Stem Cell Reviews and Reports

, Volume 9, Issue 4, pp 435–450

Generation of Mouse and Human Induced Pluripotent Stem Cells (iPSC) from Primary Somatic Cells

Authors

  • I. M. Lorenzo
    • Caubet-Cimera Foundation, Centre for Advanced Respiratory Medicine
    • Consejo Superior de Investigaciones Científicas (CSIC)
  • A. Fleischer
    • Caubet-Cimera Foundation, Centre for Advanced Respiratory Medicine
    • Caubet-Cimera Foundation, Centre for Advanced Respiratory Medicine
    • Consejo Superior de Investigaciones Científicas (CSIC)
Article

DOI: 10.1007/s12015-012-9412-5

Cite this article as:
Lorenzo, I.M., Fleischer, A. & Bachiller, D. Stem Cell Rev and Rep (2013) 9: 435. doi:10.1007/s12015-012-9412-5

Abstract

Cellular reprogramming consists of the conversion of differentiated cells into pluripotent cells; the so-called induced Pluripotent Stem Cells. iPSC are amenable to in vitro manipulation and, in theory, direct production of any differentiated cell type. Furthermore, iPSC can be obtained from sick individuals and subsequently used for disease modeling, drug discovery and regenerative treatments. iPSC production was first achieved by transducing, with the use of retroviral vectors, four specific transcription factors: Oct4, Klf4, Sox2 and c-Myc (OKSM), into primary cells in culture Takahashi and Yamanaka, (Cell 126(4):663–676, 2006). Many alternative protocols have since been proposed: repeated transfections of expression plasmids containing the four pluripotency-associated genes Okita et al. (Science 322(5903):949–953, 2008), lentiviral delivery of the four factors Sommer et al. (Stem Cells 27(3):543–549, 2009), Sendai virus delivery Fusaki et al. (Proceedings of the Japan Academy. Series B, Physical and Biological Sciences 85(8):348–362, 2009), removal of the reprogramming vectors by ‘piggyBac’ transposition Woltjen et al. (Nature 458(7239):766–770, 2009); Kaji et al. (Nature 458(7239):771–775, 2009), Cre-recombinase excisable viruses Soldner et al. (Cell 136(5):964–977, 2009), episomal vectors Yu et al. (Science 324(5928):797–801, 2009), cell-penetrating reprogramming proteins Zhou et al. (Stem Cells 4(5):381–384, 2009), mammalian artificial chromosomes Hiratsuka et al. (PLoS One 6(10):e25961, 2011) synthetically modified mRNAs Warren et al. (Scientific Reports 2:657, 2012), miRNA Anokye-Danso et al. (Cell Stem Cell 8(4):376–388, 2009); however, although some of these methods are commercially available, in general they still need to attain the reproducibility and reprogramming efficiency required for routine applications Mochiduki and Okita (Biotechnol Journal 7(6):789–797, 2012). Herein we explain, in four detailed protocols, the isolation of mouse and human somatic cells and their reprogramming into iPSC. All-encompassing instructions, not previously published in a single document, are provided for mouse and human iPSC colony isolation and derivation. Although mouse and human iPSC share similarities in the cellular reprogramming process and culture, both cell types need to be handled differently.

Keywords

Induced pluripotent stem cellsCellular reprogrammingPrimary keratinocytesPrimary mouse embryonic fibroblastsRetrovirusFeeder cellsCell derivation

Introduction

iPSC technology represents the most important breakthrough in the stem cell field in recent years. It brings new ways to improve our understanding of stem cell biology, and expands enormously the potential of these cells for applications in cell therapy and disease modelling. iPSC may be used to treat diseases using patient-specific cells, circumventing immunological and ethical obstacles.

Originally, Takahashi and Yamanaka described the direct reprogramming of Mouse Embryonic Fibroblasts (MEF) to pluripotent cells, using a cocktail of 4 transcription factors: Oct4, Sox2, Klf4, and cMyc [1]; however, alternative methods exist [213]. Depending on the expression profile of the starting somatic cell type, the composition of Yamanaka’s cocktail can be modified [14]. For example, Kim and co-workers have shown that, due to the endogenous expression of Sox2, cMyc, and Klf4 in mouse neural stem cells (NSC), the sole exogenous expression of Oct4 is sufficient to transform them into pluripotent stem cells [15]. Using fewer factors decreases the risk of insertional mutagenesis, although the reprogramming efficiency is reduced and the time required to obtain iPSC colonies increased.

More recently, advancements in reprogramming efficiency have been achieved by adding small molecules to the reprogramming cocktail. Such molecules are histone deacetylase inhibitors like valproic acid [16] or butyrate [17]; histone methyltransferase inhibitors as BIX01294 [18]; vitamin C that alleviates cell senescence [19] and a combination of Utf1, a chromatin-associated protein, and anti-p53 specific siRNA which reduces senescence and apoptosis [20, 21].

The protocols presented here describe in detail how to isolate Mouse Embryonic Fibroblasts (MEFs), as well as human primary fibroblasts and keratinocytes from skin biopsies. We also describe the procedures required to transform them into iPSC with the use of retroviral vectors (see a general scheme in Fig. 1). Mouse keratinocytes are routinely isolated and cultured for a variety of purposes [22, 23], but they are not a cell type of choice for reprogramming experiments, therefore their use is not described in this paper. In the first two protocols, we describe how to obtain primary murine and human cells suitable for cellular reprogramming, as well as feeder cells production from mitotically inactivated MEFs. Feeder cells support survival, pluripotency, and growth of iPSC in culture, but must be in proliferative arrest. The third protocol deals with the reprogramming process itself. It includes segments explaining how to produce retroviral particles, and how to infect and reprogram somatic primary cells. Finally, the fourth protocol describes the establishment and storage of permanent iPSC lines.
https://static-content.springer.com/image/art%3A10.1007%2Fs12015-012-9412-5/MediaObjects/12015_2012_9412_Fig1_HTML.gif
Fig. 1

Protocol outline. Somatic primary cells are obtained either from mouse or humans samples. A 12.5-day pregnant mouse female is sacrificed, and MEF cells obtained by protease digestion of the isolated embryos. Keratinocytes or fibroblasts are harvested from skin biopsies obtained from human donors. MEFs can be used either as feeder cells or as primary cells for reprogramming. At day −3 Phoenix packaging cells are seeded, and next day, transfected in order to produce fresh reprogramming retroviral particles. Retrovirus-containing supernatants are filtered and the different viruses mixed into a reprogramming cocktail. At day 0 primary somatic cells at early passages are infected. After infection, cells are reseeded on feeder cells and cellular reprogramming starts. Once colonies appear, they are individually picked and expanded for further freezing, culturing or characterization

Materials, Reagents and Solutions

Materials

0.45 μm-sterile PVDF membrane filters: Millipore, #SLHV033RS

100 mm polystyrene Petri dishes: Corning, #430167

2, 5, 10 and 25 ml serological pipettes: Daslab, #PN2E1, #PN5E1, #PN10E1, #PN25E1.

24-well plates: NUNC, #140675

50 ml conical tubes: Biologix, #10-9502

15 ml conical tubes: Deltalab, #429920

6-well plates: NUNC, #142475

Filter paper: Albet Labscience, #RM2534252

Cell Strainer, 100 μm Nylon mesh: Decton Dickinson, #352360

1,5 ml Eppendorf tubes: Sarstedt, #72690

Sterile long glass (150 mm) Pasteur pipettes: Normax, #5426023

Sterile surgical tools: fine tweezers, dissecting scissors, and scalpel

T-75 Flasks: NUNC, #156499

Pulled glass Pasteur pipettes for mechanical isolation of iPSC colonies (see Ref [24] for manufacturing details)

Cabinet X-Ray system (Model RX-650, Faxitron X-ray Corp, Wheeling, IL)

