We would like to acknowledge the many patients at the Colorado Center for Reproductive Medicine (CCRM) that have graciously donated their embryos for research. We would also like to thank Karen Maruniak and the clinical laboratory at CCRM for their help in processing hCG samples, as well as Sue McCormick and her clinical IVF embryology team at CCRM for their help with embryo collection, storage, tracking, and donation. Funding was provided internally by CCRM.

All human embryos have been donated with consent for use in research. Protocols for extended human embryo culture have been approved by the Western Institutional Review Board (study no. 1179872) and follow international guidelines. Any use of human embryos must be reviewed by the appropriate ethics and governing bodies associated with the research institution using this protocol.
1. Preparation
<ol>
	<li>Prepare media and recovery plates one day prior to embryo warming in a sterile laminar flow hood.
	<ol>
		<li>Prepare 2 mL of blastocyst culture media (BM) with 10% v/v serum protein substitute (SPS). 
		NOTE:&#160;Blastocyst culture media may or may not contain added protein. Here, we used a medium that must be supplemented with 10% of the indicated albumin protein. Check the manufacturer guidance for variation in this supplementation. All media should be filtered using a sterile 0.22 &#956;M filter attached to a sterile syringe before equilibration.</li>
		<li>Fill two center-well organ culture wash dishes with 500 &#956;L of BM with 10% v/v SPS covered with 500 &#956;L of embryo culture grade oil.</li>
		<li>In a 60 mm tissue culture dish, layer 8 mL of embryo culture oil and then anchor 20 &#956;L drops of BM with 10% v/v SPS to the bottom of the culture plate. Make 1 drop for each embryo warmed. 
		NOTE: More media, drops, and wash dishes may be made as needed. Drops may also be added to the dish before the oil is layered. Anchoring drops under the oil will help reduce evaporation and any subsequent changes in osmolality.</li>
		<li>Equilibrate BM with 10% v/v SPS wash dishes and recovery plate in an incubator at 37 &#176;C, 6% CO2, 5% O2 for at least 4 h.</li>
		<li>Thaw one vial of the first step of extended culture media (IVC1) in 4 &#176;C or on the bench top.</li>
		<li>Prepare one wash dish of 500 &#956;L of IVC1 with no oil overlay. Aliquot approximately 4 mL of IVC1 into a 5 mL snap cap tube.</li>
		<li>Equilibrate IVC1 wash dish and small snap cap tube of IVC1 in 37 &#176;C, 6% CO2, and atmospheric O2 for at least 4 h.</li>
	</ol>
	</li>
	<li>Prepare extended culture plates one day prior to embryo warming in a sterile laminar flow hood.
	<ol>
		<li>Dilute fibronectin from human serum in phosphate buffered saline (PBS) to 30 &#956;g/mL. 250 &#956;L of 30 &#956;g/mL fibronectin per embryo will be needed to coat the chambers.</li>
		<li>Open the 8 well chambered coverslip package under the hood while taking care not to touch the wells. Gently pipette 250 &#956;L of 30 &#956;g/mL fibronectin into each well.</li>
		<li>Return the lid to the chambered coverslip and place in 4 &#176;C for 20-24 h.</li>
	</ol>
	</li>
	<li>Prepare extended culture plate in the morning of embryo warming.
	<ol>
		<li>Retrieve the chambered coverslip with fibronectin and place in laminar flow hood. Remove the fibronectin mixture with a 1 mL pipette and discard into the waste container.</li>
		<li>Retrieve warmed, equilibrated IVC1 media from small 5 mL snap cap tube.</li>
		<li>Pipette 300 &#956;L of equilibrated IVC1 into each well. Be careful not to let any fluid touch the lid as this will increase the risk of contamination.</li>
		<li>Return the chambered coverslip with IVC1 to incubate in 37 &#176;C, 6% CO2, and atmospheric O2 until removal of the zona pellucida in step 4.</li>
	</ol>
	</li>
</ol>
2. Warming vitrified D5 human embryos
NOTE: Other manufacturers may have slightly different protocols according to their own vitrification technology. See manufacturer&#8217;s instructions for use when applicable.
<ol>
	<li>Warm 3.0 mL of thawing solution (TS) in 35 mm dish to 37 &#176;C. Bring 300 &#956;L of dilution solution (DS) and two wells of 300 &#956;L of washing solution (WS) in a 6 well plate to room temperature.</li>
