Name: feeder-free adaptation, culture and passaging of human ips cells using complete knockout serum replacement feeder-free medium
Uploaded: 2010-07-15T17:00:00.000+00:00
Duration: 8 min 29 s
Description: Watch this Scientific Journal Video about Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium at JoVE.com

Note: To maintain sterile culture conditions, all of the procedures in this protocol are carried out using sterile laboratory practices and conducted under a laminar flow hood.
Prior to starting, ensure that any media is equilibrated to 37&deg;C and appropriately gassed.
Preparing Geltrex-coated Culture Dishes
Note: see appendix for the use of CELLstart coated culture dishes
<ol>
 <li>Thaw one tube of Geltrex (1 mL) slowly at 2-8&deg;C and dilute 1:100 it in 99 mL of Knockout D-MEM/F-12. Mix the solution gently. 
 
 Note: Some iPS cell lines may require a different Geltrex dilution for optimal growth. See appendix for alternative dilutions.</li>
 <li>Cover the whole surface of each culture dish with the Geltrex solution (1 mL for a 35-mm dish, 1.5 mL for a 60-mm dish).</li>
 <li>Seal each dish with Parafilm to prevent drying and incubate the dishes for 1 hour at 37&deg;C. 
 
 Note: At this point you may store the Geltrex-coated culture dishes at 4&deg;C for up to 1 month. Seal each dish with Parafilm to prevent the Geltrex from drying out.</li>
 <li>Prior to using, transfer the Geltrex-coated dishes to a laminar flow hood and allow them to equilibrate to room temperature (about 1 hour).</li>
</ol>
Preparing Complete KnockOut SR Feeder-Free Medium
<ol>
 <li>To prepare 1 mL of 10 &mu;g/mL Basic FGF solution, aseptically mix the components listed below. Aliquot the solution and store at -20&deg;C for up to 6 months. 
 
 <table border="0">
 <tbody>
 <tr>
 <td>Basic FGF</td>
 <td>10 &mu;g</td>
 </tr>
 <tr>
 <td>D-PBS</td>
 <td>990 &mu;L</td>
 </tr>
 <tr>
 <td>10% BSA</td>
 <td>10 &mu;L</td>
 </tr>
 </tbody>
 </table>
 
 
 Note: BSA can be substituted with HSA or Knockout SR at the same concentration.</li>
 <li>To prepare 50 mL of 2 mg/mL Dispase solution, aseptically mix the components listed below. Filter sterilize the solution and store at 4&deg;C for up to 2 weeks. 
 
 <table border="0">
 <tbody>
 <tr>
 <td>Dispase</td>
 <td>100 mg</td>
 </tr>
 <tr>
 <td>D-PBS</td>
 <td>50 mL</td>
 </tr>
 </tbody>
 </table>
 </li>
 <li>To prepare 100 mL of complete KnockOut SR Feeder-Free (KSR-FF) medium aseptically combine the components listed in the table below. 
<table border="1">
 <tbody>
 <tr>
 <td>Component</td>
 <td>Stock Concentration</td>
 <td>Final Concentration</td>
 <td>Volume </td>
 </tr>
 <tr>
 <td>Knockout DMEM/F12 (Cat. no. 12660-012)</td>
 <td>-</td>
 <td>1X</td>
 <td>76.8 mL</td>
 </tr>
 <tr>
 <td>GlutaMAX -I (Cat. No. 35050-061)</td>
 <td>200 mM</td>
 <td>2 mM</td>
 <td>1 mL</td>
 </tr>
 <tr>
 <td>KnockOut SR (Cat. no. 10828-028)</td>
 <td>-</td>
 <td>20%</td>
 <td>20 mL</td>
 </tr>
 <tr>
 <td>KnockOut SR-GFC (Cat. no. A10580-01)</td>
 <td>50X</td>
 <td>1X</td>
 <td>2 mL</td>
 </tr>
 <tr>
 <td>bFGF (Cat. no. PHG0024)</td>
 <td>10 &mu;g/mL</td>
 <td>20 ng/mL</td>
 <td>200 &mu;L</td>
 </tr>
 </tbody>
</table>
You may store the KSR-FF medium at 2-8&deg;C for up to one week.</li>
<li>Just before pre-equilibrating the complete medium to temperature and gases, aseptically add the required volume of 2 mercaptoethanol (55 mM stock concentration) for a 0.1 mM final concentration. For example, to prepare 100 mL of KSR-FF medium add 182 &mu;L of 55 mM 2-mercaptoethanol (1:550 dilution) Alternatively, the 2-mercaptoethanol may be added to the 1X completed medium and stored at 2-8&deg;C for up to one week.</li>
</ol>
Adapting iPSCs to KSR-FF Medium
<ol>
 <li>Prior to starting, pre-equilibrate your Geltrex coated dishes to room temperature in the hood.</li>
 <li>Culture the iPSCs on MEF feeder cells until they are 70-80% confluent. Please refer to the GIBCO Mouse Embryonic Fibroblast handbook for MEF-based culture protocols.</li>
 <li>Pre-warm the required volume of Dispase in a 37 &deg;C water bath. Refer to Table 1 below for details on the volumes required. </li>
 <li>Pre-equilibrate the required volume of KSR-FF in a 37&deg;C water bath for15 min. Refer to Table 1 below for details on the volumes required. </li>
 <li>Aspirate the medium from the culture dishes, and add an appropriate amount of Dispase solution. Incubate the dishes at 37&deg;C for 3-5 minutes.</li>
 <li>Aspirate the Dispase solution from each culture vessel and wash off the MEF feeder cells gently with D-PBS (2 to 3 times).</li>
 <li>Add an appropriate amount of complete KnockOut SR medium to each culture vessel. Use a cell scraper or a 5 mL pipette to gently scrape the cells off the surface of culture vessel. 
 
