This work was supported by SSF (B13-0074) and VINNOVA (2016-04134) funding agents.

The collection of patient material and derivation of hiPS cells is approved by the Ethics Review Board, Stockholm, March 28, 2012, Registration number: 2012/208-31/3. Cell culture steps should be performed in biosafety cabinets unless otherwise mentioned. Always practice sterile handling techniques when working with cells. Allow media, plates and reagents to reach room temperature before starting. Incubate cells at 37 &#7506;C, 5% CO2 in high humidity.
1. Isolation of Human Fibroblast...

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B., Okamoto, S., Xiang, P., Takahashi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration-free+induced+pluripotent+stem+cells+derived+from+retinitis+pigmentosa+patient+for+disease+modeling.">Integration-free induced pluripotent stem cells derived from retinitis pigmentosa patient for disease modeling.</a> Stem Cells Transl Med. 1 (6), 503-509 (2012).</li><li>Zhou, H., Ding, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+induced+pluripotent+stem+cell+technology.">Evolution of induced pluripotent stem cell technology.</a> Curr Opin Hematol. 17 (4), 276-280 (2010).</li><li>Thomson, J. 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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeder-independent+culture+of+human+embryonic+stem+cells.">Feeder-independent culture of human embryonic stem cells.</a> Nat Methods. 3 (8), 637-646 (2006).</li><li>Rodin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+self-renewal+of+human+pluripotent+stem+cells+on+human+recombinant+laminin-511.">Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511.</a> Nat Biotech. 28 (6), 611-615 (2010).</li><li>Chen, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+defined+conditions+for+human+iPSC+derivation+and+culture.">Chemically defined conditions for human iPSC derivation and culture.</a> Nat Meth. 8 (5), 424-429 (2011).</li><li>Rodin, S., Antonsson, L., Hovatta, O., Tryggvason, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monolayer+culturing+and+cloning+of+human+pluripotent+stem+cells+on+laminin-521-based+matrices+under+xeno-free+and+chemically+defined+conditions.">Monolayer culturing and cloning of human pluripotent stem cells on laminin-521-based matrices under xeno-free and chemically defined conditions.</a> Nat Protoc. 9 (10), 2354-2368 (2014).</li><li>Rodin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+culturing+of+human+embryonic+stem+cells+on+laminin-521/E-cadherin+matrix+in+defined+and+xeno-free+environment.">Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment.</a> Nat Commun. 5, 3195 (2014).</li><li>Fusaki, N., Ban, H., Nishiyama, A., Saeki, K., Hasegawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+induction+of+transgene-free+human+pluripotent+stem+cells+using+a+vector+based+on+Sendai+virus,+an+RNA+virus+that+does+not+integrate+into+the+host+genome.">Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome.</a> Proc Jpn Acad Ser B Phys Biol Sci. 85 (8), 348-362 (2009).</li><li>Warren, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Efficient+Reprogramming+to+Pluripotency+and+Directed+Differentiation+of+Human+Cells+with+Synthetic+Modified+mRNA.">Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA.</a> Cell Stem Cell. 7 (5), 618-630 (2010).</li><li>Unger, C., Skottman, H., Blomberg, P., Dilber, M. 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Biosafety in Microbiological and Biomedical Laboratories Available from: <a target="_blank" href="https://www.cdc.gov/biosafety/publications/bmbl5/bmbl.pdf">https://www.cdc.gov/biosafety/publications/bmbl5/bmbl.pdf</a> (2009)</li><li>Itskovitz-Eldor, J., et al. <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+human+embryonic+stem+cells+into+embryoid+bodies+compromising+the+three+embryonic+germ+layers.">Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers.</a> Mol Med. 6 (2), 88-95 (2000).</li><li>Chen, K. G., Mallon, B. S., McKay, R. D., Robey, P. 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The authors have no conflicts of interests to report.

The expected result of this protocol is the successful generation of several clonally derived hiPS cell lines. Importantly, the method for the maintenance of and expansion of established hiPS cells here described is reliable and can be performed with little prior experience of stem cell culture. Enzymatic single cell passaging with ROCKi together with the LN-521 matrix is known to maintain cells as karyotypically normal, pluripotent and readily able to differentiate while avoiding induced heterogeneity that colony based ...

