Name: derivation of stem cell lines from mouse preimplantation embryos
Uploaded: 2017-08-20T13:00:00
Description: Watch this Scientific Journal Video about Derivation of Stem Cell Lines from Mouse Preimplantation Embryos at JoVE.com

We thank Jonatan Lucas for his technical assistance with feeder cell culture, Servei Estabulari de la UAB for animal care and Dom&#232;nec Mart&#237;n for recording the video. This work has been supported by Ministerio de Economia y Competitividad AGL2014-52408-R and Generalitat de Catalunya 2014 SGR-524. MVC is beneficiary of a PIF-UAB fellowship.

The use of the animals described in the sections below has been approved by the Ethics Committee on Animal and Human Research of the Universitat Aut&#242;noma de Barcelona and by the Departament d&#39;Agricultura, Ramaderia, Pesca i Alimentaci&#243; of the Generalitat de Catalunya (protocol #8741).
1. Feeder Cell Inactivation and Storage
NOTE: Human foreskin fibroblasts (HFF-1) were previously frozen in 1 mL of freezing solution consisting of 90% Fetal Bovine Serum (F...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+and+efficient+derivation+of+embryonic+stem+cells+in+the+mouse.">The origin and efficient derivation of embryonic stem cells in the mouse.</a> Proc Nat Acad Sci U S A. 94 (11), 5709-5712 (1997).</li><li>Ward, C. M., Stern, P., Willington, M. A., Flenniken, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+germline+transmission+of+mouse+embryonic+stem+cells+grown+in+synthetic+serum+in+the+absence+of+a+fibroblast+feeder+layer.">Efficient germline transmission of mouse embryonic stem cells grown in synthetic serum in the absence of a fibroblast feeder layer.</a> Lab Invest. 82 (12), 1765-1767 (2002).</li><li>Ying, Q. 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=The+ground+state+of+embryonic+stem+cell+self-renewal.">The ground state of embryonic stem cell self-renewal.</a> Nature. 453 (7194), 519-523 (2008).</li><li>Wakayama, 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=Efficient+establishment+of+mouse+embryonic+stem+cell+lines+from+single+blastomeres+and+polar+bodies.">Efficient establishment of mouse embryonic stem cell lines from single blastomeres and polar bodies.</a> Stem cells (Dayton, Ohio). 25 (4), 986-993 (2007).</li><li>Chatot, C. L., Ziomek, C. A., Bavister, B. D., Lewis, J. 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Although mESC line derivation is a well-known procedure routinely used in many laboratories, its efficiency is not always as high as expected due to the multiple factors that can disturb pluripotency maintenance. In the present article we show, step by step, how to establish mESC lines with high efficiencies using a simple and reliable protocol. These efficiencies are similar to those reported in the literature. 
To ensure maximum efficiency, it is important to control the cells daily or every...

Embryonic stem cells (ESC) are pluripotent cells derived from preimplantation embryos, which retain the ability to either self-renew or differentiate under specific conditions1. Based on these properties, ESC have become a useful tool for regenerative medicine, disease modeling, and tissue engineering studies2.
ESC were derived for the first time from preimplantation mouse embryos, originating mouse ESC (mESC) lines with low success3,4. For years, the derivation efficiency remained low due to the difficulty of maintaining pluripotency in vitro. Besides the presence of feeder cells5, which improve derivation efficiency, promote colony attachment, and enhance karyotypic stability6, several modifications of the protocol lead to an improvement in pluripotency maintenance. The most important were the addition of Leukemia Inhibitory Factor (LIF) to mESC culture medium7,8, the use of embryos from the 129S2 permissive background9 and the culture of mESC with medium supplemented with defined serum, free of potential differentiation factors10. More recently, compelling evidence has shown that addition of a cocktail of two small-molecule modulators of signaling pathways to the mESC culture medium (known as 2i) is best to maintain pluripotency. The 2i cocktail consists of a combination of the MEK inhibitor PD0325901, which reduces the differentiation signals by inhibiting the mitogen-activated protein kinase (MAPK) pathway, and the GSK3&#946; inhibitor CHIR99021, which enhances cell survival at low density by activating the Wnt pathway11. 
In the present article, we describe a simple protocol to efficiently derive mESC lines from mouse blastocysts. We follow standard procedures, culturing embryos from permissive strains on feeder cells in derivation medium supplemented with LIF and a serum replacement12. Following this protocol, mESC lines can be derived with efficiencies equivalent to those obtained in 2i medium. In spite of this, 2i can be added to avoid possible problems with pluripotency maintenance or when working with non-permissive strains.

