rpm for 3 min and aspirate the supernatant. 21. Resuspend with 300-500 mL of 1% BSA/HBSS. 22. Sort the TACSTD2 + ENPEP + cells (Figure 2).
Note: As with any cell sorting, make sure the cell sorter is clean before sorting so that contamination with other cells or microbials is avoided.
Note: Keep the collection medium cool (4 C) during cell sorting to maintain the viability of the cells.
Note: Cell sorting should be performed with a sufficiently rigorous selection to obtain a cell fraction of high purity (Figure 2). 23. Seed the TACSTD2 + ENPEP + cells at a density of 2 3 10 4 -4 3 10 4 cells/cm 2 on Laminin-E8coated dishes in ACE medium with 10 mM Y-27632 and 1% Penicillin-Streptomycin. 24. Replace the ACE medium every two days. 25. Passage the cells every 3-6 days by dissociation with Accutase for 10-15 min and seed them at a 1:3-1:4 split ratio. Add 10 mM Y-27632 for every passage.
Note: The combination of biotin-conjugated anti-TACSTD2 antibody and APC-conjugated anti-Streptavidin antibody demonstrate the sensitivity and resolution (Figures 1B and 2).
Note: If you use fluorescent dye-conjugated antibodies as primary antibodies, you can omit the secondary antibody (anti-Streptavidin, APC-conjugated antibody).

Maintenance of nCTs
Timing: 4 h; 3-6 days until next splitting nCTs can be maintained in ACE medium for more than 40 passages as cytotrophoblast stem cells (Io et al., 2021).
26. Prepare Laminin-E8-coated wells beforehand. a. Add 1.5 mg of Laminin-E8 to 1 mL of PBS and pipette up and down several times. b. Add the Laminin-E8/PBS solution to one well of 6-well dishes. c. Incubate at 37 C for at least 30 min in 5% O 2 . 27. Dissociation of nCTs.
a. Aspirate the medium from the wells and rinse with PBS two times in 21% O 2 . b. Add 500 mL Accutase and incubate at 37 C for 10-15 min in 5% O 2 . c. Add 1 mL of Wash medium and gently pipette up and down to dislodge the cells from the well in 21% O 2 . d. Transfer the cells to a 15-mL tube and add 5 mL of Wash medium in 21% O 2 . e. Centrifuge at 1,300 rpm for 3 min in 21% O 2 . 28. Add ACE medium with Y-27632 for 24 h. 29. Seed the cells at a density of 4 3 10 4 cells/cm 2 on the Laminin-E8-coated wells in 21% O 2 and incubate the cells in 5% O 2 , 5% CO 2 . 30. Replace the medium every two days ( Figure 3A). 31. Passage the cells every 3-6 days by dissociation with Accutase for 10-15 min and seed them at a 1:3-1:4 split ratio. Add 10 mM Y-27632 for every passage.
Note: nCTs should be cultured with Y-27632 for 24 h with every passage.
Pause point: Cells can be cryopreserved in Stem-Cellbanker and stored in liquid nitrogen.
32. Prepare Laminin-E8-coated wells beforehand. a. Add 1.5 mg (3 mL) of Laminin-E8 to 1 mL of PBS and pipette up and down several times. b. Add the Laminin-E8/PBS solution to one well of 6-well dishes. c. Incubate at 37 C for at least 30 min. 33. Dissociation of nCTs.
a. Aspirate the medium from the wells and rinse with PBS two times. b. Add 500 mL Accutase and incubate at 37 C for 20-30 min. c. Add 1 mL of Wash medium and gently pipette up and down to dislodge the cells from the well. d. Transfer the cells to a 15-mL tube and add 5 mL of Wash medium. e. Centrifuge at 1,300 rpm for 3 min. 34. Resuspend with ST medium. 35. Seed the cells at a density of 1 3 10 4 cells/cm 2 on the Laminin-E8-coated wells. 36. Replace the medium at day 3. 37. Observe the nSTs on day 6 ( Figure 3B).
Note: The cells are incubated in 21% O 2 , 5% CO 2 in a humidified incubator at 37 C during the induction.
Note: Since ST medium contains Y-27632, no additional Y-27632 is needed at seeding.
a. Add 1.5 mg (3 mL) of Laminin-E8 to 1 mL of PBS and pipette up and down several times. b. Add the Laminin-E8/PBS solution to one well of 6-well dishes. c. Incubate at 37 C for at least 30 min. 39. Dissociation of nCTs.
a. Aspirate the medium from the wells and rinse with PBS two times. b. Add 500 mL Accutase and incubate at 37 C for 20-30 min. c. Add 1 mL of Wash medium and gently pipette up and down to dislodge the cells from the well. d. Transfer the cells to a 15-mL tube and add 5 mL of Wash medium. e. Centrifuge at 1,300 rpm for 3 min. 40. Resuspend with EVT-1 medium. 41. Seed the cells at a density of 0.8 3 10 4 cells/cm 2 on the Laminin-E8-coated wells. 42. Add 2% of Geltrex to the medium. 43. Replace EVT-1 medium with EVT-2 medium on day 3 and add 0.5% of Geltrex to the medium. 44. Dissociate the cells into single cells with Accutase for 10-15 min on day 6 and then resuspend with EVT-3 medium. 45. Seed the cells on new Laminin-E8-coated wells at a 1:2-1:3 split ratio. 46. Add 0.5% of Geltrex to the medium. 47. Analyze the cells on day 8 ( Figure 3C).
Note: The cells are incubated in 21% O 2 , 5% CO 2 in a humidified incubator at 37 C during induction.
Note: Since EVT medium contains Y-27632, no additional Y-27632 is needed at seeding.
Optional: You may switch to EVT-3 medium without re-plating on day 6.