	<li>Using forceps, carefully remove the cryo device sleeve while ensuring that the vitrified embryo always remains under liquid nitrogen.</li>
	<li>Quickly move the cryo device from liquid nitrogen and plunge the tip of the cryo device in TS at 37 &#176;C. Set a timer for 1 min and keep the cryo device submerged until the embryo detaches into the TS. 
	NOTE: Warm embryos one at a time to keep track of embryo identities as they may relate to patient demographic information in downstream analysis.</li>
	<li>During the 1 min incubation in TS, carefully remove the cryo device and gently pick up the embryo and move to the opposite side of the TS dish. 
	NOTE: If culturing multiple embryos, be diligent to keep embryos separate to ensure proper identities are maintained.</li>
	<li>After 1 min, gently pick up the embryo with a small amount of TS, approximately 2 &#956;L. Move the embryo to the bottom of the 300 &#956;L DS well while covering the embryo in a thin layer of TS from the pipette. Set a timer for 3 min and place the 6-well plate on the bench top at room temperature.</li>
	<li>After 3 min, pick up the embryo with a small amount of DS, approximately 2 &#956;L, and move the embryo to the bottom of the next well containing 300 &#956;L of WS. Again, gently layer a small amount of DS from the pipette over the embryo. Set a timer for 5 min and return to the bench top.</li>
	<li>After 5 min, pick up the embryo with minimal volume of WS and move to the top of the final well of 300 &#956;L WS. The embryo will slowly fall and wash through the WS to the bottom. Expel any retained WS from the pipette into an empty well.</li>
	<li>Pick up the embryo with minimal volume and return to the top of the same 300 &#956;L well of WS. The embryo will once again fall to the bottom of the well. Set a timer for 1 min and return the 6-well plate to the bench top.</li>
</ol>
3. Recovery of warmed embryos
<ol>
	<li>One at a time, move warmed embryos into a center-well organ tissue dish containing 500 &#181;L of equilibrated BM with 10% v/v SPS under 500 &#181;L of equilibrated embryo culture grade oil.</li>
	<li>Wash embryos by picking up each embryo and moving them around to several areas in the dish while blowing out old media between moves. Pick up the embryo and move it to an individual 20 &#181;L culture drop of equilibrated BM with 10% v/v SPS under oil.</li>
	<li>Let the warmed embryos recover for 2 h in an incubator at 37 &#176;C, 6% CO2, 5% O2.</li>
</ol>
4. Zona removal
<ol>
	<li>After a 2 h recovery, assess embryos for re-expansion and take pictures of each embryo.</li>
	<li>Move embryos individually in 500 &#181;L of a 3-(N-morpholino)-propanesulfonic acid (MOPS)-buffered handling medium with 5% (v/v) fetal calf serum (FCS) prior to the treatment with acidic Tyrode&#8217;s solution.</li>
	<li>Move one embryo quickly through 300 &#956;L of warmed acidic Tyrode&#8217;s solution while actively watching through the microscope. The zona pellucida will visually start to dissolve. This will take approximately 5 s.</li>
	<li>Immediately move the embryo with dissolving zona into 300 &#956;L of warmed MOPS buffered medium to quench the acid Tyrode&#8217;s solution.</li>
	<li>Move the embryo with minimal volume of MOPS buffered medium into a center-well organ tissue dish containing 500 &#181;L of equilibrated BM with 10% v/v SPS under 500 &#181;L of equilibrated embryo culture grade oil to wash.</li>
	<li>Return the embryo to the 20 &#956;L recovery drop from step 3.2. 
	NOTE: Upon visual examination following zona removal, any embryos with retained zona pellucidae may be further treated with acidic Tyrode&#8217;s solution if necessary, by repeating steps 4.2-4.6. Minimizing exposure to the Tyrode&#8217;s solution is desired.</li>
</ol>
5. Blastocyst extended culture
<ol>
	<li>Move the embryos individually to the wash dish with equilibrated IVC1 media from step 1.1.6. Carefully move one embryo to one well of the chambered coverslip with equilibrated IVC1 and maintain embryo identification. 
	NOTE: This step needs to be finished as quickly as possible to minimize medium evaporation.</li>