<table border="0" cellpadding="0" cellspacing="0" width="1125">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">1 mL sryringe</td>
			<td style="width:199px;">BD&#160;</td>
			<td style="width:141px;">309628</td>
			<td style="width:524px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">2-&#946;-mercaptoethanol</td>
			<td>Life Technologies</td>
			<td>31350-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">4-well plate</td>
			<td>Thermo Scientific Nunc</td>
			<td style="width:141px;">176740</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:263px;">Acidic Tyrode&#39;s solution&#160;</td>
			<td style="width:199px;">Home-made</td>
			<td style="width:141px;"></td>
			<td style="width:524px;">See recipe in Nagy et al, 2003. For short-term storage (up to 1 month) keep it at 4&#186;C. Alternatively, for long-term storage keep it at -20&#186;C. If the efficiency of the solution diminishes, it should be acidified by the adition of a small drop of HCl 1M.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">BSA</td>
			<td>Sigma</td>
			<td style="width:141px;">A3311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:22px;width:263px;">Chicken anti-Mouse IgG, Alexa Fluor 488</td>
			<td>Molecular Probes - Invitrogen</td>
			<td style="width:141px;">A-21200</td>
			<td>1:500 dilution. Secondary antibody for Oct4, Tuj1, &#945;SMA and AFP mouse antibodies.&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:263px;">CHIR990212</td>
			<td style="width:199px;">Axon Medchem&#160;</td>
			<td style="width:141px;">1386</td>
			<td style="width:524px;">Reconstitute with 2.15 ml DMSO for each mg of powder, to obtain a 1 mM stock solution. Aliquot at desired volume and store at -20&#186;C. Avoid freeze and thaw cycles.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">CryoTube</td>
			<td>Thermo Scientific Nunc</td>
			<td style="width:141px;">3754518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">DMSO</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:141px;">41640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">DMEM</td>
			<td style="width:199px;">BioWest</td>
			<td>L0107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">FBS</td>
			<td style="width:199px;">BioWest</td>
			<td>S1810</td>
			<td>Inactivate it prior to use by heating for 30 mins at 56&#186;C. Aliquot and store at -20&#186;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Flushing needle</td>
			<td style="width:199px;">BD&#160;</td>
			<td>304000</td>
			<td>Cut the end of the needle and ground to a blunt tip on an abrasive stone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Gelatin from porcine skin</td>
			<td style="width:199px;">Fluka</td>
			<td>48724</td>
			<td>Dilute at 0,2% in destilled water and autoclave it. Each dilution can be used for a month.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Goat anti-Rabbit IgG, Alexa Fluor 594</td>
			<td>Molecular Probes - Invitrogen</td>
			<td style="width:141px;">A-11037</td>
			<td>1:500 dilution. Secondary antibody for Sox2 rabbit antibody.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">HBSS</td>
			<td style="width:199px;">BioWest</td>
			<td style="width:141px;">L0611</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Hepes-buffered CZB&#160;</td>
			<td style="width:199px;">Home-made</td>
			<td style="width:141px;"></td>
			<td>See composition in Chatot et al, 1989</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">Human chorionic gonadotropin</td>
			<td style="width:199px;">Divasa-Farmavic</td>
			<td style="width:141px;">Veterin-Corion 3000 UI</td>
			<td style="width:524px;">Dilute in NaCl 0.9% at 50 IU/ml. Aliquot it in 1 ml and store at -20&#186;C for up to two months. Once thawed do not freeze again.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human foreskin fibroblasts</td>
			<td style="width:199px;">ATCC</td>
			<td style="width:141px;">ATCCSCRC-1041</td>
			<td>For long term storage it is recommended to store them in liquid nitrogen.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:263px;">KnockOut Serum Replacement</td>
