This work was supported by a Pennsylvania Health Research Formula Fund.

NOTE: For the differentiation of either hESCs or hiPSCs into trophoblast progenitors, hPSCs grown on mouse embryonic fibroblasts (MEFs) are transitioned to feeder-free conditions for two passages before initiating differentiation with BMP4/A/P. This process eliminates the MEF contamination of differentiated cells. Here, we present a protocol for hESC differentiation, and the same protocol can be applied to hiPSCs.
1. Culture and Recovery of hESCs on Irradiated Mouse Embryonic Fibroblasts (MEFs) (Preparations)
<ol>
	<li>Gamma-irradiation of MEFs
	<ol>
		<li>Make 500 ml of medium for culturing the MEFs: DMEM with 10% FBS, 2 mM L-glutamine, 1x penicillin/streptomycin (Pen/Strep), and 0.1 mM non-essential amino acids (NEAA).</li>
		<li>Thaw one vial of primary MEFs and use MEF medium for culturing the cells. Expand the primary MEFs to 30 plates using MEF culture medium. 
		NOTE: Oxygen concentrations are not critical for this step, so either physiological (4%) or ambient (20%) conditions within the incubator can be used.</li>
		<li>Harvest the MEFS. Use 0.25% trypsin to remove the MEFs from the plates and transfer the cells to a conical tube. Place the MEFs into an irradiation instrument and expose the cells to 3,500 grays. 
		NOTE: The amount of time will depend on the activity of the source inside the irradiator unit.</li>
		<li>Resuspend the irradiated MEFs with MEF culture medium and count the number of cells using a hemocytometer.</li>
		<li>Pellet the MEFs using a centrifuge and add 50% MEF culture medium and 50% cell freezing medium (80% FBS and 20% MEF culture medium). Aliquot 1 x 106 cells per vial.</li>
		<li>Place the irradiated MEF vials in a -80 &#176;C freezer overnight, and then transfer them to liquid nitrogen for long-term storage.</li>
	</ol>
	</li>
	<li>Thawing frozen MEFs and hESCs for culture
	<ol>
		<li>Make 500 ml of hESC medium: DMEM/F-12 with 20% Serum Replacement, 0.1 mM NEAA, 2 mM L-Glutamine, 10 ng/ml bFGF, 0.1 mM &#223;-mercaptoethanol, and 1x Pen/Strep.</li>
		<li>Thaw one vial of MEFs one day before thawing the hESCs. Coat a 6-well plate with 0.1% gelatin, using 1 ml for each well. Incubate for at least 20 min at room temperature, and then remove the gelatin.</li>
		<li>Remove a vial of MEFs from storage in liquid nitrogen and immerse the vial in a 37 &#176;C water bath. Watch the vial intermittently until only small ice crystals remain. Quickly transfer the contents of the vial to 9 ml of MEF medium inside a 15 ml conical tube.</li>
		<li>Pellet the cells by centrifuging at 200 x g for 5 min. Aspirate the supernatant and resuspend the cell pellet in MEF medium. Aliquot the MEFs evenly onto a 6-well plate. Put the plate into a 37 &#176;C low-oxygen (4%) incubator overnight.</li>
	</ol>
	</li>
	<li>Routine culture of hESCs on MEFs
	<ol>
		<li>Remove an hESC vial from liquid nitrogen and quickly thaw the vial using a 37 &#176;C water bath. Gently pipette the hESCs into a 15 ml conical tube containing 9 ml of hESC medium and spin down at 200 x g for 5 min. Remove the medium and resuspend the cells with 1 ml of hESC medium.</li>
		<li>Aspirate the MEF medium from the plate prepared in step 1.2.4 and add 1 ml of hESC medium. Add Rock inhibitor Y-27632 (10 &#181;M) to the hESC medium. Transfer the hESCs onto MEFs.</li>
		<li>Place the cells into a 37 &#176;C low-oxygen (4%) incubator overnight. Replace the hESC medium daily. Scrape off differentiated cells with a Pasteur pipette, visualized under an upright microscope at 4X.</li>
		<li>Passage the hESCs every 4-6 days, depending on the cell confluency and the size of the hESC colonies. Prepare a plate of MEFs the day before passaging. When the cells are ready to passage, remove the hESC medium and wash the well with 1 ml of PBS.</li>
		<li>Add 1 mg/ml collagenase (pre-warmed to 37 &#176;C), incubate at 37 &#176;C for 5 min, and remove the collagenase. Wash the cells with PBS, add 1 ml of hESC medium per well, and manually scrape the cells into small clumps using the tip of a 5 ml pipette. Transfer the suspended cell clumps into a new MEF-coated well(s).</li>
	</ol>
	</li>
</ol>
2. Transition of hESCs from MEFs to Feeder-free Conditions on Extracellular Matrix-coated Plates
<ol>
	<li>Prepare MEF Conditioned Medium (CM)
	<ol>
		<li>Plate the irradiated MEFs in a T75 flask at 90-100% confluency; it is important that the MEFs are densely plated. Observe the cells using a microscope to determine whether they have attached to the bottom of the flask (MEFs attach approximately 6 hr after plating or the next day). 
