Work described in this manuscript was made possible by funding from the California Institute for Regenerative Medicine (CIRM) for UCR Stem Cell Core.

Our protocol begins with a method to adapt human iPSCs to feeder-free cultures, followed by protocols for transfecting human iPSCs using GeneJuice (EMD) and nucleofection of human iPSCs using an AMAXA nuclefector device.
Note: The following procedures are performed in a sterile laminar flow hood. All media and solutions are equilibrated to 37&deg;C or room temperature before starting unless otherwise specified.
1. Establishing human iPSCs on feeder-free system
Human iPSCs previously maintained on feeder cells can be split, transferred onto Geltrex-coated dish and maintained for two passages prior to feeder-free transfection.
<ol>
	<li>Thaw Geltrex overnight at 4&deg;C. To prepare Geltrex coating, dilute defrosted Geltrex 1:50 in cold DMEM. Mix the solutions gently.</li>
</ol>
Note: Geltrex, like Matrigel is a soluble form of basement membrane matrix purified from murine Engelbreth-Holm-Swarm (EHS) tumor cells. Alternatively, Matrigel can be used as an extracellular matrix to establish feeder-free human iPSC cultures.
<ol start="2">
	<li>Cover the whole surface of culture wells with Geltrex solution (1 ml for a 35-mm well). Coat wells with Geltrex at 37&deg;C incubator for 1 hour.</li>
	<li>To passage human iPSCs, add 1 ml of accutase per well and incubate at 37&deg;C for 1 min until most cells start to detach.</li>
</ol>
To passage human iPSCs, add 1 ml of accutase per well and incubate at 37&deg;C for 1 min until most cells start to detach.
<ol start="4">
	<li>Add 10-15 glass beads to the cells and gently swirl the plate. Add 2 ml of KnockOut DMEM/ F12 and gently triturate. Transfer cell suspension to a 10 ml centrifuge tube.</li>
	<li>Spin cells at 800 rpm for 3 min at room temperature.</li>
	<li>Aspirate supernatant from the tube, leaving human iPSC pellet intact. Gently flick tube to disperse cell pellet.</li>
	<li>Remove Geltrex from the coated well.</li>
	<li>Gently resuspend human iPSC pellet in an appropriate volume of STEMPRO. Distribute between wells of feeders, depending on the proliferation rate). Human iPSCs can be passaged in a split ratio of 1:2 to 1:6.</li>
	<li>Carefully place into 5% CO2 incubator, swirl the plate carefully to ensure an even distribution of cells across the wells.</li>
	<li>Feed cells daily until cells are ready to be split again (when cells reach 80% confluency).</li>
	<li>Passage human iPSCs onto new Geltrex-coated well (step 1.3 to 1.9) in a split ratio of 1:2. Small colonies should be formed and distributed evenly on Geltrex-coated well prior to transfection.</li>
</ol>
2. Transfection of human iPSCs with GeneJuice
Cells (grown on 6-well plates) should be approximately 40 -50% confluent on the day of transfection to achieve optimal transfection efficiency. It is not necessary to change the cell medium until the next day.
<ol>
	<li>Prepare 100 &mu;l KO-DMEM/F12 in a sterile 1.5 ml eppendorf tube. Add 27 &mu;l GeneJuice transfection reagent. Mix well. Incubate at room temperature for 5 mins.</li>
	<li>Add 4 &mu;g plasmid DNA. Incubate the tube at room temperature for 15 mins. The choice of plasmid is critical for optimal transfection efficiency. We use a plasmid with an enhanced green fluorescence protein (eGFP) driven by CAG promoter5 (pCAG-eGFP). CAG promoter is a strong promoter that is transcriptionally active in human iPSCs and thus can be used to drive transgene expression in these cells. In our hands, linearization of plasmid did not seem to affect transfection efficiency.</li>
	<li>Add GeneJuice-DNA mixture to the cells and swirl the plate. Spin the plate at 1200 rpm for 5 minutes ('spinoculation' method) to increase the contact of transfection mixture with human iPSCs on the well.</li>
	<li>Incubate the cells at 37&deg;C overnight.</li>
	<li>Monitor for transfection efficiency the next day.</li>
	<li>For stable transfection, change medium the next day with fresh STEMPRO. Add appropriate antibiotic selection after 24 - 48 hours.</li>
