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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#246; 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&#38;D systems</td>
			<td>MAB650</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGFR&#945; antibody</td>
			<td>R&#38;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://jove.unicartagenaproxy.elogim.com/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://jove.unicartagenaproxy.elogim.com/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://jove.unicartagenaproxy.elogim.com/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://jove.unicartagenaproxy.elogim.com/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

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

6.2K Views.  Vienna Biocenter (VBC). A protocol outlining the formation of human blastoids that efficiently, timely, and sequentially generate blastocyst-like cells.

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 ...

A Modified Trier Social Stress Test for Vulnerable Mexican American Adolescents

The Trier Social Stress Test (TSST) is a well validated and widely used social stressor that has been shown to induce a 2-4 fold increase in cortisol, the biological output produced by the Hypothalamic-Pituitary-Adrenal (HPA) axis in humans. While studies have explored how modifications to the TSST influence stress responsivity, few studies have created a modified TSST appropriate for vulnerable youth that elicits a significant cortisol stress response. Thus, the current study sought to modify or adjust the TSST in a culturally sensitive way for a vulnerable sample of 14 year-old adolescents. The present study took place within the context of a longitudinal birth cohort study of Mexican American families in California called the Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS). The CHAMACOS sample was optimal to test the effectiveness of a modified culturally appropriate TSST, as it is comprised of Mexican American youth, who are often excluded from research. These youths also have experienced significant early life adversity. Example modifications included timed prompts, alternative math tasks, use of same-ethnicity peers as confederates, debriefing immediately after the conclusion of the TSST, and using an unknown youth examiner to administer the debrief. Saliva samples were collected at baseline (after a resting phase), and then again at 15, 30, and 45 min post-TSST onset to assess cortisol concentration. A pilot study of 50 participants (50% female) have been analyzed for cortisol reaction to the TSST. Results confirmed that this modified version of the TSST was successful at eliciting a significant cortisol reaction, with a wide range of variability likely due to individual differences. Goals for modifications and ethnicity considerations are discussed. This study provides the foundation for future research to utilize a modified TSST with vulnerable youth.

Here, we present a protocol that provoked cortisol reactivity in a vulnerable adolescent Mexican American sample utilizing a modified version of the Trier Social Stress Test (TSST). Saliva samples were collected at baseline, 15, 30, and 45 min post-TSST onset. Future research could utilize this modified TSST with vulnerable youth.

The Trier Social Stress Test (TSST) is a well validated and widely used social stressor that has been shown to induce a 2-4 fold increase in cortisol, the ...

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying the human brain. One emerging technology to address this issue is the use of three dimensional (3D) neural cell cultures, termed &#39;organoids&#39; or &#39;spheroids&#39;, for long term preservation of intercellular interactions including extracellular adhesion molecules. However, these culture systems are time consuming and not systematically generated. Here, we detail a method to rapidly and consistently produce 3D cocultures of neurons and astrocytes from human pluripotent stem cells. First, pre-differentiated astrocytes and neuronal progenitors are dissociated and counted. Next, cells are combined in sphere-forming dishes with a Rho-Kinase inhibitor and at specific ratios to produce spheres of reproducible size. After several weeks of culture as floating spheres, cocultures (&#39;asteroids&#39;) are finally sectioned for immunostaining or plated upon multielectrode arrays to measure synaptic density and strength. In general, it is expected that this protocol will yield 3D neural spheres that display mature cell-type restricted markers, form functional synapses, and exhibit spontaneous synaptic network burst activity. Together, this system permits drug screening and investigations into mechanisms of disease in a more suitable model compared to monolayer cultures.

In this protocol, we aim to describe a reproducible method for combining dissociated human pluripotent stem cell derived neurons and astrocytes together into 3D sphere cocultures, maintaining these spheres in free floating conditions, and subsequently measuring synaptic circuit activity of the spheres with immunoanalysis and multielectrode array recordings.

A barrier to our understanding of how various cell types and signals contribute to synaptic circuit function is the lack of relevant models for studying ...