 Note: see appendix for alternative adaptation protocols for hard to adapt iPS cell lines,</li>
 <li>Collect the cell suspension from each culture dish into separate 15 mL conical tubes. Rinse each culture vessel with an appropriate amount of complete KnockOut SR medium, and add the D-PBS rinse medium into the 15 mL conical tubes containing the cell suspension. Be cautious not to break the cell clumps into single cells</li>
 <li>Centrifuge the 15 mL conical tubes at 200 x g for 5 minutes to pellet the iPSCs.</li>
 <li>Aspirate the supernatant from the iPSC pellet. Resuspend the pellet in an appropriate amount of KSR-FF medium according to the split ratio (Table 1). Do not break the cell clumps to a smaller size, because the smaller clumps do not attach well to the surface. 
 
 Note: We recommend a split ratio of 1:2 for the first 3 passages after the iPSCs have been passaged directly from the iPSC MEF Culture Medium to KSR-FF medium. Normally, a split ratio between 1:3 and 1:5 is appropriate, but passaging at 1:2 ensures the higher density of cells needed when adapting into a feeder-free culture.</li>
 <li>Prior to plating the iPSCs on Geltrex coated dishes, aspirate residual Geltrex solution from the pre-coated dish, and slowly add an appropriate amount of cell suspension to each culture dish. Note: do not rinse dishes prior to plating.</li>
 <li>Move the culture dish back and forth and side to side several times to disperse the cells across the surface of the dish. Gently place the culture dish in a 37&deg;C incubator with a humidified atmosphere of 4 to 6% CO2 in air. Replace the spent medium with KSR-FF every day. </li>
</ol>
Passaging human iPS cells using KSR-FF
<ol start="13">
 <li>Observe the human iPSCs growing in complete KSR-FF under the microscope to confirm that the cells are 70-80% confluent and ready to be subcultured. Refer to Figure 1. 
 