		NOTE: Unattached cells float in the medium when observed using a microscope. The next day, remove MEF medium and add 25 ml of hESC medium lacking B-FGF.</li>
		<li>Collect the conditioned medium after 24 hr of incubation. Replace with fresh medium daily for a maximum of 12 days.</li>
		<li>Filter the collected CM using a 0.22 &#181;M filter. Prior to use, add fresh B-FGF to the CM. 
		NOTE: CM can be frozen at -80 &#176;C and stored for up to 1 year. Alternatively, store the CM at 4 &#176;C for 2 weeks.</li>
	</ol>
	</li>
	<li>Transition the hESCs from culture on the MEFs to feeder-free extracellular matrix-coated plates.
	<ol>
		<li>Preparation of extracellular matrix for coating tissue culture plates
		<ol>
			<li>Thaw an aliquot of extracellular matrix on ice (about 3-4 hr). Using ice-cold tips, one cold, 50 ml conical tube, and DMEM/F12 medium, transfer 2 mg of extracellular matrix into 24 ml of ice-cold DMEM/F-12 (the dilution depends on the extracellular matrix concentration from the supplier).</li>
			<li>Immediately transfer the 50 ml conical tube to ice and store it at 4 &#176;C. For coating tissue culture plates, add the appropriate amount of diluted extracellular matrix to the well (e.g., 1 ml per well in a 6-well plate). Swirl to coat the plate and incubate at room temperature for at least 1 hr.</li>
		</ol>
		</li>
		<li>Passage hESCs from the MEFs (as in step 1.3) using CM containing B-FGF (10 ng/ml).
		<ol>
			<li>Aspirate to remove the extracellular matrix from the new plate and add CM containing B-FGF. Transfer the scraped cellular clumps from the MEF plate using a pipette and aliquot it into the extracellular matrix-coated plate. Incubate in the 37 &#176;C low-oxygen incubator overnight.</li>
			<li>Replace the CM with B-FGF medium daily; the amount of medium will depend on the size of the culture flask. Use 2 ml of medium for one 6-well well. Scrape off differentiated cells with a Pasteur pipette.</li>
			<li>Passage hESCs every 6-7 days, when colonies are bright when viewed under the microscope. 
			NOTE: hESC colonies grown on extracellular matrix-coated plates grow larger than MEF-cultured cells.</li>
			<li>When the feeder-free cells are ready to passage, remove the medium, wash by adding 1 ml of PBS using a pipette, aspirate to remove the PBS, add 0.5 mg/ml dispase (pre-warmed to 37 &#176;C) with a pipette, and incubate at 37 &#176;C for 5 min.</li>
			<li>Wash the cells with PBS by adding 2 ml of PBS per well and aspirating afterwards. Add 1 ml of CM per well and manually scrape the cells into small clumps using the tip of a 5 ml pipette.</li>
			<li>Plate the suspended cell clumps into new extracellular matrix-coated plates; the cells should adhere after 24 hr.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Differentiation of hESCs Using BMP4/A/P
<ol>
	<li>Prepare the differentiation medium. Use CM (lacking B-FGF) and add (fresh) BMP4 (50 ng/ml), A83-01 (1 &#181;M), and PD173074 (0.1 &#181;M). Add these inhibitors to the CM prior to use.</li>
	<li>Use feeder-free hESCs growing on extracellular matrix-coated plates for 1 passage, cultured inside an incubator set at 4% oxygen. After the first passage onto extracellular matrix-coated plates, let the cells attach for 24 hr. The next day, initiate differentiation. Remove the CM (containing B-FGF) and replace it with CM containing BMP4/A/P (2 ml per well in a 6-well plate). Continue culturing the cells using 4% oxygen levels.</li>
	<li>Replace the CM containing BMP4/A/P (2 ml/well for a 6-well plate) every 2 days. Aspirate to remove the old medium and add new CM using a pipette. On the second day after adding BMP4 and removing B-FGF (considered differentiation day 2), the cell morphology will change, appearing larger when observed using a microscope. 
	NOTE: Undifferentiated cells, which exhibit bright round edges, will not be present.</li>
	<li>Collect cells at desired time-points. Differentiated cells will stop dividing after around 2 weeks. Transfect cells during this time period (see the next section).</li>
</ol>
4. Transfection of Trophoblastic Cells with siRNAs or Plasmid DNA
<ol>
	<li>Prepare trophoblastic cells for transfection
	<ol>
		<li>Culture hESCs for two passages on extracellular matrix after transferring them from the MEFs (see step 2.2). Use hESC medium containing B-FGF.</li>
		<li>Split the hESCs onto a new extracellular matrix plate one day before differentiation. High cellular confluency is important, so split the cells 1:1 onto a new plate/well, which will ensure at least 50% confluency the next day. Incubate the cells overnight in the low-oxygen incubator. 
		NOTE: hESCs do not need to be counted during this step.</li>
		<li>The next day, replace the medium with the differentiation medium (CM containing BMP4/A/P and lacking B-FGF, using 2 ml/well for a 6-well plate). Remove the old medium by aspiration and transfer the new medium (2 ml) using a pipette. Place the cells in a low-oxygen incubator (4% oxygen) overnight.</li>
	</ol>
	</li>
	<li>Transfections using siRNAs for gene disruption or using plasmid DNA
	<ol>
		<li>Differentiate the cells until the desired time-point (between days 1 and 14). The day before transfection, trypsinize the cells using 0.05% trypsin for 5 min. Plate them to the desired confluency (depending on the transfection reagent protocol) onto a gelatin-coated plate (add 0.1% gelatin to a tissue culture plate for 20 min and aspirate to remove). Add CM containing BMP4/A/P (lacking B-FGF) and incubate overnight.</li>
		<li>The next day, add fresh differentiation medium to the cells. Transfect siRNAs using an siRNA transfection reagent. For plasmid DNA, use an appropriate lipofectamine reagent.</li>
		<li>Follow the product protocol as described for each transfection reagent. Transfer the siRNA-lipofectamine complexes to each well/plate of cells, mix gently, and culture the cells overnight in a low-oxygen incubator. 
		NOTE: Some transfection reagents are inhibited by antibiotics, which require CM lacking Pen/Strep.</li>
		<li>The next day, replace with fresh differentiation media.</li>
		<li>Harvest the cells at the desired time-points (e.g., 24 hr, 48 hr, and 72 hr) to check the gene disruption efficiency using quantitative RT-PCR. For harvesting cells, use 0.05% trypsin, adding 1 ml per well and incubating for 5 min at 37 &#176;C. Add 5 ml of MEF medium per well to neutralize the trypsin. Spin down the cells at 200 x g for 5 min and use the cell pellet for RNA isolation.</li>
	</ol>
	</li>
</ol>
5. Pathogen Infection of Trophoblastic Cells: Sendai Viral Infection
<ol>
	<li>Prepare hESCs for differentiation
	<ol>
		<li>Culture hESCs for two passages on extracellular matrix after the transfer from the MEFs (see step 2.2). Use hESC medium containing B-FGF.</li>
		<li>Split the hESCs onto a new extracellular matrix plate one day before differentiation. High cellular confluency is important, so split cells 1:1 onto a new plate/well, which will ensure at least 50% confluency the next day. Incubate the cells overnight in a low-oxygen incubator (4% oxygen). 
		NOTE: hESCs do not need to be counted during this step.</li>
		<li>The next day, replace the medium with the differentiation medium (CM containing BMP4/A/P and lacking B-FGF) and culture overnight in a low-oxygen incubator (4% oxygen).</li>
	</ol>
	</li>
	<li>Sendai viral infection of trophoblastic cells
	<ol>
		<li>One day before the desired differentiation day, trypsinize the differentiating cells (to single cells) using 0.05% trypsin for 5 min and transfer them to a gelatin-coated plate. Add differentiation medium and incubate overnight in a low-oxygen incubator.</li>
		<li>The next day, add medium for infection (this will vary depending on the virus). Use CM lacking Pen/Strep containing BMP4/A/P, using 0.5 ml for one well of a 6-well plate. Add Sendai virus for MOI = 1. Incubate for 8 hr in a low-oxygen incubator.</li>
		<li>If additional time-points are necessary, remove the virus-containing medium after 4 hr and replace it with BMP4/A/P CM. Collect cells for RNA isolation using a cell scraper.</li>
		<li>Determine the efficiency of viral infection by performing qRT-PCR for genes involved in the viral response18.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Rugg-Gunn, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+features+of+the+mouse+trophoblast.">Epigenetic features of the mouse trophoblast.</a> Reproductive biomedicine online. 25 (1), 21-30 (2012).</li><li>Rossant, J., Cross, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+development:+lessons+from+mouse+mutants.">Placental development: lessons from mouse mutants.</a> Nature reviews. Genetics. 2 (7), 538-548 (2001).</li><li>Hertig, A. T., Rock, J., Adams, E. C., Menkin, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thirty-four+fertilized+human+ova,+good,+bad+and+indifferent,+recovered+from+210+women+of+known+fertility;+a+study+of+biologic+wastage+in+early+human+pregnancy.">Thirty-four fertilized human ova, good, bad and indifferent, recovered from 210 women of known fertility; a study of biologic wastage in early human pregnancy.