			<td style="width:199px;">Life Technologies</td>
			<td>&#160;10828-028</td>
			<td style="width:524px;">Photosensitive. It is recommended to aliquot it in small volumes such as 10 ml and store it at -20&#186;C (check the expiration date). Avoid freeze and thaw cycles.&#160;&#160;&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">KSOMaag Evolve</td>
			<td style="width:199px;">Zenith Biotech</td>
			<td style="width:141px;">ZEKS-050</td>
			<td style="width:524px;">Supplement with 4 mg/ml BSA. Equilibrate at 37&#186;C and 5% CO2 a day prior to use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Leukemia Inhibitory Factor</td>
			<td style="width:199px;">Merk Millipore</td>
			<td>ESG1106</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:263px;">Mice&#160;</td>
			<td style="width:199px;">Charles River/Harlan</td>
			<td style="width:141px;"></td>
			<td style="width:524px;">It is recommended to use mice from a permissive strain in terms of mESC derivation (such as 129S2 or C57BL). However, inbreed strains are less efficient producing embryos. Therefore, it is recomended to use a hybrid strain, such as 129S2 x C57BL or B6CBAF1 to take advantage of the hybrid vigor.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Mineral oil</td>
			<td>Sigma</td>
			<td>M8410</td>
			<td>Embryo tested. Photosensitive.&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:263px;">Mitomycin C</td>
			<td style="width:199px;">Serva</td>
			<td>2980501</td>
			<td style="width:524px;">Reconstitute the powder with MilliQ water at 0.5 mg/ml. Store at 4&#186;C protected from light. Attention, it is harmful and suspected of causing cancer.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Mouse anti-tubulin &#946; III (Tuj1)</td>
			<td>Biolegend</td>
			<td style="width:141px;">MMS-435P</td>
			<td>1:500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Mouse monoclonal anti-&#945;SMA</td>
			<td>Sigma</td>
			<td style="width:141px;">A5228</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Mouse monoclonal anti-AFP</td>
			<td>R&#38;D Systems</td>
			<td style="width:141px;">MAB1368</td>
			<td>1:50 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Mouse monoclonal anti Oct3/4&#160;</td>
			<td>Santa cruz</td>
			<td style="width:141px;">Sc-5279</td>
			<td>1:50 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Non-Essential Amino Acids</td>
			<td>Life Technologies</td>
			<td>11140-035</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:263px;">PD0325901</td>
			<td style="width:199px;">Axon Medchem&#160;</td>
			<td style="width:141px;">1408</td>
			<td style="width:524px;">Reconstitute with 2.08 ml DMSO for each mg of powder, to obtain a 1 mM stock solution. Aliquot at desired volume and store at -20&#186;C. Avoid freeze and thaw cycles.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Petri dish (35mm)</td>
			<td>Thermo Scientific Nunc</td>
			<td style="width:141px;">153066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Petri dish (60mm)</td>
			<td>Thermo Scientific Nunc</td>
			<td>150288</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">Pregnant mare&#39;s serum gonadotropin</td>
			<td style="width:199px;">Foligon</td>
			<td>Foligon-1000 UI</td>
			<td style="width:524px;">Dilute in NaCl 0.9% at 50 IU/ml. Aliquot it in 1 ml and store at -20&#186;C for up to two months. Once thawed do not freeze again.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Rabbit policlonal anti-Sox2</td>
			<td>Merck Millipore</td>
			<td style="width:141px;">AB5603</td>
			<td>1:200 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">T75 falcon</td>
			<td style="width:199px;">Thermo Scientific</td>
			<td style="width:141px;">130190</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Trypsin-EDTA 10x</td>
			<td style="width:199px;">BioWest</td>
			<td style="width:141px;">X0930</td>
			<td>Dilute 1:10 in HBSS to obtain a 1x solution</td>
		</tr>
	</tbody>
</table>