 Note: If colonies become too dense or too large, increased differentiation occurs.</li>
 <li>Cut out and remove any differentiated iPSC colonies prior to passaging the culture. </li>
 <li>Pre-warm the required volume of Dispase in a 37 &deg;C water bath. Refer to Table 1 below for details on the volumes required. </li>
 <li>Pre-equilibrate the required volume of KSR-FF in a 37&deg;C water bath for15 min. Refer to Table 1 below for details on the volumes required. </li>
 <li>Aspirate the spent medium from the culture vessel using a pipette, and rinse the cells twice with D-PBS.</li>
 <li>Gently add pre-warmed Dispase solution to the culture vessel (e.g., 1 mL of Dispase solution per 60-mm culture dish). Swirl the culture vessel to coat the entire cell surface.</li>
 <li>Incubate the culture vessel at 37&deg;C for 3 minutes.</li>
 <li>Remove the vessel from the incubator, aspirate the Dispase solution, and gently wash the cells with D-PBS.</li>
 <li>Gently scrape the cells off the surface of the culture dish using a cell scraper, and transfer the cells to a sterile 15mL centrifuge tube.</li>
 <li>Rinse the culture dish twice with KSR-FF, gently "spraying off" any cells that have not detached. Pool the rinse medium with the cells in the 15mL tube.</li>
 <li>Centrifuge the tube at 200 x g for 5 minutes at room temperature to pellet the cells.</li>
 <li>Carefully aspirate the supernatant without disturbing the cell pellet and discard it.</li>
 <li>Gently flick the tube to fully dislodge the cell pellet from the tube bottom.</li>
 <li>Gently resuspend the cells in pre-equilibrated KSR-FF using a 5mL serological pipette. Do not triturate. Note: it is critical at the step to gently resuspend the cells without using force to avoid damage. </li>
 <li>Transfer the cells to a fresh 60-mm Geltrex-coated dish at the desired split ratio and move the culture dish back and forth and side to side several times to disperse the cells across its surface. </li>
 <li>Place the culture dish in a 37&deg;C incubator with a humidified atmosphere of 4 to 6% CO2 in air.</li>
 <li>The next day, gently replace the spent medium with KSR-FF to remove cell debris. Replace the spent medium everyday thereafter. Observe the iPS cells daily and passage them as needed (approximately every 4-5 days). Passaging is recommended when the cells reach 70-80% confluence. For iPS cell cryopreservation and thawing, refer to our protocol titled "Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium".</li>
</ol>
Expected Results
<img src="/files/ftp_upload/2236/2236fig1.jpg" alt="Figure 1" /> 
 Figure 1. The phase contrast image below shows iPSCs grown on Geltrex-coated culture dishes using complete KnockOut Serum Replacement Feeder-Free medium. The iPSCs exhibit morphology similar to hESCs, characterized by large nuclei and scant cytoplasm. (40x magnification)
<table border="1">
 <tbody>
 <tr>
 <td>Component</td>
 <td>35mm Dish</td>
 <td>60mm Dish</td>
 <td>100mm Dish</td>
 </tr>
 <tr valign="top">
 <td>Complete KnockOut SR medium</td>
 <td>2 mL</td>
 <td>4 mL</td>
 <td>10 mL</td>
 </tr>
 <tr valign="top">
 <td>Geltrex Solution</td>
 <td>1 mL</td>
 <td>1.5 mL</td>
 <td>4-5 mL</td>
 </tr>
 <tr valign="top">
 <td>Dispase</td>
 <td>0.5 mL</td>
 <td>1 mL</td>
 <td>3-4 mL</td>
 </tr>
 <tr valign="top">
 <td>D-PBS for rinsing</td>
 <td>2 mL</td>
 <td>4 mL</td>
 <td>10 mL</td>
 </tr>
 </tbody>
</table>
Table 1. Recommended Volumes
<a href="https://www.jove.com/files/ftp_upload/2236/2236appendix.pdf">Please click here to see the appendix</a>.

<ol>
	<li>Takahashi, K. <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>Takahashi, K. <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+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131, 861-872 (2007).</li><li>Yu, J. <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>Maherali, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+and+techniques+for+the+generation+of+induced+pluripotent+stem+cells.">Guidelines and techniques for the generation of induced pluripotent stem cells.</a> Cell Stem Cell. 3, 595-605 (2008).</li><li>Li, W. <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+rat+and+human+induced+pluripotent+stem+cells+by+combining+genetic+reprogramming+and+chemical+inhibitors.">Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors.</a> Cell Stem Cell. 4, 16-19 (2009).</li><li>Liao, J. <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+induced+pluripotent+stem+cell+lines+from+adult+rat+cells.">Generation of induced pluripotent stem cell lines from adult rat cells.</a> Cell Stem Cell. 4, 11-15 (2009).</li><li>Dimos, J. T. <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+cells+generated+from+patients+with+ALS+can+be+differentiated+into+motor+neurons.">Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons.</a> Science. 321, 1218-1221 (2008).</li><li>Aasen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+rapid+generation+of+induced+pluripotent+stem+cells+from+human+keratinocytes.">Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes.</a> Nat Biotechnli. 26, 1276-1284 (2008).</li><li>Park, I. H. <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+human-induced+pluripotent+stem+cells.">Generation of human-induced pluripotent stem cells.</a> Nat Protoc. 3, 1180-1186 (2008).</li></ol>

The authors of this article are employed by Life Technologies that produces reagents and instruments used in this article.

