Name: The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System (Video) | JoVE Journal
Uploaded: 2017-02-28T13:00:00.000+00:00
Duration: 8 min 24 s
Description: Learn to generate induced pluripotent stem cells from mouse amniotic fluid cells using a non-viral transposon system. This method enables safer iPSC production for regenerative medicine and patient-specific therapeutic applications.

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5 minutes

This work was supported by CARIPARO Foundation Grant number 13/04 and Fondazione Istituto di Ricerca Pediatrica Citt&#224; della Speranza Grant number 10/02. Martina Piccoli, Chiara Franzin and Michela Pozzobon are funded by Fondazione Istituto di Ricerca Pediatrica Citt&#224; della Speranza. Enrica Bertin is funded by CARIPARO Foundation Grant number 13/04. Paolo De Coppi is funded by Great Ormond Street Hospital Children's Charity.

All procedures were in accordance with Italian law. Murine AF samples were harvested from pregnant mice at 13.5 days post coitum (d.p.c.) from C57BL/6-Tg(UBC-GFP)30Scha/J mice called GFP.
1. Transposon Production
NOTE: Transposon expression vectors were generated using standard cloning procedures. The plasmid DNA for mouse AF cells transfection was prepared using commercial kits.
<ol>
	<li>Mix 10 ng of plasmid DNA with 50 &#181;l of DH5&#945; bacteria in a 1.5 ml microcentrifuge tube. Incubate the microcentrifuge tube on ice for 20 min.</li>
	<li>Heat shock at 42 &#176;C for 40 sec.</li>
	<li>Place microcentrifuge tube on ice for 2 min.</li>
	<li>Add 250 &#181;l of pre-warmed (37 &#176;C) lysogeny broth (LB) broth. Shake (300 rpm) at 37 &#176;C for 30 min.</li>
	<li>Spread 30 &#181;l of each transformation onto LB plates with 0.1 mg/ml ampicillin. Allow plates to dry and incubate inverted at 37 &#176;C overnight.</li>
	<li>The next day, in the afternoon, pick 3 colonies, using sterile 200 &#181;l tips, from each transformation and transfer onto 0.1 mg/ml LB amp.</li>
	<li>Shake (300 rpm) bacteria overnight at 37 &#176;C.</li>
	<li>For plasmid purification use commercial kits. Stock plasmids at -20 &#176;C.</li>
</ol>
2. Mouse Embryonic Fibroblast (MEF) Culture
<ol>
	<li>Coating of the 6-well Plates with 0.1% Gelatin
	<ol>
		<li>Coat plates under the hood adding 1 ml of sterile 0.1% gelatin to each of the six wells. Let the gelatin polymerize on the incubator (37 &#176;C) for 1 hr.</li>
		<li>Discard the excess, and dry the plates for 1 hr.</li>
		<li>Use parafilm to seal and store plates at room temperature.</li>
	</ol>
	</li>
	<li>Inactivation of MEF with Mitomycin C
	<ol>
		<li>Thaw a vial of 4 x 106 MEF cells using a 37 &#176;C bath.</li>
		<li>Seed MEF in the hood on a Petri dish (150 mm) with MEF medium.</li>
		<li>Cultivate cells at 37 &#176;C and 5% CO2.</li>
		<li>&#8203;(CAUTION) Add mitomycin C (5 mg/mL) when cells are confluent.</li>
		<li>Place cells into an incubator (37 &#176;C and 5% CO2) for 3 h.</li>
		<li>Remove medium with mitomycin C. Wash cells three times with 1x phosphate buffer saline (PBS).</li>
		<li>Add 2 ml of the commercial trypsin and place cells into the 37 &#176;C incubator for 5 min. After, add 18 ml of blocking medium to stop the trypsin reaction.</li>
		<li>Count the cells using a burker chamber according to manufacturer&#39;s protocol.</li>
		<li>Centrifuge cells at 145 x g for 5 min. Discard the supernatant.</li>
		<li>Seed 60,000 cells/cm2 of inactivated MEF on 0.1% gelatin coated plates.</li>
		<li>Cultivate cells for 24 h at 37 &#176;C and 5% CO2, before seeding AF and/or iPS-AF cells.</li>
	</ol>
	</li>
</ol>
3. Mouse AF Cell Isolation
<ol>
	<li>Set up timed matings and check vaginal plugs after 24 h of co-housing.</li>
	<li>Observe until 13.5 d.p.c and sacrifice pregnant dam through cervical dislocation.</li>
	<li>Clean the abdominal wall of the animal using 70% ethanol.</li>
	<li>Using scissors, perform a midline laparotomy incising the length of the abdominal wall to gain access into the abdominal cavity.</li>
	<li>Expose the uterus using forceps. Remove the uterus using scissors and transfer into a 100 mm Petri dish filled with sterile 1x PBS placed on ice.</li>
	<li>Use a stereomicroscope to collect the AF at 10X magnification.</li>
	<li>Hold the uterus with forceps and remove the uterine wall with scissors. Be careful to avoid any damage to the fetal membranes and consequent amniotic fluid leakage.</li>
	<li>Collect fetuses into a 100 mm Petri dish filled with sterile 1x PBS and place on ice.</li>
	<li>Grasp the placenta of one fetus with a fine point forceps and place in a clean 100 mm Petri dish.</li>
	<li>Remove the yolk sac using fine point forceps.</li>
	<li>Disrupt the amnion using fine point forceps and collect 30 &#181;l of AF for each fetus using an insulin syringe into a 15 ml conical vial.</li>
	<li>After AF collection, wash each fetus with sterile 1x PBS supplemented with 1% fetal bovine serum (FBS) and collect into the 15 ml conical tube containing the AF.</li>
	<li>Centrifuge AF at 145 x g for 5 min. Remove the supernatant under the hood and seed the pelleted AF cells.</li>
</ol>
4. Mouse AF Cell Culture
<ol>
	<li>Seeding of Mouse AF Cells
	<ol>
		<li>One day prior to the seeding, prepare a 0.1% gelatin-coated 6-well plate with mitomycin C-treated MEF.</li>
		<li>After AF collection, seed the cells obtained from an amniocentesis (around 1 x 107 cells obtained from 6 fetuses) onto the mitotically inactivated MEF using AF culture medium.</li>
		<li>Cultivate cells at 37 &#176;C and 5% CO2, changing the medium every other day.</li>
		<li>After 7 days of culture, transfect AF cells following the procedure described below.</li>
	</ol>
	</li>
</ol>
5. Transfection, iPS-AF Cell Generation and Culture
NOTE: Mouse AF cells were transfected with the PB-tetO2-IRES-OKMS transposon plasmid, in conjunction with a transposase expression plasmid (mPBase) and with the reverse tetracycline transactivator (PB-CAG-rtTA) transposon plasmid. All plasmids were provided by Professor Andras Nagy33.
<ol>
	<li>Mix 1 &#181;g of PB-tetO2-IRES-OKMS with 0.5 &#181;g of mPBase and 0.5 &#181;g of PB-CAG-rtTA (Total DNA: 2 &#181;g) in a 1.5 ml microcentrifuge tube.</li>
	<li>Dilute the DNA to 100 &#181;L with serum-free medium (Dulbecco&#39;s Modified Eagle Medium - DMEM).</li>
	<li>Add 8 &#181;L of the transfection reagent (e.g., FUGENE) (at the ratio of plasmid DNA:transfection agent of 2 &#181;g (DNA) to 8 &#181;L transfection agent) into the 1.5 mL microcentrifuge tube containing DNA.</li>
	<li>Incubate transfection reagent/DNA mixture for 15 min at room temperature.</li>
	<li>Add dropwise 100 &#181;L of the transfection reagent/DNA mixture per well to a 6-well plate with mouse AF cells and distribute by gentle swirling, with a final volume of 2 mL/well. Leave for 24 h.</li>
	<li>The day after, induce the expression of the Yamanaka&#39;s factors (Oct4, Klf4, cMyc, Sox2) by feeding the cells with fresh iPS-AF medium supplemented with 1.5 &#181;g/mL of doxycycline (2 mL of media for each well).</li>
	<li>Feed cells every day, without passage, with fresh dox-containing medium (maintain cells in media supplemented with doxycycline until point 5.13).</li>
	<li>Observe cells within 24 h for mCherry expression and daily for colony formation (colonies typically appear between 20 and 30 days after transfection).</li>
	<li>One day before colony picking, prepare a 96-well tissue culture plate with mitomycin C-treated MEF.</li>
	<li>Pick single colonies in 50 &#181;L of volume using a 200 &#181;L pipette, and transfer them sequentially into a 96-well plate in individual wells.</li>
	<li>The day after, dissociate every colony into single cells by adding 50 &#181;l of the commercial trypsin and incubate for 5 min at 37 &#176;C.</li>
	<li>Pipette up and down to disaggregate the colony.</li>
	<li>Transfer 50 &#181;L of the trypsin containing the dissociated colony into a new well with inactivated MEF on a 0.1% gelatin coated 96 well-plate filled with 100 &#181;L of fresh iPS-AF medium supplemented with doxycycline.</li>
	<li>When cells reach 60-70% confluence, split into a well of a 24-well plate.</li>
	<li>When cells reach 60-70% confluence, split 1:2 to two wells, one with doxycycline and the other without doxycycline, to verify if colonies appear also in the condition without doxycycline.</li>
	<li>Continue to maintain iPS-AF clones, doxycycline independent, on inactivated MEF in the iPS-AF medium.</li>
	<li>Cultivate cells at 37 &#176;C and 5% CO2, changing medium daily.</li>
</ol>
6. Alkaline Phosphatase Staining
<ol>
	<li>Perform alkaline phosphatase staining following the manufacturer&#39;s protocol whenever there is the appearance of doxycycline independent iPS-AF cells.</li>
</ol>
7. Immunofluorescence
<ol>
	<li>Whenever there is the appearance of doxycycline independent iPS-AF cells, seed 150,000 cells/cm2 on a well of a 24-well plate with inactivated MEF, and grow cells for 48 hr at 37 &#176;C and 5% CO2.</li>
	<li>(CAUTION) Fix the cells by adding 300 &#181;l per well of 4% paraformaldehyde (PFA) for 15 min at room temperature.</li>
	<li>Add 300 &#181;l of 0.1% NP-40 to permeabilize cells.</li>
	<li>Rinse cells with 300 &#181;l of 0.1% PBS-Tween 20.</li>
	<li>Add 300 &#181;l of the blocking buffer (10% horse serum in 1x PBS) for 1 hr at room temperature.</li>
	<li>Use the following primary antibodies: Oct4 (1:80), Sox2 (1:80), Klf4 (1:80), Nanog (1:100) and SSEA1 (1:80). Dilute SSEA1 with 1% bovine serum albumin (BSA), 10% normal goat serum, 0.3 M glycine in 0.1% PBS-Tween-20. Dilute all other antibodies with 10% horse serum in 1x PBS.</li>
	<li>Incubate antibodies overnight at 4 &#176;C.</li>
	<li>Rinse with 300 &#181;l of 0.1% PBS-Tween-20.</li>
	<li>Use the secondary antibodies diluted in 10% horse serum in 1x PBS: Alexa594-conjugated goat anti-mouse IgG (1:200), Alexa594-conjugated chicken anti-goat IgG (1:200), Alexa594-conjugated chicken anti-rabbit IgG (1:200), Alexa568-conjugated goat anti-mouse IgM (1:200). Incubate for 2 hr at room temperature.</li>
	<li>Rinse with 1x PBS.</li>
	<li>Stain nuclei with Hoechst solution (1:10,000) in 1x PBS.</li>
</ol>
8. In Vitro Cell Differentiation
<ol>
	<li>To form hanging drop embryoid bodies (EBs), cultivate single drops (2,000 cells/20 &#181;l) onto the lid of Petri dishes, using the differentiation medium.</li>
	<li>Incubate the dishes at 37 &#176;C and 5% CO2 for 4 days.</li>
	<li>Transfer EBs into 100 mm bacterial-grade dishes in the suspension culture for 3 days in differentiation medium.</li>
	<li>Plate EBs in 24 well-plates coated with basement membrane matrix (diluted 1:10 in DMEM) for another 14 days.</li>
	<li>For immunofluorescence analyses, use primary antibodies: T (1:100), &#945;fp (1:200) and Tubb3 (1:500)37.</li>
</ol>
9. In Vivo Teratoma Formation
<ol>
	<li>Inject 100 &#181;l of 1x PBS containing 1 x 106 iPS-AF cells into the muscle of the hindlimb of Rag2-/-&#947;c-/- mice.</li>
	<li>Sacrifice mice through cervical dislocation 6 weeks after cell injection.</li>
	<li>Remove the tumor masses, distinguished by their size, from the hindlimb of the mice for the following analyses.</li>
	<li>Cut 7-10 &#181;m thick transverse sections of the isopentane-frozen tumor using a cryostat.</li>
	<li>For Haematoxylin and Eosin (HE) Stain:
	<ol>
		<li>Stain with HE to evaluate the tumor tissue composition, following the manufacturer&#39;s protocol.</li>
	</ol>
	</li>
	<li>For immunoperoxidase Stain:
	<ol>
		<li>Fix teratoma sections with 4% PFA for 15 min at room temperature.</li>
		<li>Permeabilize with 0.1% Triton X-100.</li>
	</ol>
	</li>
	<li>Incubate with the following antibodies: Tubb3 (1:100), &#945;fp (1:50) and &#945;SMA (1:100) diluted in 1% BSA in 1x PBS. Incubate for 1 hr at 37 &#176;C (for Tubb3) or overnight at 4 &#176;C (for the other antibodies).</li>
	<li>Block peroxidase using 100% methanol for 20 min.</li>
	<li>Incubate for 45 min at 37 &#176;C with secondary antibody anti-mouse HRP-conjugated (1:150).</li>
	<li>Rinse with 1x PBS.</li>
	<li>Incubate with peroxidase (HRP) substrate (See Materials Table) for 5 min.</li>
	<li>Rinse with tap water for 5 min.</li>
	<li>Stain with Haematoxylin QS for 9 sec.</li>
	<li>Rinse with tap water.</li>
</ol>
10. RNA Extraction from Cells
<ol>
	<li>Use a commercial RNA extraction kit according to the manufacturer&#39;s recommended protocol.</li>
</ol>
11. RNA Extraction from Teratoma
<ol>
	<li>Homogenize the teratoma using a pestle and mortar and liquid nitrogen to avoid thawing the sample.</li>
	<li>Weigh 25 mg of tissue.</li>
	<li>Add 1 mL of commercial reagent for RNA extraction and 200 &#181;L of chloroform.</li>
	<li>Incubate at room temperature for 5 min.</li>
	<li>Centrifuge at 13,000 x g and 4 &#176;C for 15 min.</li>
	<li>Transfer the cleared supernatant to a new tube.</li>
	<li>To purify the RNA use a commercial RNA extraction kit according to the manufacturer&#39;s protocol.</li>
</ol>
12. Reverse Transcription-polymerase Chain Reaction Analysis
<ol>
	<li>Synthesize the first-strand cDNA using a reverse transcriptase for reverse transcription (RT)-polymerase chain reaction (PCR) and oligo (dT) according to manufacturer&#39;s protocol, to obtain 50 ng/&#181;L of cDNA. Dilute cDNA with RNase-free water at a concentration of 10 ng/&#181;L.</li>
	<li>Perform RT-PCR using the DNA polymerase according to manufacturer&#39;s protocol, utilizing 1 ng/&#181;L of cDNA.</li>
</ol>

The authors have nothing to disclose.

The method chosen to obtain the induction of pluripotency is relevant for cell clinical safety with respect to long term transplantation. Nowadays, there are several methods suitable for the reprogramming. Among the non-integrative methods, the Sendai viral (SeV) vector is an RNA virus that can produce large amounts of protein without integrating into the nucleus of the infected cells40 and could be a strategy to obtain iPS cells. SeV vectors could be an attractive candidate for the generation of ...

Prenatal diagnosis is an important clinical tool to evaluate genetic diseases (i.e. chromosomal aberrations, monogenetic or polygenetic/multifactorial diseases) and congenital malformations (i.e. congenital diaphragmatic hernia, cystic lung lesions, exomphalos, gastroschisis). Amniotic fluid (AF) cells are simple to obtain from routinely scheduled procedures during the second trimester of pregnancy (i.e. amniocentesis and amnioreduction) or caesarean sections1,2. The availability of AF cells from prenatal or neonatal patients provides the possibility of using this source for regenerative medicine, and several researchers investigated the possibility to treat different tissue damages or diseases using a stem cell population isolated from AF3,4,5,6,7,8,9,10,11,12. The possibility of easily obtaining AF cells from diseased patients, in a time window in which the disease is often stationary, opens the way to the idea of using this cell source for reprogramming purposes. Indeed, induced pluripotent stem (iPS) cells derived from AF cells could be differentiated in the cells of interest for in vitro drug testing or for tissue engineering approaches, in order to prepare an adequate patient-specific therapy before childbirth. Many studies have already demonstrated the ability of AF cells to be reprogrammed and differentiated into a wide range of cell types13,14,15,16,17,18,19,20,21,22,23,24,25,26,27.
Since the discovery by Takahashi and Yamanaka28 of reprogrammed somatic cells through the forced expression of four transcription factors (Oct4, Sox2, cMyc and Klf4), progress has been made in the field of reprogramming. Considering the different methods, we can distinguish between viral and non-viral approaches. The first expects the usage of viral vectors (retroviruses and lentiviruses), which have high efficiency but usually incomplete silencing of the retroviral transgene, with both the consequence of a partially reprogrammed cell line and the risk of insertional mutagenesis29,30,31. The non-viral method uses different strategies: i.e. plasmids, vectors, mRNA, protein, transposons. The derivation of iPS cells free of transgenic sequences aims to circumvent the potentially harmful effects of leaky transgene expression and insertional mutagenesis. Among all the above mentioned non-viral strategies, the PiggyBac (PB) transposon/transposase system requires only the inverted terminal repeats flanking a transgene and transient expression of the transposase enzyme to catalyse insertion or excision events32. The advantage in using transposons over other methods for iPS cell generation is the possibility of obtaining vector-free iPS cells with a non-viral vector approach that shows the same efficiency of retroviral vectors. This is possible by trace-less excision of the integrated transposon encoding for the reprogramming factors following a new transient expression of the transposase in the iPS cells33. Given that PB is efficient in different cell types34,35,36,37, is more suitable for a clinical approach with respect to viral vectors, and allows for the production of xeno-free iPS cells contrary to current viral production protocols that use xenobiotic conditions, this system is used to obtain iPS cells from murine AF.
Here we propose a detailed protocol following already published work to show the production of pluripotent iPS clones from mouse AF cells (iPS-AF cells)38.

