Protocol for Human Blastoids Modeling Blastocyst Development and Implantation

Research on human embryos is limited by their low availability and ethical concerns, thus obtaining individual models that faithfully replicate early human development would support scientific and medical progress. The ability of this technique to predict human development depends on its ability to reproduce the sequences of blastocyst cellular determination and morphogenesis effectively, according to the developmental sequence and pace, and such modeling would ensure the formation of cells that truly reflect the blastocyst stage. In the future, human blastoids may be used to identify therapeutic targets and contribute to pre-clinical modeling, for example, to improve a in vitro fertilization medium, or to develop non-hormonal contraceptives.

Demonstrating the procedure will be Harunobu Kagawa, Alok Javali, Heidar Heidari Khoei, Theresa Maria Sommer, and Giovanni Sestini. To begin, prepare and prewarm the PXGL media, N2B27 basal media, washing buffer, PBS, and aggregation media. Exclude MEFs from the hPSC suspension for forming blastoids.

For MEF exclusion, prepare a gelatin coated plate by adding one milliliter of 0.1%gelatin into the well of a six-well plate. Incubate the plates at 37 degrees Celsius for 30 to 90 minutes. To harvest the cells, remove the medium, and wash the cells with one millimeter of PBS.

Add 500 microliter of cell detachment solution to each well of a six well plate, and incubate at 37 degrees Celsius for five minutes. Check the cells under the microscope to follow the dissociation of colonies into single cells. Dilute the cell detachment solution with one milliliter of washing buffer.

Collect the cells from the plate by gently pipetting five to 10 times. Transfer the cell suspension into a 15 milliliter tube. Spin down the cells at 200 RCF for four minutes.

Remove the supernatant, and resuspend the cells in 1.5 milliliters of PXGL medium in each well. Seed the cells on the gelatin coated plates for MEF exclusion, and incubate the plates at 37 degrees Celsius for 60 to 90 minutes. Once the naive cells are seeded for MEF exclusion, remove the PBS from MicroWells and equilibrate the wells with 200 microliters of basal N2B27 medium for each MicroWell chip, and incubate for 60 minutes at 37 degrees Celsius.

Collect the supernatant containing the unattached naive cells. Transfer it to a 15 milliliter tube, and spin down the cells at 200 RCF for four minutes. Discard the media, and resuspend the cells in one milliliter of N2B27 basal media.

Count the cells using cell counting slides. Spin down the cells at 200 RCF for four minutes. Discard the medium, and add an appropriate amount of N2B27 media containing 10 micromolar Y27632 to obtain a cell density of 30, 000 cells per 50 microliters.

Remove the medium from equilibrated MicroWell arrays, and add 25 microliters of N2B27 media with 10 micromolar Y27632. Add 50 microliters of cell suspension, and incubate at 37 degrees Celsius for 15 minutes to allow cells to fall into the bottom of the MicroWell. Then add 125 microliters of N2B27 medium, supplemented with 10 micromolar Y27632.

Within 24 hours, aggregates of naive hPSCs will be observed on the MicroWell chip. Initiate the blastoid formation by preparing the PALY medium. Prewarm it at 37 degrees Celsius for 30 minutes.

Remove the aggregation medium, and add 200 microliters of prewarmed PALY medium to the MicroWells. Place the cell culture plate back into a hypoxic incubator at 37 degrees Celsius. Repeat the media change on day one.

On day two, remove the PALY medium, and add 200 microliters of N2B27 medium, supplemented with LPA and 10 micromolar Y27632. Repeat media change on day three. Complete blastoid formation occurs by day four.

Prepare naive hPSC cell suspension for blastoid formation as described earlier. Discard the medium, and add an appropriate amount of N2B27 media containing 10 micromolar Y27632 to obtain the cell density of 70 cells per 100 microliters of the medium. To cluster cells at the bottom of the wells, centrifuge the plate at 200 RCF for two minutes at room temperature.

Incubate the plate at 37 degrees Celsius under hypoxic culture conditions. Within 24 hours, aggregates of naive hPSCs will be observed on the wells. Add 100 microliters of freshly prepared and prewarmed two times PALY medium to the wells.

Place the cell culture plate back in a hypoxic incubator at 37 degrees Celsius. After 24 hours, discard half of the media, and replace it with 100 microliters of prewarmed PALY medium. Repeat the step till day four.

Prepare naive HPSC cell suspension for troposphere formation as described earlier. Once aggregates of naive HPSCs have formed after 24 hours, exchange the aggregation medium with PALY without LIF, supplemented with three micromolar SC144 to form trophospheres representing early trophectoderm. To form trophospheres representing mature trophectoderm, exchange the aggregation medium with PALY, supplemented with two micromolar XMUMP1.

Refresh the medium daily. Complete trophosphere formation occurs by day four. To evaluate the cell states, compare the transcriptomic data from blastoid and trophospheres with the appropriate controls, including post implantation-like human trophoblast stem cells.

Naive hPSCs cultured in PXGL were aggregated and cavitated structures that emerged between 48 to 72 hours after PALY induction, and reached a diameter of 150 to 250 micrometer within 96 hours. Based on morphometric parameters and the presence of the three lineages, the induction efficiency reached 70 to 80%Trophospheres were formed at 50 to 60%efficiency within 96 hours of induction. Blastoid formation was successful in commercially available ultra low attachment 96-well plates under optimized induction conditions.

Single cell RNA sequencing technology was used to characterize blastoid cell state further. Cells display markedly reproducibly distinct clustering profiles, regardless of the parameters used to perform the clustering and visualization of data, which allowed distinguishing the three blastocyst lineages confidently. Between 24 and 60 hours, the PXGL pluripotent stem cells form analogs of the epiblast and of the trophectoderm.

Then between 60 and 96 hours, analogs of the polar trophectoderm, and of the primitive endoderm are formed. This is the sequence of specification within the blastocyst, thus blastoids recapitulate the developmental sequence of lineage specification. The merging of blastoids datasets with the reference dataset of cells isolated from embryos at different stages of development showed that 97%of the cells from blastoids clustered with the blastocyst cells, but not with cells from post implantation embryos.

Independent analysis confirmed that over time, the stem cells formed cells that transcriptionally matched with the cells of human embryos between day five and 7.5. This is indeed the developmental stage during which the human blastocyst forms. They also confirmed that more than 95%of the cells within blastoids have a transcriptome that matches the three cell lineages of blastocysts, while 3%of the cells were off target, and matched with post implantation stages.

Of note, we observed that our initial 2D cultures also comprise about 5%of off target cells. The equivalent developmental stage can also be visualized by looking at the expression patterns of specific markers such as CDX2 for the blastocyst trophectoderm, and PRMD14 for the blastocyst epiblast. The quality of the initial PXGL culture is absolutely crucial, and the number of cells within the initial aggregate is also critical, and it can be controlled using MicroWells.

The overall size of the cellular aggregate after 24 hours should be about 50 to 70 micrometers. The efficient formation of phase full blastoids is paving the way for studying the processes of implantation and post implantation development of the human embryo.