Generation of induced pluripotent stem cells from neural stem cells

PROTOCOL

Generation of induced pluripotent stem cells from neural stem cells Jeong Beom Kim, Holm Zaehres, Marcos J Arau´zo-Bravo & Hans R Scho¨ler Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Mu¨nster, Germany. Correspondence should be addressed to H.R.S. ([email protected]).

© 2009 Nature Publishing Group http://www.nature.com/natureprotocols

Published online 17 September 2009; doi:10.1038/nprot.2009.173

The generation of induced pluripotent stem (iPS) cells from mouse and human somatic cells by expression of defined transcription factors (Oct4, Sox2, c-Myc, Klf4, Nanog and Lin28) is a powerful tool for conducting basic research and investigating the potential of these cells for replacement therapies. In our laboratory, iPS cells have been generated from adult mouse neural stem cells (NSCs) by ectopic expression of either Oct4 alone (one factor; 1F) or Oct4 plus Klf4 (two factors; 2F). Successful reprogramming of mouse NSCs by 1F or 2F depends on endogenous expression of Sox2, Klf4 and c-Myc. Direct reprogramming of somatic stem cells to 1F or 2F iPS cells avoids expression of the oncogenes Klf4 and c-Myc and, hence, the development of tumors in chimeras and offspring derived from these cells. Here we present a detailed protocol for the derivation of NSCs from adult mouse brain (which takes 4 weeks), and generation of 1F (4–5 weeks) or 2F iPS cells (2–3 weeks) from adult mouse NSCs.

INTRODUCTION Induced pluripotent stem (iPS) cells have been generated from delivering the four reprogramming proteins (Oct4, Sox2, Klf4 and mouse and human somatic cells by overexpression of defined c-Myc)17,18. Protein transduction eliminates the risks of transgene transcription factors1–9. The resultant iPS cells closely resemble integration into the host genome accompanied with the use of embryonic stem cells (ESCs) at the molecular level and with expression vectors. However, the efficiencies are significantly lower respect to their differentiation potential both in vitro and in vivo. and protein production is more tedious. Retrovirus is the most Human iPS cells can be used to produce ‘patient-specific iPS efficient way to generate iPS but carries the risks of insertional cells’ to conduct drug discovery research to alleviate disease states mutagenesis. Non-integrating expression vectors or proteins are and in regenerative medicine with the potential to cure diseases9. generating iPS ‘safer’ but are much less efficient. In this protocol, The use of oncogenes such as c-Myc in the process of iPS generation we describe NSC derivation as well as the generation of 2F or 1F iPS can lead to the formation of tumors in chimeras and in offsprings cells from NSCs with retroviral gene transfer extended from derived from these iPS cells2. Therefore, the generation of iPS cells previously published protocols19,20. must avoid the use of the c-Myc and Klf4 oncogenes to render iPS cells suitable for clinical applications. Research into this area has Experimental design made it possible to generate iPS cells without the use of c-Myc10–12. OG2–ROSA26 mice. To produce compound homozygous mice We have reported generation of iPS cells from adult mouse stem for the neo/lacZ and Oct4-green fluorescent protein (GFP) transgenes (OG2), we crossed the OG2 mouse strain with the ROSA26 cells (neural stem cells (NSCs)) by direct reprogramming with transgenic mouse strain for several generations. The use of the either two transcription factors (Oct4 plus Klf4; 2F) or one Oct4-GFP transgene construct is beneficial in the case of low transcription factor (Oct4; 1F) (Fig. 1)11,12. The reprogramming efficiency is similar or higher when generating iPS from NSC than frequencies of iPS cell formation, and is mandatory to identify from fibroblasts (see Supplementary Table 2 in ref. 11, and ref. 12). and isolate iPS colonies. We then crossed homozygous OG2– The reprogramming target population of NSCs, which endogen- ROSA26 male mice with Imprinting Control Region (ICR) female mice to produce heterozygous pups, which we used to derive the ously express Sox2, Klf4 and c-Myc, as well as AP and SSEA-1, are reprogrammed earlier and more efficiently than fibroblasts, and are NSCs from 5-d-old mice19. The protocol works for all NSCs a unique source for studying the mechanisms of iPS cells generation, derived either on postnatal day 5 or from adult cells from diverse as they can be reprogrammed by one, two, three (3F) or four (4F) genetic background of mice. factors. NSC-derived 2F or 1F iPS cells have less transgene integrations compared with Culture conditions for Oct4 or Oct4+Klf4 embryonic stem cells Comparative analyses 4F iPS cells3,13. Considering clinical applicability, reducing the number of factors decreases the chance of retroviral insertional Neural stem cells (NSCs) 1F or 2F-induced mutagenesis. Embryonic stem cells pluripotent stem cells It will be interesting to discover how efficiently iPS cells can be generated with Oct4 alone by non-retroviral expression Figure 1 | Schematic figure depicting reprogramming of neural stem cells (NSCs) into a state of vectors, such as adenovirus or plasmid pluripotency. Reprogramming NSCs (green) into a pluripotent state (violet) by transduction with either 14–16 . Two recent studies Oct4 alone (one factor (1F)) or Oct4 plus Klf4 (two factor (2F) OK) and culturing under mouse embryonic transfection described generation of iPS cells by directly stem cell (ESC) culture conditions. 1464 | VOL.4 NO.10 | 2009 | NATURE PROTOCOLS