Analysis of Epididymal Protein Synthesis and Secretion

The mammalian epididymis generates one of the most complex intraluminal fluids of any endocrine gland in order to support the post-testicular maturation and storage of spermatozoa. Such complexity arises due to the combined secretory and absorptive activity of the lining epithelial cells. Here, we describe the techniques for the analysis of epididymal protein synthesis and secretion by focusing on the model protein family of dynamin (DNM) mechanoenzymes; large GTPases that have the potential to regulate bi-directional membrane trafficking events. For the study of protein expression in epididymal tissue, we describe robust methodology for immunofluorescence labeling of target proteins in paraffin-embedded sections and the subsequent detection of the spatial distribution of these proteins via immunofluorescence microscopy. We also describe optimized methodology for the isolation and characterization of exosome like vesicles, known as epididymosomes, which are secreted into the epididymal lumen to participate in intercellular communication with maturing sperm cells. As a complementary approach, we also describe the immunofluorescence detection of target proteins in an SV40-immortalized mouse caput epididymal epithelial (mECap18) cell line. Moreover, we discuss the utility of the mECap18 cell line as a suitable in vitro model with which to explore the regulation of epididymal secretory activity. For this purpose, we describe the culturing requirements for the maintenance of the mECap18 cell line and the use of selective pharmacological inhibition regimens that are capable of influencing their secretory protein profile. The latter are readily assessed via harvesting of conditioned culture medium, concentration of secreted proteins via trichloroacetic acid/acetone precipitation and their subsequent analysis via SDS-PAGE and immunoblotting. We contend that these combined methods are suitable for the analysis of alternative epididymal protein targets as a prelude to determining their functional role in sperm maturation and/or storage.

Here, we report the immunofluorescence localization of dynamin to illustrate the protocols for the detection of proteins in paraffin-embedded mouse epididymal sections and those of an immortalized epididymal cell line (mECap18). We also describe the protocols for the isolation of secretory proteins from both epididymal fluid and conditioned cell media.

The mammalian epididymis generates one of the most complex intraluminal fluids of any endocrine gland in order to support the post-testicular maturation ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

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 ...

Generation of Multicellular Human Primary Endometrial Organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids.

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and apoptosis, development and ageing, to neuronal synaptic plasticity and regeneration have been associated with Ryanodine receptors (RyRs). Despite the importance of calcium signaling, the exact mechanism of its function in early development is unclear. As an organism with a short gestational period, the embryos of Drosophila melanogaster are prime study subjects for investigating the distribution and localization of CICR associated proteins and their regulators during development. However, because of their lipid-rich embryos and chitin-rich chorion, their utility is limited by the difficulty of mounting embryos on glass surfaces. In this work, we introduce a practical protocol that significantly enhances the attachment of Drosophila embryo onto slides and detail methods for successful histochemistry, immunohistochemistry, and in-situ hybridization. The chrome alum gelatin slide-coating method and embryo pre-embedding method dramatically increases the yield in studying Drosophila embryo protein and RNA expression. To demonstrate this approach, we studied DmFKBP12/Calstabin, a well-known regulator of RyR during early embryonic development of Drosophila melanogaster. We identified DmFKBP12 in as early as the syncytial blastoderm stage and report the dynamic expression pattern of DmFKBP12 during development: initially as an evenly distributed protein in the syncytial blastoderm, then preliminarily localizing to the basement layer of the cortex during cellular blastoderm, before distributing in the primitive neuronal and digestion architecture during the three-gem layer phase in early gastrulation. This distribution may explain the critical role RyR plays in the vital organ systems that originate in from these layers: the suboesophageal and supraesophageal ganglion, ventral nervous system, and musculoskeletal system.

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Here, we describe a protocol for detection and localization of Drosophila embryo protein and RNA from collection to pre-embedding and embedding, immunostaining, and mRNA in situ hybridization.

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and ...

Protocol for Human Blastoids Modeling Blastocyst Development and Implantation

Summary

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Synaptic Microcircuit Modeling with 3D Cocultures of Astrocytes and Neurons from Human Pluripotent Stem Cells

Analysis of Epididymal Protein Synthesis and Secretion

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Human Blastocyst Biopsy and Vitrification

Generation of Multicellular Human Primary Endometrial Organoids

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo