Name: the production of pluripotent stem cells from mouse amniotic fluid cells using a transposon system
Uploaded: 2017-02-28T13:00:00
Description: Watch this Scientific Journal Video about The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System at JoVE.com

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 ...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mm Bacterial-grade Petri Dishes&#160;</td>
			<td>BD Falcon</td>
			<td>351029</td>
			<td>For in vitro differentiation</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol&#160;</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&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21043</td>
			<td>Secondary antibody (immunofluorescence)</td>
		</tr>
		<tr>
			<td>Alexa594-conjugated chicken anti-goat IgG&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21468</td>
			<td>Secondary antibody (immunofluorescence)</td>
		</tr>
		<tr>
			<td>Alexa594-conjugated chicken anti-rabbit IgG&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21442</td>
			<td>Secondary antibody (immunofluorescence)</td>
		</tr>
		<tr>
			<td>Alexa594-conjugated goat anti-mouse IgG&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11005</td>
			<td>Secondary antibody (immunofluorescence)</td>
		</tr>
		<tr>
			<td>Alkaline Phosphatase kit&#160;</td>
			<td>Sigma</td>
			<td>85L1</td>
			<td>Alkaline Phosphatase&#160; staining</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Sigma</td>
			<td>A0166</td>
			<td>For bacterial selection</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin&#160;</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 rowspan="2">DH5&#945; cells</td>
			<td rowspan="2">Thermo Fisher Scientific</td>
			<td rowspan="2">18265-017</td>
			<td rowspan="2">Bacteria for cloning procedure</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Dulbecco&#39;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&#160;</td>
			<td>Sigma</td>
			<td>D9891</td>
			<td>For exogenous factors expression</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes (1.5 mL)&#160;</td>
			<td>Sarstedt&#160;</td>
			<td>72.706</td>
			<td>For PB production&#160;</td>
		</tr>
		<tr>
			<td>ES FBS&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>10439024</td>
			<td>For mouse AF, iPS-AF cells and differentiation medium</td>
		</tr>
		<tr>
			<td>FBS&#160;</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&#160;</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&#160;</td>
			<td>Bio-Optica</td>
			<td>04-061010</td>
			<td>Histological analysis of teratoma</td>
		</tr>
		<tr>
			<td>Hoechst&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>H3570</td>
			<td>Nuclei detection</td>
		</tr>
		<tr>
			<td>Horse Serum&#160;</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&#160;</td>
			<td>Vector Laboratories</td>
			<td>SK4805</td>
			<td>Peroxidase substrate</td>
		</tr>
		<tr>
			<td>Insulin syringe with needle (25G)</td>
			<td>Terumo</td>
			<td>SS+01H25161</td>
			<td>Amniocentesis procedure</td>
		</tr>
		<tr>
			<td>Klf4&#160;</td>
			<td>SantaCruz</td>
			<td>sc-20691</td>
			<td>Rabbit polyclonal IgG</td>
		</tr>
		<tr>
			<td>L-glutamine&#160;</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&#160;</td>
			<td>Sigma</td>
			<td>L5158</td>
			<td>For mouse AF and iPS-AF cells medium</td>
		</tr>
		<tr>
			<td>Matrigel&#160;</td>
			<td>BD</td>
			<td>354234</td>
			<td>For in vitro 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 in vitro 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&#160;</td>
			<td>ReproCELL</td>
			<td>RCAB0002P-F</td>
			<td>Rabbit polyclonal IgG</td>
		</tr>
		<tr>
			<td>Non-essential amino acids&#160;</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 rowspan="2">Oct4</td>
			<td rowspan="2">SantaCruz</td>
			<td rowspan="2">sc-5279</td>
			<td rowspan="2">Mouse monoclonal IgG2b</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Oligo (dT)&#160;</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 Ca2+/Mg2+. Diluted with sterile water to obtain PBS 1X</td>
		</tr>
		<tr>
			<td>Penicillin - Streptomycin&#160;</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 (150mm)</td>
			<td>BD Falcon</td>
			<td>353025</td>
			<td>For MEF culture, tissue culture</td>
		</tr>
		<tr>
			<td>PiggyBac transposase expression plasmid&#160;</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&#160;</td>
			<td>Provided by professor Andras Nagy laboratory</td>
			<td>-</td>
			<td>rtTA</td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit&#160;</td>
			<td>Qiagen</td>
			<td>74134</td>
			<td>For RNA extraction</td>
		</tr>
		<tr>
			<td>Sox2&#160;</td>
			<td>SantaCruz</td>
			<td>sc-17320</td>
			<td>Goat polyclonal IgG</td>
		</tr>
		<tr>
			<td>SSEA1&#160;</td>
			<td>Abcam</td>
			<td>ab16285</td>
			<td>Mouse monoclonal IgM</td>
		</tr>
		<tr>
			<td>SuperScript II Reverse Transcriptase&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>18064-014</td>
			<td>For RT-PCR</td>
		</tr>
		<tr>
			<td>T&#160;</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&#160;</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 rowspan="2">TRIzol Reagent</td>
			<td rowspan="2">Thermo Fisher Scientific</td>
			<td rowspan="2">15596-026</td>
			<td rowspan="2">For RNA extraction</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">Tubb3&#160;&#160;</td>
			<td rowspan="2">Promega&#160;</td>
			<td rowspan="2">G712A</td>
			<td rowspan="2">Mouse monoclonal IgG1</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>TWEEN-20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td>For cell permeabilization, diluted in PBS 1X</td>
		</tr>
		<tr>
			<td>&#945;fp&#160;&#160;&#160;</td>
			<td>R&#38;D Systems</td>
			<td>MAB1368</td>
			<td>Mouse Monoclonal IgG1</td>
		</tr>
		<tr>
			<td rowspan="2">&#945;SMA&#160;</td>
			<td rowspan="2">Abcam</td>
			<td rowspan="2">ab7817</td>
			<td rowspan="2">Mouse Monoclonal IgG2a</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Transfection Reagent (FuGENE HD)</td>
			<td>Promega&#160;</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&#160;</td>
		</tr>
		<tr>
			<td>200 ul tips</td>
			<td>Sarstedt&#160;</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&#160;</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)&#160;</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

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...

Watch this Scientific Journal Video about The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System at JoVE.com

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

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 ...

Research

JoVE Journal

Developmental Biology

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

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 ...

Differentiation of Atrial Cardiomyocytes from Pluripotent Stem Cells Using the BMP Antagonist Grem2

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

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

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

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

Semi-automated Production of Hepatocyte Like Cells from Pluripotent Stem Cells

Human pluripotent stem cells represent a renewable source of human tissue. Our research is focused on generating human liver tissue from human embryonic ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

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

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