Neubauer chamber for cell counting: VWR, #631-1114

Reagents

Trypsin/EDTA: 0.5 % (wt/vol) Trypsin/0.2 % (wt/vol) EDTA. PAA, #L11-003

2-Mercaptoethanol: GIBCO, #31350-100

Antifungal amphotericin B solution: Sigma, #A2942-20ML

Ascorbic acid: Sigma, #A4544-25 G

Butyrate: Sigma, #B5887

Calibrated ion chamber: Keithley, #96035

Coating Matrix: Invitrogen, #R-011-K

Collagenase IV: Invitrogen, #17104-019

D/F12: GIBCO, #21331

Dispase: Becton Dickinson, #354235

DMEM 4.5 g/l glucose: PAA, #E15-009

DMSO Hibri-max: Sigma, #D2650

DNAse: Roche, #10104159001

Dosimeter: Keithley, #35050A

Epilife Human Keratinocyte Growth Supplement (HKGS): Invitrogen, #S-001-5

Epilife Medium with 60 μM calcium: Invitrogen, #M-EPI-500-CA

Gelatine type A from porcine skin: Sigma, #G1890-100 G

GlutaMAX: Invitrogen, #35050-038

Hank’s balanced salt solution without Ca2+ and Mg2+ (HBSS): PAA, #H15-009

Heat-inactivated FBS (FBS): GIBCO, #10100-147

HEPES Buffer: Sigma, #H0887

Knockout DMEM (KO-DMEM): GIBCO, #10829

KO Serum Replacement (KSR): GIBCO, #10828

Leukemia Inhibitory Factor (LIF): Sigma, #L5158-5UG

Lipofectamine LTX and Plus Reagent: Invitrogen, #15338-100

MEFLU cells, naturally immortalized mouse embryonic fibroblasts (See reference number [19])

NIH/3 T3 cells: ATCC, #CRL 1658

Mitomycin C: Sigma, #M4287-2MG

Non-essential amino acid solution (NEAA) 100 ml, 100X: GIBCO #11140

Opti-MEM + Glutamax (Opti-MEM): GIBCO, #51985

Penicillin/Streptomycin (P/S), 100 ml, 100X: GIBCO, #15140

Phoenix amphotropic packaging cells, ATCC #SD3443

Phoenix ecotropic packaging cells, ATCC #SD3444

Phosphate-buffered saline with Ca2+ and Mg2+ (PBS + Ca): PAA, #H15-002

Phosphate-buffered saline without Ca2+ and Mg2+ (PBS): PAA, #H15-002

Retrovirus vectors: pMXs and pMSCV retrovirus vectors containing Oct4, Sox2, Klf4, and c-Myc genes, available from Addgene (e.g., 13366, 13367, 13370, 13375; 20072, 20073, 20074, 20075, respectively)

TrypLE Express Stable Trypsin Replacement: Invitrogen, #12604-039

Valproic Acid: STEMGENT, #04-0007

Y-27632 (ROCK Inhibitor): #Sigma, Y0503-5MG

Solutions

Phosphate-buffered saline with antibiotics (sPBS): 100 U/ml penicillin, and 100 mg/ml streptomycin and 25 mM HEPES buffer. Store at 4 °C for up to 2 weeks.

Hank’s balanced salt solution with antibiotics and antifungal (sHBSS-Amph): 100 U/ml penicillin, 100 mg/ml streptomycin, 25 mM HEPES buffer and 250 ng/ml Amphotericin B. Store at 4 °C for up to 2 weeks.

Hank’s balanced salt solution with antibiotics (sHBSS): 100 U/ml penicillin, 100 mg/ml streptomycin and 25 mM HEPES buffer. Store at 4 °C for up to 2 weeks.

Epilife medium: Add 5 ml HKGS to a 500 ml Epilife bottle. Supplement with 50 U/ml penicillin and 50 mg/ml streptomycin. If necessary, P/S may be omitted. Protect from light. Store at 4 °C for up to 2 weeks.

Epilife with dispase and amphotericin B: Epilife medium containing 1 % HKGS, 100 U/ml penicillin and 100 mg/ml streptomycin is supplemented with 250 ng/ml amphotericin B and 5 U/ml dispase. Epilife contains HEPES that will buffer the medium once outside the incubator. Prepare fresh each time.

Complete DMEM medium: DMEM with 10 % FBS (vol/vol), 1 mM GlutaMAX, 50 U/ml penicillin, 50 mg/ml streptomycin. Store at 4 °C for up to 2 weeks.

Complete aa-DMEM medium: Add 10 % FBS (vol/vol), 1 mM GlutaMAX, 100 μM Non-essential amino acids, 50 U/ml penicillin, 50 mg/ml streptomycin to DMEM medium. Store at 4 °C for up to 2 weeks.

Complete D/F12: Mix 1:1 DMEM and DMEM/F12 medium in order to obtain a final proportion 3:1 of

DMEM/F12. Supplement it with 10 % FBS (vol/vol), 1 mM GlutaMAX, 50 U/ml penicillin, 50 mg/ml streptomycin, and 1 % HKGS. Store in dark at 4 °C for up to 2 weeks.

Conditioned-complete D/F12: Plate 4 × 106 mitotically inactivated feeders in gelatine-coated T-75 flask with complete aa-DMEM medium, as described in steps 34–38 in protocol A. Next day, rinse with PBS and replace the medium with 13 ml of complete D/F12. For the next 5 days, collect everyday the medium and replace it with 13 ml fresh complete D/F12. Filter through 0.45 μm-sterile filter. Store the feeder cell supernatant at 4 °C for up to 1 week, or alternatively at −20 °C for long-term storage.

Mouse iPSC (m-iPSC) medium: KO-DMEM supplemented with 15 % KSR (vol/vol), 1 mM GlutaMAX, 1 μM Non-essential amino acids (vol/vol), 100 μM Mercaptoethanol, 50 U/ml penicillin, 50 mg/ml streptomycin, and 1000 U/ml LIF.

Human iPSC (h-iPSC) medium: KO-DMEM supplemented with 20 % KSR (vol/vol), 1 mM GlutaMAX, 1 μM Non-essential amino acids (vol/vol), 100 μM Mercaptoethanol, 50 U/ml penicillin, 50 mg/ml streptomycin, and 10 ng/ml bFGF.

ROCK Inhibitor stock solution: Reconstitute 5 mg Y-27632 in milli-Q H2O to 1 mM. Store at 4 °C for up to 1 week, or aliquot and store at −20 °C for longer periods.

h-iPSC medium with ROCK inhibitor: Add 10 μM Y-27632 to h-iPSC medium. Store at 4 °C for up to 1 week.

Matrigel solution: Standarize Matrigel concentration to 5 mg/ml with KO-DMEM medium. Pay attention: Matrigel polimerizes irreversibly at Room Temperature (RT). Carry out all steps using pre-cooled fungible material at −20 °C.

Gelatine solution: 0.1 % (wt/vol) gelatine type A from porcine skin in distilled water. Add 500 ml of distilled water to a 500 ml glass bottle containing 0.5 g gelatine and autoclave. Store at 4 °C up to 1 year.

Gelatine-coated culture dishes: Cover the bottom of a well plate or a petri dish with 0.1 % gelatine. E.g., use 2, 5, or 15 ml to cover each 35, 100, or 150-mm Petri dish, respectively. Move back and forth the plate or dish in order to cover equally all area. Incubate at least 30 min at 37 °C. Eliminate gelatine solution before use.

Coating matrix-coated culture dishes: Cover the bottom of a well plate or a petri dish with coating matrix solution. Use 2, 5, or 15 ml to cover each 35-, 100, or 150-mm Petri dish, respectively. Move the plate or dish in order to cover perfectly all the bottom area. Incubate at least 30 min at 37 °C. Eliminate coating matrix solution before use.

Mitomycin C stock solution: Add 2 ml of PBS into the vial to obtain a 1 mg/ml solution. This amount allows for the inactivation of thirteen 150-mm Petri dishes. Prepare the solution fresh before use, and protect it from light.

0.05 % (wt/vol) Trypsin/0,2 % (wt/vol) EDTA solution: dilute the 0.5 % Trypsin/EDTA stock solution in PBS 1:10. Keep at 4 °C for up to 1 month.

0.25 % (wt/vol) Trypsin/EDTA solution: dilute the 0.5 % Trypsin/EDTA stock solution in PBS 1:2. Keep at 4 °C for up to 1 month.

Collagenase IV stock solution: Resuspend the lyophilized powder in HBSS adjusting the enzyme concentration to 500 U/ml, pass it through 0.22 um filter to sterilize. Aliquot and store at −20 °C for up 1 year.

50 U/ml Collagenase IV solution: dilute 1:10 the stock of Collagenase IV in KO-DMEM.

Disaggregating solution: 0.25 % (wt/vol) Trypsin/EDTA + 100 Kunitz units DNAse per ml or 0,1 ug/ml DNAse.

Freezing medium: 10 % DMSO and 90 % FBS. Keep at 4 °C for 1 week.