</ol>
3. Nucleofection of human iPSCs
Human iPSCs grown on feeders or Geltrex can be used for nucleofection. However, we strongly recommend replating nucleofected human iPSCs onto mouse embryonic fibroblast (MEF) or human foreskin fibroblast (HFF) feeders to ensure high cell viability and recovery. At least 2 million cells should be used to achieve higher cell survival following nucleofection.
<ol>
	<li>Prepare feeder-cell conditioned media (CM) by incubating human iPSC KnockOut Serum Replacement (KOSR) media with feeder cells overnight. Collect CM every 24 hours.</li>
</ol>
Note: Any mouse strain used for preparing feeder layers (such as BLK6, CF1 and MF1) is suitable to be used for making CM.
<ol start="2">
	<li>On the day of nucleofection, pre-treat human iPSCs with 10 &mu;M ROCK inhibitor for at least 1 hour.</li>
	<li>Prepare 82 &mu;l of human stem cell nucleofector solution in a sterile 1.5 ml eppendorf tube. Add 18 &mu;l supplement 1. Mix well. Incubate solution at 37&deg;C for 5 mins.</li>
	<li>Pre-warm feeder cell-conditioned media (CM) and 0.25% trypsin/ EDTA to 37&deg;C. Under the hood, prelabel one sterile 15 ml conical tube.</li>
	<li>Carefully remove media from human iPSC culture to be nucleofected. Gently wash the cells with 2 ml 1X phosphate buffered solution (PBS) per well. Aspirate the PBS. Add 1 ml of 0.25% trypsin per well. Incubate cells at 37&deg;C for 3 mins.</li>
	<li>Gently triturate the cells with 1000 ml pipette tip. Wash the bottom of the well to make sure all human iPSCs are completely detached. Transfer cell suspension to the labeled 15 ml conical tube.</li>
</ol>
Note: Trypsinization into single cells should strictly be avoided. Cells should only be dislodged into small clumps of cells consisted of approximately triplets of cells. Small clumps of cells (cell triples) will be dissociated into single cells during the subsequent handling of the cells.
<ol start="7">
	<li>Add 9 ml MEF media to inactivate trypsin. Spin cells at 800 rpm for 3 mins. Carefully aspirate the supernatant and leave the cell pellet intact.</li>
	<li>Resuspend cells in prewarmed 100 &mu;l of human stem cell nucleofector solution from Step 3.3.</li>
	<li>Transfer cells to a nucleofector cuvette using a 1 ml pipette tip.</li>
	<li>Add 4 &mu;g of plasmid DNA into the cell suspension in the cuvette. Mix cells and DNA by gentle swirling. Tap the cuvette twice on the hood surface.</li>
	<li>Insert the cuvette into the nucleofector holder. Use programs B-016. Nucleofect cells by pressing button X.</li>
</ol>
&lt;<ok>&gt; will be displayed when the nucleofection process is completed (usually only takes 1-5 seconds). The use of other nucleofection programs (A-023, A-033 and U-023) yielded less than 10% of eGFP-positive cells from human iPSCs.</ok>
<ol start="12">
	<li>Retrieve nucleofected cells from the cuvette using the provided Pasteur plastic pipette. Recover cells by resuspending them in prewarmed CM and ROCK inhibitor into a sterile 1.5 ml eppendorf tube. Incubate cells at 37&deg;C for 10 mins to let cells recover.</li>
	<li>Transfer cells drop-wise onto feeder layers using 1 ml pipette tip. Incubate the cells at 37&deg;C overnight.</li>
	<li>Monitor for transfection efficiency the next day. To derive stable clones of transgenic human iPSCs that stably expressing transgene, change medium the next day with CM and ROCK inhibitor. Add appropriate antibiotic selection after 24 - 48 hours.</li>
</ol>
4. Representative Results:
<img alt="Figure 1" src="/files/ftp_upload/3110/3110fig1.jpg" /> 
Figure 1. Photomicrographs of Riv1 human iPSCs transfected with pCAG-eGFP. (A) eGFP-expressing Riv1 cells transfected using GeneJuice on Geltrex 12 hours post-transfection. Colonies of Riv1 human iPSCs plated and formed on feeders following nucleofection using B-016 (B) and A-023 (C) program. (D) Stably eGFP-expressing human iPSC colonies with ubiquitous eGFP expression derived from GeneJuice. (E) Stably-transfected Riv1 human iPSCs retained constitutive eGFP expression during embryoid body differentiation.