Isolation of human cytotrophoblasts
Timing: 6 h; 3-6 days until next splitting This protocol describes the use of primary patient material. Please confirm that you are allowed to process primary patient material with your local Ethics Committee and request informed consent from the donors. Placental tissues should be kept under sterile conditions. 48. Prepare Laminin-E8-coated wells beforehand.
a. Add 1.5 mg (3 mL) of Laminin-E8 to 1 mL of PBS and pipette up and down several times. b. Add the Laminin-E8/PBS solution to one well of 6-well dishes. c. Incubate at 37 C for at least 30 min. 49. Human chorionic villi are manually separated from the chorionic membrane and decidua. 50. Cut the chorionic villi into small pieces and transfer to a 50-mL tube. 51. Rinse the villous fragments with 40 mL of sterile PBS. 52. Centrifuge at 800 rpm (120 g) for 2 min. 53. Aspirate most of the supernatant. 54. Repeat the rinse procedure. Shake the tube vigorously until the supernatant is clear. 55. Digest three times in a solution containing 0.25% Trypsin and 1 mg/mL collagenase IV, 200 U/mL DNase (Sigma-Aldrich), 25 mM HEPES, and DMEM/F-12 medium with agitation at 37 C. 56. Filter pooled cell suspensions through a 70-mm mesh filter (Corning) to remove debris and syncytiotrophoblasts. 57. Add 30 mL of Wash medium and centrifuge at 1,300 rpm for 3 min. 58. Resuspend with 1% BSA/HBSS and incubate on ice for 30 min to reduce the non-specific binding of antibodies. 59. Centrifuge at 1,300 rpm for 3 min, aspirate the supernatant, and resuspend the pellet with 1% BSA/HBSS with an Alexa Fluor 488-conjugated anti-TACSTD2 antibody, a PE-conjugated anti-ENPEP antibody, and a biotin-conjugated, anti-SIGLEC6 antibody. 60. Incubate at 4 C for 30 min. 61. Resuspend with 1 mL of 1% BSA/HBSS. 62. Centrifuge at 1,300 rpm for 3 min and aspirate the supernatant. 63. Resuspend with 1% BSA/HBSS with anti-Streptavidin, APC-conjugated antibody. 64. Incubate at 4 C for 30 min. 65. Collect TACSTD2 + ENPEP + SIGLEC6 + cells using a cell sorter.
Note: Keep the collection medium cool (4 C) during cell sorting to maintain the viability of the cells.
Note: Cell sorting should be performed with a sufficiently rigorous selection to obtain highpurity cell fraction.
Note: Given the increased risk of microbial contamination during purification step by flow cytometry, collected cells may be fed with ACE medium supplemented with 1% Penicillin-Streptomycin. Antimicrobial-supplemented medium should be used for a minimum of 2 days.
66. Centrifuge at 1,300 rpm for 3 min. 67. Aspirate the supernatant and resuspend with ACE medium supplemented with 1% Penicillin-Streptomycin. 68. Seed the cells at a density of 4 3 10 4 cells/cm 2 on the Laminin-E8-coated wells. 69. Replace the medium every two days. The cells are passaged every 3-6 days by dissociation with Accutase for 10-15 min and seeded at a 1:3-1:4 split ratio. 10 mM Y-27632 is added with every passage.
Note: If you use fluorescent dye-conjugated antibodies as the primary antibodies, you can omit the secondary antibody reaction.
CRITICAL: To maintain human primary cytotrophoblasts, cells should be sorted from firsttrimester placenta. Cytotrophoblasts sorted from full-term placenta cannot be maintained with ACE medium.