Methods

Protocol A. Mouse Embryonic Fibroblasts Isolation

Here we describe the isolation, freezing, storage and thawing of MEFs. The first steps of the isolation protocol are the same regardless of whether MEFs are intended to serve as feeder cells or for cell reprogramming. The choice of mouse strain influences MEF quality. Usually outbred strains or F1 mattings produce more robust embryos and cells than inbred lines. However, even among inbred lines differences exist that affect the reprogramming efficiency and pluripotency of iPSC [24]. In our experiments we have successfully used embryos obtained from crossing mice of C57BL/6 and DBA-2 backgrounds. Steps 1–14 of the protocol are common for feeder and iPSC production, but from step 15 on, specific steps for each one of these two uses are detailed. Freezing and thawing protocols are common to MEF, feeder cells, primary human fibroblasts, and virus packaging (Phoenix) cells. For MEFs to be used as feeder cells it is necessary that they are in mitotic arrest. A protocol for MEF inactivation is included at the end of this section.

Primary fibroblast Isolation

  1. 1.

    Autoclave dissecting tools: tweezers, scissors, and a scalpel.

     
  2. 2.

    Sacrifice a female at 12.5 days of pregnancy and dissect the uterine horns containing the embryos.

     
  3. 3.

    Place embryos into a 100 mm-Petri dish containing 15–20 ml sPBS. Perform the following steps in a tissue culture hood.

     
  4. 4.

    Wash 3 times with 15–20 ml sPBS.

     
  5. 5.

    Open the uterine wall to release the embryos and place them into a new Petri dish with sPBS.

     
  6. 6.

    Prepare one gelatine-coated 100 mm-Petri dishes per embryo obtained as described in the ‘Solutions’ section.

     
  7. 7.

    Using fine-tipped forceps, dissect the embryos discarding the heads and all inner organs.

     
  8. 8.

    Rinse twice the remaining carcasses with 15 ml sPBS.

     
  9. 9.

    Transfer all embryos, using a slotted spoon, into a new Petri dish containing 15 ml sPBS.

     
  10. 10.

    Mince the remaining tissues (mainly body walls) into small pieces until a homogeneous suspension is formed.

     
  11. 11.

    Add to a 50 ml conical tube 1 ml of disaggregating solution per embryo.

     
  12. 12.

    Place embryos into the 50 ml conical tube and incubate at 37 °C for 25 min, tapping every 5 min. Solution should become turbid; otherwise continue the incubation at 37 °C for additional 5–10 min.

     
  13. 13.

    Add complete aa-DMEM medium to stop digestion. Use twice the volume of disaggregating solution. Pipette the cell suspension very gently 10–15 times up and down. Ideally, the resulting cell suspension should be free of any large piece of tissue and not too viscous. Viscosity is an indication of cell lysis and DNA release, and prevents the settling down of cell clumps. If needed, add 100 Kunitz units more of DNAse per ml and continue incubation at 37 °C for 10 to 20 min, tapping every 5 min. If mucous still persists, take it out sucking it with a 1000 μl pipette.

     
  14. 14.

    To remove large cell clumps, let the suspension rest for 3–4 min. Carefully transfer the top supernatant –fraction A- to a new 50 ml conical tube leaving, at the bottom, at least 3 ml containing tissue fragments and clumps. (For feeder cell production go to step 18).

     
  15. 15.

    Centrifuge fraction A at 200 g for 5 min. Cells contained in the 200 g pellet fraction are the most suitable for cellular reprogramming [25]. Discard supernatant and resuspend the pellet in 15 ml warm complete aa-DMEM medium.

     
  16. 16.

    Count resuspended viable cells with trypan blue staining and seed 5 × 106 viable cells per gelatine-coated 100 mm-Petri dish. The resulting MEFs represent passage 0 (see Note 1).

     
  17. 17.
    Once MEFs are isolated (passage 0), grow them in a humidified incubator at 37 °C, 5 % CO2 until they reach 75–90 % confluence. Usually it takes 2 to 3 days. Figure 2a shows primary MEFs in culture at 75–90 % confluence. At day 2 you should recover around 1.5–4 × 106 cells per embryo. Do not let the culture reach 100 % confluence, because cells will become senescent. Subsequently, MEFs can be expanded (go to step 21), frozen in liquid nitrogen (go to step 27) or reprogrammed (protocol C, step 12).
    https://static-content.springer.com/image/art%3A10.1007%2Fs12015-012-9412-5/MediaObjects/12015_2012_9412_Fig2_HTML.gif
    Fig. 2

    Cellular morphologies. a Primary MEF cells cultured on gelatine with complete aa-DMEM at passage 3. At low confluence, the cells spread looking bigger. Scale bar, 50 μm. b Human keratinocyte outgrowth from a skin explants at day 10 of culture. Epidermal explants are attached to the bottom with Matrigel. Explants for keratinocyte production are cultured with conditioned-complete D/F12 on Coating Matrix-covered cell culture plates. c Keratinocytes at passage 1 cultured on plastic with Epilife. Keratinocytes growing in Epilife at low confluence lose their typical polygonal-epithelial morphology and became round. d Phoenix Amphotropic cells showing the correct cell morphology, confluence, and distribution on the day of vector transfection. e Phoenix Amphotropic cells on the third day after the transfection (day 1 in the general protocol). Note that in 3 days, Phoenix cells have grown to over-confluency. f EGFP expression of cells in E. About 80 % of the cells express EGFP, enough to achieve high retrovirus titer

     
  18. 18.

    If MEFs are intended for feeder cell production, it is not necessary to select the 200 g fraction. In this case, centrifuge at 270 g for 5 min at RT and resuspend the pellet in complete aa-DMEM. 270 g are tougher for the cells than 200 g, but the yield is better and feeder cell production is less demanding than reprogramming. Count viable cells using trypan blue staining. Seed about 2–3 × 106 viable cells per 100 mm Petri dish.

     
  19. 19.

    Next day, rinse the Petri dishes containing MEFs 3 times with 10 ml PBS + Ca.

     
  20. 20.

    Add 13 ml fresh complete aa-DMEM medium per 100 mm Petri dish and grow them until 75–90 % confluence. Change medium every 2–3 days. (For mitotic inactivation of feeder cells go to step 40).

     
  21. 21.

    For MEFs expansion, rinse each 100 mm Petri dish 3 times with 10 ml of PBS. Leave cells in the last washing step for 2–5 min at RT (see Note 2).

     
  22. 22.

    Eliminate PBS and add 2 ml of 0.05 % Trypsin/EDTA to each 100-mm Petri dish. Incubate 2–3 min at RT. Tap the dish on the side to detach cells. Check if most of the cells are in suspension; otherwise continue the incubation for another 1–3 min at 37 °C.

     
  23. 23.

    Add 10 ml of complete DMEM medium per dish to stop trypsinization, and pipette gently 7–10 times up and down to break down cell clumps into single cells. Collect cells into one single 50 ml-conical tube.

     
  24. 24.

    Count viable cells using trypan blue-dye exclusion staining.

     
  25. 25.

    Centrifuge 50 ml conical tubes at 250 g for 5 min.

     
  26. 26.

    Discard supernatant, resuspend cells in aa-Complete DMEM and seed them at 11,000 cells/cm2 on gelatine-coated dishes.

     

Freezing

The procedure is similar for MEFs, feeder cells, primary human fibroblasts, and Phoenix cells.
  1. 27.

    Prepare freezing medium and keep it at 4 °C.

     
  2. 28.

    Collect MEFs following steps 21–25 above.

     
  3. 29.

    Discard supernatant and resuspend cells in freezing medium adjusting cell density to 2 × 106 viable cells/ml.

     
  4. 30.

    Immediately, aliquot 1 ml cell suspension into cryovials and place them at −20 °C for a minimum of 3 h and a maximum of 15 h.

     
  5. 31.

    Place them at −80 °C for a minimum of 15 h and a maximum of 5 days.

     
  6. 32.

    For permanent storage, transfer the frozen vials into a −150 °C freezer or a liquid nitrogen container.

     

Thawing

The procedure is very similar for all cell types, but in each case, a specific culture medium might be needed in step 34. MEFs: complete aa-DMEM; feeder cells: complete aa-DMEM; primary human keratinocytes: Epilife; primary human fibroblasts: complete aa-DMEM and Phoenix cells: complete DMEM.
  1. 33.

    Prepare a gelatine-coated cell culture plate as described in ‘Solutions’ section. Phoenix cells and keratinocytes do not require coating.

     
  2. 34.

    Place a 15 ml-conical tube containing 9 ml of the appropriate culture medium into the bath at 37 °C.

     
  3. 35.

    Place a frozen cryovial in a 37 °C water bath until it is almost thawed (around 30 s). Spray vial with 70 % ethanol and wipe it dry before placing it into the laminar flow hood.

     
  4. 36.

    Transfer the cells to the pre-warmed medium using a sterile Pasteur pipette.

