**5. Protocols for the generation of patient-specific iPSCs**

Within the last years, our group has participated in the development of protocols for the derivation of patient iPSCs for disease modelling and compound screening. Taking advantage of different basic techniques that are commonly used on a daily basis in any laboratory worldwide, we have generated simple methodologies that allow the generation of patientspecific iPSCs in a period that lasts only 50 days from the moment we get the primary samples from patients (i.e., skin biopsy, lipoaspirates, etc.). In this section, we provide two concise protocols for the derivation of patient iPSCs taking advantage of retroviruses and episomal vectors.

#### **5.1. Protocol for the generation of hiPSCs from keratinocytes derived from plucked hair samples**

The development of simple methods for the generation of hiPSCs from keratinocytes from plucked hair samples offers an unprecedented scenario for the production of patient-specific iPSCs, making use of a non-invasive procedure when collecting patient samples.

Our protocol is divided into three consecutive steps, which involve: A) Isolation of keratino‐ cytes from plucked hair samples, B) Production of retrovirus, and C) Infection of keratinocytes. The steps are detailed below. As described elsewhere, the same protocol can be applied when reprogramming cord blood stem cells, kidney tubular epithelial cells, and dermal fibroblasts [11,12,30].

**a.** Isolation of keratinocytes from plucked hair

**A.1** The day before hair isolation coat the required number of 35-mm culture dishes with Matrigel® (Becton Dickinson, S.A. cat. no. 356234) by adding 1 ml of Matrigel® and incubate overnight at 4 ºC.

**A.2** The same day of sample recovery, prepare a non-coated 100-mm bacterial plate containing HBSS (Invitrogen cat. no. 14170-088) with 1% (vol/vol) Penicillin/Streptomycin (Invitrogen cat. no. 15140-122).

**A.3** After the patient reads and signs the informed consent use tweezers to gently pull the hair out and place it on plates filled with HBSS medium. As recommended by Aasen and colleagues [89] use hair from the occipital part of the head.

**A.4** Making use of forceps submerge the hair in HBSS medium. Next, cut off the external part of the hair leaving the bulb and outer root sheath.

**A.5** As described by Aasen [89], at this stage two optional methodologies for growing kerati‐ nocytes from plucked hair are described: direct outgrowth and enzymatic digestion. In this section, we are going to detail how to get direct outgrowths of keratinocytes from plucked hair. For enzymatic digestion procedure follow Aasen recommendations [89].

**A.6** Remove the plate from 4 ºC, aspirate the Matrigel® coating and rinse the plate with 2 ml of hESCs medium: KO-DMEM (Invitrogen, cat. no. 10829-018), 20% KOSR (Invitrogen, cat. no. 10828-028), 10 ng ml-1 bFGF (Peprotech cat. No. 100-18B), 1 mM Glutamax (Invitrogen, cat. no. 35050-038), 100 µM nonessential amino acids (Invitrogen, cat. no. 11140-050), 100 µM 2 mercaptoethanol (Sigma, cat. no. M7522), and 50 U/ml (penicillin and 50 mg/ml streptomicin).

**A.7** Place gently the hair obtained from the coated culture plates.

**A.8** Add few drops of hESCs medium (0.3 mL) on top of the hair sample in order to keep the hair humid. In the next 3–4 hours, add gently fresh hESCs medium (0.3 mL). The next day carefully check under the microscope that the hair sample is still attached at the bottom of the plate. Refill the plate with more hESCs medium on top of the hair, if necessary.

**A.9** Add 1 ml of hESCs medium every following day. After 4 days, outgrowths of typical epithelial keratinocytes are visible.

**A.10** After 10–14 days, large colonies of keratinocytes (up to 1 cm of diameter) are visible. At this stage, it is advisable to split the cells for infection or subculture to avoid cells to initiate contact-dependent differentiation.

**b.** Production of retrovirus

**B.1** Seed out 4.3x106 Phoenix Amphotropic 293 cells in 10 ml of DMEM complete medium which consists in DMEM (Invitrogen, cat. no. 11965-092), 10% FBS (Invitrogen, cat. no. 10270-106), Glutamax 2 mM, Penicillin/Streptomycin (100 U/ml, 100 µg/ml) in 100-mm culture dishes and place in a 37 ºC 5% CO2 incubator.

**B.2** Next day, prepare FuGENE:DNA complex according to the manufacturer´s instructions (Roche Applied Science, cat. no. 1181509001). We recommend a ratio of 27 µl FuGENE to 9 µg plasmid DNA for every 10 cm dish. For virus production, we will make use of pMSCV-based plasmids. pMSCV-based retroviral vectors are commercially available for OCT4, SOX2, KLF4, and c-Myc in Addgene (reference numbers are: 20072, 20073, 20074, and 20075 respectively). If the infection efficiency wants to be monitored pMSCV-based retroviral vectors expressing GFP can be used (Addgene plasmid 20672).

