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 Fibroblasts from Dermal Biopsy
<ol>
	<li>Preparations of culture medium, digestive enzymes, coating of dishes and dissecting tools.
	<ol>
		<li>Prepare Fibroblast medium: 44.5 mL Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM), 5 mL Fetal bovine serum (FBS) and 500 &#181;L penicillin/streptomycin (PEST) (see Table 1), store at 4 &#176;C.</li>
		<li>Prepare digestive enzymes at a working concentration of 1 mg/mL: weigh aliquots of 4 mg of dispase and collagenase type I powder in separate 15-mL tubes and dissolve in 4 mL of DMEM/F12 + 1% PEST. Sterilize the enzymatic solution by passing it through a 0.22 &#181;m strainer. Prepare fresh enzyme solutions every time.</li>
		<li>Coat one well in a 6-well tissue culture plate (or 35-mm tissue culture dish) per biopsy dissected with 1 mL of 0.1% gelatin in phosphate buffered saline (DPBS). Incubate at room temperature for 30 min.</li>
		<li>Autoclave one set of surgical scissors and forceps per biopsy for dissection.</li>
	</ol>
	</li>
	<li>Biopsy handling 
	NOTE: Process biopsies as fast as possible after being taken. Store in PBS + 1% PEST for up to 48 h at 4 &#176;C. Biopsy should be 2 - 4 mm in diameter in size and in accordance with ethical permits.
	<ol>
		<li>Transfer the biopsy into a 35-mm dish. Move the biopsy to a new 35-mm dish and submerge the biopsy in 2 mL of 70% ethanol for 30 s.</li>
		<li>Move the biopsy to a third 35-mm dish and wash with 1 mL of sterile Hank&#39;s Balanced Salt Solution (HBSS) supplemented with 1% PEST.</li>
		<li>Transfer the biopsy to a fourth 35-mm dish, add 1 mL of freshly prepared 0.1% dispase solution.</li>
		<li>Cut the skin biopsy into 1 - 2 mm3 pieces in the dish using surgical scissors and forceps and then transfer biopsy pieces including the dispase solution into a 15-mL tube. Add an additional 2 mL of dispase.</li>
		<li>Rinse the 35-mm dish with a 1 mL dispase solution and transfer to the 15-mL tube.</li>
		<li>Incubate the biopsy at 4 &#176;C overnight.</li>
		<li>Add 4 mL of 0.1% collagenase I to the tube and incubate at 37 &#176;C for 4 h.</li>
		<li>Centrifuge the digested cells 300 x g for 3 min, aspirate the supernatant and resuspend the digest in 2 mL of fibroblast medium.</li>
		<li>Remove the gelatin solution from the 6-well tissue culture plate. Transfer the cell suspension including any remaining pieces to 6-well tissue culture plate (or 35-mm tissue culture dish) precoated with 0.1% gelatin in DPBS.</li>
		<li>Change medium every third day.</li>
	</ol>
	</li>
	<li>Expansion of human fibroblasts 
	NOTE: Fibroblasts are ready to be passaged to a T25 (25 cm2) tissue culture flask when 80% confluent (Figure 2B). Fibroblasts typically reach 80% confluency within 7 days. However, time required can vary greatly but should be no longer than 4 weeks.
	<ol>
		<li>Aspirate medium and wash once with 2.5 mL of DPBS.</li>
		<li>Remove DPBS and add 1 mL of Trypsin-EDTA (0.05%) and incubate at 37 &#176;C for 5 min or until cells display rounded edges and start to detach.</li>
		<li>Add 2 mL of fibroblast medium, if necessary rinse the cells ensuring proper disassociation of cells. Transfer the cell solution to a 15-mL tube and centrifuge at 300 x g for 3 min.</li>
		<li>Discard supernatant and resuspend the cell pellet in 5 mL of fibroblast medium and plate fibroblasts in a non-coated cell tissue culture T25 flask. When expanding fibroblasts after this step, dissociate as described above and seed 12 x 103 cells/cm2.</li>
		<li>Once confluent cells are ready to be frozen, expand or plate for reprogramming. Freeze down two rounds of fibroblast before any reprogramming is started (refer to next section for freezing procedure). Reprogramming efficiency is generally higher for lower passage fibroblasts, cells at passage 2 - 4 is optimal. However, successful reprogramming of fibroblasts up to passage 10 has been conducted using this protocol.</li>
	</ol>
	</li>
	<li>Freezing and thawing of fibroblasts
	<ol>