</a> Pediatrics. 23 (1 Part 2), 202-211 (1959).</li><li>Steptoe, P. C., Edwards, R. G., Purdy, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+blastocysts+grown+in+culture.">Human blastocysts grown in culture.</a> Nature. 229 (5280), 132-133 (1971).</li><li>Delorme-Axford, E., Sadovsky, Y., Coyne, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+placenta+as+a+barrier+to+viral+infections.">The placenta as a barrier to viral infections.</a> Annual Review of Virology. 1, 133-146 (2014).</li><li>Ji, 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=Placental+trophoblast+cell+differentiation:+physiological+regulation+and+pathological+relevance+to+preeclampsia.">Placental trophoblast cell differentiation: physiological regulation and pathological relevance to preeclampsia.</a> Molecular aspects of medicine. 34 (5), 981-1023 (2013).</li><li>Bilban, 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=Identification+of+novel+trophoblast+invasion-related+genes:+heme+oxygenase-1+controls+motility+via+peroxisome+proliferator-activated+receptor+gamma.">Identification of novel trophoblast invasion-related genes: heme oxygenase-1 controls motility via peroxisome proliferator-activated receptor gamma.</a> Endocrinology. 150 (2), 1000-1013 (2009).</li><li>Xu, R. H., 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=BMP4+initiates+human+embryonic+stem+cell+differentiation+to+trophoblast.">BMP4 initiates human embryonic stem cell differentiation to trophoblast.</a> Nature biotechnology. 20 (12), 1261-1264 (2002).</li><li>Amita, 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=Complete+and+unidirectional+conversion+of+human+embryonic+stem+cells+to+trophoblast+by+BMP4.">Complete and unidirectional conversion of human embryonic stem cells to trophoblast by BMP4.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (13), E1212-E1221 (2013).</li><li>Genbacev, O., 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=Establishment+of+human+trophoblast+progenitor+cell+lines+from+the+chorion.">Establishment of human trophoblast progenitor cell lines from the chorion.</a> Stem Cells. 29 (9), 1427-1436 (2011).</li><li>Marchand, 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=Transcriptomic+signature+of+trophoblast+differentiation+in+a+human+embryonic+stem+cell+model.">Transcriptomic signature of trophoblast differentiation in a human embryonic stem cell model.</a> Biology of reproduction. 84 (6), 1258-1271 (2011).</li><li>Hyslop, 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=Downregulation+of+NANOG+induces+differentiation+of+human+embryonic+stem+cells+to+extraembryonic+lineages.">Downregulation of NANOG induces differentiation of human embryonic stem cells to extraembryonic lineages.</a> Stem cells. 23 (8), 1035-1043 (2005).</li><li>Harun, R., 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=Cytotrophoblast+stem+cell+lines+derived+from+human+embryonic+stem+cells+and+their+capacity+to+mimic+invasive+implantation+events.">Cytotrophoblast stem cell lines derived from human embryonic stem cells and their capacity to mimic invasive implantation events.</a> Human reproduction. 21 (6), 1349-1358 (2006).</li><li>Lichtner, B., Knaus, P., Lehrach, H., Adjaye, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP10+as+a+potent+inducer+of+trophoblast+differentiation+in+human+embryonic+and+induced+pluripotent+stem+cells.">BMP10 as a potent inducer of trophoblast differentiation in human embryonic and induced pluripotent stem cells.</a> Biomaterials. 34 (38), 9789-9802 (2013).</li><li>Chen, Y., Wang, K., Chandramouli, G. V., Knott, J. G., Leach, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trophoblast+lineage+cells+derived+from+human+induced+pluripotent+stem+cells.">Trophoblast lineage cells derived from human induced pluripotent stem cells.</a> Biochemical and biophysical research communications. , (2013).</li><li>Roberts, R. 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=Differentiation+of+trophoblast+cells+from+human+embryonic+stem+cells:+to+be+or+not+to+be?.">Differentiation of trophoblast cells from human embryonic stem cells: to be or not to be?.</a> Reproduction. 147 (5), D1-D12 (2014).</li><li>Sarkar, P., 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=Activin/nodal+signaling+switches+the+terminal+fate+of+human+embryonic+stem+cell-derived+trophoblasts.">Activin/nodal signaling switches the terminal fate of human embryonic stem cell-derived trophoblasts.