	<li>Return chambered coverslip to an incubator set to 37 &#176;C, 6% CO2, atmospheric O2 for 2 days. 
	NOTE: Be sure to thaw and equilibrate media in 37 &#176;C, 6% CO2, atmospheric O2 for the media exchange 4 h in advance.</li>
	<li>At outgrowth D2 (D7 post fertilization), carefully examine the attachment of embryos under the microscope and perform media exchange.
	<ol>
		<li>Note which embryo is attached to the dish before exchanging media. Gently tap the plate and examine whether an embryo has securely attached to the plate under the microscope.</li>
		<li>Remove the lid and carefully remove 150 &#956;L of IVC1 and discard while not disturbing the attached embryo. If an embryo has not yet attached to the plate, do not exchange the media, as the serum in IVC1 will aid in embryo attachment.</li>
		<li>Pipette 150 &#956;L of equilibrated extended culture media, IVC2, slowly into each well and return the lid to the chambered coverslip. 
		NOTE: Ensure that removing the lid from the chambered coverslip is minimized to avoid medium evaporation.</li>
	</ol>
	</li>
	<li>Carefully return the chambered coverslip to the incubator without splashing any media on the lid. Repeat media exchange and attachment check every day of extended culture until embryos are ready for fixation or single cell digestion.</li>
</ol>
6. Optional collection of spent media
<ol>
	<li>Optionally, collect spent media during the exchange of culture media after D7 or any day thereafter. During step 5.3.2, rather than discarding the medium snap-freeze the 150 &#956;L of removed IVC1 into a sterile, low-bind 0.5 mL tube for future analysis.</li>
</ol>
7. Optional fixation for immunofluorescence
<ol>
	<li>Use a 200 &#956;L pipette to wash embryos with &#189; media exchanges of PBS for 3x before fixation to remove any extracellular debris. Washing away excess debris and protein will help to optimize clarity and reduce background in images obtained with immunofluorescence.</li>
	<li>Remove all media and slowly add 200 &#956;L of 4% paraformaldehyde (PFA) in PBS to the well. The embryo will want to stick to the surface of the fluid. Multiple 150 &#956;L washes with 4% PFA before removing all fluid will help to minimize any damage to the embryo.</li>
	<li>Incubate the embryos in 4% PFA for 20 min for fixation.</li>
	<li>Wash embryos 3x for 10 min per wash with 200 &#956;L of 0.1% v/v polysorbate 20 in fresh PBS.</li>
	<li>Store fixed embryos in 0.1% v/v polysorbate 20 in PBS at 4 &#176;C before proceeding with the immunofluorescence protocol.</li>
</ol>
8. Single cell digestion with Trypsin
NOTE: Fresh (not fixed) embryos are used for single cell digestion.
<ol>
	<li>Wash the embryo once with 200 &#956;L of PBS and add 200 &#956;L of trypsin solution to each well. Return the chambered coverslip to the incubator for 5 min.</li>
	<li>Remove the chambered coverslip from the incubator and examine the embryos under a stereoscope. Cells on the periphery of the embryo will start to retract and MTB should still be attached to the plate where they are remotely located from the embryo.</li>
	<li>Use a small pipette or finely pulled mouth pipette to pick up individual MTB before breaking apart the whole embryo. Skip ahead to step 9.1 to save MTB and return to step 8.4 after step 9.3.</li>
	<li>Use a handheld micromanipulation pipette or mouth pipette to gently dissociate the embryo by aspirating up and down.</li>
	<li>Cells will begin to dissociate from the whole embryo. Continue to aspirate the embryo gently and repeatedly using a smaller diameter pipette tip or mouth pipette until the embryo has been incubated for a total of 10 min in trypsin.</li>
</ol>
9. Single cell selection and sample collection
<ol>
	<li>With minimal trypsin, move the dissociated cells through three wash drops of 20 &#956;L PBS + 0.1% polyvinylpyrrolidone (PVP) under embryo culture oil with care not to lose any cells.</li>
	<li>After washing the cells, use a finely pulled glass pipette to select one cell. Carefully pipette the single cell into a sterile 0.2 mL low-bind tube with minimal volume of PBS+0.1% PVP.</li>
	<li>Snap freeze single cells in liquid nitrogen (LN2), and store in -80 &#176;C for future use.</li>
</ol>