EXPECTED OUTCOMES
We can purify trophectoderm-like cells from naive PSCs.
nCTs maintain trophoblast markers and have the capacity to differentiate to nSTs and nEVTs according to previous reports (Okae et al., 2018;Io et al., 2021) (Figure 3).
Primary human cytotrophoblasts can be maintained and differentiated to STs and EVTs by the same procedure used for nCTs.

LIMITATIONS
The induction efficiency of nTE is highly dependent on the quality of the naive PSCs. We recommend that you check the quality of your naive PSC cultures before they are used for the experiments. It is also important to use several cell lines.
Although the global gene expression profiles of nTE and nCTs are very similar to their in vivo counterparts, the epigenomes of nTE and nCTs were not evaluated because there is less epigenetic data of in vivo human trophoblast development.
We usually obtain in vivo samples of human trophoblasts at 5-7 weeks pregnancy or full-term pregnancy. We do not know if cytotrophoblast stem cells can be established at other stages. Since we cannot access E14-E21 human embryos, we do not have in vivo human data on this stage.

Potential solution
The Laminin-E8 concentration affects the efficiency. The recommended concentration is 0.10-0.25 mg/cm 2 . Another possible cause is the contamination of MEF feeder cells during the nTE induction. If fibroblasts are observed in addition to naive cell-derived cells on the day after the nTE induction, less nTE is induced. In this case, MEF feeder cells should be removed more strictly. Longer incubation times of dissociated cells under gelatin-coated dishes will help remove MEF feeder cells, but also less PSCs are harvested. Repeat the procedure several times to determine the ideal incubation time.

Problem 2
Many differentiated cells are mixed with naive cells (steps 2).

Potential solution
Differentiated cells may reduce the induction efficiency of nTE or nCTs. The longer naive cells are cultured, the more likely differentiated cells are to appear. When MEFs are removed in Step 2i, the differentiated cells also stick to the bottom of the dish to some extent and are removed. However, if there are many differentiated cells and the induction rate is low, it is better to establish new naive cells from primed cells.

Potential solution
Adding Y-27632 to cultured naive PSCs at least 30 min before the dissociation can prevent cell death during the induction.

Potential solution
Adding Y-27632 to cultured nTE at least 30 min before the dissociation can prevent cell death during the induction. Immunostaining and cell sorting should be done as quickly as possible to avoid cell damage.

Problem 5
Excessive loss of cells during the digestion procedure of human chorionic villi (steps 55-57).

Potential solution
If the amount of chorionic villi specimen is too high compared with the digestive solution, cell clumps sometimes appear, and the single-cell isolation does not work well. Using a concentrated digestive solution, the samples can be dissociated completely without sticky clumps. DNase also reduces the formation of sticky cell clumps.

Problem 6
Many red blood cells are mixed in the sample (steps 55-57)

Potential solution
The most important and simple solution is to wash with PBS repeatedly. Additionally, you can use red blood cell lysis buffer (Santa Cruz Biotechnology Cat. sc-296258) to remove red blood cells. Density gradient reagents like Debris Removal Solution (Miltenyi Biotec Cat. 130-109-398) also may remove red blood cells.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Yasuhiro Takashima (y.takashima@cira.kyoto-u.ac.jp).

Materials availability
This study did not generate new unique reagents.

Data and code availability
This study did not generate datasets and codes.