PROTOCOL


Derivation of NSCs. Mouse brain is removed from the animal and minced to produce a single-cell suspension; the cells are seeded in NSC medium and cultured for 5–7 d until neurospheres arise. Within 3–5 d, NSCs will outgrow from the attached neurospheres. It is important to characterize the NSC before the introduction of viruses. The expression levels of Oct4, Sox2, cMyc and Klf4 should be in NSCs, as shown in Figure 2. If not, the NSCs will not be successfully reprogrammed by ectopic expression of Oct4 or Oct4 plus Klf4, as the endogenous expression of Sox2, Klf4 and c-Myc complements the exogenous Oct4 expression in the process of iPS generation11. Retrovirus production. To produce retroviral expression vectors for iPS cell generation, retroviruses can either be produced as an ecotropic virus21, which can only infect mouse cells, or virus pseudotyped with the vesicular stomatitis virus surface protein (VSV-G)22, which can infect mammalian cells of all species. Advantage of ecotropic virus is the possibility to handle infections under safety level 1, whereas the VSV-G pseudotyping allows efficient infection with a small volume of concentrated virus.

MATERIALS REAGENTS Experimental animals OG2/ROSA26 mouse at postnatal day 5 ! CAUTION Experiments must comply with national and institutional regulations concerning the use of animals for research purposes. . Dulbecco’s modified Eagle medium: Nutrient Mixture F-12 (DMEM/F-12) (Invitrogen, cat. no. 11330-32) . Dulbecco’s modified Eagle medium (DMEM) (PAA Labs, cat. no. H15-005) . Heat-inactivated fetal bovine serum (FBS) (Biowest, cat. no. S1810-500) . Phosphate buffered saline (PBS), Ca2+ and Mg2+-free (PAA Labs, cat. no. H15-011) . L-Glutamine (Invitrogen, cat. no. 25030-081) . Nonessential amino acids (NEAA) solution (Invitrogen, cat. no. 11140-050) . Penicillin/streptomycin (Invitrogen, cat. no. 15070-063) . b-Mercaptoethanol (Invitrogen, cat. no. 21985-023) ! CAUTION b-Mercaptoethanol is toxic; avoid inhalation, ingestion or contact with skin. . Fibroblast growth factor 2 (FGF2) (Invitrogen, cat. no. 13256-029) . Epidermal growth factor (EGF) (R&D system, cat. no. 236-EG) . BSA solution, 7.5% (wt/vol) (Invitrogen, cat. no. 15260-037) . Trypsin/EDTA (Invitrogen; 0.25% (wt/vol), cat. no. 25200-056), 0.05% (wt/vol) cat. no. 25200-054) . Putrescine (Sigma, cat. no. P7630) . Progesterone (Sigma, cat. no. P6149) . Insulin (Sigma, cat. no. I6634) . Sodium selenite (Sigma, cat. no. S9133) . Apotransferrin (Intergen, cat. no. 4452-01) . Ultra-pure laminin (BD, cat. no. 354239) . Recombinant mouse leukemia inhibitory factor (LIF) (1,000 U ml 1 of ESGRO; Chemicon International, cat. no. ESG1107) . Poly-D-lysine hydrobromide (PDL; Sigma, cat. no. P7405) . 293T cells (American Type Culture Collection, cat. no. CRL11268)

100 Gene expression relative to day 3

Figure 2 | Quantitative real-time PCR analyses of expression levels of Oct4, Sox2, c-Myc and Klf4 in neural stem cells (NSCs) relative to embryonic stem cells (ESCs). RNA levels were determined by qRT-PCR using primers specific for endogenous transcripts. Endogenous relative expression levels of NSCs compared with those in ESCs. Transcripts levels were normalized to b-actin levels. Error bars shown are the averages with s.d. of three independent experiments. Experiments using laboratory animals complied with institutional and national guidelines.

ESCs NSCs

10 1 –1

10

10–2 10–3 10–4 10–5 10–6

Oct4

Sox2

c-Myc

Klf4

Transduction of NSC. Transduction efficiencies 50% or higher should be achieved by using a GFP virus when transducing NSCs. As the number of colonies generated with Oct4 alone or Oct4 with Klf4 or cMyc is much lower then with 3F or 4F, a sufficient transduction efficiency is mandatory. NSCs are maintained in NSC medium for up to 3 d and then on day 4 post-infection, the cells are cultured and maintained in ESC medium until iPS colonies appear. Isolation of NSC iPS. Oct4-GFP-positive iPS cell colonies composed of cells infected with Oct4 plus Klf4 (2F OK) will appear within 2–3 weeks and those infected with Oct4 alone (1F) within 4–5 weeks.

. FuGENE 6 transfection reagent (Roche Diagnostics, cat. no. 11814443001). . NSC medium (see REAGENT SETUP) . ESC medium (see REAGENT SETUP) . Mouse Embryonic Fibroblast (MEF) medium (see REAGENT SETUP) Retroviral vectors

. pMX vectors containing the cDNAs of Oct4 (Plasmid 13366) and Klf4 (Plasmid 13370) (Addgene)

. pCL-Eco packaging plasmid (Plamid 12371), retroviral gag–pol packaging plasmid (Plasmid 8449) and VSV-G expression plasmid (Plasmid 8454) (Addgene) . MX and MX-GFP (Cell Biolabs). EQUIPMENT . Tissue culture plates and dish: 6-well (SARSTEDT, cat. no. 9036082), 12-well plate (Nunc, cat. no. 098147), 24-well plate (SARSTEDT, cat. no. 7323082) and 100-mm dish (SARSTEDT, cat. no. 831802) . Filter unit, 0.45 mm (Nalgene Labware, cat. no. 165-0045) . Polypropylene conical tubes: 15 ml (SARSTEDT, cat. no. 8044901) and 50 ml (SARSTEDT, cat. no. 9041101) . Cell strainer, 40 mm (Falcon, cat. no. 352340) . Cotton-plugged glass Pasteur pipettes, 15 cm—sterilized by autoclaving . Hemocytometer, 0.1-mm deep (Hausser Scientific, cat. no. 1483) . Syringe, 1 ml (BD, cat. no. 309623) . Microdissection instruments: large forceps, 12-cm surgical scissors, 451-angled Dumont #5 forceps, 8-cm curved extra-fine Moria spring scissors and 13-cm long curved forceps (Fine Science Tools)—all sterilized by autoclaving . Stereomicroscope REAGENT SETUP NSC medium To prepare 500 ml of NSC medium, add 5 ml of L-glutamine, 5 ml of penicillin/streptomycin, 12.5 mg of insulin, 0.05 g of apotransferrin, sodium selenite at a final concentration of 30 nM, putrescine at a final concentration of 100 mM and progesterone at a final concentration of 20 nM to DMEM/F12, supplement with 10 ng ml 1

NATURE PROTOCOLS | VOL.4 NO.10 | 2009 | 1465

PROTOCOL of both FGF2 and EGF 10 ng ml 1, and bring up to a final volume of 500 ml with DMEM/F12. Store NSC medium at 4 1C for up to 1 week. ESC medium To prepare 500 ml of ESC medium, mix 409 ml of DMEM, with 75 ml of FBS, 5 ml of L-glutamine, 5 ml of penicillin/streptomycin, 5 ml of NEAA solution, 1 ml of b-mercaptoethanol and 50 ml of LIF. Store ESC medium

at 4 1C for up to 1 week. ! CAUTION b-Mercaptoethanol is toxic; avoid inhalation, ingestion or contact with skin. MEF medium To prepare 500 ml of MEF medium, mix 445 ml of DMEM, with 50 ml of FBS and 5 ml of penicillin/streptomycin. Store MEF medium at 4 1C for up to 1 week.

