Name: generation of induced pluripotent stem cells by reprogramming mouse embryonic fibroblasts with a four transcription factor, doxycycline inducible lentiviral transduction system
Uploaded: 2009-11-13T20:00:00
Duration: 11 min 48 s
Description: Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral T

1. Viral Transduction of MEFs
 <ol>
<li>The day before beginning your experiment, coat a 15 cm cell culture dish with 0.2% gelatin sterile filtered in ddH2O. Incubate the plate overnight at 37&deg;C and 5% CO2.</li>
<li>Aspirate the liquid from the gelatin coated dish, and seed MEF cells (we used Nanog-GFP/rtTA MEF cells) at a density of 4x105 cells per dish. Incubate the cells in 30 ml of MEF growth medium (450ml DMEM supplemented with 50 ml FBS, 5 ml 100x non-essential amino acids, 5 ml penicillin/streptomycin, 5 ml 200 mM L-glutamine and 0.5 ml 55mM &beta;-mercaptoethanol) for two days at 37&deg;C and 5% CO2 until approximately 80% confluent.</li>
<li>Aspirate the medium and add 30 ml of MEF growth medium supplemented with concentrated lentivirus (4 x 100 &mu;l virus stock solution + 29.6 ml growth medium). Rock the dish gently to ensure even distribution of the medium. Incubate the cells overnight at 37&deg;C and 5% CO2.</li>
</ol>
 2. Doxycycline Induced Reprogramming
 <ol>
<li>20-24 hours post-transduction, trypsinize the transduced cells, centrifuge at 200 x g for 5 minutes and resuspend in ES/iPS growth medium (450 ml Knockout DMEM supplemented with 50ml ES cell-qualified calf serum, 5 ml penicillin/streptomycin, 5 ml 200 mM L-glutamine, 0.5 ml &beta;-mercaptoethanol and 25 &mu;l LIF) . Seed transduced MEF's at an appropriate concentration for the cell culture dish size (we used 2.5 x 105 cells per 10 cm dish for reprogramming efficiency experiments and colony isolation, and 2 x 104 cells per well in 4 well plates for ICC experiments). Incubate overnight at 37&deg;C and 5% CO2. Note: any remaining transduced cells can be frozen in liquid nitrogen for future analysis.</li>
<li>Aspirate the medium and replace with ES/iPS medium prepared fresh and supplemented with doxycycline to a final concentration of 2 &mu;g/ml (we also added medium without DOX as a negative control). Incubate at 37&deg;C and 5% CO2.</li>
</ol>
 3. Immunocytochemical Analysis of Transduction Efficiency
48 hours post-doxycycline induction, determine transduction efficiency by immunohistochemistry(ICC). Carry out ICC testing on cells replated in 4 well plates. All volumes listed in the following protocol should be adjusted according to the cell culture plate size.
<ol>
<li>Wash the cells gently once with PBS (without Mg2++ or Ca2+).</li>
<li>Fix the cells with 500 &mu;l of 0.5 ml 4% paraformaldehyde in PBS for 15 minutes at room temperature.</li>
<li>Wash the cells gently two times with PBS.</li>
<li>Permeabilize cells by adding 500 &mu;l of ice-cold 0.2% Tween&reg;-20 in PBS. Incubate for 10 minutes at room temperature.</li>
<li>Wash the cells gently two times with PBS.</li>
<li>Block non-specific binding with 200 &mu;l blocking buffer for one hour at room temperature.</li>
<li>Incubate the cells with 200 &mu;l of the specific primary antibody overnight at 4&deg;C (we used Oct4, Klf4, Sox2 and c-Myc).</li>
<li>Wash the cells gently two times with PBS.</li>
<li>Incubate the cells with 200 &mu;l of secondary antibody for 1 hour at room temperature, protecting the plates from light (we used Donkey anti-Rabbit Rhodamine conjugate, Donkey anti-Goat AlexaFluor&reg; 594 conjugate, Donkey anti-Mouse Rhodamine conjugate and Donkey anti-Rabbit Rhodamine conjugate).</li>
<li>Wash the cells gently two times with PBS.</li>
<li>Add DAPI (final concentration 2 &mu;g/ml in PBS) and incubate 10 minutes to visualize nuclei.</li>
<li>Wash the cells gently with PBS.</li>
<li>Add anti-fade Aqua-Mount before imaging using an inverted fluorescent microscope.</li>
</ol>
4. Isolating and expanding iPS Cell Colonies
<ol>
<li>After initiating the reprogramming process (as described in section 2), monitor the cultures and replace medium every 48 hours. We used doxycycline containing medium to culture the cells for the first twelve days and then subsequently removed doxycycline from the medium to ensure that the iPS colonies manually picked for expansion would be DOX independent.</li>
<li>Cells should be monitored daily for morphological changes indicative of the reprogramming process; or when using cells like Nanog-GFP/rtTA MEF, monitor morphology and GFP fluorescence to identify reprogrammed colonies. Each experiment will be different, but colonies are generally large enough for isolation between 16 and 22 days. Colonies identified during this time frame can then be manually isolated and trypsinized for expansion and analysis.</li>
<li>The day before isolating and trypsinizing the reprogrammed colonies, prepare a 24-well plate by seeding with gamma-irradiated feeder layer MEFs at a density of 5 x 104 cells per well. Incubate overnight at 37&deg;C and 5% CO2.</li>