<ol>
	<li>Liew, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cells:+possibilities+for+human+cell+transplantation.">Human embryonic stem cells: possibilities for human cell transplantation.</a> Ann Med. 37, 521-532 (2005).</li><li>Eiges, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+human+embryonic+stem+cell-transfected+clones+carrying+a+marker+for+undifferentiated+cells.">Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells.</a> Curr Biol. 11, 514-518 (2001).</li><li>Zwaka, T. P., Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homogous+recombination+in+human+embryonic+stem+cells.">Homogous recombination in human embryonic stem cells.</a> Nat Biotechnol. 21, 319-321 (2003).</li><li>Siemen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleofection+of+human+embryonic+stem+cells.">Nucleofection of human embryonic stem cells.</a> Stem Cells Dev. 14, 378-383 (2005).</li><li>Liew, C. G., Draper, J. S., Walsh, J., Moore, H., Andrews, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+and+stable+transgene+expression+in+human+embryonic+stem+cells.">Transient and stable transgene expression in human embryonic stem cells.</a> Stem Cells. 25, 1521-1528 (2007).</li><li>Xia, X., Ayala, M., Thiede, B. R., Zhang, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro-+and+in+vivo-induced+transgene+expression+in+human+embryonic+stem+cells+and+derivatives.">In vitro- and in vivo-induced transgene expression in human embryonic stem cells and derivatives.</a> Stem Cells. 26, 525-533 (2008).</li><li>Braam, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+genetic+manipulation+of+human+embryonic+stem+cells.">Improved genetic manipulation of human embryonic stem cells.</a> Nat Methods. 5, 389-392 (2008).</li><li>Braam, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeder-free+culture+of+human+embryonic+stem+cells+in+conditioned+medium+for+efficient+genetic+modification.">Feeder-free culture of human embryonic stem cells in conditioned medium for efficient genetic modification.</a> Nat Protoc. 3, 1435-1443 (2008).</li><li>Hohenstein, K. A., Pyle, A. D., Chern, J. Y., Lock, L. F., Donovan, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleofection+mediates+high-efficiency+stable+gene+knockdown+and+transgene+expression+in+human+embryonic+stem+cells.">Nucleofection mediates high-efficiency stable gene knockdown and transgene expression in human embryonic stem cells.</a> Stem Cells. 26, 1436-1443 (2008).</li><li>Placantonakis, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAC+transgenesis+in+human+embryonic+stem+cells+as+a+novel+tool+to+define+the+human+neural+lineage.">BAC transgenesis in human embryonic stem cells as a novel tool to define the human neural lineage.</a> Stem Cells. 27, 521-532 (2009).</li></ol>

No conflicts of interest declared.

Our protocols result in simple, robust and highly reproducible techniques to introduce transgenes into human iPSCs without prominent toxic effect and cell death. Human iPSCs should be passaged into smaller clumps of cells (5-10 cells) and plated on Geltrex at high density (1:2) to ensure optimal transfection efficiency in numerous small colonies. For human iPSC lines that are more prone to differentiation and cell death, higher number of human iPSCs (up to 4 X 106 cells) should be used for a single nucleofection experiment. Transient transfection assay generates large numbers of transgene-expressing human iPSCs within 1 day. Stably transfected iPSC clones usually appear within 7 days, and these transgenic colonies should be ready to be picked within three weeks. The use of CAG promoter described here ensures ubiquitous expression of eGFP reporter. Under such improvements, our protocols can be fed into other applications, including overexpression, conditional induction, derivation of lineage-specific reporter lines, shRNA or siRNA knockdown, gene targeting and homologous recombination.

