Name: Derivation of Stem Cell Lines from Mouse Preimplantation Embryos (Video) | JoVE Journal
Uploaded: 2017-08-20T13:00:00.000+00:00
Duration: 12 min 59 s
Description: Learn to derive mouse embryonic stem cell lines from blastocyst-stage embryos using standard culture conditions with human foreskin fibroblast feeders. This protocol achieves high derivation efficiencies comparable to 2i modulators.

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5 minutes

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 (FBS) and 10% dimethyl sulfoxide (DMSO) and stored in liquid nitrogen until use.
<ol>
	<li>Thaw a cryogenic vial of HFF-1 (1 mL) by immersing the vial in a 37 &#176;C water bath.</li>
	<li>Add the thawed contents of the cryogenic vial to pre-warmed Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) supplemented with 10% FBS to make a 1:10 dilution.</li>
	<li>Seed the diluted cells (10 mL) in a T75 flask and culture them at 37 &#176;C and 5% CO2.</li>
	<li>The next day, change the medium to eliminate the DMSO remnants. Remove the medium and add 10 mL of pre-warmed DMEM supplemented with 10% FBS.</li>
	<li>Culture the HFFs and change the medium every 48 h until the culture reaches the confluent stage. This may take approximately 7 to 10 days.</li>
	<li>To prepare the inactivation solution, dilute mitomycin C in DMEM supplemented with 10% FBS at a final concentration of 10 &#181;g/mL. Incubate the cells with the inactivation solution for 3 h at 37 &#176;C and 5% CO2.</li>
	<li>Remove the inactivation solution and rinse the cells thrice with 2 mL of Hanks Balanced Salt Solution (HBSS) to eliminate mitomycin C remnants.</li>
	<li>To detach the inactivated cells, add 1 mL of pre-warmed Trypsin-EDTA solution and incubate for 5 min at 37 &#176;C and 5% CO2.</li>
	<li>Observe the cells under an inverted microscope to ensure they are detached (10x objective with 0.3 numerical aperture or NA). If they are not, incubate them for a couple more minutes at 37 &#176;C and 5% CO2. Check the cells again under the inverted microscope.</li>
	<li>Pipette the solution with a Pasteur pipette in order to dissociate the cell clumps.</li>
	<li>Neutralize the enzymatic solution by adding 4 mL of DMEM supplemented with 10% FBS and resuspend.</li>
	<li>Take an aliquot of the cell suspension and make a 1:100 dilution in HBSS. To determine the number of cells, load 10 &#181;L of the dilution into a Neubauer&#39;s chamber. Count the cells located in one of the big squares of the chamber under an inverted microscope (20x objective with a 0.45 NA).
	<ol>
		<li>Repeat the counting in a total of four big squares. Calculate the total number of cells (N) as N = average number of cells counted x total volume x dilution x 104.</li>
	</ol>
	</li>
	<li>Centrifuge the rest of the sample for 5 min at 200 x g. Discard the supernatant and resuspend the pellet with freezing solution at a concentration of 106 cells per mL.</li>
	<li>Aliquot the suspension in cryogenic vials with 106 cells (1 mL) each. Place the cryogenic vials in a freezing container and freeze at -80 &#176;C.</li>
	<li>The next day, store the cryogenic vials in liquid nitrogen.</li>
</ol>
2. Feeder Cell Culture
<ol>
	<li>One to four days before starting the derivation process, thaw a cryogenic vial containing 1 x 106 inactivated HFF-1 cells in a water bath at 37 &#176;C.</li>
	<li>Make a 1:6 dilution of the thawed HFF-1 in pre-warmed DMEM supplemented with 10% FBS and keep the temperature at 37 &#176;C.</li>
	<li>Fill every well of a 4-well plate with 500 &#181;L of pre-warmed 0.2% gelatin from porcine skin and keep the plate at 37 &#176;C for 10 min.</li>
