This project has received funding from the European Research Council (ERC) under the European Union&#39;s Horizon 2020 research and innovation program (ERC-Co grant agreement No.101002317 &#39;BLASTOID: a discovery platform for early human embryogenesis&#39;). H.H.K. is supported by the Austrian Science Fund (FWF), Lise Meitner Programme M3131-B. We thank Yasuhiro Takashima for sharing the H9 and H9-GFP cell lines, and Austin Smith, Peter Andrews, and Ge Guo for sharing the HNES1, Shef6, niPSC 16.2b and cR-NCRM2 cell lines. We thank Hossein Baharvand for sharing the endometrial organoids. We thank Joshua M. Brickman for sharing the RNA isolated from PrE differentiated cells and nEND cells. We thank Shankar Srinivas for sharing the single-cell RNA sequencing data of the peri-gastrulation embryo. We thank Aleksand Bykov and Luisa Cochella for technical assistance for SMARTSeq2 library preparation. We thank the NGS, Biooptic, and Stem Cell facility at IMBA for critical assistance.

The Guidelines for Stem Cell Research and Clinical Translation of the International Society for Stem Cell Research (ISSCR) recommends that research on human blastoids is permissible only after review and approval through a specialized scientific and ethics review process3,4. All the experimental procedures were conducted by following the guidelines of the human research ethics committee of the Institute of Molecular Biotechnology of the Austrian Academy of Science (IMBA) under the approval Rivron_Stellungnahme_2020-04-22. Compliance with these guidelines is necessary for publishing research results in scientific journals.
1. Culture of human naive embryonic stem cell in PXGL condition
NOTE: Naive hPSCs can be obtained from relevant laboratories. Lines used here were obtained from the laboratories of Yasuhiro Takashima (currently at CiRA, Kyoto, Japan) and of Austin Smith (currently at Living Systems Institute, Exeter, UK). Alternatively, naive hPSCs can be resetted in house from lines of primed hPSCs as described previously13,14. Naive hPSCs appear stable for multiple passages (&#62; 15) but the quality of the culture can vary over time. If the quality of naive hPSC decreases, thaw a new vial of cells or generate de novo naive hPSCs from primed PSCs. For all media compositions see Supplementary Table 1.
<ol>
	<li>Preparation of irradiated mouse embryonic feeder (MEFs) layer
	<ol>
		<li>The day before passaging of naive hPSCs, prepare a 6-well cell culture plate with irradiated MEF layers by following the steps described below.</li>
		<li>Coat a 6-well cell culture plate with 1 mL of 0.1% gelatin in PBS per well. Incubate the plate at 37 &#176;C for 30 min. Remove the gelatin solution.</li>
		<li>Prepare MEF medium at 37 &#176;C.</li>
		<li>Thaw MEFs in a water bath at 37 &#176;C until only a small ice clump is left. Dissolve the volume of the vial with 1 mL of prepared MEF medium using a P1000 pipette.</li>
		<li>Transfer the cell suspension into a 15 mL tube. Spin down the suspension at 200 x g for 4 min. Aspirate the supernatant and resuspend the MEF pellet by adding fresh MEF medium (sufficient for 1.5 mL per well).</li>
		<li>Count the cells using cell counting slides and add 300,000 cells per well and transfer the plate into a normoxy incubator at 37 &#176;C. 
		NOTE: If MEFs detach over time, fresh MEFs can be added into the PXGL medium during routine media change.</li>
	</ol>
	</li>
	<li>Passaging of human naive pluripotent stem cells
	<ol>
		<li>Before passaging hPSCs, check their morphology under the microscope. Colonies typically have a dome-shaped morphology with bright, defined borders. If the individual colonies show a flatter morphology or if differentiated colonies start to emerge, follow the instructions of step 1.2.8.</li>
		<li>Aspirate the medium and wash cells with PBS once. Add 500 &#181;L of cell detachment solution per well of a 6-well plate.</li>
		<li>Incubate the cells for 5 min at 37 &#176;C. Use a P1000 pipette and pipette the cells several times to dissociate the colonies into single cells.</li>
		<li>Collect the cells and transfer them into a 15 mL tube containing washing buffer (1 mL per well of a 6-well plate ). Spin down the cells at 200 x g for 4 min.</li>
		<li>Aspirate the supernatant and resuspend the pellet in fresh PXGL medium (sufficient for 1.5 mL per well). Consider a splitting ratio of 1:3-1:6 for routinely passaging. 
		NOTE: After every 3-4 passages or if the quality of cell culture decreases based on the cell morphology (e.g., the emergence of flat colonies in the population), add 10 &#181;M Y-27632 and growth factor basement membrane extract (5 &#181;L / well) to the medium during the first 24 h after passaging.</li>
		<li>Before replating the hPSCs, prepare the plates with fresh MEFs by aspirating the MEF medium and washing the cells once with PBS. Then, use a P1000 pipette to transfer 1.5 mL of hPSCs cell suspension per well of a 6-well plate containing the MEFs. 
		NOTE: Ensure that pipetting leads to a homogeneous seeding of the cells across the well area. This will ensure the growth of colonies with homogeneous sizes and relative synchrony of the cells.</li>
		<li>Culture hPSCs under hypoxic conditions at 37 &#176;C in a humidified environment. After 24 h, hPSCs should be attached. A high number of non-adherent (or floating) cells reflect a problem of viability or of attachment upon passaging.</li>
		<li>Change medium with 1.5 mL of PXGL medium per well daily. Passage hPSCs every 3-4 days or use them for blastoid formation experiments. 
		&#8203;NOTE: After thawing hPSCs, passage them for a minimum of three passages before starting a blastoid experiment.</li>
	</ol>
	</li>
</ol>
2. Formation of Blastoids
<ol>
	<li>Formation of naive PSC aggregates
	<ol>
		<li>Prepare and pre-warm the PXGL media, N2B27 basal media, washing buffer, PBS, and aggregation media before starting the experiment. Exclude MEFs from the hPSCs suspension for forming blastoids by following the steps described below.</li>
		<li>For MEF exclusion, prepare a gelatin-coated plate by adding 1 mL of 0.1% gelatin into the well of a 6-well plate and incubating at 37 &#176;C for 30-90 min.</li>
		<li>To harvest the cells, aspirate the medium and wash the cells with 1 mL of PBS.</li>
		<li>Add 500 &#181;L of cell detachment solution (per well of a 6-well plate) and incubate at 37 &#176;C for 5 min.</li>
		<li>Check the cells under the microscope to follow the dissociation of colonies into single cells (a few multicellular clumps can be dissociated later by gentle pipetting).</li>
		<li>Dilute the cell detachment solution with 1 mL of washing buffer. Collect the cells from the plate by gently pipetting 5 to 10 times. Transfer the cell suspension into a 15 mL tube. Spin down the cells at 200 x g for 4 min.</li>
		<li>Aspirate the supernatant, resuspend the cells in 1.5 mL of PXGL medium (per well of a 6-well plate) and seed the cells on the gelatin-coated plates for MEF exclusion and incubate at 37 &#176;C for 60-90 min.</li>
		<li>Once the naive cells are seeded for MEF exclusion, remove the PBS from microwells and equilibrate the wells with 200 &#181;L of basal N2B27 medium (per 1 microwell chip) and incubate for 60 min at 37&#160;&#176;C.</li>
		<li>Collect the supernatant containing the unattached naive cells, transfer it to a 15 mL tube, and spin down the cells at 200 x g for 4 min.</li>
		<li>Aspirate the media and resuspend the cells in 1 mL of N2B27 basal media. Count the cells using cell counting slides. Spin down the cells at 200 x g for 4 min.</li>
		<li>Aspirate the medium and add an appropriate amount of 10 &#181;M Y-27632 contained in N2B27 media to obtain a cell density of 30,000 cells per 50 &#181;L. 
		NOTE: Optimal initial seeding cell number can vary among the different cell lines. For example, to seed 50-60 cells/microwell, 30,000 cells (including surplus considering some cells fall outside of the microwell) are seeded in 1 well of 96 well plate which contains 430 microwells. An inappropriate starting cell number can result in the small aggregate formation without cavity or formation of cavitated structure reaching more than 250 &#181;m.</li>
		<li>Aspirate the medium from equilibrated microwell arrays and add 25 &#181;L of N2B27 media with 10 &#181;M Y-27632. Add 50 &#181;L of cell suspension and incubate at 37&#160;&#176;C for 15 min (until cells fall into the bottom of the microwell). Then, add 125 &#181;L of N2B27 medium supplemented with 10 &#181;M Y-27632.</li>
	</ol>
	</li>
	<li>Blastoid development
	<ol>
		<li>Within 24 h, aggregates of naive hPSCs can be observed (day 0) on the microwell chip. To initiate blastoid formation, prepare PALLY medium and follow the steps outlined below.</li>
		<li>Pre-warm PALLY medium at 37 &#176;C for 30 min. 
		NOTE: 1-Oleoyl Lysophosphatidic acid (LPA) and 10 &#181;M Y-27632 need to be added just before use. The optimal concentration of LPA varies between 0.5-5 &#181;M. This has to be titrated for individual hPSC lines used for blastoids.</li>
		<li>Aspirate the aggregation medium. Add 200 &#181;L of pre-warmed PALLY medium to the microwells. Place the cell culture plate back into a hypoxic incubator at 37 &#176;C. Repeat the media change on day 1.</li>