     
  5. 37.

    Centrifuge 5 min at 250 g and resuspend cells in 13 ml of pre-warmed culture medium, and transfer them into a T-75 flask.

     
  6. 38.

    Place the flask in a 37 °C, 5 % CO2 incubator. MEF, feeder cells, primary human fibroblasts, and Phoenix cells require medium changes every 2–3 days; keratinocytes every 3–4 days.

     

Mitotic Inactivation of Feeder Cells

There are two main methods to induce mitotic arrest in fibroblasts: Mitomycin C treatment and X-Ray irradiation. In what follows, we will explain our protocol for X-Ray inactivation, (for Mitomicyn C inactivation, please see Ref. [26]). In our lab, we produce two different types of feeder cells: NIH-3 T3 to support the growth of human primary keratinocytes, and MEFLU [27] to support both human and mouse iPSC. Other kinds of feeder cells may be used, including primary MEF cells. It is advisable to test the efficiency of different feeder cell types in supporting your iPSC during at least 3–4 serial passages. Once decided, keep always the same feeder cell to support a particular iPSC line. After inactivation, MEFs can be stored at −150 °C for several months. It is, therefore, advisable to prepare a big batch at a time.
  1. 39.

    Grow MEFs on 15 gelatine-coated 150-mm Petri dishes until they reach 80 % – 90 % confluence.

     
  2. 40.

    Trypsinize cells as described in steps 21–23 in protocol A. Use 3 ml trypsin/EDTA per 150-mm Petri dish, and inactivate each trypsinization reaction with 7 ml of aa-Complete DMEM.

     
  3. 41.

    Distribute the cell suspension into four 50-ml conical tubes.

     
  4. 42.

    Centrifuge at 250 g for 5 min.

     
  5. 43.

    Resuspend the content of each tube in 10 ml aa-Complete DMEM and collect the cells from the four tubes in one 50-ml conical tube.

     
  6. 44.

    Count viable cells and adjust the cellular concentration to 2 × 106 cells/ml.

     
  7. 45.

    Be sure the X-ray system is correctly calibrated. It is worthwhile to have the dosimetry performed by the distributor, using a dosimeter equipped with a calibrated ion chamber. The chamber is placed in the middle of the working shelf radiation field to detect dose rate during control X-ray measurements.

     
  8. 46.

    Our equipment (see Materials, Reagents and Solutions section) is set at 120 kV, 5 mA with a 0.5 mm aluminium filter.

     
  9. 47.

    In order to minimize radiation heterogeneity, place the 50-ml conical tube-containing MEFs in horizontal position at the middle of the working shelf of the cabinet.

     
  10. 48.

    Deliver a total of 40 Gy, at a dose rate of 0.83 Gy/min.

     
  11. 49.

    Centrifuge the cells at 250 g for 5 min, and resuspend them in 10 ml aa-Complete DMEM.

     
  12. 50.

    Count viable cells and distribute them into two 50-ml conical tubes for freezing.

     
  13. 51.

    Centrifuge 50 ml conical tubes at 250 g for 5 min.

     
  14. 52.

    Freeze the cells as explained in steps 29–32 above.

     

Protocol B. Isolation of Primary Keratinocytes and Fibroblasts from Human Skin

Keratinocytes and fibroblasts are obtained from the epidermal and dermal layers of the skin respectively. In this section, we explain two different methods for obtaining primary cells: (i) digestion of the epidermis, and (ii) skin explants. Digestion of epidermal pieces is better suited for large and young skin samples and for the production of keratinocytes. It generates cells in suspension that can be seeded and cultured in feeder-free conditions using Epilife, a chemically defined medium. Epilife-based culture conditions allow cells to grow in a more undifferentiated state (Fig. 2c). Explants give a better yield when starting with small biopsies or delicate samples like old donor tissues. Both fibroblasts and keratinocytes can be obtained from dermal and epidermal explants, respectively. Explants attach to the bottom of the plate rendering an outgrowth of cells. Keratinocytes obtained from explants show a more epithelial-like morphology than those obtained by digestion of the epidermis (Fig. 2b).

Skin Biopsies Processing

  1. 1.

    Cut filter paper into pieces fitting into a 100-mm Petri dish, and autoclave along with the dissecting tools.

     
  2. 2.

    Start with a skin biopsy of about 10 mm2, but keep in mind that bigger pieces will yield more cells. Place the skin into a 10 ml-conical tube with cold sHBSS-Amph and keep it at 4 °C until use. Perform subsequent steps as soon as possible in a tissue culture hood (see Note 3).

     
  3. 3.

    Place the tissues, using sterile fine tweezers, in a 100-mm Petri dish containing 15–20 ml of sHBSS-Amph. Rinse the sample three times with sHBSS-Amph for at least 5 min each. Smaller Petri dishes or other sterile containers containing abundant sHBSS-Amph may also be used. Adjust the volumes proportionally to the area.

     
  4. 4.

    In order to remove the hypodermis, transfer the skin pieces into a 100 mm Petri dish containing enough sHBSS-Amph (at least 10 ml) to cover all the pieces.

     
  5. 5.

    Using sterile tweezers, place the skin with the epidermis facing the bottom of the dish. With the help of curved scissors remove yellow subcutaneous fat and loose connective tissue (hypodermis) until the dermis appears as a white sheet. Discard unwanted materials transferring them into another sterile Petri dish containing sHBSS-Amph. Minimize the time of these manipulations.

     
  6. 6.

    Cut the remaining skin into 2–3 mm wide strips using a sterile scalpel.

     
  7. 7.

    To prepare the skin for dermis-epidermis separation, place a pre-cut autoclaved filter paper into a 100-mm Petri dish. Spread the intact strips directly onto it, dermis side down on the dry paper. Make sure edges of the skin are not rolled. Pay attention when the skin is spread. It tends to stretch covering a bigger area than initially thought.

     
  8. 8.

    Add carefully 13 ml of Epilife with dispase and Amphotericin B to the 100 mm Petri dish containing the filter paper with the skin stripes attached, and incubate it at 4 °C overnight (see Note 4).

     
  9. 9.

    Next morning, the dermal and epidermal layers are separated mechanically. Hold the tissue, and separate the epidermis (upper, thin and almost transparent layer) from the dermis (lower, thicker and white layer) using sterile tweezers. Transfer immediately all the pieces of each layer type to one 100-mm Petri dishes containing 20 ml of sHBSS-Amph.

     
  10. 10.

    Rinse the pieces of both layers two times with 20 ml subs sHBSS-Amph for at least 5 min at RT. (Go to step 11 for keratinocyte isolation by tissue digestion. Go to step 38 for keratinocyte isolation from the explants. Go to step 54 for fibroblast isolation from the explants).

     

Keratinocytes Isolation by Epidermal Digestion

  1. 11.

    Using sterile fine tweezers, place the minced epidermal pieces into a 50 ml conical tube containing 15 ml TrypLE.

     
  2. 12.

    Incubate the tube in a bath at 37 °C for 30 min tapping it firmly every 5 min.

     
  3. 13.

    Add 10 ml of TrypLE more to the 50 ml conical tube.

     
  4. 14.

    Continue the incubation at 37 °C for 25 min more. Tap firmly the tube every 5 min. This would be digestion A.

     
  5. 15.

    Transfer the epidermal pieces using sterile fine tweezers to a new 50 ml conical tube containing 15 ml TrypLE. Digestion A contains the cells already detached from the epidermal fragments. It is important to continue the incubation of the fragments in a separate tube (digestion B) in order to avoid damaging the cells already in suspension.

     
  6. 16.

    Stop digestion A by adding 20 ml of complete D/F12.

     
  7. 17.

    Keep digestion B in a bath at 37 °C for 25 min tapping firmly every 5 min.

     
  8. 18.

    Add another 10 ml of TrypLE to the 50 ml tube of digestion B.

     
  9. 19.

    Continue the incubation of digestion B in a bath at 37 °C for 20 min more, tapping firmly every 5 min.

     
  10. 20.

    Stop digestion B by adding 20 ml complete D/F12.

     
  11. 21.

    Pipette gently up and down digestions A and B about 15 times to break down all cell clumps.

     
  12. 22.

    Filter both cell suspensions through a 100 μm cell strainers. The strainers will retain undigested larger tissue pieces. Wash the cell strainer with 5 ml of complete D/F12. Collect the elutions containing the cells into 50 ml conical tubes.

     
  13. 23.

    Centrifuge both tubes at 250 g for 5 min.

     
  14. 24.

    Discard supernatant and resuspend each pellet in 10 ml of Epilife.