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The authors have nothing to disclose.

The development of the protocol to culture human embryos through implantation has allowed scientists to explore a previously uncharted time in development8,9. Here, we use an extended culture system to culture human embryos and study early trophoblast differentiation before the formation of villous placenta. The methods described here allow us to collect different TB sublineages for use in downstream single-cell analysis. This work allows the scientific community...

Human implantation and the emergence of the early placenta are historically difficult to investigate and remain largely unknown because human tissues are inaccessible at this stage when pregnancy is clinically undetectable. Animal models are inadequate, as human placentation has its own unique features compared to other eutherian mammals. For example, human placenta invades deeply into the decidua with some trophoblast cells reaching at least the inner third of the uterine myometrium while other cells remodel the uterine spiral arteries. Even our closest evolutionary ancestors, the non-human primates, show differences in placental morphology and trophoblast interactions with the maternal decidual tissues1,2,3. Obtaining pre-implantation human embryos in vitro was not possible until in the 1980&#8217;s when clinical human in vitro fertilization (IVF) started as a routine practice for treating infertility4. Now, human blastocysts can be grown in vitro to allow for the selection of more viable embryos for transfer, as well as enable safe genetic testing. Improvement in embryo culture techniques, as well as the increasing use of IVF has yielded many surplus blastocysts which remains after patient&#8217;s treatment cycles have been completed. With patient consent, IRB approval, and with certain restrictions, these blastocysts may be utilized for research studies. They have become an invaluable resource that were used for the derivation of human embryonic stem cells5, understanding the transition of inner cell mass to embryonic stem cells6,7, and more recently, have been successfully cultured until day (D) 13 to remodel human implantation8,9. By utilizing recently developed single cell omics approaches, the access to these implantation stage human embryo tissues has offered unique opportunities to describe the molecular mechanisms that regulate this highly dynamic cell differentiation process, which were previously impossible to explore10,11,12,13.
Here, we describe the methods used in our recent publication characterizing the dynamics of trophoblast differentiation during human implantation12. This protocol includes the warming of vitrified blastocysts, extended embryo culture up to D12 post IVF, enzymatic digestion of the embryo into single cells, and cell collection for downstream assays (Figure 1). This extended culture system supports peri-implantation stage human embryo development without maternal input and recapitulates trophoblast differentiation that appears consistent with the observations made from histological specimens many years ago14,15,16,17. During implantation, the trophoblast population is comprised of at least two cell types: the mononucleated progenitor-like cytotrophoblast (CTB) and the terminally differentiated, multinucleated syncytiotrophoblast (STB). Upon trypsin digestion, the CTB are small, round cells that are morphologically indistinguishable from other cell lineages (Figure 2A, left panel). Separation of CTB from other cell lineages, such as epiblast and primitive endoderm, can be achieved by their distinct transcriptomic profiles revealed by single cell RNA sequencing. Syncytiotrophoblast cells can be easily identified as irregular shaped structures that are significantly larger than the other cell types and mainly located at the periphery of the embryo (Figure 2A, middle panel; Figure 2B, left panel). Migratory trophoblast cells (MTB) are another trophoblast sublineage found during embryo extended culture and can be recognized as seemingly moving away from the main body of the embryo (Figure 2A, right panel, Figure 2B, right panel). Migratory trophoblast, although expressing many of the same markers as extra villous trophoblast (EVT), should not be referred to as EVT, since the villous structures in the placenta have not emerged at this very early stage of development.
Experimentally, we were able to collect small CTB and large STB that are easily distinguishable after embryos are digested into single cells at D8, D10, and D12 (Figure 2A,B). Migratory trophoblasts arise at the later stages of extended embryo culture and can be collected at D12 before enzymatic digestion of the whole embryo (Figure 2A,B). By phenotypically separating these three trophoblast sublineages before single cell analysis, we can identify specific transcriptomic markers and define the biological role of each cell type. Cytotrophoblast are highly proliferative and act as progenitor cells in supplying the STB and MTB differentiated lineages10,11,12,13. Syncytiotrophoblast are involved in producing placental hormones to maintain pregnancy and may be also responsible for the embryos burrowing into the endometrium10,11,12,13. Migratory trophoblast has even stronger features of an invasive, migratory phenotype and are likely responsible for deeper and more extensive colonization of the uterine endometrium10,11,12,13. After defining the transcriptomic signature of each cell type, clustering analyses have also revealed two additional subsets of cells that were morphologically indistinguishable from CTB and had transcriptomes with features of STB and MTB, respectively12. These intermediate stage cells are likely in the process of differentiating from CTB to either the MTB or STB sublineages and would have been overlooked if embryos were blindly digested and cells were separated by transcriptome alone.
The protocol described here utilizes a two-dimensional (2D) culture system and may not be optimal for supporting three-dimensional (3D) structural development, as suggested by a recent publication describing a newly developed 3D culture system13. Nevertheless, in this 2D system the differentiation of early trophoblast seems to be consistent with observations made from in vivo specimens14,15,16,17. This protocol may also be easily adapted for the use in the recently described 3D culture system13 with minimal changes. All steps are carried out with a handheld micromanipulation pipette with commercially available disposable tips or a mouth pipette from a finely pulled glass pipette attached to rubber tubing, a filter, and a mouthpiece.