	<li>Remove the 500 &#181;L of gelatin from the wells and add 500 &#181;L of the HFFs dilution. Gently rock the plate to ensure a uniform distribution of the HFFs on the bottom of the wells. Avoid circular movements, otherwise the cells will concentrate at the center of the well.</li>
	<li>Incubate the plate at least 24 h at 37 &#176;C and 5% CO2 to obtain a monolayer of inactivated HFFs in each well.</li>
	<li>The next day, observe the cells under an inverted microscope (10X objective with 0.3 NA) and check that they are attached and homogeneously distributed. Change the medium to eliminate the DMSO remnants. Remove the medium and add 500 &#181;L of pre-warmed DMEM supplemented with 10% FBS.</li>
</ol>
3. Embryo Collection and Culture
<ol>
	<li>Four days before embryo collection, administer 5 IU of pregnant mare&#39;s serum gonadotropin by intraperitoneal injection to 6-12 week old female mice. 
	NOTE: We have used females from the 129S2 or B6CFAF1 strains, though females from other mouse strains can be used. Nevertheless, when working with females from non-permissive strains (e.g. CBA), medium should be supplemented with 2i as mentioned in step 4.2.</li>
	<li>After 48 h, administer 5 IU of human chorionic gonadotropin by intraperitoneal injection, and mate the females with males, at least 8 weeks old, at a 1:1 ratio. Separate the females from the males the next day.</li>
	<li>The day before embryo collection, prepare a 35-mm cell culture Petri dish with several 40 &#181;L drops of KSOMaag medium supplemented with 4 mg/mL Bovine Serum Albumin (BSA) and fill the plate with mineral oil to fully cover the drops. Incubate the dish at 37 &#176;C and 5% CO2 to equilibrate the medium.</li>
	<li>To prepare embryo manipulation pipettes, heat the thinner part of a long-tipped glass Pasteur pipette using a Bunsen burner. Once soft, withdraw it from the flame and pull the two ends apart. Break it to the desired length. The inner diameter at the tip should be 100-150 &#181;m.</li>
	<li>Euthanize the female mice by cervical dislocation 45-48 h post-coitum (p.c.).</li>
	<li>Dissect the mice to expose the abdominal cavity by cutting the skin with curved smooth sharp scissors and the peritoneum with straight sharp scissors. Grab one uterus with forceps, cut off the oviduct using straight sharp scissors, and place it in Hepes-buffered CZB medium (HCZB)13 at 37 &#176;C. Repeat the process to remove the other oviduct.</li>
	<li>Flush the oviducts with a 1 mL syringe loaded with HCZB held in place by watchmaker forceps to collect the 2-cell embryos. 
	NOTE: To prepare the flushing needle take a 30 G hypodermic needle, cut the end, and ground to a blunt tip on an abrasive stone.</li>
	<li>Using the mouth pipette apparatus, pick up the embryos, wash them in HCZB and culture them in the previously prepared embryo culture dish at 37 &#176;C and 5% CO2 until the blastocyst stage. Up to 50 embryos can be cultured in each 40 &#181;L drop. Monitor embryo development every 24 h under a stereomicroscope. 
	NOTE: Alternatively, embryos can be collected at the blastocyst stage to shorten in vitro embryo culture. In this case, euthanize the females at day 3.5 - 4.5 p.c., dissect the uterus. and flush it with HCZB.</li>
</ol>
4. Stem Cell Derivation
<ol>
	<li>Preparation of the derivation plates
	<ol>
		<li>On the day of embryo seeding, prepare the derivation medium by supplementing DMEM medium with 100 &#181;M 2-&#946; mercaptoethanol, 1x Non-Essential Amino Acids, 20% of serum replacement, and 103 U/mL LIF.</li>