<table><tbody><tr><td>100 mm Bacterial-grade Petri Dishes&amp;nbsp;</td><td>BD Falcon</td><td>351029</td><td>For &lt;em&gt;in vitro&lt;/em&gt; differentiation</td></tr><tr><td>2-mercaptoethanol&amp;nbsp;</td><td>Sigma</td><td>M6250</td><td>For mouse AF, iPS-AF cells and differentiation medium</td></tr><tr><td>Alexa568-conjugated goat anti-mouse IgM&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A21043</td><td>Secondary antibody (immunofluorescence)</td></tr><tr><td>Alexa594-conjugated chicken anti-goat IgG&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A21468</td><td>Secondary antibody (immunofluorescence)</td></tr><tr><td>Alexa594-conjugated chicken anti-rabbit IgG&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A21442</td><td>Secondary antibody (immunofluorescence)</td></tr><tr><td>Alexa594-conjugated goat anti-mouse IgG&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A11005</td><td>Secondary antibody (immunofluorescence)</td></tr><tr><td>Alkaline Phosphatase kit&amp;nbsp;</td><td>Sigma</td><td>85L1</td><td>Alkaline Phosphatase&amp;nbsp; staining</td></tr><tr><td>Ampicillin</td><td>Sigma</td><td>A0166</td><td>For bacterial selection</td></tr><tr><td>Bovine Serum Albumin&amp;nbsp;</td><td>Sigma</td><td>A7906</td><td>BSA, for blocking solution. Diluted in PBS 1x</td></tr><tr><td>Chloroform</td><td>Sigma</td><td>C2432</td><td>For RNA extraction</td></tr><tr><td>DH5&amp;alpha; cells</td><td>Thermo Fisher Scientific</td><td>18265-017</td><td>Bacteria for cloning procedure</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Thermo Fisher Scientific</td><td>41965039</td><td>For MEF, mouse AF, iPS-AF cells and differentiation medium</td></tr><tr><td>Doxycycline&amp;nbsp;</td><td>Sigma</td><td>D9891</td><td>For exogenous factors expression</td></tr><tr><td>Microcentrifuge tubes (1.5 ml)&amp;nbsp;</td><td>Sarstedt&amp;nbsp;</td><td>72.706</td><td>For PB production&amp;nbsp;</td></tr><tr><td>ES FBS&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>10439024</td><td>For mouse AF, iPS-AF cells and differentiation medium</td></tr><tr><td>FBS&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>10270106</td><td>For MEF medium</td></tr><tr><td>Fine point forceps</td><td>F.S.T</td><td>Dumont #5&amp;nbsp;</td><td>AF isolation</td></tr><tr><td>Gelatin</td><td>J.T.Baker</td><td>131</td><td>Used 0.1%, diluted in PBS 1x</td></tr><tr><td>Glycine</td><td>Bio-Rad</td><td>161-0718</td><td>For blocking solution. Diluted in PBS 1x</td></tr><tr><td>Haematoxylin QS</td><td>Vector Laboratories</td><td>H3404</td><td>Nuclei detection</td></tr><tr><td>HE&amp;nbsp;</td><td>Bio-Optica</td><td>04-061010</td><td>Histological analysis of teratoma</td></tr><tr><td>Hoechst&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>H3570</td><td>Nuclei detection</td></tr><tr><td>Horse Serum&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>16050-122</td><td>For blocking solution</td></tr><tr><td>HRP-conjugated goat anti-mouse IgG</td><td>SantaCruz</td><td>sc2005</td><td>Secondary antibody (immunoperoxidase)</td></tr><tr><td>ImmPACT NovaRED&amp;nbsp;</td><td>Vector Laboratories</td><td>SK4805</td><td>Peroxidase substrate</td></tr><tr><td>Insulin syringe with needle (25 G)</td><td>Terumo</td><td>SS+01H25161</td><td>Amniocentesis procedure</td></tr><tr><td>Klf4&amp;nbsp;</td><td>SantaCruz</td><td>sc-20691</td><td>Rabbit polyclonal IgG</td></tr><tr><td>L-glutamine&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>25030</td><td>For mouse AF, iPS-AF cells and differentiation medium</td></tr><tr><td>LB broth (Lennox)</td><td>Sigma</td><td>L3022</td><td>For bacterial growth</td></tr><tr><td>LIF&amp;nbsp;</td><td>Sigma</td><td>L5158</td><td>For mouse AF and iPS-AF cells medium</td></tr><tr><td>Matrigel&amp;nbsp;</td><td>BD</td><td>354234</td><td>For &lt;em&gt;in vitro&lt;/em&gt; differentiation. Diluted 1:10 in DMEM</td></tr><tr><td>Methanol</td><td>Sigma</td><td>32213</td><td>Peroxidase blocking</td></tr><tr><td>MULTIWELL 24 well plate</td><td>BD Falcon</td><td>353047</td><td>For &lt;em&gt;in vitro&lt;/em&gt; differentiation</td></tr><tr><td>MULTIWELL 6 well plate</td><td>BD Falcon</td><td>353046</td><td>For MEF, mouse AF and iPS-AF cells culture</td></tr><tr><td>Nanog&amp;nbsp;</td><td>ReproCELL</td><td>RCAB0002P-F</td><td>Rabbit polyclonal