</a> The Journal of biological chemistry. 290 (14), 8834-8848 (2015).</li><li>Penkala, I., 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=lncRHOXF1,+a+Long+Noncoding+RNA+from+the+X+Chromosome+That+Suppresses+Viral+Response+Genes+during+Development+of+the+Early+Human+Placenta.">lncRHOXF1, a Long Noncoding RNA from the X Chromosome That Suppresses Viral Response Genes during Development of the Early Human Placenta.</a> Mol Cell Biol. 36 (12), 1764-1775 (2016).</li><li>Penkala, I., 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=lncRHOXF1,+a+Long+Noncoding+RNA+from+the+X+Chromosome+That+Suppresses+Viral+Response+Genes+during+Development+of+the+Early+Human+Placenta.">lncRHOXF1, a Long Noncoding RNA from the X Chromosome That Suppresses Viral Response Genes during Development of the Early Human Placenta.</a> Molecular and cellular biology. 36 (12), 1764-1775 (2016).</li><li>Hockemeyer, D., 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=Genetic+engineering+of+human+pluripotent+cells+using+TALE+nucleases.">Genetic engineering of human pluripotent cells using TALE nucleases.</a> Nature biotechnology. 29 (8), 731-734 (2011).</li><li>Bernardo, A. S., 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=BRACHYURY+and+CDX2+mediate+BMP-induced+differentiation+of+human+and+mouse+pluripotent+stem+cells+into+embryonic+and+extraembryonic+lineages.">BRACHYURY and CDX2 mediate BMP-induced differentiation of human and mouse pluripotent stem cells into embryonic and extraembryonic lineages.</a> Cell stem cell. 9 (2), 144-155 (2011).</li><li>Zhang, P., 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=Short-term+BMP-4+treatment+initiates+mesoderm+induction+in+human+embryonic+stem+cells.">Short-term BMP-4 treatment initiates mesoderm induction in human embryonic stem cells.</a> Blood. 111 (4), 1933-1941 (2008).</li><li>Vallier, 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=Early+cell+fate+decisions+of+human+embryonic+stem+cells+and+mouse+epiblast+stem+cells+are+controlled+by+the+same+signalling+pathways.">Early cell fate decisions of human embryonic stem cells and mouse epiblast stem cells are controlled by the same signalling pathways.</a> PloS one. 4 (6), e6082 (2009).</li><li>Arman, E., Haffner-Krausz, R., Chen, Y., Heath, J. K., Lonai, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+fibroblast+growth+factor+(FGF)+receptor+2+suggests+a+role+for+FGF+signaling+in+pregastrulation+mammalian+development.">Targeted disruption of fibroblast growth factor (FGF) receptor 2 suggests a role for FGF signaling in pregastrulation mammalian development.</a> Proceedings of the National Academy of Sciences of the United States of America. 95 (9), 5082-5087 (1998).</li><li>Yu, P., Pan, G., Yu, J., Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF2+sustains+NANOG+and+switches+the+outcome+of+BMP4-induced+human+embryonic+stem+cell+differentiation.">FGF2 sustains NANOG and switches the outcome of BMP4-induced human embryonic stem cell differentiation.</a> Cell stem cell. 8 (3), 326-334 (2011).</li><li>Sudheer, S., Bhushan, R., Fauler, B., Lehrach, H., Adjaye, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF+inhibition+directs+BMP4-mediated+differentiation+of+human+embryonic+stem+cells+to+syncytiotrophoblast.">FGF inhibition directs BMP4-mediated differentiation of human embryonic stem cells to syncytiotrophoblast.</a> Stem cells and development. 21 (16), 2987-3000 (2012).</li><li>Bischof, P., Irminger-Finger, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+cytotrophoblastic+cell,+a+mononuclear+chameleon.">The human cytotrophoblastic cell, a mononuclear chameleon.</a> The international journal of biochemistry &amp; cell biology. 37 (1), 1-16 (2005).</li><li>Cole, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperglycosylated+hCG,+a+review.">Hyperglycosylated hCG, a review.</a> Placenta. 31 (8), 653-664 (2010).</li><li>Apps, R., 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=Human+leucocyte+antigen+(HLA)+expression+of+primary+trophoblast+cells+and+placental+cell+lines,+determined+using+single+antigen+beads+to+characterize+allotype+specificities+of+anti-HLA+antibodies.">Human leucocyte antigen (HLA) expression of primary trophoblast cells and placental cell lines, determined using single antigen beads to characterize allotype specificities of anti-HLA antibodies.</a> Immunology. 127 (1), 26-39 (2009).</li></ol>