<table><tbody><tr><td>Knockout DMEM/F12</td><td></td><td>Cat. no. 12660-012</td><td>Note: see appendix for the use of alternative DMEM products</td></tr><tr><td>GlutaMAX-I</td><td></td><td>Cat. No. 35050-061</td><td></td></tr><tr><td>KnockOut Serum Replacement</td><td></td><td>KnockOut SR, Cat. no. 10828-028</td><td></td></tr><tr><td>KnockOut Serum Replacement Growth Factor Cocktail</td><td></td><td>KnockOut SR-GFC, Cat. no. A10580-01</td><td></td></tr><tr><td>FGF-basic, human recombinant protein, 10 &amp;mu;g</td><td></td><td>bFGF, Cat. no. PHG0024</td><td>Note: see appendix for alternative bFGF pack sizes.</td></tr><tr><td>2-Mercapt&amp;#8211;thanol</td><td></td><td>Cat. no. 21985-023</td><td></td></tr><tr><td>Dispase</td><td></td><td>Cat. no. 17105-041</td><td>Note: see appendix for alternative passaging methods.</td></tr><tr><td>Geltrex hESC-Qualified Reduced Growth Factor Basement Matrix, 1 mL</td><td></td><td>Cat. no. A10480-01</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline (D-PBS) without calcium and magnesium </td><td></td><td>Cat. no. 14190-144</td><td></td></tr><tr><td>Sterile Tissue Culture Hood</td><td></td><td></td><td></td></tr><tr><td>Incubator set at 37&amp;#x00B0;C</td><td></td><td></td><td></td></tr><tr><td>Pipette-Aid</td><td></td><td></td><td></td></tr><tr><td>Water Bath set at 37&amp;#x00B0;C</td><td></td><td></td><td></td></tr><tr><td>Sterile serological pipettes (5 mL, 10 mL)</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td></td><td></td><td></td></tr><tr><td>15 mL centrifuge tubes</td><td></td><td></td><td></td></tr><tr><td>60mm Tissue Culture treated dishes</td><td></td><td></td><td></td></tr></tbody></table>

feeder-free adaptation, culture and passaging of human ips cells using complete knockout serum replacement feeder-free medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS cell cultures to feeder-free while still maintaining your use of Knockout SR.

Watch this Scientific Journal Video about Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium at JoVE.com

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem ...

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Biology

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Video: Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of interest. Nevertheless, an ideal humanized ex vivo feeder-free propagation method for hPSCs has not been developed; currently, additional exogenous substrates including basic fibroblast growth factor (bFGF), a master hPSC-sustaining factor, is added to all of culture media and synthetic substrata such as Matrigel or laminin are used in all feeder-free cultures. Recently, our group developed a simple and efficient protocol for the propagation of hPSCs using only conditioned media derived from the human placenta on a gelatin-coated dish without additional exogenous supplementation or synthetic substrata specific to hPSCs. This protocol has not been reported previously and might enable researchers to propagate hPSCs efficiently in humanized culture conditions. Additionally, this model obviates hPSC contamination risks by animal products such as viruses or unknown proteins. Furthermore, this system facilitates easy mass production of hPSCs using the gelatin coating, which is simple to handle, dramatically decreases the overall costs of ex vivo hPSC maintenance.

This protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

The propagation of human pluripotent stem cells (hPSCs) in conditioned medium derived from human cells in feeder-free culture conditions has been of ...

Developmental Biology

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

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

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

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

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Neuroscience

Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells

Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson&#8217;s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.

We, based on knowledge from developmental biology and published research, developed an optimized protocol to efficiently generate A9 midbrain dopaminergic neurons from both human embryonic stem cells and human induced pluripotent stem cells, which would be useful for disease modeling and cell replacement therapy for Parkinson&#8217;s disease.

Dopaminergic (DA) neurons in the substantia nigra pars compacta  (also known as A9 DA neurons) are the specific cell type that is lost in ...

Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

The discovery in 2006 that human and mouse fibroblasts could be reprogrammed to generate iPS cells 1-3 with qualities remarkably similar to embryonic stem cells has created a valuable new source of pluripotent cells for drug discovery, cell therapy, and basic research.
GIBCO media and reagents have been at the forefront of pluripotent stem cell research for years. Knockout DMEM supplemented with Knockout Serum Replacement is the media of choice for embryonic stem cell growth and now iPS cell culture 3-9. This gold standard media system can now be used for feeder-free culture with the addition of Knockout SR Growth Factor Cocktail.
Traditional human ES and iPS cell culture methods require the use of mouse or human fibroblast feeder layers, or feeder-conditioned medium. These culture methods are labor-intensive, hard to scale and it is difficult to maintain hiPS cells undifferentiated due to the undefined conditions. Invitrogen has developed Knockout SR Growth Factor Cocktail to allow you to easily transition your hiPS and hES cell cultures to feeder-free while still maintaining your use of Knockout SR.

This protocol describes the detailed procedure for cryopreserving human iPS cells in KnockOut SR cryopreservation medium and recovering these cells in complete KnockOut SR Feeder Free (KSR-FF) medium or feeder-based KnockOut SR medium.

Induced Pluripotent Stem Cell Differentiation Using an Automated Culture System

An Automated Culture System for Maintaining and Differentiating Human-Induced Pluripotent Stem Cells

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, including the elucidation of rare disease pathologies, the development of new medicines, and regenerative medicine aiming to restore damaged organs. Despite this, the social implementation of hiPSCs is still limited. This is partly because of the difficulty of reproducing differentiation in culture, even with advanced knowledge and sophisticated technical skills, due to the high sensitivity of iPSCs to minute environmental changes. The application of an automated culture system can solve this issue. Experiments with high reproducibility independent of a researcher's skill can be expected according to a shared procedure across various institutes. Although several automated culture systems that can maintain iPSC cultures and induce differentiation have been developed previously, these systems are heavy, large, and costly because they make use of humanized, multi-articulated robotic arms. To improve on the above issues, we developed a new system using a simple x-y-z axis slide rail system, allowing it to be more compact, lighter, and cheaper. Furthermore, the user can easily modify parameters in the new system to develop new handling tasks. Once a task is established, all the user needs to do is prepare the iPSC, supply the reagents and consumables needed for the desired task in advance, select the task number, and specify the time. We confirmed that the system could maintain iPSCs in an undifferentiated state through several passages without feeder cells and differentiate into various cell types, including cardiomyocytes, hepatocytes, neural progenitors, and keratinocytes. The system will enable highly reproducible experiments across institutions without the need for skilled researchers and will facilitate the social implementation of hiPSCs in a wider range of research fields by diminishing the obstacles for new entries.

Author Spotlight: Automating iPSC Culture for Enhanced Reproducibility 

Here, we present a protocol for an automated cell culture system. This automated culture system reduces labor and benefits the users, including researchers unfamiliar with handling induced pluripotent stem (iPS) cells, from the maintenance of iPS cells to differentiation into various types of cells.

Human induced pluripotent stem cells (hiPSCs) with infinite self-proliferating ability have been expected to have applications in numerous fields, ...

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 ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Advances in Human Induced Pluripotent Stem Cell-Derived Chimeric Antigen Receptor-Expressing Natural Killer Cells

Natural killer (NK) cells are innate immune cells that play a crucial role in the body's defense against tumors and viral infections. The generation of human induced pluripotent stem cell (iPSC)-derived chimeric antigen receptor (CAR) expressing NK cells has emerged as a promising avenue for "off the shelf" cancer immunotherapy. Here, we utilized an NK cell-optimized CAR construct that includes the transmembrane domain of NKG2D, the 2B4 co-stimulatory domain, and the CD3&#950; signaling domain, which has been demonstrated to stimulate robust antigen-specific NK cell-mediated antitumor activity. The use of iPSCs for CAR NK cell generation offers several advantages, including homogenous CAR expression, scalability, reproducibility, and the potential for clinical application. This detailed step-by-step protocol from cell engineering to differentiation enables the generation of NK cell-optimized iPSC-derived CAR-expressing NK cells, providing a standardized and targeted cancer immunotherapy with improved antitumor activity and highlighting their potential as a promising treatment option for various malignancies.

Here, we present a method to differentiate and expand human iPSC-derived chimeric antigen receptor (CAR)-expressing natural killer cells with improved killing against various malignancies. This protocol demonstrates the differentiation and expansion of natural killer (NK) optimized iPSC-derived CAR-NK cells and the measurement of antitumor activity against various tumor cell lines.

Natural killer (NK) cells are innate immune cells that play a crucial role in the body's defense against tumors and viral infections. The generation of ...