**Acknowledgements**

**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.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

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

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

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

**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

**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

**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.1** Follow Human MSC Nucleofector® Kit recommendations (http://bio.lonza.com/filead‐

**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

min/groups/marketing/Downloads/Protocols/Generated/Optimized\_Protocol\_90.pd).

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

derivation of hiPSCs from mesenchymal stem cells from adipose tissue.

only 20 to 22 days from the moment the reprogramming experiment starts.

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

specifications detailed before by others [12,29,30].

**from adipose tissue**

348 Muscle Cell and Tissue

**a.** Before nucleofection

at 4°C.

(Plasmid #27080).

**b.** Nucleofection and iPSCs generation

L.O and E.G were partially supported by La Fundació Privada La Marató de TV3, 121430/31/32. EM and JS were partially supported by funds from the Spanish Ministry of Economy and Competitiveness (Ref PLE 2009-0164) and the Commission for Universities and Research of the Department of Innovation, Universities, and Enterprise of the Generalitat de Catalunya (2014 SGR 1442). This work was also supported by funds from the "Ministerio de Ciencia e Innovación" (grants AGL2010-17324 to E.C., AGL2011-24961 to I.N. and AGL2012-39768 to J.G.) and the Catalonian Government (grant 2009SGR-00402).N.M was partially supported by La Fundació Privada La Marató de TV3, 121430/31/32, the Spanish Ministry of Economy and Competitiveness (Ref PLE 2009-0164) and from the Catalonian Government (2014SGR 1442). CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund.
