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 ...

The overall goal of this procedure is to isolate and reprogram murine amniotic fluid cells using a transposon system. The main advantage of this technique is that it is easily performed. In a therapeutic setting, cells from human disease fetuses can be differentiated to cells of interest for drug testing or tissue engineering, in order to prepare another way the patient specific therapy before childbirth.This method can help answer key questions of programming. The transposon system is efficient in different cell types. It's more suitable for clinical approach with respect to viral vectors and allow the production of oxino-free IPS cells.To begin the isolation, clean the abdominal wall of the dam using 70 percent ethanol. Using scissors, perform a midline laparotomy, incising the length of the abdominal wall to gain access to the abdominal cavity. Use forceps to expose the uterus and then excise it using scissors.Transfer the uterus into a 100 millimeter dish filled with sterile 1xPBS and keep on ice. Place the dish under a stereomicroscope at 10X magnification and hold the uterus with forceps whilst removing the uterine wall with scissors, taking care to avoid damaging the fetal membranes. Collect the fetuses into a 100 millimeter dish filled with sterile 1XPBS and place on ice.Grasp the placenta of one fetus with the fine point forceps and move it to a clean 100 millimeter dish. Here, carefully remove the yolk sack. Disrupt the amnion using the forceps and using an insulin syringe, collect 30 microliters of amniotic fluid.Transfer the fluid to a 15 milliliter conical vial. Repeat this process for each fetus, collecting the fluid into the same vial each time. Next, wash each fetus with sterile 1XPBS supplemented with one percent fetal bovine serum and collect this into the 15 milliliter conical tube along with the amniotic fluid.Centrifuge the tube at 145 times G for five minutes and then under a laminar flow hood, remove the supernatant. Re-suspend the pelleted cells in two milliliters of amniotic fluid culture medium. Take a zero point one percent gelatin-coated six well plate containing myomycin-C treated mouse embryonic fibroblasts or MEF and then seed two milliliters of cells onto the plate.Cultivate the cells at 37 degrees Celsius and five percent carbon dioxide, changing the medium every other day. Continue culture for seven days until the cells are ready for transfection. To begin transfection, in a one point five millimiter microcentrifuge tube, mix one microgram of transposon plasmid with zero point five micrograms of transposase expression plasmid and zero point five micrograms of reverse tetracycline transactivator plasmid.Dilute to 100 microliters using d-man. Add eight microliters of transfection reagent into the microcentrifuge tube containing the DNA. Incubate this mix for 15 minutes at room temperature.Taking the six well plate with mouse AF cells, dropwise add 100 microliters of the transfection agent DNA mixture per well, distributing by gentle swirling with a final volume of two milliliters per well. Leave cells for 24 hours at 37 degrees Celsius in five percent carbon dioxide. The following day, remove the culture medium and then feed each well of cells with two milliliters of fresh iPS AF medium supplemented with one point five micrograms per milliliter of doxycycline to induce the expression of Yamanaka's factors.Feed the cells daily with fresh dox-containing medium without passage from this point forward. Observe the cells under the florescence microscope within 24 hours of the first doxycylcine treatment to look for any mCherry expression and daily from this point on for colony formation. Once colony formation is observed, pick single colonies into 50 microliters of cell culture medium.Next, transfer them sequentially into separate wells of a 96 well tissue culture plate with mytomycin c treated MEF. The following day, add 50 microliters of trypsin to each well and incubate for five minutes at 37 degrees Celsius to dissociate the colonies into single cells. Following incubation, pipette the liquid up and down to further disaggregate the colonies.Transfer 50 microliters of the trypsin cell mix into a new well containing inactivated MEF and then return the cells to the incubator. When cells reach 60 to 70 percent confluence, split them into a new well of a 24 well plate and then continue incubation at room temperature. Once these cells reach 60 to 70 percent confluence, flip the cells one to two into two wells, one containing doxycycline and one without to verify that colonies will also grow in the absence of doxycycline.Continue to maintain the IPS AF colonies and doxycycline independent medium on inactivated MEF at 37 degrees Celsius in five percent carbon dioxide. Change the medium daily until cells are ready for staining, imaging, and RNA extraction. Here, the stable doxycycline independent induced pleuripotent mouse amniotic GFP positive cells express the pleuripotency markers required to confirm successful reprogramming.RTPCR analysis using R1 ES cells as a control showed expression of the induced pleuripotency markers in the presence or absence of doxycycline. The expression of the housekeeping gene, beta two microglobulin, was also observed in all three cell types. Teratomas isolated from mice injected with the induced pleuropotent cells were immunostained.Detection of alpha fetal protein, alpha smooth muscle actin, and beta three tubulin in the masses confirm cell differentiation into all three germ layers, ectoderm, mesoderm, and endoderm. Further, RTPCR analysis of in vitro embryoid bodies and in vivo teratoma cells confirm the expression of germ layer specific marker genes for the mesoderm, endoderm, and ectoderm. After watching this video, you should have a good understanding of how to isolate and reprogram murine amniotic fluid cells.This is a simple, safe procedure that can be applied also to human amniotic fluid cells from a sample obtained from routine diagnoses or cesarean sections.