		<li>On day 2, remove PALLY medium and add 200 &#181;L of N2B27 medium supplemented with LPA and 10 &#181;M Y-27632. 
		NOTE: On day 2 the majority of aggregates continue to grow. However, some aggregates form small cavities. Continuously culture blastoids in PALLY until day 4 or in in vitro culture medium (IVC1) from day 2 onwards. However, following this media change enhances the formation of PrE in mature blastoids.</li>
		<li>Repeat media change on day 3. Complete blastoid formation occurs by day 4. 
		&#8203;NOTE: Blastoids are considered to have fully developed when they have undergone complete morphogenesis based on morphometrics of day 7 human blastocysts (e.g., a diameter range from 150 -250 &#181;m; one inner cluster surrounded by an epithelium of trophectoderm-like cells) and have formed polar trophectoderm-like cells (NR2F2+/CDX2-) and PrE-like cells (GATA4+). This can be assessed using immunofluorescence staining or fluorescence-activated cell sorting (FACS).</li>
	</ol>
	</li>
</ol>
3. Formation of blastoids in 96-well ultra low attachment microplates
<ol>
	<li>Prepare naive hPSCs cell suspension for blastoid formation by following the steps described above from 2.1.1 - 2.1.11.</li>
	<li>Aspirate the medium and add an appropriate amount of N2B27 media containing 10 &#181;M Y-27632 to obtain the cell density of 70 cells per 100 &#181;L of the medium. 
	NOTE: Optimal initial seeding cell number can vary among different cell lines. For example, 70 cells/well can be the optimal cell number for most cell lines.</li>
	<li>Centrifuge the plate at 200 x g for 2 min at room temperature to cluster cells at the bottom of the wells.</li>
	<li>Incubate the plate in an incubator at 37 &#176;C under hypoxic culture conditions. Within 24 h aggregates of naive hPSCs can be observed (day 0) on the wells.</li>
	<li>Prepare 2x PALLY medium. Add 100 &#181;L of prewarmed 2x PALLY medium to the wells.</li>
	<li>Place the cell culture plate back in a hypoxic incubator at 37 &#176;C. After 24 h, aspirate half of the media (100 &#181;L) and replace it with 100 &#181;L of prewarmed PALLY medium. Repeat the step till day 4. Ensure not to aspirate the aggregates. 
	&#8203;NOTE: On day 2, the majority of aggregates continue to grow. However, some aggregates have small liquid-filled cavities. On day 4, the majority of the cavitated structures undergo complete morphogenesis to form blastocyst-like structures.</li>
</ol>
4. Formation of trophospheres
<ol>
	<li>For trophosphere formation, follow the blastoid formation protocol from step 2.1.1 (MEF exclusion) until step 2.1.12 (last step of the seeding protocol).</li>
	<li>Once aggregates of naive hPSCs have formed after 24 h, exchange the aggregation medium with PALY (without LIF) supplemented with 3 &#181;M SC-144 for the formation of trophospheres representing early trophectoderm and PALLY supplemented with 2 &#181;M XMU-MP-1 for the formation of trophospheres representing mature trophectoderm.</li>
	<li>Refresh the medium daily. Complete trophosphere formation occurs by day 4.</li>
</ol>
5. Analysis of the blastoid cells state and its reflected stage using scRNAseq
<ol>
	<li>To pick up blastoids and perform dissociation, warm up the shaking incubator to 37 &#176;C and set it to 100 rpm.</li>
	<li>Collect blastoids from the initial 96-well plate and transfer them to multiple wells of a U-bottom 96-well plate using a mouth pipet equipped with a glass capillary. 
	NOTE: Blastoids (&#62; 70%) should be selected based on the morphometric criteria (size = 150-250 &#181;m with a unique inner cluster) in order to avoid contamination with non-blastoid structures (&#60; 30%).</li>
	<li>Wash once with 200 &#181;L of PBS using a P200 by viewing under a stereomicroscope. Transfer to a well containing 50 &#181;L of collagenase and incubate for 30 min in the shaking incubator.</li>
	<li>Transfer the blastoids to a well with 100 &#181;L of 10x trypsin-EDTA and mix well. Incubate for 20 min in the shaking incubator.</li>
	<li>Dissociate the blastoids into single cell by using P200 pipette. Transfer the cells into a 15 mL tube with FACS buffer (1% FBS in PBS).</li>
	<li>To capture specific ratios of the analogs of the three lineages, stain the TE and PrE analogs with TROP2 and PDGFRa antibodies respectively. 
	NOTE: The number of PrE cells in human blastocysts is less as compared to mouse blastocysts, which might reflect developmental defects of blastocysts formed through in vitro fertilization (IVF) or a species difference. In blastoids, the PrE analogs are less abundant than the TE and EPI analogs and represent 7.4% of the cells upon counting of GATA4+ cells by immunofluorescence imaging. Also, the dissociation process might induce biases in the proportions of the different cell types as PrE analogs to represent 1%-2% of the cells upon blastoid dissociation, PDGFRa tagging and, FACS analysis.</li>
	<li>FACS-sort cells from all three lineage analogs into 384-plates containing a lysis buffer for smart-seq2 analysis. Exclude dead cells marked by DAPI staining (performed according to the manufacturer&#39;s instructions).</li>
	<li>To evaluate the cell states (cell type and developmental stage), compare the transcriptomic data from blastoid with the appropriate controls.</li>
</ol>
6. Extended culture to assess blastoid developmental progression
<ol>
	<li>Culture human blastoids on basement membrane matrix-coated plates (glass bottom).
	<ol>
		<li>Coat the plate with basement membrane matrix.</li>
		<li>Visually inspect the blastoids to assess and record morphology. 
		NOTE: Only blastoids that display the classic hollow-ball blastocyst morphology with compact ICM have the potential of growing and developing further.</li>
		<li>Add 100 &#181;L of CMRL medium-1 per one well of the 96-well plate, and place the plate in the incubator at least 2 h before blastoids transfer for equilibration.</li>
		<li>Using a stereomicroscope, visually identify the blastoids with good morphology, transfer the selected blastoids in a well of 96-well plate containing 100 &#181;l of CMRL medium-1 to remove the traces of blastoid media.</li>
		<li>Transfer the blastoids into the well containing pre-equilibrated CMRL media-1. Place the plate in the incubator and incubate at 37&#176;C overnight. 
		NOTE: Upto 5 blastoids can be cultured in one well of 96 well plates. Having too many blastoids in a single well may lead to the formation of aggregates of multiple blastoids.</li>
		<li>The next day, visually inspect the blastoids under a microscope. If the blastoids are attached, add 100 &#181;L of pre-equilibrated CMRL media-1 supplemented with 5% basement membrane matrix. Place the plate in the incubator and incubate at 37 &#176;C overnight.</li>
		<li>The next day, monitor the blastoids under a microscope. Remove half of the media (100 &#181;L) and replace it with 100 &#181;L of pre-equilibrated CMRL media-2 supplemented with 5% basement membrane matrix.</li>
		<li>On the following days, replace half of the media (100 &#181;L) with pre-equilibrated CMRL media-3 supplemented with 5% basement membrane matrix. Place the plate in the incubator and incubate at 37 &#176;C overnight. Repeat every day for up to 4-6 days of in vitro culture. 
		NOTE: We have cultured blastoids for up to 6 days in extended culture conditions which corresponds to the time equivalent of day 13 of in vitro cultured human embryos.</li>
	</ol>
	</li>
</ol>
7. Immunostaining blastoids
<ol>
	<li>Aspirate the medium. Wash samples 3x with PBS for 5 min.</li>
	<li>Add 200 &#181;L of cold 4% paraformaldehyde (PFA) in PBS and fix samples for 30 min at room temperature. Remove PFA solution and wash samples 3x with PBS for 10 min. 
	NOTE: If blastoids were cultured on microwell chips, transfer the blastoids from the chip into 96-well U-bottom plates for the following steps.</li>
	<li>Permeabilize and block the blastoids in 100 &#181;L of blocking solution per well (PBS containing 0.3% Triton-X 100 and 10% normal donkey serum) for 60 min. 
	NOTE: Depending on the host species of the antibodies, adapt serum accordingly.</li>
	<li>Remove the blocking solution. Add 100 &#181;L primary antibodies diluted in fresh blocking solution and incubate samples overnight at 4&#176;C. 
	NOTE: The concentrations of primary antibodies must be determined based on the manufacturer&#39;s instructions.</li>
	<li>Wash samples 3x with 0.1% Triton-X 100 in PBS (washing solution) for 10 min. Add 100 &#181;L of secondary antibodies in blocking solution together with 20 &#181;g/mL Hoechst nuclear stain and incubate samples for 1 h at room temperature. Protect samples from light. 
	NOTE: The concentrations of secondary antibodies must be determined based on the manufacturer&#39;s instructions.</li>
	<li>Wash samples 3x with washing solution for 10 min. For imaging, transfer the samples onto the glass bottom &#181;-slide in PBS. 
	NOTE: The mounting medium should be selected based on the objective used for the imaging. For example, 80% glycerol in PBS is possible to use for mounting the samples while using oil objectives.</li>
</ol>