     
  15. 25.

    Collect the resuspended pellets (reactions A and B) into one tube.

     
  16. 26.

    Count the cells determining cell number and viability by trypan blue exclusion staining.

     
  17. 27.

    Centrifuge at 250 g for 5 min.

     
  18. 28.

    Resuspend the pellet in Epilife. Get a final density of about 40,000 viable cells per cm2 and seed in 100-mm Petri dishes. Dishes should be pre-coated with Coating Matrix.

     
  19. 29.

    Incubate at 37 °C, 5 % CO2.

     
  20. 30.

    Change media 3 days after seeding, and subsequently every 3 days until they reach 70–75 % confluency. Primary Keratinocytes spontaneously differentiate above this level.

     
  21. 31.

    Rinse twice the 100-mm Petri dish with 13 ml PBS and leave the second wash for 5 min at 37 °C.

     
  22. 32.

    Remove PBS, add 1.5 ml TrypLE, and incubate at 37 °C for 5 to 10 min (see Note 5).

     
  23. 33.

    Tap the dish gently and add 10 ml Epilife medium.

     
  24. 34.

    Transfer the cell suspension into a 15 ml conical tube and centrifuge at 250 g for 5 min.

     
  25. 35.

    At this stage, cells can be seeded or frozen. (For keratinocyte freezing go to steps 29–32 in protocol A).

     
  26. 36.

    For seeding, resuspend the cells in Epilife medium and seed them at 5000 cells/cm2 into T-75 flasks without coating.

     
  27. 37.

    Change media next day with 13 ml Epilife and subsequently every 3 days.

     

Keratinocytes Isolation from Epidermal Explants

  1. 38.

    (From step 10). The day before seeding the explant, prepare one 6-well plate coated with Coating Matrix. Add 2 ml of autoclaved water in the space between the wells.

     
  2. 39.

    Place epidermis in a new 100-mm Petri dish containing 20 ml sHBSS-Amph.

     
  3. 40.

    Remove excess of sHBSS-Amph until the epidermis touches the bottom of the dish. Cut it into small pieces of about 1 mm2 using a scalpel. Make sure that all the cuts are precisely done and minimize epidermis handling.

     
  4. 41.

    Place and spread the minced epidermal pieces into the wells of a Coating Matrix-covered 6-well plate prepared in step 38. Place no more than 4 pieces per well of the 6-well plate (10 cm2).

     
  5. 42.

    Add one drop of 5 mg/ml Matrigel onto each epidermal piece covering it completely. Use a 100 μl-pipette with a tip pre-cooled at 4 °C. Matrigel solidifies easily at RT. Keep Matrigel at 4 °C and handle it quickly to glue the epidermal pieces to the bottom of the wells.

     
  6. 43.

    Before Matrigel solidifies, remove a little bit of the drop with the 100 μl-pipette. This will help to flatten the epidermal piece against the bottom surface of the well.

     
  7. 44.

    Incubate the plate 15 min at 37 °C.

     
  8. 45.

    Add 2 ml of conditioned-complete D/F12 per well of the 6-well plate (see Note 6).

     
  9. 46.

    Remove any floating epidermal tissue with sterile fine tweezers and, in order to recover more primary cells, explant them again in a new well following steps 41–45.

     
  10. 47.

    Incubate in a humidified incubator at 37 °C and 5 % CO2 changing media every 3–4 days.

     
  11. 48.

    After 10 to 20 days in culture, cells will reach 70–75 % confluence and should be passed.

     
  12. 49.

    Follow steps 31–34 in protocol B.

     
  13. 50.

    Resuspend in complete Epilife at about 40,000 cells/ml. After replacing complete D/F12 with Epilife, keratinocytes loose their cuboidal-like shape and become round. See Fig. 2b and c.

     
  14. 51.

    Seed 400,000 keratinocytes per 100-mm Petri dish pre-coated with Coating Matrix.

     
  15. 52.

    Culture cells at 37 °C and 5 % CO2, changing medium every 3 days until they reach 70–75 % confluence. Remember that primary Keratinocytes spontaneously differentiate above this level.

     
  16. 53.

    Keep expanding keratinocytes as described, or freeze them according to steps 31–35 in protocol B.

     

Fibroblasts Isolation from Epidermal Explants

  1. 54.

    (From step 10). To obtain human fibroblasts for reprogramming purposes cut dermis into 1–2 mm2 pieces with a scalpel. Place the pieces on gelatine-coated 100 mm Petri dishes without medium and leave them for 20 to 30 min at RT. Place 1 piece of tissue per 2–3 cm2 of dish surface.

     
  2. 55.

    Cover carefully dermal pieces with one drop of complete aa-DMEM medium, and incubate overnight at 37 °C, 5 % CO2.

     
  3. 56.

    Next day, triple the volume of each explant-containing drop with extra aa-DMEM medium and incubate overnight at 37 °C, 5 % CO2.

     
  4. 57.

    The following morning add 8 ml more of complete aa-DMEM to the 100 mm Petri dish, and maintain the culture until fibroblast outgrowths appear. Replace medium with 10 ml complete aa-DMEM every 4–5 days.

     
  5. 58.

    Pass outgrowing fibroblasts when they reach 75–80 % confluence as described in steps 21–26 in protocol A. Continue with the expansion if needed, or freeze and store the cells in liquid nitrogen.

     

Protocol C. Cellular Reprogramming

Cellular reprogramming is achieved by infecting primary cells with a mix of species–specific retroviral particles carrying the four Yamanaka transcription factors: Oct4, Klf4, Sox2 and c-Myc. Each virus is produced separately in Phoenix packaging cells, and then mixed with the other three, prior to infection of the primary cell cultures. Phoenix cells produce and release to the medium retroviral particles that are collected and added to the primary cell culture. After two consecutive rounds of infection, the reprogramming process starts, and somatic cells initiate a pluripotent program (see Fig. 3 for a diagram). During the process, the composition of the culture media has to be modified in order to facilitate reprogramming and to select pluripotent cell colonies. Primary cells passage number is critical for reprogramming efficiency. We strongly recommend using cells at passage 3 or less.
https://static-content.springer.com/image/art%3A10.1007%2Fs12015-012-9412-5/MediaObjects/12015_2012_9412_Fig3_HTML.gif
Fig. 3

Reprogramming timetable. The diagram starts with the seeding of Phoenix packaging cells and ends with the harvesting of iPS colonies. a Timetable of mouse iPSC production from MEFs. Phoenix ecotropic cells produce retrovirus particles into complete aa-DMEM medium. MEF transduction with the reprogramming factors is carried out in 2 rounds of infection on the same day. On day 1, the infected cells are reseeded on feeder cells. Next day, treatment with small molecules begins. VPA treatment lasts for 6 days, while Vit C treatment is maintained until colony picking. On the day 5, infected MEFs culture medium is replaced by m-iPSC medium. b Timetable of human iPSC production from keratinocytes. Phoenix amphotropic cells produce retrovirus particles into conditioned-complete D/F12. Human keratinocytes are infected twice on days 0 and 1. After infection in conditioned-complete D/F12, keratinocytes are washed and cultured again with Epilife medium. On the day 2 they are reseeded on feeder cells. Two days after (day 4), the culture media is replaced with h-iPSC medium containing small molecules. Butyrate treatment is carried out for 6 days, and the Vit C treatment lasts until colony picking

Phoenix packaging cells are second-generation retrovirus-producing cell lines for the generation of helper-free ecotropic and amphotropic retroviruses [28]. Ecotropic packaging cells produce mouse-specific retroviruses. Amphotropic packaging cells produce retroviruses able to infect most mammalian dividing cells, including mouse and human. Phoenix-ecotropic cells are transfected with pMXs vectors encoding for mouse transgenes [1]. Phoenix-amphotropic cells are transfected with pMSCV vectors coding for flagged human transgenes [29]. Other cell types may be also used for viral production [30] like Plat-E cells [25, 31], or HEK-293 T cells co-transfected with helper plasmids expressing the gag-pol and env genes [32].

Mouse Primary Cells Reprogramming

Retrovirus production, primary cell infection, and cellular reprogramming.

Ecotropic Retrovirus Production

  1. 1.

    One week before transfection thaw one cryovial of Phoenix cells (as described in steps 33–38, protocol A).

     
  2. 2.

    Place the flask in a humidified incubator at 37 °C, 5 % CO2. Change medium every 2 days. Pass Phoenix cells twice before transfecting them for retrovirus production. Always split at 1:3 to 1:4. (see Note 7).

     
  3. 3.