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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 development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

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 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 ...

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 phenotypes. Researchers interested in pursuing studies involving specific myocyte subtypes require a more directed differentiation approach. By treating mouse embryonic stem (ES) cells with Grem2, a secreted BMP antagonist that is necessary for atrial chamber formation in vivo, a large number of cardiac cells with an atrial phenotype can be generated. Use of the engineered Myh6-DSRed-Nuc pluripotent stem cell line allows for identification, selection, and purification of cardiomyocytes. In this protocol embryoid bodies are generated from Myh6-DSRed-Nuc cells using the hanging drop method and kept in suspension until differentiation day 4 (d4). At d4 cells are treated with Grem2 and plated onto gelatin coated plates. Between d8-d10 large contracting areas are observed in the cultures and continue to expand and mature through d14. Molecular, histological and electrophysiogical analyses indicate cells in Grem2-treated cells acquire atrial-like characteristics providing an in vitro model to study the biology of atrial cardiomyocytes and their response to various pharmacological agents.

Generating cardiomyocytes from pluripotent stem cells in vitro allows access to large amounts of cardiac tissue in vitro for basic science and clinical applications. This protocol uses the atrializing factor Grem2 to both increase the numbers of cardiomyocytes obtained and to generate cardiomyocytes with an atrial phenotype.

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 methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

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 state. Now, reprogramming is done with various types of adult somatic cells: keratinocytes, urine cells, fibroblasts, etc. Early experiments were usually done with dermal fibroblasts. However, this required an invasive surgical procedure to obtain fibroblasts from the patients. Therefore, suspension cells, such as blood and urine cells, were considered ideal for reprogramming because of the convenience of obtaining the primary cells. Here, we report an efficient protocol for iPSC generation from peripheral blood mononuclear cells (PBMCs). By plating the transduced PBMCs serially to a new, matrix-coated plate using centrifugation, this protocol can easily provide iPSC colonies. This method is also applicable to umbilical cord blood mononuclear cells (CBMCs). This study presents a simple and efficient protocol for the reprogramming of PBMCs and CBMCs.

We propose a protocol for reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). By plating the transduced blood cells onto matrix-coated plates with centrifugation, iPSCs are successfully induced from floating cells. This technique suggests a simple and effective reprogramming protocol for cells such as PBMCs and CBMCs.

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 microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

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

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

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

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

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

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 stem cells (hESCs) and induced pluripotent stem cells (iPSCs). Current differentiation procedures generate human hepatocyte-like cells (HLCs) displaying a mixture of fetal and adult traits. To improve cell phenotype, we have fully defined our differentiation procedure and the cell niche, resulting in the generation of cell populations which display improved gene expression and&#160;function. While these studies mark progress, the ability to generate large quantities of multi well plates for screening has been limited by labour intensive procedures and batch to batch variation. To tackle this issue, we have developed a semi-automated platform to differentiate pluripotent stem cells into HLCs. Stem cell seeding and differentiation were performed using liquid handling and automatic pipetting systems in&#160;96-well plate format. Following the differentiation, cell phenotype was analyzed using automated microscopy and a multi well luminometer.

This protocol describes a semi-automated approach to produce hepatocyte-like cells from human pluripotent stem cells in a 96 well plate format. This process is rapid and cost-effective, allowing the production of quality assured batches of hepatocyte-like cells for basic and applied human research.

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 functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

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

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

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