<table><tbody><tr><td>NORM JECT Luer Lock sterile syringe</td><td>VWR</td><td>53548-019</td><td>Pack of 100</td></tr><tr><td>3130 or 3110 Forma Series II water-jacketed CO2 incubator</td><td>Thermo Fisher Scientific</td><td>13-998-078</td><td></td></tr><tr><td>35 mm Corning Primaria tissue culture dish</td><td>VWR</td><td>62406-038</td><td>Case of 500</td></tr><tr><td>5 mL snap cap tube</td><td>VWR</td><td>60819-295</td><td>Pack of 25</td></tr><tr><td>60 mm Corning Primaria tissue culture dish</td><td>VWR</td><td>25382-687</td><td>Case of 200</td></tr><tr><td>6-well dish</td><td>Agtech Inc.</td><td>D18</td><td>Pack of 1, 10, or 50</td></tr><tr><td>Acidic Tyrode&#039;s solution</td><td>Millipore Sigma</td><td>T1788</td><td>100 mL</td></tr><tr><td>Biotix 1250 &amp;micro;L pipette tips</td><td>VWR</td><td>76322-156</td><td>Pack of 960</td></tr><tr><td>Blast, blastocyst culture media</td><td>Origio</td><td>83060010</td><td>10 mL</td></tr><tr><td>Dilution Solution</td><td>Kitazato</td><td>VT802</td><td>1 x 4 mL</td></tr><tr><td>Disposable Borosilicate Glass Pasteur Pipets</td><td>Thermo Fisher Scientific</td><td>1367820D</td><td>5.75 in. (146mm); 720/Cs</td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>Millipore Sigma</td><td>D8537</td><td></td></tr><tr><td>Embryo culture paraffin oil OvOil</td><td>Vitrolife</td><td>10029</td><td>100 mL</td></tr><tr><td>Eppendorf PCR tubes 0.2 mL</td><td>VWR</td><td>47730-598</td><td>Pack of 1,000</td></tr><tr><td>Eppendorf PCR tubes 0.5 mL</td><td>VWR</td><td>89130-980</td><td>Case of 500</td></tr><tr><td>Fibronectin from human plasma. Liquid .1% solution</td><td>Millipore Sigma</td><td>F0895</td><td>1 mg</td></tr><tr><td>Gilson 1 mL Pipetteman</td><td>Thermo Fisher Scientific</td><td>F123602G</td><td>1 Pipetteman 200-1000 &amp;micro;L</td></tr><tr><td>Gilson 20 &amp;micro;L Pipetteman</td><td>Thermo Fisher Scientific</td><td>F123602G</td><td>1 Pipetteman 2-20 &amp;micro;L</td></tr><tr><td>Gilson 200 &amp;micro;L