derivation of stem cell lines from mouse preimplantation embryos

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

Following this protocol, over 95% of outgrowth formation should be achieved after the first week of culture (Figure 1A). Establishment of mESC lines after six passages is variable among experimental replicates, with an average success of 60-80%. The addition of 2i does not significantly improve the results when working with permissive mouse strains, such as those with a 129S2 background, but it is necessary when working with non-permissive strains.
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Watch this Scientific Journal Video about Derivation of Stem Cell Lines from Mouse Preimplantation Embryos at JoVE.com

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

This article describes a protocol to efficiently derive and culture pluripotent stem cell lines from mouse embryos at the blastocyst stage.

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

Stem cells are pluripotent and specialized cells characterized by their long-term self-renewal and their ability to give rise to differentiated cells from the three germ layers under certain conditions. Due to these properties, stem cells have a great potential in regenerative medicine, disease modeling, and animal engineering. For years, embryonic stem cell lines destined to research purposes have been mainly derived from mouse embryos, but in general the derivation efficiencies achieved have been low.In this video, we show a simple protocol to derive mouse embryonic stem cell lines using standard conditions that provides reasonably high derivation efficiencies comparable to the efficiencies obtained when using modulators of signaling pathways, such as 2i. In this protocol, we'll human foreskin fibroblasts as feeder cells for ESC derivation and culture. Before starting the ESC derivation process, it's necessary to inactivate the feeder cells.First, take a cryogenic vial of human foreskin fibroblasts stored in liquid nitrogen, and thaw it by immersing the vial in a water bath at 37 degrees C.Once thawed, dilute the content in DMEM supplemented with fetal bovine serum. Seed the dilution in a Falcon flask, and culture it until reaching the confluence stage. Next, to inactivate the fibroblasts, dilute mitomycin C in DMEM supplemented with fetal bovine serum and incubate the cells for three hours in this inactivation solution.Then, remove the inactivation solution and rinse the cells three times with Hanks'Balanced Salt Solution to eliminate mitomycin C remnants. To detach the inactivated feeder cells, add one milliliter of trypsin-EDTA solution and incubate the cells for five minutes. After that, observe the cells under an inverted microscope to ensure they are detached from the culture flask.Then, pipette the cells with a Pasteur pipette to dissociate the cell clumps and neutralize the enzymatic solution by adding four milliliters of DMEM supplemented with fetal bovine serum. Take a small aliquot, dilute it in saline solution, and determine the number of cells using a Neubauer chamber. In parallel, centrifuge the rest of the sample.Discard the supernatant, and dilute the pellet in freezing solution at a final concentration of one million cells per milliliter. Aliquot the suspension in cryogenic vials. And finally, place the cryogenic vials in a freezing container and put them at minus 80 degrees C.The next day, store them in liquid nitrogen.One to four days before starting the derivation process, take a cryotube of inactivated feeder cells stored in liquid nitrogen and thaw it in a water bath at 37 degrees C.Then, dilute the cells in pre-warmed DMEM supplemented with fetal bovine serum and pipette gently to ensure a homogeneous mixture. Add 500 microliters of pre-warmed gelatin solution to each well of a four-well plate, and keep it at 37 degrees C for 10 minutes. Right after, remove the gelatin from each well.Add 500 microliters of the feeder cells dilution to each well, and gently rock the plate to ensure a uniform distribution of the feeder cells on the bottom of the wells. Avoid making circular movements, as this would concentrate the cells in the center of the well. Then, place the plate in the incubator at 37 degrees and 5%carbon dioxide for at least 24 hours.The next day, observe the cells under an inverted microscope and check they're attached and thoroughly distributed. Finally, change the medium of the feeder cells with pre-warmed DMEM supplemented with fetal bovine serum to remove DMSO remnants. Before embryo collection, induce mouse female superovulation by intraperitoneal injection of five international units of pregnant mare serum gonadotropin and human chorionic gonadotropin 48 hours apart.Mate the females with males, and separate them 24 hours later. To collect two-cell stage embryos, euthanize the females 48 hours post coitum, dissect the female mice to obtain the oviducts and a short portion of the uterine horns, and place them in a dish with HEPES-buffered CZB medium. Then, one by one, flush the oviducts with HEPES-buffered CZB medium under the stereo microscope to collect the embryos.Transfer the two-cell embryos to a culture dish prepared the previous day with several drops of embryo culture medium covered with mineral oil. Keep the embryos in the incubator until the blastocyst stage. Monitor the embryo development every 24 hours.Embryos are expected to reach the blastocyst