The Institute for Molecular Biotechnology, Austrian Academy of Sciences has filed a patent application EP21151455.9 describing the protocols for human blastoid formation and the blastoid-endometrium interaction assay. HK, AJ, HHK, and NR are the inventors of this patent. All other authors declare no competing interests.

In the present study, we show, step by step, how to establish human blastoids with high efficiencies using a simple and robust protocol. Upon aggregation of naive PXGL hPSCs and their triple inhibition, blastoids form efficiently (&#62; 70%) and sequentially generate the 3 blastocyst analogs within 4 days. Limitations in the efficiency and quality of the blastoids (e.g., presence of off-target cells) can occur if the initial state is sub-optimal. Of note, we have measured that PXGL hPSCs contains about 5% of cells reflecting the post-implantation stages. These cells might limit the formation of high-quality blastoids. Beyond the initial naive PXGL state that reflects the blastocyst epiblast, another pivotal factor is the medium used for blastoid formation. In order to rapidly form blastocyst-like cells and prevent the formation of off-target, post-implantation-like cells, we propose that triple pathways inhibition (Hippo, ERK, TGF-&#946;) is essential. While different cell lines give different yields of blastoid upon ERK/TGF-&#946; inhibition (generally around 10%-20%), exposure to LPA results in the formation of equally high blastoid yield across all cell lines, while using strict morphometric and lineage specification criteria. LPA possibly acts on the inhibition of the Hippo pathway, which plays a critical role in the first lineage segregation between epiblast and trophectoderm lineages in mouse and human8,51. The significant improvement of the blastoid efficiency by LPA suggests that the Hippo pathway-mediated inner-outer cell specification mechanisms at play in the blastocyst are co-opted during blastoid formation. A current limitation lies in the fact that, due to a sub-optimality of the protocols used to culture human blastocyst or blastoids at time-equivalent day 7-13 (after blastocyst/blastoid formation), we are not capable of assessing to which extent we can properly model post-implantation development.
Analyzing the transcriptomic state of the blastoid cells can easily be achieved using scRNAseq, adequate reference maps, and bioinformatic methods. Previously, the transcriptomic analysis showed that hPSCs cultured in PXGL are more similar to the blastocyst epiblast as compared to the primed state. Limitations in the analysis of the data can occur if the reference map only comprises blastocyst-stage cells. The reference map should include cells originating from post-implantation embryos in order to assess the presence of potential off-target cells. In the future, in order to benchmark blastoid cells, a reference map including all the tissues of the pre-and post-implantation human conceptus would be extremely valuable. In addition, multi-omics single cell reference maps, for example including transcriptome, chromatin accessibility, and DNA methylation, would further help. Finally, standardized bioinformatic methods to quantitatively assess the similarities between cells from embryo models and reference concepti, and to positively identify off-target cells would further help to unbiasedly analyze and compare results.
Altogether, blastoids formed by triple inhibition of Hippo, TGF-&#946;, and ERK pathways possess the four features of 1) highly efficient morphogenesis, 2) correct sequence of lineage specification, 3) high purity of blastocyst-like cells at the transcriptome level, 4) capacity to model peri-implantation development. These features of blastoids will facilitate building hypotheses on blastocyst development and implantation, however, they do not recapitulate earlier stages of embryonic development. In contrast to the limited accessibility and versatility of human blastocyst, blastoids are amenable to genetic and drug screens for the functional investigations of blastocyst development and implantation. In the future, such basic knowledge could contribute to improving IVF media formulation, developing post-fertilization contraceptives, and better managing early pregnancy.

