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) 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>
Preparing Freezing / Cryopreservation Medium
The cryopreservation medium consists of two media; Freezing Medium A and Freezing Medium B. They are added to the cells at different times and must remain separated. They are each 50% of the total volume of Cryopreservation Medium needed. The total volume of Cryopreservation Medium needed is 1 mL for each vial to be frozen. A 90% confluent 60mm dish of hESC can be used to bank 2 vials of hESC in cryopreservation media.
Prepare enough volume of each Freezing Medium with an extra 2-5mL to ensure overage.
<table border="1">
 <tbody>
 <tr>
 <td>Freezing Medium A (50% of final volume):</td>
 <td>50% DMEM/F12</td>
 <td>50% KSR</td>
 </tr>
 <tr>
 <td>Freezing Medium B (50% of final volume):</td>
 <td>80% DMEM/F12</td>
 <td>20% DMSO</td>
 </tr>
 </tbody>
</table>
Example:
If you are freezing 20 vials of cells, you will need 20mL of Cryopreservation Medium. Add 4mL for overage for a total of 24mL Cryopreservation Medium. That means you will need 12mL of Freezing Medium A (50% of 24mL) and 12mL of Freezing Medium B (50% of 24mL).
<table border="1">
 <tbody>
 <tr>
 <td>Freezing Medium A (12 mL):</td>
 <td>6 mL DMEM/F12</td>
 <td>6 mL KSR</td>
 </tr>
 <tr>
 <td>Freezing Medium B (12 mL):</td>
 <td>9.6 mL DMEM/F12</td>
 <td>2.4 mL DMSO</td>
 </tr>
 </tbody>
</table>
The final composition of the Cryopreservation Medium is 65% DMEM/F12, 25% KSR, and 10% DMSO.
Cryopreserving iPSCs
<ol>
 <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 1below 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>Prepare a sufficient quantity of cryovials. Once the cells are in contact with DMSO, they should be aliquoted and frozen within 2-3 minutes.</li>
 <li>Gently flick the tube, to fully dislodge the cell pellet from the tube bottom and re-suspend the cells in Freezing Medium A. [by gently pipetting up and down using a 5-mL serological pipette]. Following uniform suspension of clumps, add equal volume of Freezing Medium B to it, in a drop wise manner, while gently swirling the cell suspension to mix. 
 