PROCEDURE Derivation of mouse NSCs TIMING 4 weeks 1| Obtain the whole brain of an adult mouse using appropriate methods in accordance with the institution’s regulations. ! CAUTION Experiments must comply with national and institutional regulations concerning the use of animals for research purposes. 2| Transfer the brain into a 100-mm tissue culture dish containing 10 ml of PBS. Transfer the brain into a 50-ml conical tube containing 5 ml of 0.25% (wt/vol) trypsin/EDTA, pipette the mixture up and down a few times with a 5-ml disposable pipette to dissociate the tissue, and incubate in a water bath for 10 min at 37 1C. 3| Triturate the undissociated tissue B30 times to achieve a single-cell suspension and then add 35 ml of pre-warm MEF medium. m CRITICAL STEP Avoid generating bubbles when triturating the tissue, as this will result in reduced cell viability. 4| Centrifuge the cell suspension for 5 min at 100g, room temperature (20 1C–25 1C), aspirate the supernatant and re-suspend the cells with 30 ml of fresh NSC medium by triturating them. Plate the cells into three 100-mm tissue culture dishes (10 ml per dish) and incubate the dishes at 37 1C, 5% CO2 for 5–7 d. The cells usually form neurospheres after 5–7 d in culture (Fig. 3a). 5| Transfer the neurospheres growing in suspension into a new 50-ml conical tube and dissociate the neurospheres by pipetting up and down into single cells. Centrifuge for 5 min at 100g, room temperature to remove the debris, discard the supernatant and re-suspend the cells with 30 ml of fresh NSC medium. Plate the cells into three 100-mm tissue culture dishes (10 ml per dish) and incubate at 37 1C, 5% CO2 for 5–7 d. Repeat this step 3–4 times. m CRITICAL STEP Adherent cells should remain on the tissue culture dishes to purify the NSCs under these culture conditions in each repeating passage. 6| Collect the neurospheres, and then place a single neurosphere into a single PDL/laminin double-coated 24-well plate23. Within 3–5 d, NSCs will be growing out from the attached neurospheres (Fig. 3b). Mechanically isolate the outgrowing NSCs from adherent neurosphere colonies (Fig. 3c). m CRITICAL STEP When you transfer the outgrowing NSCs from adherent neurospheres into the new plate, the adherent neurospheres should remain on the tissue culture dishes. ? TROUBLESHOOTING 7| Resuspend the cells (2 106) with 10 ml of NSC medium and transfer into a PDL/laminin double-coated 100-mm tissue culture dish. Incubate the cells at 37 1C, 5% CO2. We usually achieve a monolayer of cells with 70%–80% confluency within 3–5 d.

1466 | VOL.4 NO.10 | 2009 | NATURE PROTOCOLS

n

s

br ai e

SC N

Figure 3 | Derivation and characterization of a b c e neural stem cells (NSCs) isolated from an OG2–ROSA26 transgenic mouse brain. (a) Cells Oct4 isolated from mouse brain tissue, form Nanog neurospheres in NSC medium within 7 d. (b) Neurospheres attach to the culture dish Sox2 within 2–3 d of culture; outgrowing cells with an 100 μm 100 μm 100 μm Pax6 extensive lattice. (c) Bipolar morphology of Nestin NSCs expanding into the NSC medium d Tuj1 GFAP O4 Olig2 containing Fibroblast growth factor 2 (FGF2) Blbp and epidermal growth factor (EGF). (d) In vitro Emx2 differentiation of NSCs into neurons β-actin (Tuj1), astrocytes (GFAP) and oligodendrocytes (O4). Scale bars, 100 mm. (e) RT-PCR 100 μm 100 μm 100 μm analysis of expression levels of NSC marker genes and b-actin (control) in embryonic stem cells (ESCs), NSCs and embryonic brain (e brain). Experiments using laboratory animals complied with institutional and national guidelines.

s

8| Characterize the cells for their in vitro differentiation (Fig. 3d), expression level of NSC marker genes (Fig. 3e) and expression level of the four transcription factors (Fig. 2) refer to reference 11.