The authors declare that they have no competing financial interests.

We presented the basic steps for differentiating hESCs into trophoblast progenitors. This protocol has recently been optimized to rapidly differentiate hESCs with the addition of Activin/Nodal signaling inhibitors, increasing the differentiation to trophoblastic cells and avoiding the generation of mesoderm progenitors, which are typically observed with BMP4 treatment alone. The BMP4 model system allows for the examination of the earliest stages of human trophoblast lineage specification and expansion. In addition, this ...

The placenta is required for the growth and survival of the fetus during pregnancy and facilitates the exchange of gases, nutrients, waste products, and hormones between maternal and fetal circulation. The first organ formed during mammalian embryogenesis is the placenta, which begins developing 6-7 days post-conception in humans and 3.5-4.5 days in mice1,2,3,4. Trophoblastic cells are the most important cells of the placenta, and these cells represent one of the earliest lineage differentiation events of the mammalian embryo. They arise from the outer extra-embryonic trophectoderm cells of the preimplantation blastocyst. Our knowledge of the early stages of placental development is limited by ethical and logistical restrictions on modeling early human development.
During embryonic implantation, trophoblasts invade the maternal epithelium and differentiate into specialized progenitor cells5. Cytotrophoblasts (CTBs) are mononucleated, undifferentiated progenitors that fuse and differentiate into syncytiotrophoblasts (SYNs) and extravillous invasive trophoblasts (EVTs), which anchor the placenta to the uterus. SYNs are multinucleated, terminally differentiated cells that synthesize hormones necessary for sustaining pregnancy. The early differentiation events that generate EVTs and SYNs are essential for placental formation, as impairments in trophoblastic cells result in miscarriage, pre-eclampsia, and intrauterine growth restriction1. The types of human trophoblast cell lines that have been developed include immortalized CTBs and choriocarcinomas, which produce placental hormones and display invasive properties6. Primary trophoblastic cells from human first-trimester placentas can be isolated, but the cells quickly differentiate and stop proliferating in vitro. Importantly, transformed and primary cell lines have different gene expression profiles, indicating that tumorigenic and immortalized trophoblast cell lines may not accurately represent primary trophoblasts7. Additionally, these lines are unlikely to resemble placental trophoblast stem cell progenitors because they are derived from later-stage first through third trimesters.
There is a need for a robust in vitro culture system of early-stage human trophoblasts in order to study the early events of placental formation and function. Human embryonic stem cells (hESCs), which share properties with the inner cell mass of the preimplantation embryo, are frequently used to model early human development, including the formation of the early placenta. Both human induced pluripotent stem cells (hiPSCs) and hESCs can be differentiated into trophoblasts in vitro using Bone Morphogenic Protein 4 (BMP4)8,9,10,11,12,13,14,15. This conversion of pluripotent cells to trophoblastic cells using BMP4 is specific for human cells and is widely used to study the development of the early human placenta because it does not require access to early human embryos9,16. Recently, it was discovered that the addition of the inhibitors A83-01 (A) and PD173074 (P), which block the SMAD2/3 and MEK1/2 signaling pathways, increases the efficiency of hPSC differentiation into trophectoderm-like progenitors, mainly SYNs and EVTs, without the extensive generation of mesoderm, endoderm, or ectoderm cells9,17. Using these medium conditions, hESCs differentiated for 12 days have similar gene expression profiles as trophectoderm cells isolated from human blastocyst-stage embryos and secrete various placental-specific growth hormones, supporting the validity of this in vitro model system9,11. Here, we present a detailed protocol for the in vitro differentiation of hPSCs into human trophoblast progenitors using BMP4/A/P culture medium. These conditions produce abundant numbers of cells for a wide variety of applications, including RNA sequencing, gene disruption using siRNAs, pathogen infections, and genetic modification using lipofection-mediated transfection.