Pipetteman</td><td>Thermo Fisher Scientific</td><td>F123602G</td><td>1 Pipetteman 50-200 &amp;micro;L</td></tr><tr><td>G-MOPS handling media</td><td>Vitrolife</td><td>10129</td><td>125 mL</td></tr><tr><td>Handling media</td><td>Origio</td><td>83100060</td><td>60 mL</td></tr><tr><td>Ibidi 8 well chambered coverslip</td><td>Ibidi</td><td>80826</td><td>15 slides per box</td></tr><tr><td>IVC1/IVC2</td><td>Cell Guidance Systems</td><td>M11-25/ M12-25</td><td>5-5mL aliquots</td></tr><tr><td>K System T47 Warming Plate</td><td>Cooper Surgical</td><td>23054</td><td></td></tr><tr><td>MilliporeSigma Millex Sterile Syringe Filters with Durapore PVFD Membrane</td><td>Fisher Scientific</td><td>SLGVR33RS</td><td>Pack of 50</td></tr><tr><td>Mouth pieces</td><td>IVF Store</td><td>MP-001-Y</td><td>100 pieces</td></tr><tr><td>Oosafe center well dish</td><td>Oosafe</td><td>OOPW-CW05-1</td><td>Case of 500</td></tr><tr><td>Quinn&#039;s Advantage SPS</td><td>Origio</td><td>ART-3010</td><td>12x 12 mL</td></tr><tr><td>Rubber latex tubing for mouth pieces</td><td>IVF Store</td><td>IVFS-NRL-B-5</td><td>5 ft.</td></tr><tr><td>Stereomicroscope</td><td>Nikon</td><td>SMZ1270</td><td></td></tr><tr><td>Stripper tips</td><td>Cooper Surgical</td><td>MXL3-275</td><td>20/pk 275 &amp;micro;m</td></tr><tr><td>Thawing Solution</td><td>Kitazato</td><td>VT802</td><td>2 x 4 mL</td></tr><tr><td>The Stripper Micropipetter</td><td>Cooper Surgical</td><td>MXL3-STR</td><td></td></tr><tr><td>TrypLE Express Enzyme (1X), no phenol red</td><td>Thermo Fisher Scientific</td><td>12604013</td><td>1 x 100 mL</td></tr><tr><td>Tween20</td><td>Millipore Sigma</td><td>P1379-25ML</td><td>25 mL bottle</td></tr><tr><td>VWR 1-20 &amp;micro;L pipette tips</td><td>VWR</td><td>76322-134</td><td>Pack of 960</td></tr><tr><td>VWR 1-200 &amp;micro;L pipette tips</td><td>VWR</td><td>89174-526</td><td>Pack of 960</td></tr><tr><td>Washing Solution</td><td>Kitazato</td><td>VT802</td><td>1 x 4 mL</td></tr></tbody></table>