ES C


PROTOCOL


Retrovirus production TIMING 1 week 9| Plate 293T cells in MEF medium in 100-mm tissue culture dishes 24 h before transfection, so that the cells will have grown to 70%–80% confluency at the time of transfection. ! CAUTION When producing the VSV-G pseudotyped retrovirus, refer to the local biosafety guidelines of the institution and handle virus production under S2/BL2 conditions. 10| Produce either option A, an ecotropic virus; or option B, a VSV-G pseudotyped virus. (A) Ecotropic virus (i) Co-transfect 293T cells with 3 mg of retroviral vector, 3 mg of pCL-Eco and 18 ml of FuGENE 6 (Roche, Indianapolis, IN, USA) per plate, according to the supplier’s instructions. (B) VSV-G pseudotyped virus (i) Co-transfect 293T cells with 3-mg retroviral vector, 2-mg retroviral gag–pol packaging plasmid, 1-mg VSV-G and 18 ml of FuGENE 6 (Roche) per plate, according to the supplier’s instructions. 11| Collect the virus supernatant from every plate 48 h after transfection using plastic pipettes and filter the supernatant through a 0.45-mm filter. 12| Handle the filtered supernatant of ecotropic viruses using option A or VSV-G pseudotyped viruses using option B in the following manner: (A) Ecotropic virus (i) The filtered supernatant is ready to use, freeze in 1 ml aliquots at 80 1C until use. Avoid repeated freeze–thaw cycles. (B) VSV-G pseudotyped virus (i) Transfer the filtered supernatant to ultracentrifugation tubes and centrifuge at 80,000g for 90 min at 4 1C in a Beckmann SW28 swinging bucket rotor (Beckmann, Fullerton, CA, USA). Carefully aspirate the supernatant. An opaque pellet should be visible at the bottom center of the tube. Add 1 ml of DMEM to the tube, re-suspend the pellet, and aliquot the viral suspension into ten freezing vials and store at 80 1C until use. Avoid repeated freeze–thaw cycles. 13| For cell titer determination, plate 1 105 293T cells per well in MEF medium in a 6-well plate and after 6 h in culture add 1 ml of ecotropic supernatant or 100 ml of concentrated VSV-G pseudotyped virus. After 24 h, aspirate the medium, wash the cells with 2 ml of PBS three times and supplement with fresh medium. At 48 h after infection, analyze the cells for GFP expression using a fluorescence activated cell sorter. The titer is represented as infectious units (IU) per ml and can be calculated as: the (percentage of GFP+ cells) 105 the dilution factor. For vectors without a reporter gene, quantitative real-time PCR can be used to determine the number of vector DNA molecules per ml24.

Retroviral transduction of NSCs TIMING 1 d 14| Infection day 1: seed NSCs from Step 7 at 5 104 (2 ml) per well for a 6-well plate with NSC medium and incubate at 37 1C, 5% CO2 for 6 h. 15| Add virus supernatant: Oct4 alone or Oct4 plus Klf4 (equal amounts). You can use a defined multiplicity of infection (MOI) (number of infectious units used per cell to be infected) and/or a GFP control virus to calculate transduction efficiencies in parallel. Use 250 ml of unconcentrated virus or 100 ml of concentrated virus (from four 100-mm dishes) or a defined MOI of 20. Add 6-mg protamine sulfate per ml of culture to enhance the likelihood that the virus will bind to the cells. 16| Incubate at 37 1C, 5% CO2 for 24 h.

Culturing transduced NSCs TIMING 2F OK: 2–3 weeks and 1F: 4–5 weeks 17| Day 2: aspirate the medium, wash the NSCs with 3 ml of PBS three times and add 2 ml of fresh NSC medium. 18| Day 4: aspirate the NSC medium and add 2 ml of fresh ESC medium. 19| Change the medium, as in Step 19, every other day and maintain the cells in culture until the emergence of iPS cell colonies that are big enough to be picked. ? TROUBLESHOOTING

Picking and expanding iPS cell colonies TIMING 5 d 20| Aspirate the medium and add 3 ml of PBS per plate. 21| Aspirate the PBS and add 2 ml of ESC medium per plate.