		<li>Prepare fresh fibroblast freezing medium: 90% FBS + 10% dimethylsulfoxide (DMSO). Keep at 4 &#176;C (Table 1). Once confluent, the T25 tissue culture flask can be frozen into three cryovials. Prepare 500 &#181;L of fibroblast freezing medium per vial frozen.</li>
		<li>To freeze cells, dissociate cells as described in Step 1.3, count cells using a hemocytometer and transfer portions of 3 x 105 cells to 15-mL tubes. Spin tubes at 300 x g for 3 min. Aspirate supernatant.</li>
		<li>Resuspend cell pellets in 500 &#181;L of 4 &#730;C fibroblast freezing medium and transfer into cryovials. Place the vials in a freezing container and incubate cells at -80 &#176;C for 24 h before transferring them to liquid nitrogen for long term storage.</li>
		<li>To thaw the cells, submerge the bottom part of the cryovial in 37 &#176;C water. Once liquefied, transfer the cell-suspension to a 15-mL tube and add 5 mL of fibroblast medium. Spin cells 300 x g for 3 min.</li>
		<li>Remove the supernatant and resuspend the cell pellet in 5 mL of fibroblast medium. Transfer cells to a non-coated T25 tissue culture flask and incubate in 37 &#176;C.</li>
	</ol>
	</li>
</ol>
2. SeV Vector Reprogramming of Fibroblasts
<ol>
	<li>Preparation of vector aliquots 
	CAUTION: Handle the SeV under biosafety level 2 containment. Refer to manufacturer&#39;s protocol and local guidelines17. Virus titer varies between batches.
	<ol>
		<li>Before preparing aliquots calculate the amount of vector needed for 5 x 104 cells according to manufacturer&#39;s protocol (e.g., CytoTune 2.0). The virus titer generally varies from 8 x 107 - 1.5 x 108. Thaw and combine vectors 5:5:3 (KOS MOI = 5, hc-myc MOI = 5 and hKlf4 = MOI 3). Portion the amount calculated for the reprogramming of 5 x 104 cells into autoclaved 1.5 mL tubes. Dilute virus aliquots with fibroblast medium to a final volume of 250 &#181;L for reprogramming. 
		NOTE: Each vial is valid for reprogramming of one well of a 24-well tissue culture plate with 5 x 104 fibroblasts. Store virus aliquots at -80 &#176;C.</li>
	</ol>
	</li>
	<li>SeV Vector transduction
	<ol>
		<li>Aspirate cell culture medium and wash with 1 mL of DPBS. Aspirate DPBS and add 1 mL of Trypsin-EDTA (0.05%). Incubate at 37 &#176;C for 5 min or until cells are detached.</li>
		<li>Add 2 mL of fibroblast medium and resuspend the cells and transfer to a 15-mL tube. Centrifuge 300 x g for 3 min.</li>
		<li>Aspirate the supernatant and resuspend the pellet in 2 mL of fibroblast medium.</li>
		<li>Count fibroblasts by using a hemocytometer and seed 5 x 104 cells in one well of a 24-well tissue culture plate. Incubate cells at 37 &#176;C overnight.</li>
		<li>Aspirate cell culture medium, add 250 &#181;L of the SeV solution and incubate at 37 &#176;C overnight.</li>
		<li>Change medium daily to fresh fibroblast medium. Allow transduced cells to grow for 7 days before being passaged to LN-521 coated plates. Prepare LN-521 plates the day before passage. Refer to 2.3.1 for coating procedure.</li>
	</ol>
	</li>
	<li>Replating of transduced fibroblasts
	<ol>
		<li>Preparation of LN-521 coated dishes: Dilute LN-521 in DPBS to a final concentration of 0.63 &#181;g/cm2. Pipette 2 mL of the LN-521 solution per 60-mm tissue culture dish. Seal the 60-mm tissue culture dish using parafilm. Incubate overnight at 4 &#176;C.</li>
		<li>Prepare Essential 8 medium (E8) aliquots for easier handling: 48.5 mL of E8 medium, 1 mL of E8 supplement and 500 &#181;L of PEST (Table 1).</li>
		<li>7 days after transduction, passage the cells to a LN-521 coated 60 mm tissue culture dish. Add 250 &#181;L of Trypsin-EDTA (0.05%) and incubate at 37 &#176;C for 5 min or until cells have detached. Add 2 mL of fibroblast medium and centrifuge cells for 3 min at 300 x g.</li>
		<li>Aspirate the supernatant. Resuspend pellet in 2 mL of fibroblast medium and count cells in a hemocytometer. Aspirate LN-521 solution from the precoated plate and seed 4 x 104 cells in 5ml of fibroblast medium in the LN-521 precoated 60 mm dish. Add Rho-kinase inhibitor Y-27632 (ROCKi) to a final concentration of 5 &#181;M. Incubate in 37 &#176;C overnight.</li>
		<li>Importantly, the next day, change medium to E8 medium. Change E8 medium daily. hiPS cell colonies will emerge within 2 - 3 weeks.</li>