		<li>If desired, also add 1 &#181;M of the MEK inhibitor PD0325901 and 3 &#181;M of the GSK3 inhibitor CHIR99021 to obtain a 2i medium.</li>
		<li>Take the 4-well plate with the monolayer of inactivated HFFs (step 2.6) from the incubator. Remove the medium and add 800 &#181;L of pre-warmed derivation medium to each well. Return the plate to the incubator.</li>
	</ol>
	</li>
	<li>Zona pellucida removal and embryo seeding
	<ol>
		<li>Prepare a 35 mm cell culture Petri dish with three 30 &#181;L drops of acidic Tyrode&#39;s solution14 and three 30 &#181;L drops of derivation medium. Cover the drops with mineral oil and keep the dish at 37 &#176;C.</li>
		<li>Take the embryo culture dish from the incubator. One by one, transfer the blastocysts from the culture drops to an acidic Tyrode&#39;s solution drop using an embryo manipulation pipette. If possible, select only the expanded blastocysts to start the derivation process.</li>
		<li>Monitor the zona pellucida digestion under the stereomicroscope. As soon as the zona pellucida disappears, transfer the blastocyst to a derivation medium drop in order to wash out the acidic Tyrode&#39;s solution.</li>
		<li>Take the 4-well derivation plate from the incubator and seed one zona-free blastocyst on the feeder cells in each well.</li>
		<li>Return the 4-well derivation plate to the incubator and culture at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
	<li>Monitoring stem cell growth and medium change
	<ol>
		<li>Monitor the cultures every 24 h under an inverted microscope (20X objective with 0.45 NA). It is important to note whether there are signs of stem cell differentiation, such as swollen refractive cells surrounding the outgrowth or even pushing the feeder cells aside.</li>
		<li>Every other day, change the medium of the culture. Take the 4-well plate from the incubator, remove the medium from each well, and add 800 &#181;L of pre-warmed derivation medium supplemented with LIF and 2i, if used. Maintain the culture until outgrowths are observed (6-8 days).</li>
	</ol>
	</li>
</ol>
5. Stem Cell Culture and Maintenance
<ol>
	<li>Prepare Kolle-like handles and stem cell manipulation pipettes. Follow step 3.4 to prepare stem cell manipulation pipettes with an inner diameter at the tip of 250-300 &#181;m. Use the other part of the pulled Pasteur pipette to prepare the Kolle-like handle. Heat and shape it until obtaining a hook.</li>
	<li>Take the 4-well culture plate out of the incubator and locate the outgrowth under a stereomicroscope. Use the handle to push away the differentiated cells and the feeders that surround the outgrowth. 
	NOTE: To avoid eventual differentiation of the outgrowth, it is important to remove all the differentiated cells from the edge of the outgrowth.</li>
	<li>Aspirate the outgrowth with a stem cell manipulation pipette and place it in a new dish in a 50 &#181;L trypsin-EDTA drop.</li>
	<li>Use a scalpel to cut the outgrowth in several parts in order to disaggregate it both mechanically and enzymatically.</li>
	<li>Straight away, transfer the outgrowth fragments into a fresh drop of derivation medium to neutralize trypsin action.</li>
	<li>Transfer the outgrowth fragments to a new 4-well plate with a monolayer of feeders. Transfer up to 10 fragments per well and culture them with derivation medium at 37 &#176;C and 5% CO2.</li>
	<li>Maintain the stem cell-like cultures for at least six weeks before considering the mESC line established. Change the medium every other day and subculture them once a week.</li>
</ol>