<li>Manually pick each iPS colony and trypsinize to dissociate the cell aggregates. Re-plate the iPS cells in ES/iPS medium in individual wells of the 24-well plate pre-seeded with gamma-irradiated feeder layer MEFs. Wells should be seeded at a density of 2 x 105 cells/well. Incubate at 37&deg;C and 5% CO2. Change media every 24 hours.</li>
<li>Monitor iPS colonies daily for growth and GFP fluorescence. We incubate our cultures for 6 days before passaging.</li>
<li>Determine which wells of the 24-well plate are uniformly expressing GFP. Wells that have good GFP expression can be trypsinized and passaged 1:8 into 4-well plates that have been pre-seeded with gamma-irradiated feeder layer MEFs. These plates can be used for pluripotent marker analysis by ICC and AP staining.</li>
</ol>
5. Immunocytochemical Analysis for Pluripotency
Our ICC testing was carried out on cells expanded in 4 well plates. All volumes listed in the following protocol should be adjusted according to the cell culture plate size.
<ol>
 <li>Wash the cells gently twice with PBS (without Mg2++ or Ca2+).</li>
<li>Incubate in 0.5 ml of ice-cold 0.2 % Tween 20 in PBS per well for 10 minutes.</li>
<li>Wash the cells gently three times with PBS.</li>
<li>Block non-specific binding with 200 &mu;l blocking buffer for one hour at room temperature.</li>
<li>Incubate the cells with 200 &mu;l of the specific primary antibody overnight at 4&deg;C (we used SSEA-1, Nanog and Oct4).</li>
<li>Wash the cells gently two times with PBS.</li>
<li>Incubate the cells with 200 &mu;l of secondary antibody for 1 hour at room temperature, keeping away from light (we used Donkey anti-Mouse Rhodamine conjugate and Donkey anti-Rabbit Rhodamine conjugate).</li>
<li>Wash the cells gently two times with PBS.</li>
<li>Add DAPI (final concentration 2 &mu;g/ml in PBS) and incubate 10 minutes to visualize nuclei.</li>
<li>Wash the cells gently with PBS</li>
<li>Add anti-fade Aqua-Mount before imaging using an inverted fluorescent microscope.</li>
</ol>
6. Alkaline Phosphatase (AP) Staining of iPS Cell Colonies
<ol>
<li>iPS colonies from section 4 can also be analyzed for AP activity utilizing commercially available kits and following the manufacturer&#x2019;s protocol.</li>
</ol>
Part 7. Representative Results
The Stemgent DOX Inducible Mouse TF Lentivirus Set can be used to reprogram MEFs to iPS cells. After transduction of the MEFs, expression of transcription factors Oct4, Sox2, Klf4 and c-Myc can be detected in cells treated with doxycycline (DOX+), but little or no expression can be detected in untreated (DOX-) cells (figure 1). Morphological changes will progress over time (12 days of DOX treatment in this example) to generate larger, more ES cell-like colonies with defined colony edges and three dimensional growth (figure 2a). When DOX is removed, there is a noticeable reversion of cellular morphology for some ES cell-like colonies, however, many of the colonies maintained their iPS morphology (figure 2a). These iPS colonies, when picked and passaged, display typical pluripotency marker expression of Alkaline Phosphatase (AP), Nanog, Oct4 and SSEA-1 (figure 3). The type of MEF cells used in this experiment (Nanog-GFP/rtTA MEF cells) express GFP from the endogenous Nanog locus when reprogrammed to the pluripotency state. GFP expression can therefore be used as a preliminary indicator for successful reprogramming (figure 3).
<img src="/files/ftp_upload/1447/1447fig1.jpg" alt="Figure 1" /> Figure 1: Immunocytochemistry (ICC) analysis 48 hours post-DOX induction. The far left panel (-DOX) is a representative negative control for expression of the four transduction factors without DOX induction. Correctly expressed transcription factors were confirmed by corresponding antibodies (shown in red), stained with DAPI to visualize the nucleus.
<img src="/files/ftp_upload/1447/1447fig2.jpg" alt="Figure 2" /> Figure 2: Morphological conversion of Nanog-GFP/rtTA MEFs to the iPS cell state. A) 100x phase contrast imaging of cells demonstrating the compaction and conversion of MEFs into iPS colonies over time from day 3 (D3+Dox) to day 22 (D22). DOX was withdrawn on day 12. Upper left panel: 20x negative control image from &#x2013;DOX plate. B) 200x phase-contrast and GFP fluorescence images of highlighted day 18 post-DOX induction iPS colony. C) 200x phase-contrast and GFP fluorescence images of additional day 18 post-DOX induction iPS colony.
<img src="/files/ftp_upload/1447/1447fig3.jpg" alt="Figure 3" /> Figure 3: Analysis of iPS colonies. (A) Phase contrast microscopy and AP staining of an iPS colony (200x). (B) Pluripotency marker analysis: Left panel shows phase contrast overlay with GFP reprogramming reporter expression. GFP expression reflects the endogenous Nanog expression level. Middle panel shows ICC staining for pluripotency markers. Right panel shows DAPI staining to visualize the nuclei (100x).