	</ol>
	</li>
</ol>
3. Picking of Colonies and Expansion of hiPS Cells
NOTE: The following steps are done outside of the biosafety cabinet under a stereo microscope. The use of hairnet and procedure masks are recommended. Work carefully not to contaminate the cell culture.
<ol>
	<li>Prepare LN-521 coated tissue culture 24-well plates the day before picking. Dilute LN-521 in DPBS 0.63 &#181;g/cm2. Pipette 250 &#181;L of the LN-521 solution into one well of a 24-well tissue culture plate. Seal plates using parafilm. Incubate overnight at 4 &#176;C. 
	NOTE: Colonies are ready to be picked when they reach a size &#62; 1 mm in diameter. Colonies should display sharp edges and grow in homogeneous monolayers (Figure 2D).</li>
	<li>Aspirate the LN-521 solution and add 500 &#181;L of E8 into one well of a 24-well tissue culture plate.</li>
	<li>Cut colonies into smaller pieces with a scalpel in a grid like pattern under a stereomicroscope. Mechanically scrape the cell sheets from the dish and transfer the sheets using a 200 &#181;L pipette to the prepared well (Figure 2D-2E). Pick colonies into separate wells.</li>
	<li>Allow cells to attach without changing medium for 48 h. Thereafter, change E8 medium daily.</li>
	<li>When the colonies have reached a size of &#62; 5 mm, enzymatically passage cells as single cells to a new well of a LN-521 coated plate.</li>
	<li>Aspirate the cell culture medium from cells and wash with 250 &#181;L of DPBS.</li>
	<li>Aspirate DPBS. Add 250 &#181;L of dissociation reagent and incubate for 3 min at 37 &#176;C.</li>
	<li>Observe that the cells have started to round up. Detach cells from the plate by rinsing the cells with dissociation reagent a few times.</li>
	<li>Add 500 &#181;L of E8 medium to a 15-mL tube. Transfer the cells in the dissociation reagent to the tube and centrifuge for 3 min at 300 x g.</li>
	<li>Aspirate the LN-521 solution from the LN-521 precoated well and add 500 &#181;L room temperature E8 medium.</li>
	<li>Aspirate the supernatant and resuspend the cell pellet in 500 &#181;L of E8 medium.</li>
	<li>Count the cells using a hemocytometer and seed 2.5 x 104 - 5 x 104 cells in the prepared LN-521 precoated well (1.25 x 104 -&#160; 2.5 x 104 cells/cm2). Cell culture format can easily be upscaled to suit the intended purpose.</li>
	<li>Add ROCKi to a final concentration of 10 &#181;M. Incubate cells at 37 &#176;C.</li>
	<li>Change E8 medium daily. Cells are usually ready to passage after 4 - 6 days. Enzymatically passage cells as single cells as described above when about 90% confluent. 
	NOTE: The reprogramming vectors are progressively diluted during hiPS cell expansion and not detectable after passage 12. Cells that are impartially reprogrammed commonly cease to proliferate around passage 6 - 10 or lose their characteristic pluripotent stem cell morphology (Figure 3B).</li>
	<li>Freezing and thawing of hiPS cells
	<ol>
		<li>To freeze cells, enzymatically dissociate cells as single cells as described above, count cells in a hemocytometer and transfer 2.5 x 105 cells to a 15-mL tube containing 1 mL of E8 medium. Centrifuge at 300 x g for 3 min. Aspirate the supernatant.</li>
		<li>Resuspend cell pellet in 250 &#181;L of 4 &#176;C PSC cryomedium and transfer to a cryovial. Place the vial in a freezing container and incubate cells at -80 &#176;C for 24 h before transferring them to liquid nitrogen for long term storage.</li>
		<li>To thaw cells, prepare a precoated LN-521 well of a 24-well tissue culture plate by aspirating the LN-521 solution and adding 250 &#181;L of E8 medium. Submerge the bottom part of the cryovial in 37 &#176;C water until only a small icicle remains.</li>
		<li>Add 250 &#181;L of E8 Medium to the cryovial and transfer the cell-suspension to a 15-mL tube and add 1 mL of E8 medium. Spin cells 300 x g for 3 min.</li>
		<li>Remove the supernatant and resuspend the cell pellet in 250 &#181;L of room temperature E8 medium. Transfer the cell suspension to the prepared well and add of 1% cell viability supplement or 10 &#181;M ROCKi. Incubate the tissue culture plate at 37 &#176;C.</li>
	</ol>
	</li>
</ol>

