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.

すべての手順は、イタリアの法律に従いました。マウスAFサンプルをGFPと呼ばれるC57BL / 6-Tgの（UBC-GFP）30Scha / Jマウスからの13.5日後交尾（DPC）で妊娠したマウスから採取しました。 1.トランスポゾン生産注：トランスポゾン発現ベクターは、標準的なクローニング手順を用いて作製しました。マウスAF細胞のトランスフェクション用のプラスミドDNAを?...

多能性の誘導を得るために選択された方法は、長期移植に関して細胞臨床的安全性に関連します。今日では、再プログラミングに適したいくつかの方法があります。非組込み方法のうち、センダイウイルス（センダイウイルス）ベクターは、感染した細胞40の核内に統合することなく、タンパク質を大量に生産することができ、iPS細胞を得るための戦略であり得るRNAウイル?...

出生前診断は、遺伝性疾患（ すなわち染色体異常、単成または複成/多因子疾患）と先天性奇形（ すなわち先天性横隔膜ヘルニア、嚢胞性肺病変、臍帯ヘルニア、胃壁破裂）を評価するための重要な臨床的ツールです。羊水（AF）細胞（ すなわち 、羊水穿刺およびamnioreduction）または帝王切開1、2妊娠中期の間に定期的にスケジュールされた手順から取得するのは簡単です。出生前または新生児の患者からのAF細胞の利用可能性は、再生医療のためのこのソースを使用する可能性を提供し、いくつかの研究は、AF 3、4、5、6から単離された幹細胞集団を使用して、異なる組織の損傷または疾患を治療する可能性を検討しました、7、8、9、10、11、12。簡単に罹患した患者からのAF細胞を得る可能性は、疾患はしばしば静止している時間ウィンドウ内で、再プログラミングのために、この細胞源を使用するというアイデアに道を開きます。実際、AF細胞由来の人工多能性幹（iPS）細胞は、出産前の適切な患者特有の治療を製造するために、 インビトロでの薬物試験のために、または組織工学的アプローチのための目的の細胞に分化することができます。多くの研究は、既に細胞型13、14、15、16、17 &lt;広範囲に再プログラムと区別されるAF細胞の能力を実証しています/ SUP&gt;、18、19、20、21、22、23、24、25、26、27。 4転写因子（Oct4の、Sox2の、CMYCおよびKlf4の）の強制発現を介して、再プログラミングされた体細胞の高橋と山中28によって発見されて以来、進展が再プログラミングの分野で行われています。異なる方法を考慮すると、我々は、ウイルスおよび非ウイルス性のアプローチを区別することができます。最初は、部分的に再プログラムされた細胞株の結果とのリスクの両方で、高効率レトロウイルス導入遺伝子の通常不完全なサイレンシングを有するウイルスベクター（レトロウイルスおよびレンチウイルス）の使用を想定し挿入突然変異29、30、31。 すなわちプラスミド、ベクター、mRNA、タンパク質、トランスポゾン：非ウイルス法は、異なる戦略を使用しています。トランスジェニック配列を含まiPS細胞の誘導は、漏出性導入遺伝子発現および挿入突然変異の潜在的に有害な影響を回避することを目指しています。上記の全ての非ウイルス戦略の中で、のpiggyBac（PB）トランスポゾン/トランスポザーゼシステムは挿入または切除イベント32を触媒する導入遺伝子およびトランスポザーゼ酵素の一過性発現に隣接する唯一の逆方向末端反復を必要とします。 iPS細胞の生成のための他の方法を介してトランスポゾンを使用する利点は、レトロウイルスベクターの同じ効率を示す非ウイルスベクターアプローチとベクトルフリーiPS細胞を得る可能性があります。これはreprogrのための統合されたトランスポゾンエンコーディングのトレースレス切除することにより可能ですiPS細胞33内のトランスポザーゼの新たな一過性の発現以下の要因をamming。 PBは、異なる細胞型34、35、36、37に効率的であることを考えると、ウイルスベクターに対する臨床的アプローチに適している、および生体異物を使用し、現在のウイルス産生プロトコルに反して異種非含有のiPS細胞の産生を可能にします条件は、このシステムは、マウスAFからiPS細胞を得るために使用されます。 ここでは、マウスのAF細胞（IPS-AF細胞）38からの多能性のiPSクローンの生産を表示するには、すでに発表された研究を、以下の詳細なプロトコルを提案します。

<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.

再プログラミングの能力を評価するために、マウスAF細胞をGFPマウスの胎児から採取しました。細胞は、PB-（ドキシサイクリン誘導方法でmCherryを蛍光タンパク質に連結山中因子（Oct4の、Sox2の、CMYCおよびKlf4の）を発現円形トランスポゾンプラスミドPB-tetO2-IRES-OKMSでトランスフェクション、およびテトラサイクリントランスを逆にしました。 CAG-のrtTA）トランスポザー...

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.

トランスポゾンのシステムを用いたマウスの羊水細胞から多能性幹細胞の作製

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.

Research

JoVE Journal

Developmental Biology

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発生生物学

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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

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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

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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

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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

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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

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