<ol>
	<li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126, 663-676 (2006).</li><li>Aoi, T., Yae, K., Nakagawa, M., Ichisaka, T., Okita, K., Takahashi, K., Chiba, T., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+pluripotent+stem+cells+from+adult+mouse+liver+and+stomach+cells.">Generation of pluripotent stem cells from adult mouse liver and stomach cells.</a> Science. 321, 699-702 (2008).</li><li>Yu, J., Vodyanik, M. A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J. L., Tian, S., Nie, J., Jonsdottir, G. A., Ruotti, V., Stewart, R., Slukvin, I. I., 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=Induced+pluripotent+stem+cell+lines+derived+from+human+somatic+cells.">Induced pluripotent stem cell lines derived from human somatic cells.</a> Science. 318, 1917-1920 (2007).</li><li>Murry, C. E., Keller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+embryonic+stem+cells+to+clinically+relevant+populations:+lessons+from+embryonic+development.">Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development.</a> Cell. 132, 661-680 (2008).</li><li>Lerou, P. H., Daley, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+embryonic+stem+cells.">Therapeutic potential of embryonic stem cells.</a> Blood Rev. 19, 321-331 (2005).</li><li>Wernig, M., Meissner, A., Foreman, R., Brambrink, T., Ku, M., Hochedlinger, K., Bernstein, B. E., Jaenisch, R. <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+reprogramming+of+fibroblasts+into+a+pluripotent+ES-cell-like+state.">In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state.</a> Nature. 448, 318-324 (2007).</li></ol>

The author, Brad Hamilton, is employed by Stemgent that produces reagents and instruments used in this article.

These results demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set can be used to efficiently generate iPS colonies by inducing the ectopic expression of transduced transcription factors in mouse embryonic fibroblasts. When designing reprogramming experiments, several variables should be considered to optimize the efficiency of reprogramming. First, it is possible to modify the active virus-to-target cell ratio (i.e. M.O.I.) during the primary infection step to increase or decrease the transduction efficiency, thereby affecting the number of integrated viruses in the target cell population. Second, adjusting the length of time the cells are exposed to DOX can affect the efficiency of iPS cell colony generation. Third, the proliferative capacity of the target cells can impact reprogramming, as cells which are actively growing and dividing are more amenable to reprogramming. Lastly, when modifying the protocol for different cell numbers or different size tissue culture dishes, it is recommended that target cell numbers be adjusted proportionally to the surface area of the culture dish.

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generation of induced pluripotent stem cells by reprogramming mouse embryonic fibroblasts with a four transcription factor, doxycycline inducible lentiviral transduction system

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral T

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

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

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

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Research

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Biology

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

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 ...

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.

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 ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

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 ...