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>

<ol>
	<li>Carter, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+human+placentation--a+review.">Animal models of human placentation--a review.</a> Placenta. , 41-47 (2007).</li><li>Carter, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+placentation+and+animal+models:+patterns+of+trophoblast+invasion+--+a+workshop+report.">Comparative placentation and animal models: patterns of trophoblast invasion -- a workshop report.</a> Placenta. 27, 30-33 (2006).</li><li>Pijnenborg, R., Robertson, W. B., Brosens, I., Dixon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+article:+trophoblast+invasion+and+the+establishment+of+haemochorial+placentation+in+man+and+laboratory+animals.">Review article: trophoblast invasion and the establishment of haemochorial placentation in man and laboratory animals.</a> Placenta. 2, 71-91 (1981).</li><li>Edwards, R. G., Bavister, B. D., Steptoe, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+stages+of+fertilization+in+vitro+of+human+oocytes+matured+in+vitro.">Early stages of fertilization in vitro of human oocytes matured in vitro.</a> Nature. 221 (5181), 632-635 (1969).</li><li>Thomson, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282 (5391), 1145-1147 (1998).</li><li>O'Leary, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+the+progression+of+the+human+inner+cell+mass+during+embryonic+stem+cell+derivation.">Tracking the progression of the human inner cell mass during embryonic stem cell derivation.</a> Nature Biotechnology. 30 (3), 278-282 (2012).</li><li>O'Leary, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+human+embryonic+stem+cells+using+a+post-inner+cell+mass+intermediate.">Derivation of human embryonic stem cells using a post-inner cell mass intermediate.</a> Nature Protocols. 8 (2), 254-264 (2013).</li><li>Shahbazi, M. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-organization+of+the+human+embryo+in+the+absence+of+maternal+tissues.">Self-organization of the human embryo in the absence of maternal tissues.</a> Nature Cell Biology. 18 (6), 700-708 (2016).</li><li>Deglincerti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-organization+of+the+in+vitro+attached+human+embryo.">Self-organization of the in vitro attached human embryo.</a> Nature. 533 (7602), 251-254 (2016).</li><li>Zhou, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituting+the+transcriptome+and+DNA+methylome+landscapes+of+human+implantation.">Reconstituting the transcriptome and DNA methylome landscapes of human implantation.</a> Nature. 572 (7771), 660-664 (2019).</li><li>Lv, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+RNA+sequencing+reveals+regulatory+mechanism+or+trophoblast+cell-fate+divergence+in+human+peri-implantation+conceptuses.">Single cell RNA sequencing reveals regulatory mechanism or trophoblast cell-fate divergence in human peri-implantation conceptuses.</a> PLoS Biology. 17 (10), 3000187 (2019).</li><li>West, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+trophoblast+differentiation+in+peri-implantation-stage+human+embryos.">Dynamics of trophoblast differentiation in peri-implantation-stage human embryos.</a> Proceedings of the Nationals Academy of Sciences U.S.A. 116 (45), 22635-22644 (2019).</li><li>Xiang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+developmental+landscape+of+3D-cultured+human+pre-gastrulation+embryos.">A developmental landscape of 3D-cultured human pre-gastrulation embryos.</a> Nature. 577 (7791), 537-542 (2020).</li><li>Mall, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Report+upon+the+collection+of+human+embryos+at+the+johns+hopkins+university.">Report upon the collection of human embryos at the johns hopkins university.</a> The Anatomical Record. 5 (7), 343-357 (1911).</li><li>Buettner, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Franklin+Paine+Mall+(1862-1917).">Franklin Paine Mall (1862-1917).</a> Embryo Project Encyclopedia. , (2007).</li><li>Carnegie Institution of Washington. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Contributions to embryology. , (1915).</li><li>Hertig, A. T., Rock, J., Adams, E. 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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>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&#39;s solution</td>
			<td>Millipore Sigma</td>
			<td>T1788</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Biotix 1250 &#181;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&#39;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 &#181;L</td>
		</tr>
		<tr>
			<td>Gilson 20 &#181;L Pipetteman</td>
			<td>Thermo Fisher Scientific</td>
			<td>F123602G</td>
			<td>1 Pipetteman 2-20 &#181;L</td>
		</tr>
		<tr>
			<td>Gilson 200 &#181;L Pipetteman</td>
			<td>Thermo Fisher Scientific</td>
			<td>F123602G</td>
			<td>1 Pipetteman 50-200 &#181;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&#39;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 &#181;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 &#181;L pipette tips</td>
			<td>VWR</td>
			<td>76322-134</td>
			<td>Pack of 960</td>
		</tr>
		<tr>
			<td>VWR 1-200 &#181;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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JoVE Journal

Biology

6.1K Views.  Colorado Center for Reproductive Medicine. 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.

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

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

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 ...

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

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 ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...