**B.3** Add the FuGENE:DNA complex solution dropwise onto media (gently). Move the plate carefully in order to distribute the transfection reaction homogenously.

**B.4** Place the transfected cells at 37 ºC, with 5% CO2 overnight.

**B.5** Next day, change DMEM complete medium gently (10 ml/plate) and incubate the plates overnight at 32 ºC in a 5% CO2 incubator.

**B.6** Collect viral supernatants and add fresh complete DMEM medium to the different plates. Take care to avoid cells detaching from the tissue culture plates.

**B.7** Every following day, for 2 days, repeat steps B.5 and B.6 in order to collect more viral supernatants.

**B.8** Filter the viral supernatant through a 0.45 µm PVDF filter (Millipore® SLHV033NK) to remove any contaminant cells.

**B.9** Add 1 µl polybrene (10 mg/ml; Chemicon cat. no. TR-1003-6) for each ml viral supernatant needed (final polybrene concentration of 10 µg/ml).

**c.** Infection of keratinocytes derived from plucked hair samples

**C.1** Wash obtained keratinocyte colonies growing from a hair in hESCs medium as described in section (A) with PBS (Invitrogen, cat. no. 10010-056) and trypsinize them using 1 ml 0.25% Trypsin/EDTA (Invitrogen, cat. no. 25200-056).

**C.2** After 5–8 minutes, when cells are released from the plastic surface resuspend them with 10 ml hESCs medium.

**C.3** Centrifuge at 200g for 5 min.

The steps are detailed below. As described elsewhere, the same protocol can be applied when reprogramming cord blood stem cells, kidney tubular epithelial cells, and dermal fibroblasts

**A.1** The day before hair isolation coat the required number of 35-mm culture dishes with Matrigel® (Becton Dickinson, S.A. cat. no. 356234) by adding 1 ml of Matrigel® and incubate

**A.2** The same day of sample recovery, prepare a non-coated 100-mm bacterial plate containing HBSS (Invitrogen cat. no. 14170-088) with 1% (vol/vol) Penicillin/Streptomycin (Invitrogen cat.

**A.3** After the patient reads and signs the informed consent use tweezers to gently pull the hair out and place it on plates filled with HBSS medium. As recommended by Aasen and colleagues

**A.4** Making use of forceps submerge the hair in HBSS medium. Next, cut off the external part

**A.5** As described by Aasen [89], at this stage two optional methodologies for growing kerati‐ nocytes from plucked hair are described: direct outgrowth and enzymatic digestion. In this section, we are going to detail how to get direct outgrowths of keratinocytes from plucked

**A.6** Remove the plate from 4 ºC, aspirate the Matrigel® coating and rinse the plate with 2 ml of hESCs medium: KO-DMEM (Invitrogen, cat. no. 10829-018), 20% KOSR (Invitrogen, cat. no. 10828-028), 10 ng ml-1 bFGF (Peprotech cat. No. 100-18B), 1 mM Glutamax (Invitrogen, cat. no. 35050-038), 100 µM nonessential amino acids (Invitrogen, cat. no. 11140-050), 100 µM 2 mercaptoethanol (Sigma, cat. no. M7522), and 50 U/ml (penicillin and 50 mg/ml streptomicin).

**A.8** Add few drops of hESCs medium (0.3 mL) on top of the hair sample in order to keep the hair humid. In the next 3–4 hours, add gently fresh hESCs medium (0.3 mL). The next day carefully check under the microscope that the hair sample is still attached at the bottom of the

**A.9** Add 1 ml of hESCs medium every following day. After 4 days, outgrowths of typical

**A.10** After 10–14 days, large colonies of keratinocytes (up to 1 cm of diameter) are visible. At this stage, it is advisable to split the cells for infection or subculture to avoid cells to initiate

which consists in DMEM (Invitrogen, cat. no. 11965-092), 10% FBS (Invitrogen, cat. no.

Phoenix Amphotropic 293 cells in 10 ml of DMEM complete medium

hair. For enzymatic digestion procedure follow Aasen recommendations [89].

plate. Refill the plate with more hESCs medium on top of the hair, if necessary.

**A.7** Place gently the hair obtained from the coated culture plates.

[11,12,30].

346 Muscle Cell and Tissue

overnight at 4 ºC.

no. 15140-122).

**a.** Isolation of keratinocytes from plucked hair

[89] use hair from the occipital part of the head.

of the hair leaving the bulb and outer root sheath.

epithelial keratinocytes are visible.

contact-dependent differentiation.

