Sep 04, 2025
- Yake Gao1,
- Mingying Li1,
- Jian Zhang1
- 1State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Center for Life Sciences, School of Life Sciences, Yunnan University, Kunming 650500, PR China
External link: https://doi.org/10.1038/s41556-025-01732-8
Protocol Citation: Yake Gao, Mingying Li, Jian Zhang 2025. MTSC-Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbkjkogpk/v1
Manuscript citation:
Yake Gao, Mingying Li, Wei Guan, Wei Guo, Shu Wei, Fang Yan, Wenrui Han, Xueting Zhang, Tong Yin, Yunkun Dang, Huanhuan Li, José C. R. Silva, Jian Zhang (2025) Mouse trophectoderm stem cells generated with morula signalling inducers capture an early trophectoderm state.Nature Cell Biology doi: 10.1038/s41556-025-01732-8
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
forms.status.working_default
Created: June 17, 2025
Last Modified: September 04, 2025
Protocol Integer ID: 220409
Keywords: morula-derived trophectoderm stem cell, 32-cell embryos, MTSC-organoids, protocol successful derivation of mouse trophoblast stem cell, mouse trophoblast stem cell, derived trophectoderm stem cell line, trophectoderm stem cell, trophectoderm stem cell line, uniformed trophoblast core gene expression, embryonic stem cell, cell embryo, understanding of early trophectoderm differentiation, early trophectoderm differentiation, trophoblast development, understanding of trophoblast differentiation, studying trophoblast development, cell lineage, cell lineages of the placenta, early blastocyst trophectoderm state, trophoblast differentiation, earlier trophectoderm state, placenta organoid, escs into mtsc, implant uterus, blastoid, differentiated mtsc, placenta, like cell
Abstract
Successful derivation of mouse trophoblast stem cells (TSCs) and trophectoderm stem cells (TESCs) greatly facilitates understanding of trophoblast differentiation. However, understanding of early trophectoderm differentiation remains incomplete. Here, we report establishment of a morula-derived trophectoderm stem cell line (MTSCs) from 32-cell embryos that show enhanced and uniformed trophoblast core gene expression. Importantly, distinct from TSCs or TESCs, MTSCs represent a much earlier trophectoderm state (E3.5) than those of TSCs(E5.5-6.5) and TESCs(E4.5-5.5). MTSCs can robustly integrate into all cell lineages of the placenta. Moreover, MTSCs can self-organize to form placenta organoids. When partially differentiated MTSCs aggregated with embryonic stem cells (ESCs), they form blastoids that efficiently implant uteruses. Finally, MSTC medium can efficiently convert ESCs into MTSC-like cells. Thus, MTSCs capture an early blastocyst trophectoderm state and provide a research model for studying trophoblast development.
Materials
| A | B | C | |
| REAGENT or RESOURCE | SOURCE | IDENTIFIER | |
| Chemicals, peptides, and recombinant proteins | |||
| RPMI 1640 | Gibco | 61870036 | |
| DMEM/F-12 | Gibco | 10565018 | |
| Neurobasal | Gibco | 21103049 | |
| GlutaMAXTM | Gibco | 35050038 | |
| KnockOut‱Serum Replacement | Gibco | 10828-028 | |
| MEM NEAA | Gibco | 2281490 | |
| Sodium Pyruvate | Gibco | 11360070 | |
| B-27 supplement | Gibco | 17504-044 | |
| N-2 supplement | Gibco | 17502-048 | |
| Acid Tyrode's Solution | Sigma | T1788 | |
| Hyaluronidase | Sigma | H3506 | |
| Trypsin-EDTA | Gibco | 25200056 | |
| TryPLETM Express | Gibco | 12605010 | |
| Gelatin | SARTORIUS | 01-944-1B | |
| OVOILTM | Vitrolife | 10029 | |
| 2-mercaptoethanol | Gibco | 31350010 | |
| L-ascorbic acid 2-phosphate | Sigma | A8960 | |
| Penicillin/streptomycin | Gibco | 1114050 | |
| Insulin-Transferrin-Selenium | Gibco | 41400-045 | |
| rhFGF-4 | R&D system | 235-F4 | |