<table><tbody><tr><td>DMEM/F12</td><td>Invitrogen</td><td>11330-057</td><td></td></tr><tr><td>Knock Out Serum Replacement</td><td>Invitrogen</td><td>10828-028</td><td>This is referred to as &quot;serum replacement&quot; in this protocol.</td></tr><tr><td>NEAA</td><td>Invitrogen</td><td>11140-050</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>16000-044</td><td></td></tr><tr><td>L-Glutamine</td><td>Invitrogen</td><td>10828-028</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Invitrogen</td><td>15140-155</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma</td><td>M-7522</td><td></td></tr><tr><td>B-FGF</td><td>Millipore</td><td>GF-003</td><td></td></tr><tr><td>DMEM</td><td>Invitrogen</td><td>11965-118</td><td></td></tr><tr><td>Dispase</td><td>Invitrogen</td><td>17105-041</td><td></td></tr><tr><td>Collagenase Type IV</td><td>Invitrogen</td><td>17104-019</td><td></td></tr><tr><td>Rock inhibitor Y27632</td><td>Calbiochem</td><td>688000</td><td></td></tr><tr><td>Irradiated CF1 MEFs</td><td>GlobalStem</td><td>6001G</td><td>MEFs can be generated from embryonic day 13.5 embyos and irradiated.</td></tr><tr><td>0.22 &amp;micro;m syringe filter</td><td>Millipore</td><td>SLGS033SS</td><td></td></tr><tr><td>Heracell 150i low oxygen incubator</td><td>Heracell/VWR</td><td>89187-192</td><td>Any tissue culture incubator with capacity to regulate oxygen concentrations is sufficient.</td></tr><tr><td>BMP4</td><td>R&amp;amp;D Systems</td><td>314-BP-01M</td><td></td></tr><tr><td>A 83-01</td><td>R&amp;amp;D Systems</td><td>2939/10</td><td></td></tr><tr><td>PD173074</td><td>R&amp;amp;D Systems</td><td>3044/10</td><td></td></tr><tr><td>RNAiMax</td><td>Invitrogen</td><td>13778150</td><td></td></tr><tr><td>Trizol</td><td>ThermoFisher</td><td>15596026</td><td>Trizol is used to isolate total RNA.</td></tr><tr><td>X-tremeGENE 9</td><td>Roche</td><td>6365779001</td><td></td></tr><tr><td>Matrigel</td><td>Corning</td><td>356231</td><td>This is referred to as &quot;extracellular matrix&quot; in this protocol.</td></tr></tbody></table>