Since the first derivation of hiPS cell lines by Takahashi et al.1,2, hiPS cells have provided a useful tool for disease modeling, drug discovery and as source material for generating cell therapies in regenerative medicine3. hiPS cell culture has long been dependent on co-culture with fibroblast feeder cells4,5 or on Matrigel6 and with media formulations containing fetal bovine serum (FBS). Batch-to-batch variances are a common consequence of the undefined nature of these culture conditions, resulting in unpredictable variations, which is a major contributor to the unreliability of these protocols7. The development of defined medium such as Essential 8 (E8)8 and defined cell culture matrices for instance LN-5219, allows for the establishment of highly reproducible protocols and aid in the robust generation and maintenance of homogenous hiPS cells7,8,9,10.
Development of integration free reprogramming techniques have been a leap forward. Originally, reprogramming depended on retroviral vectors which randomly integrated into the genome with disruptive effects on genomic integrity11. Advances in reprogramming methodologies includes the development of RNA based vectors. RNA vectors have an advantage over the DNA based reprogramming method as unintended integration through genomic recombination is not possible12. SeV vectors provide high and transient expression of exogenous factors through single-stranded RNA without a DNA-phase11. The reprogramming vectors delivered by the SeV are diluted throughout cell expansion and eventually shed from culture providing a foot-print free way of reprogramming. Thereafter, maintenance of pluripotency is dependent on endogenous expression of the pluripotency genes2.
As pioneering hiPS cell based therapies are beginning to move into clinical trials, the demands for standardized batches, reproducibility, and safety are essential issues to address13. Therefore, products of animal origin should be avoided. For instance, the use of xenogeneic products has been associated with risk of nonhuman pathogen contamination. Also, cells cultured in the presence of animal derived culture components have been shown to incorporate nonhuman siliac acids into cell membranes which threatens to render derived cells immunogenic14. Hence, the need to eliminate xenogeneic products is necessary to any future clinical pursuits. This protocol applies xeno-free and defined culture in the maintenance of hiPS cells moving cells closer to clinical compliance.
This protocol describes a consistent, highly reproducible and easy-to-use method that generates standardized hiPS cells from fibroblasts. It also offers a user-friendly culture system for the maintenance of established hiPS cells. This protocol has been used to derive more than 300 hiPS cell lines in the Swedish national human iPS Core facility at Karolinska Institutet of which some lines have previously been described15,16.

<table border="0" cellpadding="0" cellspacing="0" width="711">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Dispase</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:119px;">171105-041</td>
			<td style="width:223px;">Biopsy digestion</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase</td>
			<td>Sigma</td>
			<td>C0130</td>
			<td>Biopsy digestion</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gelatin</td>
			<td>Sigma</td>
			<td>G1393</td>
			<td>Fibroblast matrix</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IMDM</td>
			<td>Life Technologies</td>
			<td>21980002-032</td>
			<td>Fibroblast medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS</td>
			<td>Invitrogen</td>
			<td>10270106</td>
			<td>Fibroblast medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PenicillinStreptomycin</td>
			<td>Life Technologies</td>
			<td>15070-063</td>
			<td>Antibiotic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Essential 8 media</td>
			<td>ThermFisher Scientific</td>
			<td>A1517001</td>
			<td>iPS cell culture media</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LN-521</td>
			<td>Biolamina</td>
			<td>LN521-03</td>
			<td>iPS cell culture matrix</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TrypLE select 1X</td>
			<td>ThermoFisher Scientific</td>
			<td>12563011</td>
			<td>Dissociation reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rho-kinase inhibitor Y27632</td>
			<td>Millipore</td>
			<td>SCM075</td>
			<td>Rho-kinase inhibitor (ROCKi)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CytoTune &#8211; iPS 2.0 reprogramming kit</td>
			<td>Life Technologies</td>
			<td>A1377801</td>
			<td>Sendai virus reprogramming vector</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">35 mm tissue culture dish</td>
			<td>Sarstedt</td>
			<td>83.3900.300</td>
			<td>Cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">60 mm tissue culture dishes</td>
			<td>Sarstedt</td>
			<td>83.3901.300</td>
			<td>Cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">24 well tissue culture plates</td>
			<td>Sarstedt</td>
			<td>83.3922.300</td>
			<td>Cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T25 tissue culture flasks</td>
			<td>VWR</td>
			<td>734-2311</td>
			<td>Cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 ml tubes</td>
			<td>Corning</td>
			<td>430791</td>
			<td>Centrifuge tubes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5 ml tube</td>
			<td>Eppendorf</td>
			<td>0030123.301</td>
			<td>1.5 ml tube</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CoolCell cell LX</td>
			<td>Biocision</td>
			<td>BCS-405</td>
			<td>Freezing container</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMSO</td>
			<td>Sigma</td>
			<td>D2650-100ml</td>
			<td>Fibroblast freezing</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;">Cryovials 1.8 ml</td>
			<td>VWR</td>
			<td>479-6847</td>
			<td>Cryovials</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PSC Cryopreservation Kit</td>
			<td>Gibco</td>
			<td>A2644601</td>
			<td>PSC CryomediumRevitaCell supplement</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA (0.05%)</td>
			<td>ThermoFisherScientific</td>
			<td>25300054</td>
			<td>Fibroblast dissociation enzyme</td>
		</tr>
		<tr height="20">
			<td height="40" style="height:40px;">DMEM/F12</td>
			<td>Life Technologies</td>
			<td>31331-028</td>
			<td>Digestive enzymes dilutant</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DPBS</td>
			<td>Life Technologies</td>
			<td>14190-094</td>
			<td>PBS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS</td>
			<td>ThermoFIsher Scientific</td>
			<td>14025-050</td>
			<td>Biopsy preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Haemocytometer</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">BRAND, 718920</td>
			<td>Cell counting</td>
		</tr>
	</tbody>
</table>