stage 48 hours after the start of the in vitro culture. If possible, select only expanded blastocysts to start the derivation process. On the day of embryo seeding, prepare the medium for stem cell derivation.Add leukemia inhibitory factor, or LIF, to DMEM supplemented with 2-beta-mercaptoethanol non-essential amino acids and KnockOut serum replacement. If desired, add an MAPK inhibitor and a GSK-3 inhibitor to obtain a 2i medium. Take the four-well plate with the monolayer of feeder cells from the incubator.Remove the medium, and add 800 microliters of the freshly prepared derivation medium. Before seeding the blastocysts on the feeder cells, the zona pellucida must be removed. To do so, transfer one embryo from the culture plate to a drop of acidic Tyrode's solution and keep it there until the zona pellucida is digested.Make sure that the zona pellucida has been completely removed by adjusting the focus and the contrast of the stereo microscope. Then, rinse the zona-free embryo in several drops of derivation medium and seed it on a monolayer of feeder cells. Repeat the process with the rest of the embryos.Culture the plate at 37 degrees C and 5%carbon dioxide. Monitor the cultures every 24 hours under an inverted microscope to assess their growth and to identify potential early signs of differentiation, such as swollen refractive cells surrounding the outgrowth or even pushing the feeder cells aside. Change the medium of the culture every other day.To do so, take the four-well plate from the incubator, remove the medium from each well, and add 800 microliters of pre-warmed derivation medium supplemented with LIF and 2i if used. Return the plates to the incubator, and maintain the culture until outgrowths are observed. Six to eight days after blastocyst seeding, an outgrowth should be observed, and it must be subcultured.For this procedure, cull-like handles and stem cells manipulation pipettes are required. To make them, take a long-tip Pasteur pipette and heat its thinner part using a Bunsen burner. Once soft, withdraw the pipette from the flame and pull the two ends apart until they separate.Use the thicker part as the stem cell manipulation pipette. Heat the tip of the thinner part, and shape it until obtaining a hook to prepare cull-like handle. To subculture the outgrowth, locate it under the stereo microscope.With the help of the handle, push away the differentiated cells that surround the outgrowth. To avoid an eventual differentiation, it's important to be strict when removing the differentiated cells from the edge of the outgrowth. Using a stem cell manipulation pipette, aspirate the outgrowth and place it in a drop of pre-warmed trypsin-EDTA solution.With the help of a scalpel, cut the outgrowth in several pieces to disaggregate it mechanically and enzymatically. Recover the outgrowth fragments, and place them in a drop of pre-warmed derivation medium in order to neutralize the trypsin action. Finally, seed the outgrowth fragments into the four-well plate with a monolayer of feeder cells and culture them in a derivation medium at 37 degrees C and 5%carbon dioxide.Stem cell-like cultures must be maintained for at least six weeks before an ESC line can be considered as established. During this period, it's necessary to change the medium every other day and subculture them once a week. As passage numbers increase, the morphology of the colonies progressively changes from a dome shape to a more elongated and flattened morphology.After six passages, mouse ESC lines constitutively express pluripotency markers, such as Oct 4 and Sox 2. Moreover, after inducing ESC in vitro differentiation by culturing them in DMEM supplemented with 10%fetal bovine serum and without feeder cells, differentiation markers representative of the three germ layers can be detected by immunofluorescence. Here, we show the detection of the anti-tubulin beta-3 protein as a marker for ectoderm, alpha-smooth muscle actin for mesoderm, and alpha-fetoprotein for endoderm.Following this simple procedure, embryonic stem cells can be derived from mouse blastocysts with efficiencies ranging from 60%to 78%While attempting this procedure, it's important to control the cells daily or every other day because the timings provided here are just illustrative. If embryonic stem cell colonies overgrow, they become darker because of cell accumulation and tend to differentiate more easily. After watching this video, you should have a good understanding of mouse embryonic stem cell derivation and culture using standard conditions.

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Research

JoVE Journal

Developmental Biology

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/&#946;-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes ...

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell&#39;s endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent&#160;stem cells.

Genome editing of human pluripotent stem cells (hPSCs) can be done quickly and efficiently. Presented here is a robust experimental procedure to genetically engineer hPSCs as exemplified by editing the AAVS1 safe harbor locus to express EGFP and introduce antibiotic resistance.

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-&#946; receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate 125I-TGF-&#946;2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.

A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...