IgG</td></tr><tr><td>Non-essential amino acids&amp;nbsp;</td><td>Sigma</td><td>M7145</td><td>For mouse AF, iPS-AF cells and differentiation medium</td></tr><tr><td>Normal Goat Serum</td><td>Vector Laboratories</td><td>S2000</td><td>For blocking solution. Diluted in PBS 1x</td></tr><tr><td>NP-40</td><td>Sigma</td><td>12087-87-0</td><td>For cell permeabilization. Diluted in PBS 1x</td></tr><tr><td>Oct4</td><td>SantaCruz</td><td>sc-5279</td><td>Mouse monoclonal IgG2b</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Oligo (dT)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>18418012</td><td>For RT-PCR</td></tr><tr><td>Paraformaldehyde (solution)</td><td>Sigma</td><td>441244</td><td>PFA, fixative, diluted in PBS</td></tr><tr><td>PBS 10x</td><td>Thermo Fisher Scientific</td><td>14200-067</td><td>D-PBS, free of Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;. Diluted with sterile water to obtain PBS 1x</td></tr><tr><td>Penicillin - Streptomycin&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>15070063</td><td>For MEF, mouse AF, iPS-AF cells and differentiation medium</td></tr><tr><td>Petri Dish (150 mm)</td><td>BD Falcon</td><td>353025</td><td>For MEF culture, tissue culture</td></tr><tr><td>PiggyBac transposase expression plasmid&amp;nbsp;</td><td>Provided by professor Andras Nagy laboratory</td><td>-</td><td>mPBase</td></tr><tr><td>PiggyBac-tetO2-IRES-OKMS transposon plasmid</td><td>Provided by professor Andras Nagy laboratory</td><td>-</td><td>PB-tetO2-IRES-OKMS</td></tr><tr><td>QIAprep Spin Maxiprep Kit</td><td>Qiagen</td><td>12663</td><td>For plasmids purification</td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>Qiagen</td><td>27106</td><td>For plasmids purification</td></tr><tr><td>Reverse tetracycline transactivator transposon plasmid&amp;nbsp;</td><td>Provided by professor Andras Nagy laboratory</td><td>-</td><td>rtTA</td></tr><tr><td>RNeasy Mini Kit&amp;nbsp;</td><td>Qiagen</td><td>74134</td><td>For RNA extraction</td></tr><tr><td>Sox2&amp;nbsp;</td><td>SantaCruz</td><td>sc-17320</td><td>Goat polyclonal IgG</td></tr><tr><td>SSEA1&amp;nbsp;</td><td>Abcam</td><td>ab16285</td><td>Mouse monoclonal IgM</td></tr><tr><td>SuperScript II Reverse Transcriptase&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>18064-014</td><td>For RT-PCR</td></tr><tr><td>T&amp;nbsp;</td><td>Abcam</td><td>ab20680</td><td>Rabbit polyclonal IgG</td></tr><tr><td>Taq DNA Polymerase</td><td>Thermo Fisher Scientific</td><td>10342020</td><td>PCR</td></tr><tr><td>Trypsin&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>25300-054</td><td>Cell culture passaging</td></tr><tr><td>Triton X-100</td><td>Bio-Rad</td><td>161-047</td><td>For cell permeabilization, diluted in PBS 1x</td></tr><tr><td>TRIzol Reagent</td><td>Thermo Fisher Scientific</td><td>15596-026</td><td>For RNA extraction</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Tubb3&amp;nbsp;&amp;nbsp;</td><td>Promega&amp;nbsp;</td><td>G712A</td><td>Mouse monoclonal IgG1</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>TWEEN-20</td><td>Sigma</td><td>P1379</td><td>For cell permeabilization, diluted in PBS 1x</td></tr><tr><td>&amp;alpha;fp&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>R&amp;amp;D Systems</td><td>MAB1368</td><td>Mouse Monoclonal IgG1</td></tr><tr><td>&amp;alpha;SMA&amp;nbsp;</td><td>Abcam</td><td>ab7817</td><td>Mouse Monoclonal IgG2a</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Transfection Reagent (FuGENE HD)</td><td>Promega&amp;nbsp;</td><td>E2311</td><td>For AF cells transfection</td></tr><tr><td>Stereomicroscope</td><td>Nikon</td><td>SM2645</td><td>To perform amniocentesis&amp;nbsp;</td></tr><tr><td>200 &amp;mu;l tips</td><td>Sarstedt&amp;nbsp;</td><td>70.760012</td><td>To pick bacteria colonies</td></tr><tr><td>Scissor</td><td>F.S.T</td><td>14094-11 stainless 25U</td><td>To perform amniocentesis&amp;nbsp;</td></tr><tr><td>Ethanol</td><td>Sigma</td><td>2860</td><td>To clean the abdominal wall of the pregnant dam</td></tr><tr><td>Tissue culture petri dish (150 mm)&amp;nbsp;</td><td>BD Falcon</td><td>353025</td><td>For MEF expansion</td></tr><tr><td>Mitomycin C</td><td>Sigma</td><td>M4287-2MG</td><td>For MEF inactivation</td></tr><tr><td>MULTIWELL 96 well plate</td><td>BD Falcon</td><td>353071</td><td>For iPS-AF culture</td></tr></tbody></table>

