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>

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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 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 ...

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8.5K 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

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

This protocol introduces a simple two-step method for differentiating corneal limbal epithelial stem cells from human pluripotent stem cells under xeno- and feeder cell-free culture conditions. The cell culture methods presented here enable cost-efficient, large-scale production of clinical quality cells applicable to corneal cell therapy use.

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Transient Treatment of Human Pluripotent Stem Cells with DMSO to Promote Differentiation

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and iPSCs) across various lineages remain. Numerous differentiation protocols have been developed, yet variability across cell lines and low rates of differentiation impart challenges in successfully implementing these protocols. Described here is an easy and inexpensive means to enhance the differentiation capacity of PSCs. It has been previously shown that treatment of stem cells with a low concentration of dimethyl sulfoxide (DMSO) significantly increases the propensity of a variety of PSCs to differentiate to different cell types following directed differentiation. This technique has now been shown to be effective across different species (e.g., mouse, primate, and human) into multiple lineages, ranging from neurons and cortical spheroids to smooth muscle cells and hepatocytes. The DMSO pretreatment improves PSC differentiation by regulating the cell cycle and priming stem cells to be more responsive to differentiation signals. Provided here is the detailed methodology for using this simple tool as a reproducible and widely applicable means to more efficiently differentiate PSCs to any lineage of choice.

Generating differentiated cell types from human pluripotent stem cells (hPSCs) holds great therapeutic promise but remains challenging. PSCs often exhibit an inherent inability to differentiate even when stimulated with a proper set of signals. Described here is a simple tool to enhance multilineage differentiation across a variety of PSC lines.

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and ...

In vitro Neuromuscular Junction Induced from Human Induced Pluripotent Stem Cells

The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical movement. The architecture of the NMJ structure influences the functions of the neuron, the muscle and the mutual interaction. Previous studies have reported many strategies by co-culturing the motor neurons and myotubes to generate NMJ in vitro with complex induction process and long culture period but have struggled to recapitulate mature NMJ morphology and function. Our in vitro NMJ induction system is constructed by differentiating human iPSC in a single culture dish. By switching the myogenic and neurogenic induction medium for induction, the resulting NMJ contained pre- and post- synaptic components, including motor neurons, skeletal muscle and Schwann cells in the one month culture. The functional assay of NMJ also showed that the myotubes contraction can be triggered by Ca++ then inhibited by curare, an acetylcholine receptor (AChR) inhibitor, in which the stimulating signal is transmitted through NMJ. This simple and robust approach successfully derived the complex structure of NMJ with functional connectivity. This in vitro human NMJ, with its integrated structures and function, has promising potential for studying pathological mechanisms and compound screening.

Here we provide a protocol to generate in vitro NMJs from human induced pluripotent stem cells (iPSCs). This method can induce NMJs with mature morphology and function in 1 month in a single well. The resulting NMJs could potentially be used to model related diseases, to study pathological mechanisms or to screen drug compounds for therapy.