A lack of experimental models has limited the understanding of early human embryogenesis. Current knowledge of human-specific aspects of embryonic development is derived from surplus in vitro fertilization (IVF) embryos donated for research. However, the limited availability, difficulties of experimental manipulations, and variable quality of the embryos hinder scientific investigations. On the contrary, a faithful in vitro model of human embryos would allow for complex experimental manipulations, thus providing an ethical opportunity to complement the research on human embryos1,2,3,4. A previously developed model of mouse blastocysts combined mouse embryonic stem cells and trophoblast stem cells5. In this detailed protocol, a method to generate a model of the human blastocyst from naive pluripotent stem cells that is faithful to elemental blastocyst criteria is described6.
Four criteria for human blastoids. Here, in an attempt to establish a standardized definition of human blastoids, we propose four minimal criteria. Although not exhaustive, these criteria might serve as a basis to evaluate the parameters that permit the formation of human blastoids (Figure 1A). (1) Blastoids should form efficiently in terms of morphology and of generation of the analogs of the three lineages namely, epiblast (Epi), trophectoderm (TE), and primitive endoderm (PrE). Inefficiency is likely to point at an inadequate initial cell state or/and culture condition (e.g., blastoid medium). (2) Blastoids should generate analogs of the three lineages according to the developmental sequence (Epi/TE first, PrE/polarTE last)7,8 and timing (induction ~ 3 days; embryonic days 5-7)7,9. (3) Blastoids should form analogs of the blastocyst stage, but not of post-implantation stages (e.g., post-implantation epiblast, trophoblast, or amnion cells). (4) Finally, blastoids should be capable of recapitulating functional features of blastocyst implantation and development. Using this protocol, human blastoids form efficiently using multiple cell lines (&#62;70%), are able to generate the blastocyst cellular analogs sequentially and within 4 days, and the analogs are transcriptionally similar to the blastocyst stage (&#62;96% based on several analysis)6,10,11. Finally, blastoids robustly generate the embryonic-abembryonic axis, which allows them to interact with hormonally stimulated endometrial cells through the polar region, and robustly expand the lineages upon extended culture (time-equivalent: embryonic day 13).
The importance of the initial cell state. Human pluripotent stem cells (hPSCs) can be stabilized in different states that attempt to capture precise developmental stages. These states are sustained by culture conditions which, although still suboptimal, constrain cells in a pre-implantation- (~&#160;embryonic days 5-7) or post-implantation-like (~ embryonic days 8-14) epiblast stage12. Transcriptomic analysis showed that hPSCs cultured in PD0325901, XAV939, G&#246;6983, and leukemia inhibitory factor (LIF; termed PXGL naive hPSCs)13,14&#160;are more similar to the blastocyst epiblast as compared to hPSCs cultured in fibroblast growth factor (FGF) 2 and activin15&#160;(termed primed hPSCs12) and to human extended pluripotent stem cells (hEPSCs)16&#160;(see analysis in references17,18,19). Accordingly, the transcriptome of primed hPSCs best matches with a post-implantation/pre-gastrulation cynomolgus monkey epiblast20. Additional molecular criteria, like transposon expression, DNA methylation, and X chromosome state, confirmed that variations of the naive state more closely resemble the blastocyst epiblast as compared to the primed state17,21. Finally, lines of naive hPSCs have been successfully derived directly from blastocysts using PXGL culture conditions22.
Human early blastocyst cells are not yet committed. Murine lineage specification occurs from the morula stage that precedes the blastocyst stage23. On the contrary, dissociation and re-aggregation experiments have shown that the human trophectoderm cells of early blastocysts are not yet committed24. Accordingly, analysis of the cells of human blastocysts by single-cell RNA sequencing (scRNAseq) has shown that the first lineage specification (trophoblast/epiblast) occurs after the formation of the blastocyst cavity7. This deferred human specification correlates with observations that hPSCs are potent to form trophoblasts25,26,27&#160;when mouse PSCs are largely committed to the epiblast lineage. These combined observations led to the possibility that naive hPSCs reflect a blastocyst stage and retain the potential to form the three blastocyst lineages. Lately, the potency of hPSCs to specify extraembryonic analogs has been proposed to shift from trophectoderm to amnion during progression from naive to primed state27. Thus, naive hPSCs are more similar to the pre-implantation stage17,18,21&#160;and have an enhanced capacity to form trophoblasts as compared to primed hPSCs27, hEPSCs16, or intermediate reprogrammed states28, which are prone to form post-implantation analogs10&#160;(Figure 1B). The initial cell state is thus crucial to forming the appropriate extraembryonic analogs. Although a thorough side-by-side analysis of converted trophectoderm analogs remains to be done, a PXGL naive state reflecting the early blastocyst appears important to form high-fidelity blastoids.
Prompting specification and morphogenesis by signaling pathways inhibition. The inhibition of the Hippo signaling pathway is a conserved mechanism driving trophoblast specification in mice, cows, and humans9,29,30. Also, since 2013, it is known that the inhibition of the NODAL (A83-01) and the extracellular signal-regulated kinase (ERK; PD0325901 or equivalent) and the activation of the bone morphogenetic protein (BMP) signaling pathways triggers primed hPSCs to activate the transcriptional network associated with the trophoblast lineage25,31,32,33,34. Moreover, recently several reports also confirmed that the inhibition of both NODAL and ERK pathway and activation of BMP facilitate the trophoblast differentiation from naive hPSCs25,31,32,33,34. Finally, if trophoblast specification is triggered from a naive state, cells recapitulate aspects of the developmental progression of the trophectoderm26. However, self-renewing lines reflecting the blastocyst trophectoderm haven&#39;t been stabilized in vitro. Following trophoblast specification, induction of the epidermal growth factor (EGF) and Wnt signaling pathways along with HDAC inhibition might facilitate trophoblast developmental progression34,35&#160;and stabilize cells into lines of human trophoblast stem cells (hTSCs) reflecting post-implantation cytotrophoblasts18,35. Such lines can be derived both from blastocysts and placental tissues35.
The second extraembryonic lineage, termed PrE, is specified after trophoblasts and originates from the epiblast7,9. Contrary to murine PrE36, the human counterpart is thought to be independent of FGF signaling37,38. Lines reflecting the extraembryonic endoderm (termed nEnd) were established from naive hPSCs by induction of signaling pathways using activin A, Wnt, and LIF39. Inconsistent with embryo inhibition experiments, ERK inhibition has been shown to prevent the formation of such nEND cells in vitro39. Until now, such lines have not been derived directly from blastocysts.
Lately, models of the early embryo have been formed by combining variations of the media previously developed for hTSCs35&#160;and nEND cells39&#160;thus using activators of the transforming growth factor- &#946; (TGF-&#946;), EGF, and Wnt signaling pathways28,40. These embryo models form at low efficiency (10%-20%) and form cells resembling the post- rather than pre-implantation stage10, including analogs of the post-implantation epiblast, trophoblast, amnion, gastrula, mesodermal tissues (~ embryonic day 14), and cytotrophoblasts10. On the contrary, a triple inhibition of the Hippo, ERK, and TGF-&#946;&#160;pathways&#160;efficiently guides the formation of blastoids comprising blastocyst-like cells41. Along with the initial cell state, we propose that triple pathways inhibition (Hippo, ERK, TGF-&#946;) is the second essential parameter to form high-fidelity blastoids (Figure 1B).
Evaluating the cell state and reflected stage using scRNAseq. The states of cells composing blastoids can be evaluated through scRNAseq analysis. Their transcriptional similarity to specific embryonic stages can be measured using blastoid cells alone and by comparison with primed hPSCs or hTSCs that reflect post-implantation stages20,35. Performing cluster analyses using different levels of definition reveals how subpopulations progressively merge when definition decreases, thus revealing clusters&#39; similarities. Although optimality in the number of clusters can be measured42, high-resolution clustering also informs on the eventual presence of small abnormal subpopulations, for example reflecting the post-implantation stages10. The genes differentially expressed between clusters can provide information on their analogs in the development process by assessing the expression levels of reference gene sets that define stage-specific lineages. This allows measuring the enrichment of blastoid subpopulations either through unsupervised distance maps (e.g., using top enriched genes) or by gene set enrichment analysis (GSEA)43. Using this blastoid protocol, only three main clusters form that transcriptionally reflect the three blastocyst lineages. One cluster includes both the initial naive hPSCs and the epiblast analog of the blastoids. Analyzing cells at different time points showed the sequential nature of lineages specification (trophoblasts start to specify within 24 h, and primitive endoderm cells within 60 h). A high-resolution clustering captured a subpopulation of cells (3.2%) expressing genes specific to gastrulation stage embryos (possibly mesoderm or amnion). Of note, the initial naive hPSCs also comprised 5% of post-implantation-like cells, as previously described44. In a second analysis, blastoid cells can be merged in silico with reference cells isolated from concepti at different stages45,46,47&#160;in order to infer stage equivalence. Here, cells isolated from pre-implantation concepti45,46, in vitro cultured blastocysts45, and gastrulation-stage embryos47&#160;were used as reference points. Using this protocol, it was quantified that the mismatched blastoid cells revealed by high-resolution clustering indeed cluster with post-implantation mesoderm and amnion. In future steps, transcriptome benchmarking should be complemented with analysis of transposon expression, DNA methylation, and of the X chromosome status that also provide landmarks of developmental stages21.
Evaluating axis formation and other functionalities of human blastoids. A mature blastocyst is characterized by the formation of the embryonic-abembryonic axis patterning trophoblasts for implantation. Using this blastoid protocol, an axis robustly forms exemplified by a maturation of the proximal trophoblasts (e.g., NR2F2+/CDX2-) that acquire the capacity of attaching to endometrial organoid cells only when they are hormonally-stimulated48,49. Comparison with trophospheres that do not form the epiblast shows that these inner cells induce abutting trophoblasts to mature so as to mediate the initial attachment to the endometrium. When cultured in an extended culture medium designed for cynomolgus monkey blastocysts50, all three lineages from the blastoid consistently expand for six additional days (time-equivalent of day 13) although their organization doesn&#39;t reflect that developmental stage.
The implication of high-efficiency and high-fidelity human blastoids. The conservation of developmental principles that were discovered in model organisms is inherently difficult to test in the human conceptus due to the restricted access and to the technical difficulties in genetically and physically manipulating it. A high-efficiency and high-fidelity blastoid model would allow for high-throughput genetic and drug screens, which are at the basis of scientific and biomedical discoveries. In addition, the incorporation of complex genetic modifications to alter and record biological processes would complement such studies. Overall, we propose that the triple inhibition (Hippo, TGF-&#946;, ERK) of naive PXGL hPSCs is conductive for the efficient formation of high-fidelity human blastoids complying with the four minimal criteria. The scalable and versatile nature of this protocol makes it suitable to generate targeted hypotheses that can then be validated using human blastocysts. As such, human blastoids will not replace the use of human conceptus for in vitro research but might act as a powerful way to funnel research through previously inaccessible experimental approaches at the heart of the scientific and biomedical discovery process. The protocol shows how to form human blastoids and also how to analyze the cells that are contained within the blastoid.