PROTOCOL 22| Pick the desired colonies from each plate using fire-polished Pasteur pipettes. Transfer each colony into one well of a 12-well plate containing an MEF feeder layer25, add 1 ml of ESC medium per well, and incubate the plate at 37 1C, 5% CO2. m CRITICAL STEP To prevent cross contamination with clones that may have originated in the same well, the medium should be discarded after picking the single colony and the whole well must be washed with PBS. Pick each colony with a new pipette. 23| Change the medium every other day until the cells reach 70%–80% confluency. 24| Aspirate the medium and wash each well with 2 ml of PBS.


25| Add 0.5 ml of 0.05 % (wt/vol) trypsin/EDTA per well and incubate the plates at 37 1C, 5% CO2 for 5 min. 26| Add 0.5 ml of MEF medium per well and carefully pipette up and down to obtain a single-cell suspension. 27| Transfer the cell suspension to a 15-ml conical tube containing 4.5 ml of MEF medium. Centrifuge the tube at 200g for 4 min, room temperature and aspirate the supernatant. 28| Re-plate the cells in 6-well plates containing an MEF feeder layer, add 2 ml ESC medium, and incubate the plates at 37 1C, 5% CO2. Within 3–4 days (70–80% confluency) after re-plating, iPS cells can be maintained like mouse ESCs.

Freezing of iPS cells TIMING 1 h 29| Aspirate the medium and wash the cells with 2 ml of PBS. 30| Aspirate the PBS, add 0.5 ml of 0.05 % (wt/vol) trypsin/EDTA, and incubate at 37 1C for 5 min. 31| Add 0.5 ml of MEF medium and carefully pipette up and down to obtain a single-cell suspension. 32| Transfer the cell suspension to a 15-ml conical tube containing 4.5 ml of MEF medium. Centrifuge the tube for 4 min at 200g, room temperature and aspirate the supernatant. 33| Re-suspend the cells with ESC medium (containing 10% (vol/vol) dimethyl sulfoxide (DMSO)) at a final concentration of 1 106 cells per ml and aliquot 1 ml of cells per freezing vial. 34| Keep the vials in a cell-freezing container overnight at 80 1C and then transfer them into a liquid nitrogen tank the next day. ! CAUTION Liquid nitrogen is hazardous; use personal protective clothing.

TIMING Steps 1–8, Derivation of mouse NSCs: 4 weeks Steps 9–13, Retrovirus production: 1 week Steps 14–16, Retroviral transduction of NSCs: 1 d Steps 17–19, Culturing transduced NSCs: 2F OK: 2–3 weeks and 1F: 4–5 weeks Steps 20–28, Picking and expanding iPS cell colonies: 5 d Steps 29–34, Freezing of iPS cells: 1 h ? TROUBLESHOOTING Troubleshooting advice can be found in Table 1. TABLE 1 | Troubleshooting table. Step 6

Problem Heterogenous outgrowing cells

Possible reason Not enough purification in Step 5

Solution Repeat Step 5 until the appearance of homogenous neural stem cells (NSCs)

19

No induced pluripotent stem (iPS) cell colonies appear

Quality of Retrovirus or quality of the feeder layers

The infectivity of Retrovirus is critical for the successful generation of iPS cells. A 50% transduction efficiency should be attained with a GFP virus, when transducing NSCs. Make fresh virus from a new batch of frozen 293T cells. Avoid repeated freeze–thaw cycles. Use good quality MEF feeder cells