| Heparin | Sigma | H3149 | |
| rhTGF-β1 | R&D system | 7754-BH-025 | |
| rhBMP4 | R&D system | 314-BP-010/CF | |
| Activin A | PeproTech | 120-14E | |
| CHIR99021 | Tocris | 4423/10 | |
| PD0325901 | Tocris | 4192/10 | |
| mLIF (ESGRO) | Merck | ESG1107 | |
| XAV939 | Sigma | X3004 | |
| A-83-01 | Tocris | 2939/10 | |
| Lats-IN-1 | TargetMol | T9053 | |
| Y27632 | Tocris | 1254 | |
| FBS | Vivacell | C04002-500 | |
| FBS | Gibco | 16141-079 | |
| BSA | Sigma | A1933 | |
| Mitomycin C | Sigma | M4287 | |
| Sodium selenite | Sigma | S5261 | |
| Transferrin | Sigma | T8158 | |
| 8-Br cAMP | BIOLOG | B 007-500 | |
| rmIL-11 | PeproTech | 220-11 | |
| Sodium Bicarbonate | Sigma | S5761 | |
| Anti-adherence rinsing solution | STEM Technologies | 07010 | |
| Gelatin Solution | SARTORIUS | 01-944-1B |
Troubleshooting
Derivation of MTSCs Protocol
Preparation of mice
The C57BL/6J and ICR mice were from the Laboratory Animal Center of Yunnan University. All mice were placed in a specific pathogen-free environment, with the temperature controlled (22-24°C) and the light/dark cycle for 12 hours. C57BL/6J mice were used to obtain embryos, and ICR mice were used to prepare pseudopregnant surrogates.
Collection of mouse embryos
At 6-7 PM, 6-8 week-old female mice were given an intraperitoneal injection of 8 IU of PMSG, followed 48 h later by 8 IU of HCG. Subsequently, the female mice were mated 1:1 with the male mice with normal fertility (the female mice were placed in the male cage), and the formation of vaginal plugs was checked and marked at 8-9 am the next day. At 2 p.m., the female mice with vaginal plugs were sacrificed by cervical vertebra removal, and the mice ovary and fallopian tube tissues were obtained from the ultra-clean bench.
They were washed twice with PBS buffer, and the enlarged ampulla of the fallopian tube was gently scratched under a stereoscopic microscope with a sterile 1 mL syringe needle to allow the fertilized eggs to overflow naturally. Fertilized eggs were transferred to hyaluronidase solution using a homemade glass Pasteurized pipette for digestion to remove residual cumulus granulosa cells.
In vitro culture of mouse embryos
Fertilized eggs were transferred to KSOM medium and cultured to the 32-cell stage, and the medium was covered with mineral oil. The culture environment was 37 ° C, 5% CO2 and 5% O2, and near saturated humidity.
Preparation of MTSC medium
MTSC medium consisted of RPMI-1640 basal medium supplemented with 20% FBS (VivaCell), 100× Sodium Pyruvate additive, 200× MEM NEAA additive, 25 ng/mL rhFGF4, 1 mg/mL heparin, 0.1 mM The mixture consisted of β-Mercaptoethanol, 1 μM XAV939, 2 μM Lars-IN-1, 10 μM Y27632, 10 ng/mL rhBMP4, 20 ng/mL Activin-A, and 100× penicillin/streptomycin. A 0.22 μm syringe filter was used to filter the medium, and the prepared medium was stored at 4 ° C. After storage for more than 10 days, the medium was discarded and fresh medium was prepared.
Derivation of MTSC
The zona pellucida of 32-cell stage embryos was removed with acidic Tyrode solution and placed in a 96-well culture plate preseeded with Feeder cells, in which one embryo was placed per well. 150 μL of MTSC medium was added to each well, and the medium was changed every 2 days.
After 24 hours, the Outgrowth attached to the feeder layer and began to proliferate outward. After 4-5 days, dark cells in the middle of the derivative were carefully removed with a homemade bent glass needle, the remaining cells were dissociated with TrypLE for 6 min, and the derivative was gently separated into a number of cell clumps using a homemade glass needle, which were passed into new wells for continued culture.