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.

Healthy embryos exhibited continued proliferation over the course of extended culture (Figure 2B). Abnormal embryos began to retract from their outer edges and disintegrate (Figure 2C). From our experience, approximately 75% of embryos were attached to the bottom of the fibronectin coated dish at 48 h and the attachment increased to approximately 90% by 72 h in culture. The success of embryo attachment may be largely impacted by the initial quality of the blasto...

Watch this Scientific Journal Video about Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos at JoVE.com

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

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 ...

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Research

JoVE Journal

Biology

6.2K Views.  Colorado Center for Reproductive Medicine. Veel zwangerschappen mislukken vroeg in de ontwikkeling van menselijke embryo's rond de zeven of acht post-bevruchting. Dit protocol biedt mogelijkheden voor onderzoekers om menselijke conceptors in vitro te laten groeien tijdens dit mysterieuze implantatievenster. Dit stelt ons in staat om de mechanismen te begrijpen die ten grondslag liggen aan het succes van menselijke implantatie en vroege placentavorming.Het gebruik van een uitgebreid kweeksysteem om peri-implantatiefase menselijk...

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

Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining the ability to self-renew and to differentiate into all subtypes of the trophoblast lineage, equivalent to the in vivo stem cell population giving rise to the fetal portion of the placenta. Therefore, TSCs offer a unique model to study placental development and embryonic versus extra-embryonic cell fate decision in vitro. From the blastocyst stage onwards, a distinct epigenetic barrier consisting of DNA methylation and histone modifications tightly separates both lineages. Here, we describe a protocol to fully overcome this lineage barrier by transient over-expression of trophoblast key regulators Tfap2c, Gata3, Eomes and Ets2 in murine embryonic fibroblasts. The induced trophoblast stem cells are able to self-renew and are almost identical to blastocyst derived trophoblast stem cells in terms of morphology, marker gene expression and methylation pattern. Functional in vitro and in vivo assays confirm that these cells are able to differentiate along the trophoblast lineage generating polyploid trophoblast giant cells and chimerizing the placenta when injected into blastocysts. The induction of trophoblast stem cells from somatic tissue opens new avenues to study genetic and epigenetic characteristics of this extra-embryonic lineage and offers the possibility to generate trophoblast stem cell lines without destroying the respective embryo.

Here we present a protocol for the direct conversion of murine embryonic fibroblasts into fully functional and stable trophoblast stem cells by ten day over-expression of Tfap2c, Gata3, Eomes and Ets2.

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining ...

Developmental Biology

Transcriptional Analysis by Nascent RNA FISH of In Vivo Trophoblast Giant Cells or In Vitro Short-term Cultures of Ectoplacental Cone Explants

The placenta derives from one extra-embryonic lineage, the trophectoderm. In the peri-implantation murine blastocyst, mural trophectoderm cells differentiate into primary trophoblast giant cells (TGCs) while the polar trophectoderm overlying the inner cell mass continues to proliferate later differentiating into secondary TGCs. TGCs play a key role in developing placenta and are essential for a successful pregnancy. Investigation of transcriptional regulation of specific genes during post-implantation development can give insights into TGCs development. Cells of the ectoplacental cone (EPC) from embryos at 7-7.5 days of gestation (E7-7.5), derived from the polar trophectoderm, differentiate into secondary TGCs1. TGCs can be studied in situ, on cryostat sections of embryos at E7 although the number of TGCs is very low at this stage. An alternative means of analyzing secondary TGCs is to use short-term cultures of individual EPCs from E7 embryos. We propose a technique to investigate the transcriptional status of genes of interest both in vivo and in vitro at the single-cell level using fluorescent in situ hybridization (RNA FISH) to visualize nascent transcripts. This technique provides a direct readout of gene expression and enables assessment of the chromosomal status of TGCs, which are large endoreplicating cells. Indeed, a key feature of terminal differentiation of TGCs is that they exit the cell cycle and undergo multiple rounds of endoreplication.This approach can be applied to detect expression of any gene expressed from autosomes and/or sex chromosomes and can provide important information into developmental mechanisms as well as placental diseases.

Transcriptional  Analysis by Nascent RNA FISH of In Vivo Trophoblast Giant Cells or In Vitro Short-term Cultures of Ectoplacental Cone Explants

Trophoblast giant cells (TGCs) play a key role in the placenta to ensure a healthy pregnancy. We present a protocol for assessing the transcriptional status of genes in TGCs by nascent fluorescent in situ hybridization on cryostat sections of post-implantation embryos or short-term cultures of embryonic day 7 ectoplacental cones.

The placenta derives from one extra-embryonic lineage, the trophectoderm. In the peri-implantation murine blastocyst, mural trophectoderm cells ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration and invasion is demonstrated by pregnancy disorders such as pre-eclampsia and intrauterine growth restriction, which are associated with inadequate trophoblast invasion of the maternal vasculature. Unfortunately, our understanding of the mechanisms by which the placenta develops from migrating trophoblasts is limited. In vitro analysis of cell migration via the scratch assay is a useful tool in identifying factors that regulate trophoblast migratory capacity. However, this assay alone does not define the cellular changes that can result in altered cell migration. This protocol describes three different in vitro assays that are used collectively to evaluate trophoblast cell movement: the scratch assay, the invasion assay, and the proliferation assay. The protocols described here may also be modified for use in other cell lines to quantify cell movement in response to stimuli. These methods allow investigators to identify individual factors that contribute to the cell movement and provide a thorough examination of potential mechanisms underlying apparent changes in cell migration.