<table><tbody><tr><td>Neurobasal media</td><td>in house</td><td></td><td></td></tr><tr><td>DMEM/F12</td><td>in house</td><td></td><td></td></tr><tr><td>100X N2 supplemen</td><td>Gibco</td><td>17502048</td><td></td></tr><tr><td>50X B27 supplement</td><td>Gibco</td><td>17504044</td><td></td></tr><tr><td>100X Glutamax</td><td>Gibco</td><td>35050038</td><td></td></tr><tr><td>100 mM Sodium Pyruvate</td><td>Gibco</td><td>11360039</td><td></td></tr><tr><td>MEM-Non-essential amino acids</td><td>Gibco</td><td>11140050</td><td></td></tr><tr><td>1 M Hepes</td><td>in house</td><td></td><td></td></tr><tr><td>50 mM 2-Mercaptoethanol</td><td>Thermofisher</td><td>31350010</td><td></td></tr><tr><td>100X Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P0781</td><td></td></tr><tr><td>Bovine Serum Albumin solution</td><td>Sigma-Aldrich</td><td>A7979</td><td></td></tr><tr><td>PD0325901</td><td>Medchem express</td><td>HY-10254</td><td></td></tr><tr><td>XAV-939</td><td>Medchem express</td><td>HY-15147</td><td></td></tr><tr><td>G&amp;ouml; 6983</td><td>Medchem express</td><td>HY-13689</td><td></td></tr><tr><td>Human recombinant Leukemia Inhibitory Factor</td><td>in house</td><td></td><td></td></tr><tr><td>A83-01</td><td>Medchem express</td><td>HY-10432</td><td></td></tr><tr><td>1-Oleoyl Lysophosphatidic acid (LPA)</td><td>Peprotech</td><td>2256236</td><td></td></tr><tr><td>Y-27632</td><td>Medchem express</td><td>HY-10583</td><td></td></tr><tr><td>CMRL medium</td><td>Gibco</td><td>21530027</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma-Aldrich</td><td>F7524</td><td></td></tr><tr><td>KnockOut Serum Replacement (KSR)</td><td>Thermofisher</td><td>10-828-028</td><td></td></tr><tr><td>Accutase</td><td>Biozym</td><td>B423201</td><td>cell detachment solution</td></tr><tr><td>Geltrex</td><td>Thermofisher</td><td>A1413302</td><td>growth factor basement membrane extract</td></tr><tr><td>TROP2 antibody</td><td>R&amp;amp;D systems</td><td>MAB650</td><td></td></tr><tr><td>PDGFR&amp;alpha; antibody</td><td>R&amp;amp;D systems</td><td>AF307</td><td></td></tr><tr><td>SC-144</td><td>Axon</td><td>2324</td><td></td></tr><tr><td>XMU-MP-1</td><td>Med Chem Express</td><td>HY-100526</td><td></td></tr><tr><td>Matrigel</td><td></td><td></td><td>basement membrane matrix</td></tr><tr><td>Countess cell counting chamber slides</td><td>Thermo fisher</td><td></td><td>cell counting slides</td></tr><tr><td>DAPI Staining Solution</td><td>Miltenyi Biotec</td><td>130-111-570</td><td></td></tr></tbody></table>

protocol for human blastoids modeling blastocyst development and implantation

A model of the human blastocyst formed from stem cells (blastoid) would support scientific and medical advances. However, its predictive power will depend on its ability to efficiently, timely, and faithfully recapitulate the sequences of blastocyst development (morphogenesis, specification, patterning), and to form cells reflecting the blastocyst stage. Here we show that na&#239;ve human pluripotent stem cells cultured in PXGL conditions and then triply inhibited for the Hippo, transforming growth factor- &#946;, and extracellular signal-regulated kinase pathways efficiently undergo morphogenesis to form blastoids (&gt;70%). Matching with developmental timing (~4 days), blastoids unroll the blastocyst sequence of specification by producing analogs of the trophoblast and epiblast, followed by the formation of analogs of the primitive endoderm and the polar trophoblasts. This results in the formation of cells transcriptionally similar to the blastocyst (&gt;96%) and a minority of post-implantation analogs. Blastoids efficiently pattern by forming the embryonic-abembryonic axis marked by the maturation of the polar region (NR2F2+), which acquires the specific potential to directionally attach to hormonally stimulated endometrial cells, as in utero. Such a human blastoid is a scalable, versatile, and ethical model to study human development and implantation in vitro.

Typically, naive hPSCs cultured in PXGL (Figure 2A) are aggregated and cavitated structures that emerge between 48 to 72 h after PALLY induction and reach a diameter of 150-250 &#181;m within 96 h (Figure 2B). Using optimal (1) seeding cell numbers, (2) duration of pre-culture aggregation with N2B27 (0 to 24 h), (3) concentration of individual chemical components (especially LPA), and (4) duration of PALLY treatment, the induction efficiency reaches 70%-80% as defined based on morphometric parameters (overall size of 150-250 &#181;m, single regular cavity, single inner cell cluster; Figure 2C,D) and the presence of the three lineages. A suboptimal initial cell state and/or induction conditions will result in less efficient or no blastoid formation. To ensure maximum efficiency and to only form pre-implantation analogs, it is crucial to use a high-quality culture of naive PXGL hPSCs. This can be assessed by measuring by FACS the percentage of cells positive for the surface markers SUSD2 (naive state) and CD24 (primed state). Additional surface markers specific to the off-target extraembryonic lineages (e.g., amnion, extraembryonic mesoderm) would be useful as well but, to the best of our knowledge, are currently not available. If the formation efficiency obtained is lower than the results reported, it is important to carefully check all the components of the blastoid medium, especially LPA that is reconstituted in PBS and that, as a GPCR ligand, can be more unstable as compared to synthetic molecules reconstituted in DMSO. In most cases, even if the yield is not maximal, the cavitated structures are still composed of the three blastocyst lineages. The emergence of three blastocyst lineages and formation of embryonic-abembryonic axis can be confirmed by the immunofluorescence staining of markers (EPI: NANOG, OCT4, TE: GATA3, Polar-TE NR2F2, Mural-TE: CDX2, PrE: GATA4; Figure 2E,G). Trophospheres, which are only composed of TE, help to further dissect the role of intercellular communication. Trophospheres can form at 50%-60 % efficiency within 96 h of induction (Figure 2H,I). Blastoid formation can be performed not only in homemade microwell arrays but also in commercially available ultra-low attachment 96 well-plates with optimization of induction conditions (see Protocol and Figure 2J). Blastoids also have the capacity to further develop for additional 6 days, which is time-equivalent of day 13 embryo, with in vitro differentiation protocol (Figure 2K).
In order to further characterize the cell state of blastoid cells, single-cell RNA sequencing technology must be used. UMAP is commonly applied to visualize a distribution of cell states and unsupervised clustering analysis is performed on it to evaluate the proximity of individual cell states. Different parameters in the single-cell data analysis can affect how cells are displayed in the UMAPs, thus leading to clusters with different spatial and relative positions and shapes (Figure 3A). However, in this analysis, cells display markedly reproducibly distinct clustering profiles regardless of the parameters used to perform the clustering and the visualization of the data, which allows distinguishing with high confidence the three blastocyst lineages. We used cells from embryos harvested at different developmental stages as a reference. The merging of these datasets shows that the majority of the trophectoderm analog from blastoid clustered with pre-implantation trophectoderm but not with post-implantation trophoblasts (Figure 3B). These results were also confirmed by an independent consortium10.
When Carnegie stage 7 (CS7) gastrulating embryos are introduced in the reference map, a small population of blastoid cells (3%) clustered with the mesoderm and amnion lineages of these embryos (Figure 3C). When amnion-like cells are introduced in the reference map, a small population of blastoid cells (&#60; 2%) clustered with such amnion-like cells.
Overall, only the structures comprising a single regular cavity, a single inner cell cluster, an overall size ranging from 150-250 &#181;m, comprising transcriptomic analogs of the three blastocyst lineages, and largely devoid of other lineages (e.g., amnion, mesoderm, extraembryonic mesoderm) are considered as human blastoids.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63388/63388fig01.jpg" /> 
Figure 1: Four features and two approaches for generating high-fidelity blastoids. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63388/63388fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63388/63388fig02.jpg" /> 
Figure 2: Blastoid and trophospheres derived from aggregates of Na&#239;ve hPSC. (A) Phase-contrast images showing naive hPSCs cultured in PXGL medium co-cultured with irradiated MEF. Scale bar: 50 &#181;m. (B)&#160;Phase-contrast images showing the morphological change of naive hPSCs aggregates cultured on a non-adherent hydrogel microwell array with 500 nM LPA (PALLY medium). Scale bar: 200 &#181;m. (C)&#160;Human blastoids formed on a microwell array after 96 h. Scale bars: 400 &#181;m. (D)&#160;Quantification of the percentage of microwells containing a human blastoid induced by PALLY culture condition with optimized LPA concentration from different naive hPSC lines (n= 3 microwell arrays). (E) Immunofluorescence staining of human blastoids with epiblast (EPI) markers (yellow) NANOG and OCT4; the TE markers (cyan) CDX2 and GATA3; and the primitive endoderm marker (magenta) SOX17 and GATA4. Scale bar: 100 &#181;m. (H-I)&#160;Quantification of the cell number (left) and percentage of cells (right) belonging to each lineage in blastoid (96 h) based on immunofluorescence staining of OCT4, GATA3, and GATA4. (G)&#160;Immunofluorescence staining of human blastoids for CDX2 (cyan) and NR2F2 (magenta). (F)&#160;Phase-contrast images of early and late-stage trophospheres on microwell array induced by addition of 3 &#181;M SC144 (H) or 2 &#181;M XMU-MP-1 (I), respectively. (J)&#160;Phase-contrast images showing the morphological change of naive hPSCs aggregates cultured in ultra-low attachment 96 well plate with 500 nM LPA (PALLY medium). (K)&#160;Phase-contrast image (left) and immunofluorescence staining (right) for OCT4 (yellow), GATA3 (cyan), and GATA4 (magenta) in blastoid grown in postimplantation culture condition for 6 days. Scale bar: 100 &#181;m. This figure is adapted from6,10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63388/63388fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63388/63388fig03.jpg" /> 
Figure 3: Characterization of the composition of blastoids by single cell sequencing. (A) Unsupervised clustering analysis with different parameters on UMAP of the transcriptome of single cells derived from the different time points of blastoid (24 h, 60 h, 96 h), naive hPSCs, primed hPSCs, and hTSC (represent the post-implantation cytotrophoblast). (B)&#160;UMAP of the transcriptome of cells derived from blastoid (96 h), naive hPSCs, and primed hPSCs integrated with published data sets from human embryos of pre-implantation, peri-implantation (in vitro cultured blastocysts), and gastrulation (Carnegie stage 7, i.e., between E16-19) stages. Individual cells are colored based on their origin in human embryos (left), blastoid-derived cells or stem cells (middle), and the result of unsupervised clustering analysis (right). (C)&#160;UMAPs of the transcriptome of cells derived from blastoids, naive hPSCs, primed hPSCs, and integrated with published data set from gastrulation (Carnegie stage 7, i.e., between E16-19) stage embryo. Individual cells are colored based on their origin in human embryos (left), blastoid-derived cells or stem cells (middle), and the result of unsupervised clustering analysis (right). This figure is adapted from6. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63388/63388fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Table 1: All media composition used in this study. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63388/Supplementary Table 1 _RE.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Protocol for Human Blastoids Modeling Blastocyst Development and Implantation at JoVE.com

Protocol for Human Blastoids Modeling Blastocyst Development and Implantation

A protocol outlining the formation of human blastoids that efficiently, timely, and sequentially generate blastocyst-like cells.

A model of the human blastocyst formed from stem cells (blastoid) would support scientific and medical advances. However, its predictive power will depend ...

protocol-for-human-blastoids-modeling-blastocyst-development

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

6.3K Views.  Vienna Biocenter (VBC). Onderzoek op menselijke embryo's wordt beperkt door hun lage beschikbaarheid en ethische zorgen, dus het verkrijgen van individuele modellen die de vroege menselijke ontwikkeling getrouw repliceren, zou wetenschappelijke en medische vooruitgang ondersteunen. Het vermogen van deze techniek om de menselijke ontwikkeling te voorspellen hangt af van het vermogen om de sequenties van blastocyst cellulaire bepaling en morfogenese effectief te reproduceren, volgens de ontwikkelingssequentie en het t...

Video: Protocol for Human Blastoids Modeling Blastocyst Development and Implantation

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Modified MicroSecure Vitrification: A Safe, Simple and Highly Effective Cryopreservation Procedure for Human Blastocysts

Clinical embryo vitrification evolved with the development of unique vitrification devices in the 21st century and with the misconception that ultra-rapid cooling in an &#34;open&#34; system (i.e., direct LN2 contact) was a necessity to optimize vitrification success. The dogma surrounding the importance of cooling rates led to unsafe practices subject to technical variation and to the creation of vitrification devices that disregarded important quality-control factors (e.g., ease of use, repeatability, reliability, labeling security, and storage safety). Understanding the quality-control flaws of other devices allowed for the development of a safe, secure, repeatable, and reliable &#181;S-VTF method aimed to minimize intra- and inter-technician variation. Equally important, it combined the availability of two existing FDA-compliant devices: 1) a 0.3-mL ionomeric resin embryo straw with internalized, dual-colored, tamper-proof labeling with repeatable weld seal potential; and 2) shortened, commonly-used, 300-&#181;m ID sterile flexipettes to directly load the embryo(s) in order to create a highly-effective global vitrification device. Like other aseptic, closed vitrification systems (e.g., High Security Vitrification (HSV), Rapid-i, and VitriSafe) effectively used in reproductive medicine, microSecure Vitrification (&#181;S-VTF) has proven that it can achieve high post-warming survival and pregnancy outcomes with its attention to simplicity, and reduced technical variation. Although the 0.3-mL embryo straw containing an internal hydrophobic plug was commercially replaced with a standard semen straw possessing cotton-polyvinyl pyrrolidone (PVP) plugs, it maintained its ionomeric resin composition to ensure weld sealing. However, the cotton plugs can wick out the fluid-embryo contents of the flexipettes upon contact. A modified &#181;S-VTF method was adapted to include an additional internal weld seal before the plug on the device loading side. The added technical step to the &#181;S-VTF procedure has not affected its successful application, as high survival rates (&#62; 95%) and pregnancy rates continue today.

MicroSecure Vitrification was developed as a non-commercial, aseptic closed vitrification device system that is compliant with FDA good manufacturing and tissue-handling practices. Due to the withdrawal of hydrophobic plugged embryo straws from the industry, the vitrification procedure was modified to include an inner seal before the standard internal cotton plug.

Clinical embryo vitrification evolved with the development of unique vitrification devices in the 21st century and with the misconception that ultra-rapid ...

Human Blastocyst Biopsy and Vitrification

Blastocyst biopsy is performed to obtain a reliable genetic diagnosis during IVF cycles with preimplantation genetic testing. Then, the ideal workflow entails a safe and efficient vitrification protocol, due to the turnaround time of the diagnostic techniques and to transfer the selected embryo(s) on a physiological endometrium in a following natural cycle. A biopsy approach encompassing the sequential opening of the zona pellucida and retrieval of 5-10 trophectoderm cells (ideally 7-8) limits both the number of manipulations required and the exposure of the embryo to sub-optimal environmental conditions. After proper training, the technique was reproducible across different operators in terms of timing of biopsy (~8 min, ranging 3-22 min based on the number of embryos to biopsy per dish), conclusive diagnoses obtained (~97.5%) and live birth rates after vitrified-warmed euploid blastocyst transfer (&#62;40%). The survival rate after biopsy, vitrification and warming was as high as 99.8%. The re-expansion rate at 1.5 h from warming was as high as 97%, largely dependent on the timing between biopsy and vitrification (ideally &#8804;30 min), blastocyst morphological quality and day of biopsy. In general, it is better to vitrify a collapsed blastocyst; therefore, in non-PGT cycles, laser-assisted artificial shrinkage might be performed to induce embryo collapse prior to cryopreservation. The most promising future perspective is the non-invasive analysis of the IVF culture media after blastocyst culture as a putative source of embryonic DNA. However, this potential avant-garde is still under investigation and a reliable protocol yet needs to be defined and validated.

Blastocyst biopsy and vitrification are required to efficiently conduct preimplantation genetic testing. An approach entailing the sequential opening of the zona pellucida and retrieval of 7-8 trophectoderm cells in day 5-7 post-insemination limits both the number of manipulations required and the exposure of the embryo to sub-optimal environmental conditions.