    The day before transfection (day −3, Fig. 3a), harvest cells and seed them on four wells of a 6-well plate, at 720,000 cells per well. Transfecting each well with one of the four transcription factor (OKMS) will yield enough viral particles to infect all the primary cells plated in a 6 well plate. Perform all the steps in a biosafety 2 laminar flow hood (see Note 8).

     
  4. 4.

    Next day (day −2), carefully replace medium of each well of 6-well plates with 2 ml of complete DMEM without Penicillin/Streptomycin (P/S). Prepare the Lipofectamine-DNA mix in Opti-MEM without P/S. Dilute 3.750 ng of each TF plasmid in 0.5 ml Opti-MEM in a 1.5 ml-eppendorf tube and mix thoroughly. An additional GFP-carrying virus may be used as internal control for transfection efficiency and subsequent retroviral expression. Alternatively, construct transfection may be mediated either by FuGene reagent [33] or calcium phosphate [34].

     
  5. 5.

    Add 3.75 μl PLUS Reagent to each eppendorf, mix gently and incubate 5 min at RT.

     
  6. 6.

    Add 7.5 μl Lipofectamine, mix thoroughly and incubate 30 min at RT.

     
  7. 7.

    Add the Lipofectamine-DNA mix to each 2 ml DMEM-containing well to initiate transfection.

     
  8. 8.

    Incubate overnight at 37 °C, 5 % CO2 in a humidified incubator.

     
  9. 9.

    Next day (day −1), discard medium and add carefully 2.5 ml complete aa-DMEM medium.

     
  10. 10.

    Incubate at 32 °C, 5 % CO2 overnight in a humidified incubator. Incubation at 32 °C improves retrovirus production and stability. Alternatively, retrovirus production may be carried out at 37 °C, although with a lower yield. Retrovirus particles are stable for 3 h to 6 h at 37 °C. Supernatant from this culture will be used for primary cell infection.

     
  11. 11.

    If a GFP-carrying virus has been added to estimate transfection efficiency, check for its expression late on the day. Around 70 % of the cells should be transfected in order to produce a high enough virus titter (Fig. 2f). Smaller titters will not work efficiently in reprogramming primary cells.

     

MEF Infection and Reprogramming

Day 0 is the day of infection. Be aware that viruses are biological hazards for both research personnel, and environment. Please, work under a biosafety 2 laminar flow hood and follow safety regulations. Reprogramming requires two consecutive infections of the same primary cell culture. To obtain viral particles for both infections, viruses are harvested twice from the Phoenix cells in culture.
  1. 12.

    The day before virus infection (day −1), seed 125,000 MEF cells in 2.5 ml of aa-DMEM medium in each of the wells of a gelatine-coated 6-well plate (see Note 9).

     
  2. 13.

    Label 50 ml-conical tubes with the name of each of the TF-containing retroviruses (plus GFP, if it is the case). Open the tubes and attach to each one of them a 0.45 μm sterile filter of PVDF membrane. Connect 5 ml syringes, without the plungers, to the filters.

     
  3. 14.

    Collect separately the virion-containing supernatant from each one of the 6-well plates from step 11 in protocol C.

     
  4. 15.

    Add 10 μl of 1 mg/ml polybrene solution to each syringe followed by 2.5 ml of the corresponding supernatant. Adjust polybrene volumes to the volume of supernatant to be filtered (see Note 10).

     
  5. 16.

    Refill gently each well of the 6-well plate with 2.5 ml of complete aa-DMEM, and place it back in the incubator. This culture will produce the virion-containing supernatants required for the second round of infection. Phoenix cells must be confluent at this step; if this is not the case, increase cell number at step 3 in protocol C.

     
  6. 17.

    Place the plungers into the syringes and filter the virus-containing supernatants to eliminate any Phoenix cell contamination.

     
  7. 18.

    Prepare the infection inoculum by mixing the different virus-containing supernatants in equal proportions.

     
  8. 19.

    Add 1 ml sterile H2O between the wells of the 6-well plate containing the somatic cells (step 12), in order to minimize media evaporation.

     
  9. 20.

    Discard culture medium, and rinse MEFs twice with 2.5 ml PBS.

     
  10. 21.

    Infect MEFs with 1 ml of infective inoculum per well of a 6-well plate (see Note 11).

     
  11. 22.

    Incubate MEFs at 37 °C, 5 % CO2 in a humidified incubator until the next round of infection. The second infection should be performed between 8 and 12 h (overnight) after the first one.

     
  12. 23.

    Eight hours after initiating the second round of retrovirus-producing cultures (step 16), harvest the new viruses and repeat the infection as it is described above. This time, discard the Phoenix cells by bleaching and autoclaving all the materials.

     
  13. 24.

    Incubate infected MEFs at 37 °C, 5 % CO2 overnight in a humidified incubator.

     
  14. 25.

    On the next morning (day 1), stop infection by adding 2 ml complete aa-DMEM medium, and place the plate back into the incubator at 37 °C, 5 % CO2 while you prepare reagents and materials for passing the cells to feeder-containing wells.

     
  15. 26.

    Split infected cells at a ratio 1:4 on MEFLU-containing 6-well plates with complete aa-DMEM. This ratio will give rise to approximately to 20–25 iPSC colonies per well when infecting with four reprogramming factors. Higher densities will make difficult picking individual colonies.

     
  16. 27.

    At days 2 and 4, replace media with 2.5 ml complete aa-DMEM medium containing 25 μg/ml ascorbic acid, and 1 mM valproic acid.

     
  17. 28.

    From day 5 to day 7, replace media daily with 2.5 ml m-iPSC medium containing 25 μg/ml ascorbic acid, and 1 mM valproic acid.

     
  18. 29.

    From day 8 onwards, change media daily with m-iPSC medium with 25 μg/ml ascorbic acid, but without valproic acid.

     

Human Primary Cells Reprogramming

Retrovirus production, infection, and cellular reprogramming.

Human fibroblast reprogramming is the most common method of human iPSC production. There are several highly reliable protocols describing it in the literature [3537]; therefore we will not explain it here. The protocol for Amphotropic retrovirus production and Human keratinocyte infection and reprogramming is very similar to the one described in the previous section for Mouse primary cells reprogramming, but with some modifications. Phoenix-amphitropic cells are used in virus production from step 1 on. Complete D/F12 medium without HKGS is used in steps 12 and 16. The day before virus infection (see step 12), seed 50,000 human keratinocytes in 2.5 ml Epilife in each of the wells of a matrix-coated 6-well plate. There are also changes regarding polybrene dose and time. In step 15, add 12.5 μl of 1 mg/ml polybrene in each syringe. The final polybrene concentration for human keratinocyte should be 5 μg/ml. In step 18, add 1 μl of HKGS per 1 ml infection inoculum to supplement the medium for keratinocytes. Other differences are:
  1. 30.

    Infect keratinocytes with 1 ml of infective inoculum per well of a 6-well plate.

     
  2. 31.

    Incubate keratinocytes at 37 °C, 5 % CO2 for 3 h in a humidified incubator. Longer periods of infection may cause differentiation because, at this point, keratinocytes are still cultured in complete D/F12 medium without feeders.

     
  3. 32.

    Rinse keratinocytes twice with 3 ml PBS, and add 2 ml Epilife.

     
  4. 33.

    Repeat the infection next day as described. This time, discard virus-producing Phoenix cells by bleaching, and autoclaving all the material.

     
  5. 34.

    Incubate keratinocytes at 37 °C, 5 % CO2 in a humidified incubator for 3 h.

     
  6. 35.

    Rinse twice with 2.5 ml PBS per well.

     
  7. 36.

    Add 2.5 ml Epilife per well.

     
  8. 37.

    Incubate at 37 °C, 5 % CO2 in a humidified incubator.

     
  9. 38.

    At day 2, split infected keratinocytes at a 1:3 ratio. Tripsinize cells using TrypLE, resuspend in Epilife, and seed in a volume of 10 ml per 100-mm Petri dish on MEFLU feeders-containing plates.

     
  10. 39.

    Incubate at 37 °C, 5 % CO2 in a humidified incubator for 2 days.

     
  11. 40.

    At day 4, change media with 2.5 ml h-iPSC medium containing 25 μg/ml ascorbic acid, and 0.5 μM butyrate.

     
  12. 41.

    From day 5 to day 9, change medium daily with 2.5 ml m-iPSC medium containing 25 μg/ml ascorbic acid and 0.5 μM butyrate.

     
  13. 42.

    From day 10 onwards, change medium daily with 2.5 ml m-iPSC medium without butyrate. Maintain the ascorbic acid in the medium until picking the colonies.

