Name: in vitro differentiation of human pluripotent stem cells into trophoblastic cells
Uploaded: 2017-03-16T12:30:00
Description: Watch this Scientific Journal Video about In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells at JoVE.com

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)
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<ol>
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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#34;serum replacement&#34; 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 um 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&#38;D Systems</td>
			<td>314-BP-01M</td>
			<td></td>
		</tr>
		<tr>
			<td>A 83-01</td>
			<td>R&#38;D Systems</td>
			<td>2939/10</td>
			<td></td>
		</tr>
		<tr>
			<td>PD173074</td>
			<td>R&#38;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 &#34;extracellular matrix&#34; 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 ...

The overall goal of this protocol is to differentiate human pluripotent stem cells into human trophoblastic cells using bone morphogenic 4 and inhibitors of the active and nodal signaling pathways to study the early events of human placental formation and function. This method can help answer key questions in the study of trophoblastic cell functions and how these cells are formed. The main advantage of this technique is that it can produce abundant trophoblastic cells for a wide variety of applications including genetics and allow cell function assays.To begin the experiment, thaw an aliquot of extracellular matrix on ice for three to four hours. Using ice cold tips, one cold 50 milliliter conical tube and Dulbecco's modified equal medium, or DMEM F12 medium, transfer two milligrams of extracellular matrix into 24 milliliters of ice cold DMEM F12. Immediately transfer the 50 milliliter conical tube to ice and store it at four degrees Celsius.To coat tissue culture plates, add the one milliliter of diluted extracellular matrix per well of a six-well plate. Swirl the plate to coat the wells and incubate the plate at room temperature for at least one hour. Aspirate the extracellular matrix from the new plate and add mouse embryonic fibroblast-conditioned medium or MEFCM containing bFGF.Use a pipette to transfer the scraped cellular clamps from the MEF plate and aliquot them into the extracellular matrix-coated plate. Then incubate the plate. Scrape off differentiated cells with a Pasteur pipette before replacing the MEFCM with bFGF medium daily using two milliliters of medium for one well.When colonies appear big and bright under the microscope, passage hESCs every six to seven days. When the feeder-free cells are ready to passage, remove the medium and wash the cells by adding one milliliter of PBS using a pipette. Aspirate the PBS and replace it with 0.5 milligrams per milliliter of pre-warmed dispase with a pipette.Incubate the cells at 37 degrees Celsius for five minutes. After incubation, wash the cells with PBS by adding two milliliters of PBS per well and aspirate the PBS. Add one milliliter of CM per well and manually scrape the cells into small clumps using the tip of a five milliliter pipette.Next, plate the suspended cell clumps into new extracellular matrix-coated plates. Prepare the differentiation medium. Use CM without bFGF and add fresh inhibitors prior to use.Use feeder-free hESCs growing on extracellular matrix-coated plates for one passage cultured inside an incubator set at 5%oxygen. After the first passage onto the extracellular matrix-coated plates, let the cells attach for 24 hours. The next day, initiate differentiation by removing the CM containing bFGF and replacing it with CM containing BMP4/A/P with two milliliters per well in a six-well plate.Continue culturing the cells using 4%oxygen levels. Replace the CM containing BMP4/A/P every two days by aspirating the old medium and adding new medium containing BMP4/A/P. Collect cells at desired time points.Culture hESCs for two passages on extracellular matrix after transferring them from the MEFs with hESC medium containing bFGF. High cellular confluency is important so split the hESCs one-to-one onto a new plate which will ensure at least 50%confluency the next day. Incubate the cells overnight in the low oxygen incubator.The next day, replace the medium with two milliliters per well of differentiation medium. Place the cells in a low oxygen incubator overnight. Before transfection, gelatin coat a dish adding 0.1%gelatin to a dish.Swirl the dish to coat the plate, then leave the plate at room temp. After 20 minutes, remove the gelatin. Differentiate the cells until the desired time point.The day before transfection, trypsinize the cells using 0.05%trypsin for five minutes. Plate the cells to the desired confluency onto the gelatin-coated plate. Add CM containing BMP4/A/P lacking bFGF and incubate the culture overnight.The next day, add fresh differentiation medium to the cells. Transfect siRNAs using an siRNA transfection reagent. For plasma DNA, use an appropriate lipofectamine reagent.Follow the product protocol as described for each transfection reagent. Transfer the siRNA lipofectamine complexes to each well or plate of cells and mix gently. Then culture the cells overnight in a low oxygen incubator.The next day, replace the media with fresh differentiation media. Harvest the cells at the desired time points to check the gene disruption efficiency using quantitative RT-PCR. Finally, to harvest cells, add one milliliter of 0.05%trypsin per well and incubate the cells.Add five milliliters of MEF medium per well to neutralize the trypsin. Spin down the cells at 200 times g for five minutes and use the cell pellet for RNA isolation. Using this technique, morphological changes are clearly seen.Cells on differentiation day one are larger than the cells from day zero. As differentiation proceeds, the cells divide and expand out from the center of the colony, growing in an outward direction. The individual colonies merge together by differentiation day five with cells growing on top of each other.QRT-PCR analysis reveals that the relative expression levels of the pluripotent C-marker NANOG are reduced by 75%after one day of differentiation when cells are cultured with BMP4 and reduced by 90%in the presence of BMP4/A/P. By differentiation day two, the levels of NANOG are very low for cells cultured in BMP4 alone or in BMP4/A/P compared to those in hESC CM medium containing beta FGF. Transfections performed using a GFP plasmid and introduced into BMP4/A/P treated cells on differentiation day one and day three obtained 30 to 50%transfection efficiency at 24 hours post-transfection.After watching this video, you should have a good understanding of how to differentiate human pluripotent cells into trophoblastic cells in vitro and how to introduce DNA constructs for manipulating gene expression.

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