Blastocyst biopsy is performed to obtain a reliable genetic diagnosis during IVF cycles with preimplantation genetic testing. Then, the ideal workflow ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

Biology

Morphometric Protocol for the Objective Assessment of Blastocyst Behavior During Vitrification and Warming Steps

This article describes the noninvasive method of blastocyst morphometry based on time-lapse microphotography for the accurate monitoring of a blastocyst&#39;s volume changing during individual phases before and after vitrification. The method can be useful in searching for the most optimal timing of blastocyst exposure to different concentrations of cryoprotectants by observing blastocyst shrinkage and re-expansion in different pre- and post-vitrification phases. With this methodology, the blastocyst vitrification protocol can be optimized. For a better demonstration of the usefulness of this morphometric method, two different blastocyst preparation protocols for vitrification are compared; one with using an artificial blastocoel collapsing and one without this intervention before vitrification. Both blastocysts&#39; volume changes are followed by time-lapse microphotography and measured by photo-editing software tools. The measurements are taken every 20 seconds in previtrification phases and every 5 minutes in the post-warming period. The changes of the blastocyst dimensions per time unit are presented graphically in line diagrams. The results show a long equilibration previtrification phase in which the intact blastocyst first shrinks and then slowly refills the blastocoel, entering vitrification with a fluid-filled blastocoel. The artificially collapsed blastocyst remains in its shrunken stage through the entire equilibration phase. During the vitrification phase, it also does not change its volume. Since the blastocyst morphometry shows a constant volume of the artificially collapsed blastocysts during the previtrification step, it seems that this stage could be shorter. The described protocol provides many additional comparative parameters of blastocyst behavior during and after cryopreservation on the basis of the speed and intensity of the volume changes, the number of partial blastocoel contractions or total blastocyst collapses, and the time to a total blastocoel re-expansion or the time to hatching.

Here we present a time-lapse morphometric protocol to follow the intensity of blastocyst shrinkage and re-expansion during previtrification interventions and post-warming recovery. The application of the protocol is possible in in vitro fertilization laboratories equipped with time-lapse microscopes and is recommended in the development of an optimal blastocyst vitrification method.

This article describes the noninvasive method of blastocyst morphometry based on time-lapse microphotography for the accurate monitoring of a ...

Bioengineering

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a critical yet enigmatic biological event that has been historically difficult to study due to technical and ethical limitations. Implantation is initiated by the development of the trophectoderm to early trophoblast and subsequent differentiation into distinct trophoblast sublineages. Aberrant early trophoblast differentiation may lead to implantation failure, placental pathologies, fetal abnormalities, and miscarriage. Recently, methods have been developed to allow human embryos to grow until day 13 post-fertilization in vitro in the absence of maternal tissues, a time-period that encompasses the implantation period in humans. This has given researchers the opportunity to investigate human implantation and recapitulate the dynamics of trophoblast differentiation during this critical period without confounding maternal influences and avoiding inherent obstacles to study early embryo differentiation events in vivo. To characterize different trophoblast sublineages during implantation, we have adopted existing two-dimensional (2D) extended culture methods and developed a procedure to enzymatically digest and isolate different types of trophoblast cells for downstream assays. Embryos cultured in 2D conditions have a relatively flattened morphology and may be suboptimal in modeling in vivo three-dimensional (3D) embryonic architectures. However, trophoblast differentiation seems to be less affected as demonstrated by anticipated morphology and gene expression changes over the course of extended culture. Different trophoblast sublineages, including cytotrophoblast, syncytiotrophoblast and migratory trophoblast can be separated by size, location, and temporal emergence, and used for further characterization or experimentation. Investigation of these early trophoblast cells may be instrumental in understanding human implantation, treating common placental pathologies, and mitigating the incidence of pregnancy loss.

Here, we describe a method for warming vitrified human blastocysts, culturing them through the implantation period in vitro, digesting them into single cells and collecting early trophoblast cells for further investigation.

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a ...

Evaluating Embryo Adhesion in an In Vitro Co-Culture Model with Ishikawa Cells

Establishment of an Embryo Implantation Model In Vitro

Embryo implantation is the first step in the establishment of a successful pregnancy. An in vitro model for embryo implantation is critical for basic biological research, drug development, and screening. This paper presents a simple, rapid, and highly efficient in vitro model for embryo implantation. In this protocol, we first introduce mouse blastocyst acquisition and human endometrial adenocarcinoma cells (Ishikawa)&#160;preparation for implantation, followed by the co-culture method for mouse embryos and Ishikawa cells. Finally, we conducted a study to assess the impact of varying concentrations of 17-&#946;-estradiol (E2) and progesterone (P4) on embryo adhesion rates based on this model. Our findings revealed that high concentrations of E2 significantly reduced embryo adhesion, whereas the addition of progesterone could restore the adhesion rate. This model offers a simple and fast platform for evaluating and screening molecules involved in the adhesion process, such as cytokines, drugs, and transcription factors controlling implantation and endometrial receptivity.

Author Spotlight: Advancing Therapeutic Strategies for Improving Pregnancy Rates by Analyzing Embryo-Endometrium Interactions

This protocol describes methodologies to establish simple and efficient embryo implantation in vitro model for evaluating the relevant molecules affecting the embryo implantation process.

Embryo implantation is the first step in the establishment of a successful pregnancy. An in vitro model for embryo implantation is critical for basic ...

Cleavage and Blastulation

After a large-single-celled zygote is produced via fertilization, the process of cleavage occurs while zygotes travel through the uterine tube. Cleavage is a mitotic cell division that does not result in growth. With each round of successive cell division, daughter cells get increasingly smaller.

<h4>Zygotic Genome Activation</h4>

At the beginning of embryogenesis, maternal mRNAs control development. However, by the eight-cell stage of cleavage, embryonic genes become activated in a process called zygotic genome activation (ZGA). As a result, maternal mRNAs get degraded, and ZGA causes a transition from maternal to zygotic genetic control of developing an embryo. Although maternal mRNAs get degraded, previously translated proteins may remain in the embryo through later stages of development.

<h4>Cleavage Pattern</h4>

Cleavage patterns vary between organisms depending on the presence and distribution of egg yolk amongst other factors. For example, mammals have a holoblastic rotational cleavage pattern. They are holoblastic because they have sparse, but evenly distributed yolk and therefore end up with a cleavage furrow that extends through the entire embryo as opposed to being meroblastic where the cleavage furrow does not extend through the yolk-dense portion of the cytoplasm.

At the onset of cleavage, rotational cleavage begins when the zygote first divides to form two smaller daughter cells called blastomeres. During this first cleavage event, division occurs in an austral fashion. The two daughter blastomeres then go through mitosis to each form two new blastomeres. During this second cleavage event, one daughter blastomere cleaves in an austral manner while the second cleaves equatorially. This pattern continues so that the resultant blastomeres end up being smaller than their respective parent cells.

<h4>Compaction</h4>

At the eight-blastomere stage, compaction starts to occur&mdash;blastomeres tightly push against each other and appear to be one cell where individual cells are indistinguishable from one another. To stabilize the tightly packed blastomeres, tight junctions are formed among the exterior blastomeres while the interior blastomeres form gap junctions that allow the movement of ions and small molecules in between cells. E-cadherin, a calcium-dependent adhesion molecule, helps to further adhere blastomeres to each other.

<h4>Morula Formation</h4>

Once there are approximately thirty-two blastomeres, the zygote becomes a morula. Morula formation marks the end of cleavage. The morula then becomes a blastula that goes through further differentiation during the subsequent stages of development.

Embryogenesis: Cleavage and Blastulation

After a large-single-celled zygote is produced via fertilization, the process of cleavage occurs while zygotes travel through the uterine tube. Cleavage ...

Protocol for Human Blastoids Modeling Blastocyst Development and Implantation

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