<ol><li>Biswas, A., Hutchins, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Embryonic+stem+cells%5BTitle%5D)+AND+%22Stem+Cell+Dev%22%5BJournal%5D)">Embryonic stem cells.</a> Stem Cell Dev. 16 (2), 213-221 (2007).</li>
<li>Weinberger, L., Ayyash, M., Novershtern, N., Hanna, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+stem+cell+states%3A+naive+to+primed+pluripotency+in+rodents+and+humans%5BTitle%5D)+AND+%22Nat+Rev+Mol+Cell+Biol%22%5BJournal%5D)">Dynamic stem cell states: naive to primed pluripotency in rodents and humans.</a> Nat Rev Mol Cell Biol. 17 (3), 155-169 (2016).</li>
<li>Evans, M. J., Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishment+in+culture+of+pluripotential+cells+from+mouse+embryos%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Establishment in culture of pluripotential cells from mouse embryos.</a> Nature. 292, 154-156 (1981).</li>
<li>Martin, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+a+pluripotent+cell+line+from+early+mouse+embryos+cultured+in+medium+conditioned+by+teratocarcinoma+stem+cells%5BTitle%5D)+AND+%22Proc+Nat+Acad+Sci+U+S+A%22%5BJournal%5D)">Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.</a> Proc Nat Acad Sci U S A. 78 (12), 7634-7638 (1981).</li>
<li>Martin, G. R., Evans, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differentiation+of+clonal+lines+of+teratocarcinoma+cells%3A+formation+of+embryoid+bodies+in+vitro%5BTitle%5D)+AND+%22PNAS%22%5BJournal%5D)">Differentiation of clonal lines of teratocarcinoma cells: formation of embryoid bodies in vitro.</a> PNAS. 72 (4), 1441-1445 (1976).</li>
<li>Czechanski, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Derivation+and+characterization+of+mouse+embryonic+stem+cells+from+permissive+and+nonpermissive+strains%5BTitle%5D)+AND+%22Nat+Prot%22%5BJournal%5D)">Derivation and characterization of mouse embryonic stem cells from permissive and nonpermissive strains.</a> Nat Prot. 9 (3), 559-574 (2014).</li>
<li>Smith, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibition+of+pluripotential+embryonic+stem+cell+differentiation+by+purified+polypeptides%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides.</a> Nature. 336 (6200), 688-690 (1988).</li>
<li>Williams, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myeloid+leukaemia+inhibitory+factor+maintains+the+developmental+potential+of+embryonic+stem+cells%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells.</a> Nature. 336 (6200), 684-687 (1988).</li>
<li>Brook, F. A., Gardner, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+origin+and+efficient+derivation+of+embryonic+stem+cells+in+the+mouse%5BTitle%5D)+AND+%22Proc+Nat+Acad+Sci+U+S+A%22%5BJournal%5D)">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/pubmed?term=((Efficient+germline+transmission+of+mouse+embryonic+stem+cells+grown+in+synthetic+serum+in+the+absence+of+a+fibroblast+feeder+layer%5BTitle%5D)+AND+%22Lab+Invest%22%5BJournal%5D)">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/pubmed?term=((The+ground+state+of+embryonic+stem+cell+self-renewal%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Efficient+establishment+of+mouse+embryonic+stem+cell+lines+from+single+blastomeres+and+polar+bodies%5BTitle%5D)+AND+%22Stem+cells+%28Dayton%2C+Ohio%29%22%5BJournal%5D)">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. L., Torres, I. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/2760894">An improved culture medium support development of random-bred 1-cell mouse embryos in vitro.</a> J Reprod Fertil. 86, 679-688 (1989).</li>
<li>Nagy, A., Vintersten, K., Behringer, R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aNagy%2C+A+%22Manipulating+the+Mouse+Embryo%3A+A+Laboratory+Manual%22">Manipulating the Mouse Embryo: A Laboratory Manual</a>. , 3rd, Cold Spring Harbor Lab Press. New York. (2003).</li>
<li>Lee, K. H. <a target="_blank" href="https://www.intechopen.com/books/pluripotent-stem-cells/conditions-and-techniques-for-mouse-embryonic-stem-cell-derivation-and-culture">Conditions and techniques for mouse embryonic stem cell derivation and culture.</a> Pluripotent Stem Cells. , 85-115 (2013).</li>
<li>Ma, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+foreskin+fibroblast+produces+interleukin-6+to+support+derivation+and+self-renewal+of+mouse+embryonic+stem+cells%5BTitle%5D)+AND+%22Stem+Cell+Res+Ther%22%5BJournal%5D)">Human foreskin fibroblast produces interleukin-6 to support derivation and self-renewal of mouse embryonic stem cells.</a> Stem Cell Res Ther. 3 (29), (2012).</li>
<li>Cheng, J., Dutra, A., Takesono, A., Garrett-Beal, L., Schwartzberg, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+generation+of+C57BL%2F6J+mouse+embryonic+stem+cells+in+a+defined+serum-free+media%5BTitle%5D)+AND+%22Genesis%22%5BJournal%5D)">Improved generation of C57BL/6J mouse embryonic stem cells in a defined serum-free media.</a> Genesis. 39 (2), 100-104 (2004).</li>
<li>Chaudhry, M. A., Vitalis, T. Z., Bowen, B. D., Piret, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Basal+medium+composition+and+serum+or+serum+replacement+concentration+influences+on+the+maintenance+of+murine+embryonic+stem+cells%5BTitle%5D)+AND+%22Cytotechnology%22%5BJournal%5D)">Basal medium composition and serum or serum replacement concentration influences on the maintenance of murine embryonic stem cells.</a> Cytotechnology. 58 (3), 173-179 (2008).</li>
<li>Kawase, E., Suemori, H., Takahashi, N., Okazaki, K., Hashimoto, K., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Strain+difference+in+establishment+of+mouse+embryonic+stem+%28ES%29+cell+lines%5BTitle%5D)+AND+%22Int.+J.+Dev.+Biol%22%5BJournal%5D)">Strain difference in establishment of mouse embryonic stem (ES) cell lines.</a> Int. J. Dev. Biol. 38 (2), 385-390 (1994).</li>
<li>Gonzalez, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Embryonic+stem+cell+culture+conditions+support+distinct+states+associated+with+different+developmental+stages+and+potency%5BTitle%5D)+AND+%22Stem+Cell+Rep%22%5BJournal%5D)">Embryonic stem cell culture conditions support distinct states associated with different developmental stages and potency.</a> Stem Cell Rep. 7 (2), 177-191 (2016).</li>
</ol>