 Note: At this point, cells are in contact with DMSO, and work must be performed efficiently. Once cells are in contact with DMSO, they should be aliquoted and frozen within 2-3 minutes.</li>
 <li>Aliquot 1 ml of the cell suspension into each cryovial.</li>
 <li>Quickly place the vials in a Mr. Frosty [to rapidly freeze] and transfer it to -80&deg;C overnight.</li>
 <li>After overnight storage at -80&deg;C, transfer the cells to a liquid nitrogen tank for long term storage.</li>
</ol>
iPSC vial thaw and cell recovery
Note: KSR-FF may be replaced with KSR-containing MEF-conditioned medium, or KSR medium for use with feeders. See appendix for instructions for media preparation. 
Pre-warm 10mL of KSR-FF medium in a 50 mL tube.
<ol>
 <li>Equilibrate the appropriate quantity of Geltrex-coated dishes to room temperature and aspirate any liquid from the dishes just before plating your cells.</li>
 <li>Carefully remove the hESC (or iPSC) vial from Liquid Nitrogen Storage Tank.</li>
 <li>Rapidly thaw frozen vial of cells in a 37&deg;C water bath, until just a small frozen chunk remains in the vial.</li>
 <li>Spray vial with 70% isopropanol to decontaminate it and transfer it to the sterile tissue culture hood.</li>
 <li>Aseptically transfer the entire contents of the vial into a 15 mL conical tube using a 5 mL pipette.</li>
 <li>Rinse the vial with 1 mL of KSR-FF and add this to the 15 mL centrifuge tube.</li>
 <li>Slowly, add 4 mL of KSR-FF drop-wise to cells in the 15mL conical tube. While adding the medium, gently move the tube back and forth to mix the cells. (This reduces osmotic shock to the cells).</li>
 <li>Centrifuge the 15 mL tube with the cell suspension at 1000rpm (200 x g) for 2 minutes at room temperature.</li>
 <li>Carefully aspirate and discard the supernatant without disturbing the cell pellet.</li>
 <li>Re-suspend the cell pellet in pre-warmed KSR-FF (e.g. 4mL/60 mm dish) using a 5 mL pipette and gently pipette the cells up and down until cell pellet is fully dispersed. A viability of greater than 70% is expected, however if the viability is less than 70%, it may take longer for the cells to reach confluence.</li>
 <li>Using the same pipette, transfer the cell suspension drop-wise to Geltrex-coated culture dish pre-equilibrated to room temperature.</li>
 <li>Place the culture dish in a 37&deg;C incubator, with a humidified atmosphere of 4 to 6% CO2 in air. Carefully swirl vessel in a north to south, east to west pattern to evenly distribute the cells.</li>
 <li>Gently fluid-change the culture dish 24 hrs post-thaw and daily thereafter to remove cell debris and to provide fresh nutrients until the dish is approximately 70-80% confluent. For continued culture and passaging of your iPS cells, refer to our protocol titled "Feeder-Free Culture and Passaging of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium"</li>
</ol>
Expected Results
The cells should be roughly 70-80% confluent prior to cryopreservation. Dispase digestion results in curling up and folding of the colonies along the colony margins. After harvesting the cells they are frozen as small clumps in cryopreservation media. Once the vial is thawed, cells recover and attach to the pre-coated dish as small clumps. They grow rapidly leading to a confluent dish in 5-6 days.
Table 1 - Recommended Volumes
<table border="1">
 <tbody>
 <tr>
 <td>Component</td>
 <td>35mm Dish</td>
 <td>60mm Dish</td>
 <td>100mm Dish</td>
 </tr>
 <tr>
 <td>Complete KnockOut SR medium</td>
 <td>2 mL</td>
 <td>4 mL</td>
 <td>10 mL</td>
 </tr>
 <tr>
 <td>Geltrex Solution</td>
 <td>1 mL</td>
 <td>1.5 mL</td>
 <td>4-5 mL</td>
 </tr>
 <tr>
 <td>Dispase</td>
 <td>0.5 mL</td>
 <td>1 mL</td>
 <td>3-4 mL</td>
 </tr>
 <tr>
 <td>D-PBS for rinsing</td>
 <td>2 mL</td>
 <td>4 mL</td>
 <td>10 mL</td>
 </tr>
 </tbody>
</table>
Please <a href="https://www.jove.com/files/ftp_upload/2237/2237_appendix.pdf">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.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Knockout DMEM/F12</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>12660-012</td>
<td>Note: see appendix for the use of alternative DMEM products GlutaMAX-I (Cat. No. 35050-061)</td>
</tr>
<tr>
<td>KnockOut Serum Replacement</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>10828-028</td>
<td>&nbsp;</td>
<tr>
<td>KnockOut Serum Replacement Growth Factor Cocktail</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>A10580-01</td>
<td>&nbsp;</td>
<tr>
<td>FGF-basic, human recombinant protein, 10 &mu;g</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>PHG0024</td>
<td>Note: see appendix for alternative bFGF pack sizes.</td>
</tr>
<tr>
<td>2-Mercaptoethanol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>21985-023</td>
<td>&nbsp;</td>
<tr>
<td>Dispase</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>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>&nbsp;</td>
<td>&nbsp;</td>
<td>A10480-01</td>
<td>&nbsp;</td>
<tr>
<td>Dulbecco&#x2019;s Phosphate Buffered Saline (D-PBS) without calcium and magnesium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>14190-144</td>
<td>&nbsp;</td>
<tr>
<td>DMSO, 500mL</td>
<td>&nbsp;</td>
<td>JT Baker</td>
<td>JT9224-1</td>
<td>&nbsp;</td>
<tr>
<td>Sterile Tissue Culture Hood</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Incubator set at 37&deg;C</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pipette-Aid</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Water Bath set at 37&deg;C</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sterile serological pipettes (5 mL, 10 mL)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>15 mL centrifuge tubes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>60 mm Tissue Culture treated dishes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Liquid nitrogen storage tank</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isopropanol freezing containers</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1.5 mL Cryovials</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium at JoVE.com

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

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.

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

Predicting the Effectiveness of Population Replacement Strategy Using Mathematical Modeling

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy.  Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies.  The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy. Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies. The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.

Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy.  ...

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006; Wernig, et al., 2007; Okita, et al., 2007; Maherali, et al., 2007). We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors (Brambrink et al., 2008). Using these inducible constructs, we can derive induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). In this video, we demonstrate the procedure for the generation of inducible lentiviruses that express the four transcription factors and show how to infect MEFs with these viruses in order to produce iPS cells. By using inducible lentiviruses, the expression of the four factors in controlled by the addition of doxycyline to the culture medium. The advantage of this system over the traditional retroviral infection is the ability to turn the genes on and off so that the kinetics of reprogramming and gene expression requirements can be analyzed in detail.

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

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.

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.

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 ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 and minimum levels of 0.03 ng ml&#8722;1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

The proposed work assesses the diagnostic potential of direct and amplified surface plasmon resonance imaging (SPRi) assays, particularly for the detection of recombinant human growth hormone in spiked human serum, by comparing SPRi results directly with commercially available enzyme-linked immunosorbent assay (ELISA) kit.

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...