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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><tbody><tr><td>Dispase</td><td>Life Technologies</td><td>171105-041</td><td>Biopsy digestion</td></tr><tr><td>Collagenase</td><td>Sigma</td><td>C0130</td><td>Biopsy digestion</td></tr><tr><td>Gelatin</td><td>Sigma</td><td>G1393</td><td>Fibroblast matrix</td></tr><tr><td>IMDM</td><td>Life Technologies</td><td>21980002-032</td><td>Fibroblast medium</td></tr><tr><td>FBS</td><td>Invitrogen</td><td>10270106</td><td>Fibroblast medium</td></tr><tr><td>Penicillin/Streptomycin</td><td>Life Technologies</td><td>15070-063</td><td>Antibiotic</td></tr><tr><td>Essential 8 media</td><td>ThermFisher Scientific</td><td>A1517001</td><td>iPS cell culture media</td></tr><tr><td>LN-521</td><td>Biolamina</td><td>LN521-03</td><td>iPS cell culture matrix</td></tr><tr><td>TrypLE select 1X</td><td>ThermoFisher Scientific</td><td>12563011</td><td>Dissociation reagent</td></tr><tr><td>Rho-kinase inhibitor Y27632</td><td>Millipore</td><td>SCM075</td><td>Rho-kinase inhibitor (ROCKi)</td></tr><tr><td>CytoTune &amp;ndash; iPS 2.0 reprogramming kit</td><td>Life Technologies</td><td>A1377801</td><td>Sendai virus reprogramming vector</td></tr><tr><td>35 mm tissue culture dish</td><td>Sarstedt</td><td>83.3900.300</td><td>Cell culture</td></tr><tr><td>60 mm tissue culture dishes</td><td>Sarstedt</td><td>83.3901.300</td><td>Cell culture</td></tr><tr><td>24 well tissue culture plates</td><td>Sarstedt</td><td>83.3922.300</td><td>Cell culture</td></tr><tr><td>T25 tissue culture flasks</td><td>VWR</td><td>734-2311</td><td>Cell culture</td></tr><tr><td>15 mL tubes</td><td>Corning</td><td>430791</td><td>Centrifuge tubes</td></tr><tr><td>1.5 mL tube</td><td>Eppendorf</td><td>0030123.301</td><td>1.5 mL tube</td></tr><tr><td>CoolCell cell LX</td><td>Biocision</td><td>BCS-405</td><td>Freezing container</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D2650-100ml</td><td>Fibroblast freezing</td></tr><tr><td>Cryovials 1.8 mL</td><td>VWR</td><td>479-6847</td><td>Cryovials</td></tr><tr><td>PSC Cryopreservation Kit</td><td>Gibco</td><td>A2644601</td><td>PSC CryomediumRevitaCell supplement</td></tr><tr><td>Trypsin-EDTA (0.05%)</td><td>ThermoFisherScientific</td><td>25300054</td><td>Fibroblast dissociation enzyme</td></tr><tr><td>DMEM/F12</td><td>Life Technologies</td><td>31331-028</td><td>Digestive enzymes dilutant</td></tr><tr><td>DPBS</td><td>Life Technologies</td><td>14190-094</td><td>PBS</td></tr><tr><td>HBSS</td><td>ThermoFIsher Scientific</td><td>14025-050</td><td>Biopsy preparation</td></tr><tr><td>Haemocytometer</td><td>Sigma</td><td>BRAND, 718920</td><td>Cell counting</td></tr><tr><td>Parafilm</td><td>VWR</td><td>291-1213</td><td>Sealing plates for storage</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-...