The authors have nothing to disclose.

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><tbody><tr><td>1 mL sryringe</td><td>BD&amp;nbsp;</td><td>309628</td><td></td></tr><tr><td>2-&amp;beta;-mercaptoethanol</td><td>Life Technologies</td><td>31350-010</td><td></td></tr><tr><td>4-well plate</td><td>Thermo Scientific Nunc</td><td>176740</td><td></td></tr><tr><td>Acidic Tyrode&#039;s solution&amp;nbsp;</td><td>Home-made</td><td></td><td>See recipe in Nagy et al, 2003. For short-term storage (up to 1 month) keep it at 4&amp;ordm;C. Alternatively, for long-term storage keep it at -20&amp;ordm;C. If the efficiency of the solution diminishes, it should be acidified by the adition of a small drop of HCl 1M.&amp;nbsp;</td></tr><tr><td>BSA</td><td>Sigma</td><td>A3311</td><td></td></tr><tr><td>Chicken anti-Mouse IgG, Alexa Fluor 488</td><td>Molecular Probes - Invitrogen</td><td>A-21200</td><td>1:500 dilution. Secondary antibody for Oct4, Tuj1, &amp;alpha;SMA and AFP mouse antibodies.&amp;nbsp;</td></tr><tr><td>CHIR990212</td><td>Axon Medchem&amp;nbsp;</td><td>1386</td><td>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&amp;ordm;C. Avoid freeze and thaw cycles.&amp;nbsp;</td></tr><tr><td>CryoTube</td><td>Thermo Scientific Nunc</td><td>3754518</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>41640</td><td></td></tr><tr><td>DMEM</td><td>BioWest</td><td>L0107</td><td></td></tr><tr><td>FBS</td><td>BioWest</td><td>S1810</td><td>Inactivate it prior to use by heating for 30 mins at 56&amp;ordm;C. Aliquot and store at -20&amp;ordm;C</td></tr><tr><td>Flushing needle</td><td>BD&amp;nbsp;</td><td>304000</td><td>Cut the end of the needle and ground to a blunt tip on an abrasive stone</td></tr><tr><td>Gelatin from porcine skin</td><td>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><td>Goat anti-Rabbit IgG, Alexa Fluor 594</td><td>Molecular Probes - Invitrogen</td><td>A-11037</td><td>1:500 dilution. Secondary antibody for Sox2 rabbit antibody.&amp;nbsp;</td></tr><tr><td>HBSS</td><td>BioWest</td><td>L0611</td><td></td></tr><tr><td>Hepes-buffered CZB&amp;nbsp;</td><td>Home-made</td><td></td><td>See composition in Chatot et al, 1989</td></tr><tr><td>Human chorionic gonadotropin</td><td>Divasa-Farmavic</td><td>Veterin-Corion 3000 UI</td><td>Dilute in NaCl 0.9% at 50 IU/ml. Aliquot it in 1 ml and store at -20&amp;ordm;C for up to two months. Once thawed do not freeze again.&amp;nbsp;</td></tr><tr><td>Human foreskin fibroblasts</td><td>ATCC</td><td>ATCCSCRC-1041</td><td>For long term storage it is recommended to store them in liquid nitrogen.</td></tr><tr><td>KnockOut Serum Replacement</td><td>Life Technologies</td><td>&amp;nbsp;10828-028</td><td>Photosensitive. It is recommended to aliquot it in small volumes such as 10 ml and store it at -20&amp;ordm;C (check the expiration date). Avoid freeze and thaw cycles.&amp;nbsp;&amp;nbsp;&amp;nbsp;</td></tr><tr><td>KSOMaag Evolve</td><td>Zenith Biotech</td><td>ZEKS-050</td><td>Supplement with 4 mg/ml BSA. Equilibrate at 37&amp;ordm;C and 5% CO2 a day prior to use.