2F iPS-3 2F iPS-2 2F iPS-1 ESC-2 ESC-3 ESC-1 1F iPS-2 1F iPS-3 1F iPS-1 NSC-2 NSC-3 NSC-1 NSC 1F-2 NSC 1F-3 NSC 1F-1 700

c

600

500

16 Lin28

14

400

300

Oct4 Sox2 Rex1 Utf1 Fgf4 Cripto Eras Esg1 β-actin

16

Klf4 Oct4

Nanog

14

c-Myc

12

10 Sox2 Nanog

8 6

Klf4 Oct4 Sox2 Lin28

10 8 6 4

4 2

C2 C3 C4

Nanog

200

12 c-Myc

d

1F iPS

b

ES C N SC

a

1F iPS

Figure 4 | Generation of two factor (2F) and one factor (1F) induced pluripotent stem (iPS) cells from mouse neural stem cells (NSCs). (a) Hierarchical clustering analysis of global gene expression in pluripotent cell populations by cDNA microarrays. (b) The pluripotent cell populations (embryonic stem cells (ESCs), 2F and 1F iPS cells) are connected with red branches and the somatic cell populations with blue branches (donor NSCs and 1F-derived NSCs). Each sample is shown in triplicate. RT-PCR analysis of expression levels of pluripotency marker genes and b-actin (control) in 1F iPS cell clones (Clone2: C2, Clone3: C3 and Clone4: C4), ESCs (positive control), and NSCs (negative control). (c) Scatter plots of the global gene expression pattern of 1F iPS cells compared with ESCs (left panel) and with 2F iPS cells (right panel) by cDNA microarrays. (d) Black lines indicate twofold changes in gene expression levels. Upregulated and downregulated genes in 1F iPS compared with ESCs or 2F iPS are shown in blue or red, respectively. The positions of the pluripotency genes Oct4, Nanog, Sox2, c-Myc, Klf4 and Lin28 are indicated in scatter plots. Haematoxylin–eosin stained sections of teratomas derived from 1F iPS cells (clone 2; 1106 cells) in a nude mouse host after 4 weeks. Teratomas of 1F iPS cells (clone 2) contain all three embryonic germ layers: neural rosettes (ectoderm), epithelium (endoderm) and muscle (mesoderm). Scale bars, 100 mm. Experiments using laboratory animals complied with institutional and national guidelines. Figure reproduced with permission12.

1F iPS


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2 2

4

6

8 10 ESC

Neural rosette

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Cuboidal epithelium

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Muscle

ANTICIPATED RESULTS This protocol should reliably yield enriched populations of NSCs from neurospheres. Neurospheres originating from an adult mouse brain will form within 7 d in culture in the presence of FGF2 and EGF in NSC medium (Fig. 3a)19. Approximately, 1 105 cells will form 20–30 neurospheres depending on cell density or culture conditions, which do not support the differentiation and long-term existence of the majority of the cells. Single neurospheres are used to form about 30–40 neurospheres during the repeat passaging procedure in Step 5. This is a selection step to obtain the purified NSCs. The protocol allows purification of NSCs (without the need for genetic manipulation or drug selection) by morphology. NSCs can be isolated and expanded from neurospheres as a monolayer (self-renewal). They can differentiate into Glial fibrillary acidic protein (GFAP)-positive astrocytes, Neuronal class III b-tubulin (Tuj1)-positive neurons and Oligodendrocyte marker (O4)-positive oligodendrocytes (Fig. 3d). The established NSCs will express the representative NSC markers but not the pluripotent markers Oct4 and Nanog (Fig. 3e). Compared with ESCs, endogenous expression of Sox2 and c-Myc should be higher in NSCs, and Klf4 should be expressed at about an eightfold lower level in NSCs, (Fig. 2). Oct4–GFP-positive iPS cell colonies will appear in cells infected with Oct4 plus Klf4 (2F OK) within 2–3 weeks of culture and in those infected with Oct4 alone (1F) within 4–5 weeks of culture. We have shown a reprogramming efficiency of 0.11% (11 iPS colonies out of 50,000 starting cells, 46% transduction efficiency for each factor) for the Oct4, Klf4 approach and of 0.014% (3 iPS colonies out of 50,000 starting cells) for the Oct4 approach11,12. Hierarchical clustering of global gene expression analysis revealed that the pluripotent populations (2F iPS, ESCs and 1F iPS; red branch) clustered distantly from the somatic populations (NSCs and NSC 1F: 1F-derived NSCs; blue branch) (Fig. 4a). All three 1F clones expressed typical ESC marker genes (Fig. 4b). We also found a similar gene expression pattern between 1F iPS cells and ESC or 2F iPS cells by scatter plots (Fig. 4c). When injected into mice the 1F iPS derived teratoma contained tissues of all three germ layers (Fig. 4d). NSC-derived 1F iPS cells have a developmental potential similar to mouse ESCs both in vitro and in vivo11,12.

Note: Supplementary information is available via the HTML version of this article. ACKNOWLEDGMENTS We thank Martin Zenke, RWTH Aachen for conducting microarrays and Jeanine Mu¨ller-Keuker for illustrations. This work has been supported in part by the Deutsche Forschungsgemeinschaft DFG grant SCHO 340/4-1 and the German Federal Ministry of Education and Research BMBF grant S01GN 0811. AUTHOR CONTRIBUTIONS J.B.K.: protocol design, generation and characterization of NSC and iPS cells, preparation of manuscript, H.Z.: protocol design, generation

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