     

Protocol D. iPS Cell Derivation

Depending on the species of the donor and the type of starting primary cell, the difficulties and time involved in producing iPSC from somatic cells vary [11, 38]. Typically, reprogramming MEFs takes less time than human keratinocytes; MEF cells take approximately 10–12 days to complete the process (Fig. 3a), while human keratinocytes require one more week (Fig. 3b). In due time after viral infection, colonies resembling Embryonic Stem (ES) cells appear in the culture. Those with better morphology are picked up mechanically, cultured individually, expanded and frozen for further evaluation (Fig. 1). It does not exist a canonical set of properties for iPSC lines to fulfill in order to be considered suitable for experimentation, but the most common standards currently used in the literature are: Immunodetection of pluripotency markers, accompanied by loss of somatic cell markers [39]; silencing and integration of retroviral transgenes [39, 41]; in vitro and in vivo differentiation assays by embryoid body and teratoma formation [36, 39]; functional assays like tetraploid complementation [40]; karyotyping [41]; copy number variations (CNV) [42]; transcriptional profile analysis [39] and epigenetic analysis of DNA methylation, histone modifications or X-chromosome inactivation [41, 42].

During the reprogramming process cells become progressively more ES-like (Figs. 4a, b, 5a, and b). But several non-completely reprogrammed and bad-quality colonies also appear (Figs. 4d, e, 5d and e). Such colonies should be discarded. Mouse iPSC colonies have smooth well-defined edges, high nucleus to cytoplasm ratio, and homogenous appearance. Specifically, good mouse pluripotent colonies show (i) shiny appearance, (ii) a well-defined surrounding membrane, and (iii) compact appearance built out of densely packed cells [10] (Fig. 4c). By contrast, human pluripotent colonies are less packed, have a cobblestone appearance, less proliferative potential and less clonogenicity [10, 13] (Fig. 5c). After reprogramming is completed, most iPSC colonies in the culture are healthy and undifferentiated. Nevertheless, differentiated iPSC colonies may be occasionally found in culture (Figs. 4f and 5f).
https://static-content.springer.com/image/art%3A10.1007%2Fs12015-012-9412-5/MediaObjects/12015_2012_9412_Fig4_HTML.gif
Fig. 4

Mouse iPSC colony morphologies. a and b show the appearance of newly reprogrammed m-iPSC colonies at days 4 and 10 respectively, (passage 1) cultured on MEFLU with m-iPSC medium. cBona fide mouse iPSC. The picture shows the colony at day 4 after colony picking (passage 2). d Bad-quality mouse colony at day 4 of reprogramming. Weakly attached cells compose the core of the colony. It does not have smooth edges. e Non-completely reprogrammed colony at day 10 of cellular reprogramming. Cells are loosely attached and without brightness. The colony shows different types of cells, some are round and proliferate attached to each other forming a 3-D aggregate, while others are more elongated and spread over the plate. f Differentiated m-iPSC colony. Cells have cobblestone-like morphology and lack both well-defined edges and compactness. Scale bar, 50 μm

https://static-content.springer.com/image/art%3A10.1007%2Fs12015-012-9412-5/MediaObjects/12015_2012_9412_Fig5_HTML.gif
Fig. 5

Human iPSC colony morphologies. a and b show the evolution from day 6 to day 14 of a newly reprogrammed h-iPSC colony (passage 1), cultured on MEFLU cells with h-iPSC medium. c Good h-iPSC colony. The h-iPSC colony shown in A and B was transferred at day 15 to a new well. The picture shows the colony at day 7 after colony picking (passage 2). d Bad-quality human colony at day 6 of reprogramming. Weakly attached cells compose the core of the small colony. It does not have smooth edges. e Bad-quality colony at day 14 of cellular reprogramming. The colony is compact, and dull, without any brightness. The cells do not spread on the plate, but proliferate upwards forming a non-adherent structure, resembling an in vitro cavitating embryoid body. f Differentiated h-iPSC colony. The border of the colony is not well defined and the surface looks coarse. Scale bar, 50 μm

Mouse iPSC Derivation

  1. 1.

    Two days before picking colonies, seed 30,000 MEFLU feeder cells/cm2 on gelatine-coated 24-well plates as described in ‘Solutions’ section. Use one well of a 24-well plate per each colony picked.

     
  2. 2.

    The day before collecting the colonies, examine the cultures under a microscope and preselect the colonies to be picked. Use a marker to signal the location of the colonies at the bottom of the plate.

     
  3. 3.

    Rinse with 3 ml HBSS the MEFLU feeder cells seeded the previous day.

     
  4. 4.

    Add 800 μl of m-iPSC medium to the MEFLUs and place the plate back in the incubator.

     
  5. 5.

    The day of the picking, rinse twice the plates containing colonies with 3 ml of HBSS per well.

     
  6. 6.

    Add 2 ml m-iPSC medium to each well of the 6-well plates.

     
  7. 7.

    Under a stereoscope inside the hood, scrape one of the colonies using a pulled Pasteur pipette (see Ref [20]).

     
  8. 8.

    To avoid cell mixing, soak the pipette in 70 % ethanol after picking each one of the colonies. Let it air-dry before reusing it.

     
  9. 9.

    Recover immediately the scraped colony with an automatic 100 μl pipette.

     
  10. 10.

    Place the colony in a well of a 96-well plate containing 100 μl m-iPSC medium.

     
  11. 11.

    Pipette gently up and down 5–7 times to disaggregate the colony.

     
  12. 12.

    Collect the whole volume (~200 μl) containing the cell suspension, and transfer it into an individual well of the 24-well plates pre-seeded with MEFLU in 800 μl m-iPSC medium.

     
  13. 13.

    Shake the plate back and forth to homogenously distribute the cells.

     
  14. 14.

    Pick up the next colony. To minimize cell mixing, rinse the well containing the colonies with 2.5 ml HBSS, and repeat colony picking as described in steps 7–13.

     
  15. 15.

    Once the 24-well plate is full, place it into a humidified incubator at 37 °C, 5 % CO2, and do not disturb it for the next 48 h.

     
  16. 16.

    After all the preselected colonies have been collected from the reprogramming plate, replace HBSS with m-iPSC medium and keep it in the incubator as a backup.

     
  17. 17.

    48 h after picking, replace the medium of the 24-well plate with 1 ml m-iPSC medium per well, and keep doing it daily. Each single colony after been seeded on a well, will produce a number of derived colonies that should be considered as one clone.

     
  18. 18.

    Around 5 to 7 days after the seeding, each clone should reach the time to be passed (see Note 12).

     
  19. 19.

    Two days before passing the colonies, seed 30,000 MEFLU feeder cells/cm2 on gelatine-coated 6-well plates as described in ‘Solutions’ section. Use one well of a 6-well plate per clone.

     
  20. 20.

    Next day, rinse the MEFLUs seeded the previous day with 3 ml HBSS.

     
  21. 21.

    Add 2 ml of m-iPSC medium to the MEFLUs and place the plate back in the incubator.

     
  22. 22.

    The day of the picking, rinse twice the plates containing colonies with 0.5 ml of HBSS per well.

     
  23. 23.

    Add 0.5 ml m-iPSC medium to each well of the 24-well plates.

     
  24. 24.

    If there are more than 15 % of differentiated colonies in a given well, pick up manually the undifferentiated ones and plate them together. Otherwise, colony selection is not required, and iPSC from each clone can by collected in mass by scratching the bottom of the plate with a 1000 μl pipette.

     
  25. 25.

    Harvest all the detached colonies from each clone in 500 μl m-iPSC medium and transfer them to a well of a 24-well plate. Soak the 1000 μl tip in medium before picking the colonies, so they do not attach to its walls.

     
  26. 26.

    Gently pipette 5–7 times up and down with the pipette to disaggregate the colonies.

     
  27. 27.

    Split 1:3 to 1:6 into wells of a 6-well plate containing 2 ml m-iPSC and MEFLU feeder cells.

     
  28. 28.

    Shake the plate back and forth to homogenously distribute the m-iPS colonies.

     
  29. 29.

    Place the plate in the incubator and do not disturb for 48 h, afterwards, change medium every day.

     
  30. 30.

    Once independent clones are established on 6-well plates, they can be frozen, expanded, or further characterized.

     

Mouse iPSC Freezing

  1. 31.

    Cells have to be healthy, undifferentiated and in the growing phase before freezing.

     
  2. 32.

    Prepare freezing medium and keep it at 4 °C. Label cryovials and keep them at −20 °C until needed.

     
  3. 33.