</td></tr><tr><td>Leukemia Inhibitory Factor</td><td>Merk Millipore</td><td>ESG1106</td><td></td></tr><tr><td>Mice&amp;nbsp;</td><td>Charles River/Harlan</td><td></td><td>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.&amp;nbsp;</td></tr><tr><td>Mineral oil</td><td>Sigma</td><td>M8410</td><td>Embryo tested. Photosensitive.&amp;nbsp;</td></tr><tr><td>Mitomycin C</td><td>Serva</td><td>2980501</td><td>Reconstitute the powder with MilliQ water at 0.5 mg/ml. Store at 4&amp;ordm;C protected from light. Attention, it is harmful and suspected of causing cancer.&amp;nbsp;</td></tr><tr><td>Mouse anti-tubulin &amp;beta; III (Tuj1)</td><td>Biolegend</td><td>MMS-435P</td><td>1:500 dilution</td></tr><tr><td>Mouse monoclonal anti-&amp;alpha;SMA</td><td>Sigma</td><td>A5228</td><td>1:200 dilution</td></tr><tr><td>Mouse monoclonal anti-AFP</td><td>R&amp;amp;D Systems</td><td>MAB1368</td><td>1:50 dilution</td></tr><tr><td>Mouse monoclonal anti Oct3/4&amp;nbsp;</td><td>Santa cruz</td><td>Sc-5279</td><td>1:50 dilution</td></tr><tr><td>Non-Essential Amino Acids</td><td>Life Technologies</td><td>11140-035</td><td></td></tr><tr><td>PD0325901</td><td>Axon Medchem&amp;nbsp;</td><td>1408</td><td>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&amp;ordm;C. Avoid freeze and thaw cycles.</td></tr><tr><td>Petri dish (35mm)</td><td>Thermo Scientific Nunc</td><td>153066</td><td></td></tr><tr><td>Petri dish (60mm)</td><td>Thermo Scientific Nunc</td><td>150288</td><td></td></tr><tr><td>Pregnant mare&#039;s serum gonadotropin</td><td>Foligon</td><td>Foligon-1000 UI</td><td>Dilute in NaCl 0.9% at 50 IU/ml. Aliquot it in 1 ml and store at -20&amp;ordm;C for up to two months. Once thawed do not freeze again.&amp;nbsp;</td></tr><tr><td>Rabbit policlonal anti-Sox2</td><td>Merck Millipore</td><td>AB5603</td><td>1:200 dilution</td></tr><tr><td>T75 falcon</td><td>Thermo Scientific</td><td>130190</td><td></td></tr><tr><td>Trypsin-EDTA 10x</td><td>BioWest</td><td>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

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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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

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Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

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Derivation of Human Embryonic Stem Cells by Immunosurgery

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Isolation and Derivation of Mouse Embryonic Germinal Cells

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Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

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Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm

Learn to derive mouse embryonic stem cell lines from blastocyst-stage embryos using standard culture conditions with human foreskin fibroblast feeders. This protocol achieves high derivation efficiencies comparable to 2i modulators.

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