Here, we present a highly accessible protocol for evaluating the cell movement in human trophoblast cells using three in vitro assays: the scratch assay, the transwell invasion assay, and the cell proliferation assay.

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration ...

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 ...

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.

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 ...

Analyzing Angiogenesis: In Vitro Assay for Tube Formation on BM Matrix

Trophoblast Cell Recovery from Angiogenesis-Tube Formation Assay for Differentiation Marker Expression Analysis

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of mesenchymal to endothelial-like transition. Traditionally, this process is evaluated by an in vitro tube-formation assay, where the cells organize themselves into tube-like structures when seeded over a polymerized basement membrane preparation. Although several structural features can be measured in photomicrographs of the structures, to assess the real commitment of EVT to the endothelial-type phenotype, biochemical analysis of cell extracts is required. Scraping the cells from the culture dish to obtain RNA and/or protein extracts is not an alternative since the tube-like structures are severely contaminated by the bulk of proteins from the polymerized basement membrane. Thus, a strategy to separate the cells from the basement membrane proteins prior to the preparation of cell extracts is needed. Here, a simple, fast, and cost-effective method to recover the tube-like structures from the in vitro tube-formation assay and the subsequent analysis by biochemical techniques is presented. Tube-like structures formed by HTR8/SVneo cells, an EVT cell line, were liberated from the polymerized basement membrane by a short incubation with PBS supplemented with ethylenediaminetetraacetic acid (EDTA). After serial washes, a ready-to-use pellet of purified tube-like structures can be obtained. This pellet can be subsequently processed to obtain RNA and protein extracts. qPCR analysis evidenced the induced expression of VE-cadherin and alphav-integrin, two endothelial cell markers, in EVT-derived tube-like structures compared to control cells, which was consistent with the induction of the endothelial cell marker, CD31, evaluated by immunofluorescence. Western blot analysis of the tube-like structures' protein extracts revealed the overexpression of RECK in transfected HTR8/SVneo cells. Thus, this simple method allows to obtain cell extracts from the in vitro tube-formation assay for the subsequent analysis of RNAs and protein expression.

The present protocol allows the recovery of tube-like structures formed in the classical in vitro angiogenesis-tube formation assay where cells, such as trophoblast cells, organize in capillary-like structures over a layer of extracellular basement membrane matrix. The recovered structures are used for RNA and protein isolation for various biochemical analyses.

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of ...

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be attributed to, in part, the dysregulation of autophagy. Aberrant changes in the expression of autophagy regulators in the placentas from obese pregnancies may be regulated by inflammatory processes associated with both obesity and pregnancy. Described here is a protocol for sampling of villous tissue and isolation of villous cytotrophoblasts from the term human placenta for primary cell culture. This is followed by a method for simulating the inflammatory milieu in the obese intrauterine environment by treating primary trophoblasts from lean pregnancies with tumor necrosis factor alpha (TNF&#945;), a proinflammatory cytokine that is elevated in obesity and in pregnancy. Through the implementation of the protocol described here, it is found that exposure to exogenous TNF&#945; regulates the expression of Rubicon, a negative regulator of autophagy, in trophoblasts from lean pregnancies with female fetuses. While a variety of biological factors in the obese intrauterine environment maintain the potential to modulate critical pathways in trophoblasts, this ex vivo system is especially useful for determining if expression patterns observed in vivo in human placentas with maternal obesity are a direct result of TNF&#945; signaling. Ultimately, this approach affords the opportunity to parse out the regulatory and molecular implications of inflammation associated with maternal obesity on autophagy and other critical cellular pathways in trophoblasts that have the potential to impact placental function.

Presented here is a protocol for sampling of human placental villous tissue followed by isolation of cytotrophoblasts for primary cell culture. Treatment of trophoblasts with TNF&#945; recapitulates inflammation in the obese intrauterine environment and facilitates the discovery of molecular targets regulated by inflammation in placentas with maternal obesity.

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be ...

Medicine

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

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

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