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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) ...

integration-free-derivation-human-induced-pluripotent-stem-cells

Research

JoVE Journal

Developmental Biology

8.5K Views.  Karolinska Institutet. 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. 

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

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Neuroscience

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

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

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

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

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

Biology

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

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

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

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

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Bioengineering

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic genes remaining in actively transcribed regions of the genome, limit their potential for use in human therapeutic applications1. Additionally, non-human antigens derived from stem cell reprogramming or differentiation into therapeutically relevant derivatives preclude these hiPSCs from being used in a human clinical context2. In this video, we present a procedure for reprogramming and analyzing factor-free hiPSCs free of exogenous transgenes. These hiPSCs then can be analyzed for gene expression abnormalities in the specific intron containing the lentivirus. This analysis may be conducted using sensitive quantitative polymerase chain reaction (PCR), which has an advantage over less sensitive techniques previously used to detect gene expression differences3. Full conversion into clinical-grade good manufacturing practice (GMP) conditions, allows human clinical relevance. Our protocol offers another methodology&#8212;provided that current safe-harbor criteria will expand and include factor-free characterized hiPSC-based derivatives for human therapeutic applications&#8212;for deriving GMP-grade hiPSCs, which should eliminate any immunogenicity risk due to non-human antigens. This protocol is broadly applicable to lentiviral reprogrammed cells of any type and provides a reproducible method for converting reprogrammed cells into GMP-grade conditions.

We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions.

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic ...

Measuring the Confluence of iPSCs Using an Automated Imaging System

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence in real time has been established without the need to count cells in single cell suspension to avoid any growth perturbation. A high-content image analysis system was used to assess iPCS confluence on 4 different ECMs over time in an automated manner. Different analysis settings were used to assess cell confluence of adherent iPSCs and only a slight difference (at 24 and 48 hours with laminin) has been observed whether a 60, 80 or 100% mask was applied. We also show that laminin lead to the best confluence compared to Matrigel, vitronectin and fibronectin.

The goal of the protocol is to compare different extracellular matrix (ECM) coating conditions to assess how differential coating affects the growth rate of induced pluripotent stem cells (iPSCs). In particular, we aim to set up conditions to obtain optimal growth of iPSC cultures.

This study focuses on understanding how growing iPSCs on different ECM coating substrates can affect cell confluence. A protocol to assess iPSC confluence ...