Three days later, a single stem cell colony was selected for expansion, and purified MTSC cell lines were obtained.
During subculture, when the confluence degree of MTSC cells reached 80-90%, the cells were washed twice with PBS and then digested with appropriate Accutase cell dissociation solution or TrypLE trypsin replacement solution for 8-10 minutes. The cells were recovered by centrifugation and resuspended, and subcultured according to the ratio of 1:4.
The feeder layer cells in MTSC need to be removed first when performing embryo chimeric experiments or embryo-like reconstruction experiments. The resuspended cells were seeded in a culture plate precoated with 0.1% gelatin and incubated in an incubator for 30-40 min, at which point most Feeder cells had attached to the bottom of the dish. The plates were gently shaken, the cell suspension was collected and centrifuged, and subsequently MTSC containing almost no Feeder cells was obtained.
Culturing of MTSC organoids
MTSCs were dissociated with 0.05% Trypsin-EDTA (Gibco, 25200056) for 5 min, and resuspended in growth factor reduced matrigel (Corning, 356231) at the liquid state on ice.
One 30 μL drop containing 8000-10000 cells was plated per well onto a 24-well culture plate, set at 37°C for 10 min, and overlaid with 500 μL maintenance medium and cultured in a humidified incubator supplemented with 5% CO2 at 37°C. The medium was replaced every 2 days.
MTSC organoids were passaged after 7-8 days with 1:8-10 dilution. Organoids were removed from matrigel by cell recovery solution(Corning, 354253) at 37°C, and dissociated by TryPLETM Express(Gibco, 12605010) for 8 min, gently pipetted every 2-3 min.
To differentiate MTSC organoids, the organoids were first cultured in a maintenance medium for 4 days and changed to a differentiation medium for another 4 days, and the differentiation medium was changed daily.
Maintenance and differentiation media were prepared according to the reference 1.
Preparation of ESC medium
ESC medium consisted of N2B27 medium supplemented with 10 ng/mL rhLIF, 1 μM PD0325901, and 3 μM CHIR99021. The N2B27 medium consisted of a 1:1 mixture of DMEM/F-12 basal medium and Neurobasal basal medium. 100× N2 additive (17502-048), 50× B27 additive, 0.1 mM β-Mercaptoethanol, 100× MEM NEAA additive, 100× GlutaMAX additive, and 5% KnockOut Serum were added The mixture consisted of Replacement and 100× penicillin/streptomycin.
A 0.22 μm syringe filter was used to filter the medium, and the prepared medium was stored at 4 ° C. After storage for more than 10 days, the medium was discarded and fresh medium was prepared.
ESC to MTSC-Like conversion
When the growth density of Naive ESCs reached about 60% (about 48 hours after passage), the ESC medium was removed by aspiration and replaced with MTSC medium for induction. The induction medium was changed daily.
During subculture, when the confluence degree of the cells reached 80-90%, the cells were washed twice with PBS and then digested with appropriate Accutase cell dissociation solution or TrypLE trypsin replacement solution for 8-10 minutes. The cells were recovered by centrifugation and resuspended, and subcultured according to the ratio of 1:4.
There is no need to screen the cells in the subculture process, and all the dissociated cells can be subcultured. The induction medium was changed daily.
After 3-4 weeks of subculture, almost all of the cells no longer expressed the ESC marker OCT4, but expressed CDX2. ESC-derived MTSC-like cells could be subcultured and maintained similar cell characteristics to MTSC.
Protocol references
- MAO Q, YE Q, XU Y, et al. 2023. Murine trophoblast organoids as a model for trophoblast development and CRISPR-Cas9 screening. Developmental Cell [J].58(24): 92-108. DOI: 10.1016/j.devcel.2023.11.007
Acknowledgements
This work was supported by the Scienceand Technology Leading Talent Project Grant, Yunnan Province (202005AB160006 to J.Z.), the National Key Research Program Project Grant (2018YFC1003304 to J.Z.).