integration free derivation of human induced pluripotent stem cells using laminin 521 matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

From biopsy to hiPS cells
The entire process from biopsy to established hiPS cells, clear of reprogramming vector and ready for characterization, takes approximately 16 weeks (Figure 1). A more detailed timeline is specified in Figure 2A. Approximately 4 weeks is needed to establish and expand fibroblast cultures. The first hiPS cell colonies started to emerge about three-...

Watch this Scientific Journal Video about Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix at JoVE.com

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

The overall goal of this protocol is to derive homogenous human induced pluripotent stem cells. This method can be used to derive human induced pluripotent stem cells for many purposes including disease modeling, drug screening, or to derive cells for preclinical cell therapy trials. The advantage of this protocol is that it's robust and generates homogenous cells that are maintained in a user-friendly, Xeno-free, and fully chemically defined culture system.Since human iPS cells recapitulate the genetic background of the donor tissue and can potentially be differentiated into any disease-relevant cell type, they provide excellent tools for thesis modeling. The biopsy dissection and colony picking are intricate procedures. Seeing them performed with experienced hands assists in the understanding of the procedures and makes it easier to repeat.Demonstrating the procedure today will be Harriet RonnHolm and Kelly Day, two highly experienced laboratory technicians from the iPS Core Facility at Karolinska Institutet. Skin biopsy samples can be stored in PBS plus 1%penicillin streptomycin for up to 48 hours at four degrees Celsius, but should be processed as fast as possible after being obtained. Transfer the biopsy to a 35 millimeter dish.Submerge the skin biopsy in a 35 millimeter dish with two milliliters of 70%ethanol for 30 seconds. Add one milliliter of sterile Hank's Balanced Salt Solution supplemented with 1%penicillin streptomycin to a new 35 millimeter dish and transfer the biopsy to the new dish. Transfer the biopsy to a four 35 millimeter dish filled with one milliliter of freshly-prepared 0.1%dispase solution.Using sterile surgical scalpels and forceps, cut the skin biopsy into one to two cubic millimeter pieces. Transfer the biopsy pieces with the dispase solution into a 15 milliliter tube and add an additional two milliliters of dispase. Rinse the 35 millimeter dish with one milliliter of dispase solution and transfer the rinsed solution to the 15 milliliter tube.Incubate the biopsy at four degrees Celsius overnight. On the following morning, add four milliliters of 0.1%collagenase I to the tube with the biopsy pieces and incubate at 37 degrees Celsius for four hours. Thirty minutes prior to seeding the cells, coat one well in a six-well tissue culture plate with one milliliter of 0.1%gelatin in DPBS.Incubate at room temperature for 30 minutes. After four hours of incubation with collagenase, centrifuge the digested cells at 300 times g for three minutes at room temperature. Aspirate the supernatant and resuspend the digest in two milliliters of fibroblast medium.Remove the gelatin solution from the previously prepared six-well tissue culture plate. Transfer the cell suspension including any remaining tissue pieces to the six-well plate. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide in high humidity.Fibroblasts are ready to be passaged to a T25 tissue culture flask when 80%confluent. Fibroblasts at passage two to four are optimal for reprogramming since reprogramming efficiency is generally higher for lower passage fibroblasts. Aspirate the cell culture medium from the flask and wash the cells with one milliliter of DPBS.Aspirate the DPBS and add one milliliter of 0.5%Trypsin EDTA. Incubate at 37 degrees Celsius for five minutes or until cells are detached. Add two milliliters of fibroblast medium, resuspend the cells, and transfer the cell suspension to a 15 milliliter tube.Centrifuge at 300 times g for three minutes at room temperature. Aspirate the supernatant and resuspend the pellet in two milliliters of fibroblast medium. After counting the fibroblasts using a hemacytometer, seed fives times 10 to the fourth cells in one well of a 24-well tissue culture plate.Incubate cells at 37 degrees Celsius overnight. On the following day, because Sendai virus will be handled, continue the procedure in a designated BSL-2 laboratory while using the appropriate personal protective equipment. Aspirate the cell culture medium and add 250 