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.

Overview of In Vitro Differentiation of hPSCs
This in vitro differentiation protocol begins with undifferentiated hESCs grown on MEFs that are transitioned to feeder-free conditions for one passage (Figure 1A). While we described the differentiation of hESCs in this protocol, we used this protocol to successfully differentiate hiPSCs into trophoblastic cells. The transition to extracellular matrix...

Watch this Scientific Journal Video about In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells at JoVE.com

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.

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 ...

in-vitro-differentiation-human-pluripotent-stem-cells-into

Research

Education

JoVE Core

JoVE Journal

Anatomy and Physiology

Developmental Biology

Unit 1

Essential Cellular Processes

SCIENTISTS IN ACTION

8.6K Views.  University of Pennsylvania. 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.

Video: In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Bioengineering

Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density centrifugation, media gradient isolation and immunomagnetic purification. As observed in vivo, mononucleated villous cytotrophoblast cells in primary culture differentiate into multinucleated syncytiotrophoblast cells after 72 hr. Compared to normoxia (8% O2), villous cytotrophoblast cells that undergo hypoxia/reoxygenation (0.5% / 8% O2) undergo increased oxidative stress and intrinsic apoptosis, similar to that observed in vivo in pregnancy complications such as preeclampsia, preterm birth, and intrauterine growth restriction. In this context, primary villous trophoblasts cultured under hypoxia/reoxygenation conditions represent a unique experimental system to better understand the mechanisms and signalling pathways that are altered in human placenta and facilitate the search for effective drugs that protect against certain pregnancy disorders. Human villous trophoblasts produce melatonin and express its synthesizing enzymes and receptors. Melatonin has been suggested as a treatment for preeclampsia and intrauterine growth restriction because of its protective antioxidant effects. In the primary villous cytotrophoblast cell model described in this paper, melatonin has no effect on trophoblast cells in normoxic state but restores the redox balance of syncytiotrophoblast cells disrupted by hypoxia/reoxygenation. Thus, human villous trophoblast cells in primary culture are an excellent approach to study the mechanisms behind the protective effects of melatonin on placental function during hypoxia/reoxygenation.

This manuscript presents a unique in vitro model of immunopurified human villous cytotrophoblast cells cultured under hypoxia/reoxygenation. This model is suitable to study the protective effects of promising treatments, such as melatonin, on pregnancy complications associated with increased oxidative stress and altered placental function.

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density ...

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 ...

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 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 ...

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Neuroscience

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

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

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

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

Biology

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 ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Samenvatting

Meer video's verkennen