<table border="1">
	<tbody>
		<tr>
			<td>Name of reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>0.25% Trypsin with EDTA</td>
			<td>Invitrogen</td>
			<td>25200056</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Invitrogen</td>
			<td>21985-023</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>3 mm glass beads</td>
			<td>Fisher Scientific</td>
			<td>11-312A</td>
			<td>Glass beads should be washed with hydrochloric acid (HCl) overnight, rinsed off with sodium hydroxide (NaOH) and distilled water, and sterilized by autoclave before use. </td>
		</tr>
		<tr>
			<td>Accutase cell dissociation reagent</td>
			<td>Invitrogen</td>
			<td>A11105-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Basic fibroblast growth factor (bFGF)</td>
			<td>Invitrogen</td>
			<td>PHG0263</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM (high glucose)</td>
			<td>Lonza</td>
			<td>12-741 F</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal calf serum</td>
			<td>Invitrogen</td>
			<td>16000044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Geltrex</td>
			<td>Invitrogen</td>
			<td>12760013</td>
			<td>Growth factor reduced</td>
		</tr>
		<tr>
			<td>GeneJuice transfection reagent</td>
			<td>EMD Biosciences</td>
			<td>70967</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glutamax-I (100X)</td>
			<td>Invitrogen</td>
			<td>35050061</td>
			<td>L-Glutamine (Invitrogen, 25030081) can also be used instead.</td>
		</tr>
		<tr>
			<td>Human iPSC KOSR media</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>500 ml human ES media consists of 390 ml KnockOut DMEM/ F12, 100 ml KnockOut Serum Replacer, 5ml Glutamax-I (100X), 5ml NEAA (100X), 500 &mu;l 2-Mercaptoethanol (55mM) and supplemented with 10 ng/ml bFGF.</td>
		</tr>
		<tr>
			<td>KnockOut DMEM/F12</td>
			<td>Invitrogen</td>
			<td>12660-012</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement (KOSR)</td>
			<td>Invitrogen</td>
			<td>10828-028</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mouse embryonic fibroblast media</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>MEF media consists of 90% FBS, 1X NEAA, 1X Glutamax-I and 1X sodium pyruvate in DMEM (high glucose)</td>
		</tr>
		<tr>
			<td>Non essential amino acid, NEAA?(100X) </td>
			<td>Invitrogen</td>
			<td>11140050</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Human stem cell nucleofector solution 1 with supplement</td>
			<td>Lonza</td>
			<td>VAPH-5012</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline without Ca2+ and Mg2+</td>
			<td>Invitrogen</td>
			<td>10010023</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium pyruvate (100X) </td>
			<td>Invitrogen</td>
			<td>11360070</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>STEMPRO medium kit</td>
			<td>Invitrogen</td>
			<td>A1000701</td>
			<td>500 ml STEMPRO media consists of 454 ml KnockOut DMEM/ F12, 10 ml STEMPRO serum-free growth supplement (50X), 5ml Glutamax-I (100X), 5ml NEAA (100X), 36 ml BSA (Bovine serum albumin, 25%), 909 &mu;l 2-Mercaptoethanol (55mM) and supplemented with 10 ng/ml bFGF.</td>
		</tr>
	</tbody>
</table>

transfecting and nucleofecting human induced pluripotent stem cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Watch this Scientific Journal Video about Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells at JoVE.com

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

transfecting-and-nucleofecting-human-induced-pluripotent-stem-cells

Research

JoVE Journal

Biology

21.4K 조회수.  University of California Riverside. 유전자 변형의 최근 발전에도 불구하고, 인간 배아 줄기 세포 (HESCs)의 transfection은 변덕 과정 남아있다. 우리의 지식, 인간 유도 만능 줄기 세포를 (iPSCs) transfect하는 체계적이고 효율적인 방법은보고되지 않았습니다. 여기, 우리는 효율적으로 transfect과 인간 iPSCs를 nucleofect하는 강력한 프로토콜을 설명합니다.