microliters of Sendai virus solution.Incubate the plate at 37 degrees Celsius. One day before picking the colonies, prepare LN-521 coated 24-well tissue culture plates. Pipette 250 microliters of LN-521 solution into one well of a 24-well plate.Seal the plate using parafilm and incubate at four degrees Celsius overnight. On the following day, aspirate the LN-521 solution from the one well of the 24-well plate and add 500 microliters of E8 into the well. The use of a hairnet and procedure mask is recommended while picking colonies under the stereo microscope.Colonies are ready to be picked when they reach a size of greater than one millimeter in diameter, display sharp edges, and are growing in homogenous monolayers. Do not pick colonies with fuzzy borders and large uncompact heterogeneous cell masses. Cut each colony with a scalpel into smaller pieces in a grid-like pattern.Use a 200 microliter pipette to mechanically scrape the cell sheets from the dish and then transfer the sheet to a well of a 24-well tissue culture plate previously coated with LN-521. Colony selection is critical for the success of this protocol. Picking a few additional colonies will ensure attachment in sufficient numbers.Allow the cells to attach without changing the E8 medium for 48 hours. Enzymatically passage the cells when the colonies have reached a size of greater than five millimeters. Aspirate the cell culture medium from the cells and wash with 250 microliters of DPBS.Aspirate the DPBS and add 250 microliters of dissociation reagent. Incubate at 37 degrees Celsius for three minutes. Observe that the cells have started to round up.Detach cells from the plate by rinsing the cells with dissociation reagent. Add 500 microliters of E8 medium to a 15 milliliter tube and transfer the cells and the dissociation reagent to the tube. Centrifuge at 300 times g for three minutes.Aspirate the supernatant from the 15 milliliter tube and resuspend the cell pellet in 500 microliters of E8 medium. Count the cells using a hemacytometer and seed 2.5 to five times 10 to the fourth cells in another previously prepared LN-521 precoated well containing 500 microliters of room temperature E8 medium. Add ROCK inhibitor to a final concentration of 10 micromolar.Incubate the cells at 37 degrees Celsius. Using this protocol, human induced pluripotent stem cell colonies emerge from transduced fibroblasts at about day 12 post-transduction. Colonies were ready to be picked at about three to four weeks post-transduction.Preferred features include flattened compact cell masses, growth as homogenous monolayers, and sharp edges. These bright field images show an early passage homogenous fully reprogrammed human iPS cell line compared to a highly heterogeneous cell line. The loss of the Sendai virus vector was confirmed by the absence of Sendai virus vector-specific markers in human iPS cells at passage 12 in contrast to human iPS cells at passage three.Immunocytochemistry staining of derived cells for pluripotency markers OCT4, SSEA-4, and NANOG yielded a homogeneously positive result. mRNA expression of endogenous pluripotency genes was shown by reverse transcriptase PCR. Cell cultures containing partially reprogrammed cells that did not express pluripotency factors were discarded.The differentiation potential of human iPS cells was assessed by formation of embryoid bodies. Real-time PCR after 21 days of embryoid body differentiation detected mRNA expression of endoderm, mesoderm, and ectoderm-specific markers which confirmed the derived human iPS cells were capable of differentiation to the three germ layers. Once mastered, this technique can be done with about 22 hours of hands-on time during a time period of approximately 16 weeks.The derived human induced pluripotent stem cells can be differentiated into any cell type of the adult human body to answer any questions relevant to your research field. After watching this video, we hope that you will have a good understanding of how to perform the steps necessary to derive induced pluripotent stem cells from skin biopsies.

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JoVE Journal

Developmental Biology

8.5K 조회수.  Karolinska Institutet. The overall goal of this protocol is to derive homogenous human induced pluripotent stem cells. This method can be used to derive human induced pluripotent stem cells for many purposes including disease modeling, drug screening, or to derive cells for preclinical cell therapy trials. The advantage of this protocol is that it's robust and generates homogenous cells that are maintained in a user-friendly, Xeno-free, and fully chemically defined culture system.Since human iPS cells recapi...