the production of pluripotent stem cells from mouse amniotic fluid cells using a transposon system

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.

To evaluate the capacity of reprogramming, mouse AF cells were collected from fetuses of GFP mice. Cells were transfected with the circular transposon plasmid PB-tetO2-IRES-OKMS, which expresses the Yamanaka factors (Oct4, Sox2, cMyc and Klf4) linked to the mCherry fluorescent protein in a doxycycline-inducible manner, and reverse tetracycline transactivator (PB-CAG-rtTA) plasmids together with the transposase expression plasmid (mPBase). Mouse AF cells were transfected with Oct4, Klf4, c...

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 ...

the-production-pluripotent-stem-cells-from-mouse-amniotic-fluid-cells

Research

JoVE Journal

Developmental Biology

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells.  Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generation of Mice Derived from Induced Pluripotent Stem Cells

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Mouse embryonic fibroblast can be reprogrammed into induced pluripotent stem cells at low efficiency by the forced expression of transcription factors Oct-4, Sox-2, Klf-4, c-Myc. The rare intermediates of the reprogramming reaction are FACS isolated via labeling with antibodies against cell surface makers Thy-1.2, Ssea-1, and Epcam.

Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

This protocol describes the reprogramming of primary amniotic fluid and membrane mesenchymal stem cells into induced pluripotent stem cells using a non-integrating episomal approach in fully chemically defined conditions. Procedures of extraction, culture, reprogramming, and characterization of the resulting induced pluripotent stem cells by stringent methods are detailed.

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

Here, we describe a detailed method for seamless gene editing in human pluripotent stem cells using a piggyBac-based donor plasmid and the Cas9 nickase mutant. Two point mutations were introduced into exon 8 of the hepatocyte nuclear factor 4 alpha (HNF4α) locus in human embryonic stem cells (hESCs).

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Learn to generate induced pluripotent stem cells from mouse amniotic fluid cells using a non-viral transposon system. This method enables safer iPSC production for regenerative medicine and patient-specific therapeutic applications.

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

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