비디오: Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection reagents has often resulted in poor cell viability and low transfection efficiency.  Other mechanical methods of introducing a gene of interest, such as a &#8220;gene gun&#8221; or microinjection, are also limited by poor cell viability and low numbers of transfected cells.  The strategy of using viral constructs to introduce an exogenous gene into primary cells has been constrained by both the amount of time and labor required to create viral vectors and potential safety concerns.  We describe here a step-by-step protocol for transfecting hNSPCs using Amaxa's Nucleofector device and technology with electrical current parameters and buffer solutions specifically optimized for transfecting neural stem cells.  Using this protocol, we have achieved initial transfection efficiencies of ~35% and ~70% after stable transfection.  The protocol entails combining a high number of hNSPCs with the DNA to be transfected in the appropriate buffer followed by electroporation with the Nucleofector device.

Introducing a gene of interest into a cell is a powerful method for elucidating its function in vivo. This protocol describes an efficient method of transfecting a culture of human neural stem/precursor cells (hNSPCs) using the Nucleofector electroporation apparatus made by Amaxa.

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection ...

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

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.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

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

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

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.

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Episomal Reprogramming of Dermal Fibroblasts to Generate Human-Induced Pluripotent Stem Cells (hiPSCs)

Episomal Reprogramming of Dermal Fibroblasts to Generate Human-Induced Pluripotent Stem Cells (HiPSCs)  

To begin, take a flask of cultured fibroblast cells, coat a 10-centimeter cell culture plate suitable for hiPSC culture with four milliliters of cold coating solution per plate, and incubate the plate for one hour at 37 degrees Celsius. Afterward, remove the media from the fibroblasts and wash them with four milliliters of pre-warmed PBS per flask. Aspirate the PBS and associate the fibroblasts with four milliliters of Trypsin-EDTA per flask by incubating for five to seven minutes at 37 degrees Celsius.Monitor cell detachment using a phase contrast microscope, and if needed, tap against the side of the flask to detach the cells from the plastic surface. Transfer the detached cells to a 50-milliliter conical tube and wash the empty flasks with six milliliters of pre-warmed fibroblast medium. Collect and pool the remaining cells in the conical tube.Then centrifuge the cells at 200 g for five minutes at 20 degrees Celsius. Aspirate the supernatant and re-suspend the cell pellet in three milliliters of fresh, pre-warmed PBS. Count the cells, transfer 1.5 times 10 to the sixth fibroblasts into a new conical tube and fill up with PBS to the five millimeter mark.Centrifuge the tube at 200 g for five minutes at 20 degrees Celsius. Discard the supernatant, re-suspend the cell pellet in five milliliters of electroporation medium, and centrifuge again. In the meantime, aspirate the coating solution from the coated 10-centimeter cell culture plate and add seven milliliters of pre-warmed fibroblast medium.At the end of the centrifugation, discard the supernatant and re-suspend the cell pellet in electroporation medium with a concentration of 1.5 times 10 to the sixth cells in 250 microliters. Transfer the 250 microliter cell suspension to an electroporation cuvette with a four millimeter gap distance. Next, prepare a plasma transfection mix by adding four micrograms of each plasmid to a total volume of 50 microliters of electroporation medium.Transfer the transfection mix to the cuvette, mix gently by flicking, and electroporate with one pulse at 280 volts. Finally, transfer 150 microliters of the electroporated fibroblasts to the prepared 10-centimeter cell culture plate. Agitate the plate thrice in all directions to distribute the cells, and incubate overnight.Fibroblasts present in a long, spindle-like phenotype, the size of 150 to 300 nanometers. After reprogramming, colonies with 50 micrometer small hiPSCs occur displaying distinct borders. The hiPSCs are distinguished by a high nuclei to body ratio and increased proliferation rate.