    Wash twice the cells on the plate with KO-DMEM.

     
  4. 34.

    Eliminate KO-DMEM and add 0.5 ml 50 U/ml collagenase IV solution per well of a 6-well plate. Although most protocols recommend the use of trypsin [43], in our hands collagenase treatment preserves better the colonies as single entities without disaggregation. Colonies frozen as such show a higher recovery after thawing.

     
  5. 35.

    Incubate 5 to 7 min at 37 °C. Check that the border of the colonies starts to detach. If the colony folds back onto itself, go to next step; otherwise incubate 2–3 min more at 37 °C until most of colonies start to detach.

     
  6. 36.

    Very carefully, discard the collagenase solution without losing any colony.

     
  7. 37.

    Add 2 ml of complete m-iPSC medium, and gently pipette up and down 5 to 7 times to scrape attached colonies. Transfer the colonies to a 15 ml conical tube. Frozen iPSC are more viable when aggregated in colonies than as single cells.

     
  8. 38.

    Add another 2 ml of complete m-iPSC medium to the well and repeat the procedure to recover any remaining colony. Load colonies from up to three wells into one single 15 ml tube.

     
  9. 39.

    Add complete m-iPSC medium to the conical tube, adjusting the final volume to 15 ml.

     
  10. 40.

    Centrifuge at 200 g for 5 min.

     
  11. 41.

    Discard supernatant and resuspend cells in 3 ml cold freezing medium per original well.

     
  12. 42.

    Quickly aliquot the cellular suspension at 1 ml per cryovial, and immediately place them at −80 °C.

     
  13. 43.

    On the next day, store the frozen vials in a −150 °C freezer or in liquid nitrogen.

     

Mouse iPSC Thawing

  1. 44.

    Prepare feeder cells as described in steps 1–4 on protocol D, but instead of using 24-well plates, use 6-well plates. The day before thawing, rinse the cells with 1 ml HBSS and replace the medium in the plates with 2 ml m-iPSC medium per well.

     
  2. 45.

    Place a frozen cryovial containing m-iPSC cells in a 37 °C water bath (about 30 s) until it is almost thawed.

     
  3. 46.

    Spray the cryovial with 70 % ethanol and wipe it dry before placing it into the laminar flow hood.

     
  4. 47.

    Transfer the cells into a conical tube containing 10 ml of m-iPSC medium.

     
  5. 48.

    Centrifuge 5 min at 200 g.

     
  6. 49.

    Discard supernatant, tap the tube to dislodge the pellet and gently resuspend the cells in 1 ml pre-warmed m-iPSC medium.

     
  7. 50.

    Seed the iPSC suspension onto one well of a 6-well plate-containing MEFLU feeder cells.

     
  8. 51.

    Incubate the cells in a humidified chamber at 37 °C, 5 % CO2 for the next 2 days.

     
  9. 52.

    Change medium with 2.5 ml fresh m-iPSC medium every day.

     

Human iPSC Colony Derivation, Freezing and Thawing

The protocol for human iPSC derivation is very similar to the one already described for mouse iPSC derivation, but with several modifications: i) use h-iPSC medium instead of m-iPSC medium. ii) to compensate for the higher propensity of human iPSC to enter apoptosis, at the day of picking (step 4 in protocol D), add 100 μl of 100 μM ROCK inhibitor (10X) to each well of 24-well plate containing MEFLU feeder cells. ROCK inhibitor diminishes dissociation-induced apoptosis and increases cloning efficiency. In this context, it is also important to transfer directly the picked human colonies to the 24-well plate without any colony disaggregation. iii) detaching human iPSC colonies prior to freezing, requires longer incubation times with collagenase IV (20–30 min at 37 °C) (steps 34–35 protocol D). iv) during h-iPSC thawing (step 49 in protocol D), resuspend cells in 1 ml of h-iPSC medium containing 30 μM ROCK inhibitor (3X) and seed on feeder cells containing 2 ml h-iPSC medium. v) h-iPSC splitting ratio varies between 1:2 to 1:4, whereas m-iPSC splitting ratio fluctuates between 1:3 to 1:6.

Notes

  1. Note 1

    There are many cell types in the cell suspension; a good number of them are erythrocytes that are likely to be counted but will not attach to the plate and will be washed away the next day. Others like blood, nerve, or cartilage cells do attach, but will die off on the following days.

     
  2. Note 2

    Cell detachment by trypsinization requires the removal of all traces of serum and extracellular calcium ions from the medium. Therefore, if there is any problem in detaching the cells, wash again with PBS, add another 10 ml of PBS, and leave the plate at 37 °C for another 2–5 min.

     
  3. Note 3

    Skin samples have to be collected by a trained physician. Skin should be processed as soon as possible, ideally within few hours from extraction; otherwise, it will result in a serious reduction in cell production. It is very important that all tissue manipulations are carefully performed.

     
  4. Note 4

    Small pieces may be incubated in 60-mm Petri dishes containing about 2.5 ml Epilife with dispase. Alternatively, Epilife may be replaced by sHBSS-Amph.

     
  5. Note 5

    Keratinocytes are difficult to detach by TrypLE. After 10 min trysinization at 37 °C, some cells may remain attached. In this case, take out the supernatant-containing cells and inactivate it by diluting 1:10 with complete DMEM/F-12 medium. Add 10 ml TrypLE to the dish and incubate for another 5–10 min at 37 °C.

     
  6. Note 6

    When handling delicate explants from old individuals or from donors carrying diseases, use feeder cells. Once the explant is attached as described, add 20,000 NIH-3 T3 feeder cells in conditioned-complete D/F12 keratinocyte medium per cm2 of dish.

     
  7. Note 7

    Phoenix cells detach easily. Be careful when replacing medium. To assure the presence of the proteins required for virus production, culture Phoenix cells under selection: 300 μg/ml hygromicin (resistance in gag-pol construct), and 1ug/ml Diphtheria toxin (resistance in the env construct) for at least 1 week every few months.

     
  8. Note 8

    Cells to be transfected must be growing exponentially and close to confluency (Fig. 2e). Each well of a 6-well plate containing Phoenix cells transfected with one of the four reprogramming factors (MKOS) will produce, at least, 2 ml of filtered virus-containing supernatant. Mixing the supernatants of four wells (one per factor) will yield enough viral cocktail for reprogramming one 6 well plate of primary cells. If you are planning to infect more than one 6-well plate of primary cells, scale up the number of transfected Phoenix cells. Successful production of iPSC with only three transcription factors (OKS) has been reported [44]. If you are considering this strategy, it is enough to seed Phoenix cells in 3 wells of 6-well plate. The production of the three wells will suffice to infect one 6-wells plate containing primary cells.

     
  9. Note 9

    Other murine somatic cell types are also suitable for cellular reprogramming. Each cell type may have a different optimal density for infection, but usually they vary between 30 % to 40 % confluence. Make sure that target cells are in exponential growth during infection.

     
  10. Note 10

    Polybrene is a poly-cationic molecule that facilitates the interaction between virus particles and the cell membrane by neutralizing the negative charges on it. It also helps to reduce virus attachment to the filter membrane. The final polybrene concentration for MEF infection should be 4 μg/ml.

     
  11. Note 11

    Retrovirus should be used while they are still fresh. Do not freeze, ultracentrifuge or dilute retrovirus particles, otherwise you will seriously compromise the reprogramming efficiency. A high retroviral titter is key for iPSC generation. Some authors have suggested that centrifuging the mix of primary cells and viruses right after infection increases efficiency [45], but others prefer to skip this step [28].

     
  12. Note 12

    The time limit for iPSC passing is defined by the following criteria: (i) iPSC can not be kept on feeders older than 2 weeks, (ii) iPS colonies can not be more than 700 μm in diameter, (iii) colonies can not exceed 70 % confluency and (iv) colonies should exhibit very low levels of differentiation.

     

Acknowledgments

This work was supported by the MICINN-JDC and MICINN PLE2009-0091 and IPT-2011-1402-900000 grants. We are grateful to J.C. Izpisúa-Belmonte and T. Aasen from the Center of Regenerative Medicine in Barcelona (CRMB) for their help in establishing iPSC technology in our lab, as well as for the gift of human OKSM retroviral plasmids and Ecotropic Phoenix cells. We also thank S. Yamanaka for the gift of mouse OKSM retroviral plasmids, and M.V. Camarasa for technical advise on stem cell culture. We also thank the Balearic Islands University Institute for Biomedical Research (IUNICS) for the use of their facilities.

Conflict of interest

The authors declare no potential conflicts of interest.

Copyright information

© Springer Science+Business Media New York 2012