**b.** Production of retrovirus

**B.1** Seed out 4.3x106

**C.4** Resuspend the pellets in 4 ml of hESCs medium.

**C.5** Plate cell suspensions in the desired wells of six-well plates (Corning, cat. no. 153516) previously pre-coated with Matrigel® as explained in step A1. Seed 80.000 keratinocytes/well.

**C.6** Next day add 1 ml of every single viral suspension obtained as described in steps B6-B9 for OCT4, SOX2, KLF4, and cMYC. Perform viral transduction in the same manner for GFP in order to monitor the efficiency of viral infection.

**C.7** Centrifuge plates at 650g for 45 min.

**C.8** Replace with 2 ml fresh hESCs medium (within 4–5 hours).

**C.9** Next day, repeat steps **C.7**-**C.8** to infect cells a second time.

**C.10** Change media daily with hESCs medium.

**C.11** After 1–2 weeks large colonies are visible and can be picked mechanically and transferred onto irradiated human fibroblasts feeder layer (iHFF) and cultured as normal iPSCs following specifications detailed before by others [12,29,30].

#### **5.2. Protocol for the generation of hiPSCs from mesenchymal stem cells (MSCs) derived from adipose tissue**

The possibility to generate iPSCs by means of non-integrative strategies paves the way for the development of clinical grade iPSCs from patients. Here, we detail a specific protocol for the derivation of hiPSCs from mesenchymal stem cells from adipose tissue.

Our protocol makes use of commercial episomal plasmids generated by Okita and colleagues [16]. Our method offers the possibility to generate patient-specific iPSCs in a period that last only 20 to 22 days from the moment the reprogramming experiment starts.

**a.** Before nucleofection

**A.1** Following the Human MSC Nucleofector® Kit (DPE-1001, Amaxa) recommendations the solution for nucleofection is prepared by adding 0.5 ml of Supplement to 2.25 ml Nucleofector Solution. Human MSC Nucleofector Solution is now ready to use and is stable for 3 months at 4°C.

**A.2** Under the culture hood prepare plasmid mixture by mixing 1 µg of each pCLXE episomal based plasmid (i.e., if we want to reprogram three different samples 3 µg of each pCXLE plasmid will be added to the final mixture). Plasmids are commercially available in Addgene: pCXLE-hOCT3/4-shp53-F (Plasmid #27077), pCXLE-hSK (Plasmid #27078), pCXLE-hUL (Plasmid #27080).

**A.3** Pre-warm the complete Human MSC Nucleofector Solution to room temperature.

**A.4** Pre-warm an aliquot mesenchymal stem cells culture medium [DMEM (Invitrogen, cat. no. 11965-092), 10% FBS (Invitrogen, cat. no. 10270-106), Glutamax 2 mM, Penicillin/Strepto‐ mycin (100 U/ml, 100 µg/ml)] at 37 °C in a 50 ml tube (500 µl per sample; 1.5 ml for 3 samples).

**A.5** Prepare 6-well plates by filling the appropriate number of wells with 1 ml of culture medium containing mesenchymal stem cells culture medium and pre-incubate plates in a humidified 37 °C/5% CO2 incubator. Prepare 2 wells/sample (i.e., 6 wells for 3 samples)

**b.** Nucleofection and iPSCs generation

**B.1** Follow Human MSC Nucleofector® Kit recommendations (http://bio.lonza.com/filead‐ min/groups/marketing/Downloads/Protocols/Generated/Optimized\_Protocol\_90.pd).

**B.2** After nucleofection transfer the nucleofected cells from the cuvettes using the plastic pipettes provided by the kit to prevent damage and loss of cells distributing the total amount of cell suspensions into 2 wells containing the pre-warmed mesenchymal stem cells culture medium. Incubate the cells in a humidified 37 °C/5% CO2 incubator.

**B.3** After 4 days transfer, wash nucleofected samples with PBS (Invitrogen, cat. no.10010-056) and trypsinize them using 1 ml 0.25% Trypsin/EDTA (Invitrogen, cat. no. 25200-056).

**B.4** Transfer the nucleofected cells into six-well plates (Corning, cat. no. 153516) containing irradiated murine fibroblasts (iMEF;C57BL/6 MEF 4M IRR; Global Stem) in hESCs medium: KO-DMEM (Invitrogen, cat. no. 10829-018), 20% KOSR (Invitrogen, cat. no. 10828-028), 10 ng ml-1 bFGF (Peprotech cat. No. 100-18B), 1 mM Glutamax (Invitrogen, cat. no. 35050-038), 100 µM nonessential amino acids (Invitrogen, cat. no. 11140-050), 100 µM 2-mercaptoethanol (Sigma, cat. no. M7522), 50 U/ml (penicillin and 50 mg/ml streptomicin). Change hES media every other day.

**B.5** After 20 days, large colonies are visible and can be picked mechanically and transferred onto iMEF and cultured as normal iPSCs following specifications detailed before by others [12,29,30].
