Project description:In the past years, transdifferentiation from one lineage to another has become an important research field. In that context, we recently reported the direct reprogramming of mouse embryonic fibroblasts (MEFs) into induced neural stem cells (iNSCs). In this study, we, for the first time, successfully reprogrammed these iNSCs into induced pluripotent stem cells (iPSCs), which we termed iNSC-derived iPSCs (iNdiPSCs). iNdiPSCs proved to be truly pluripotent, as shown by a pluripotent gene expression profile and the ability to differentiate into all three germ layers both, in vitro and in vivo. We could further show that iNdiPSCs do not retain an epigenetic memory neither from iNSCs nor MEFs. In conclusion, our data validate the stable somatic stem cell status of iNSCs and demonstrate their potential to give rise to bona fide iPSCs that are indistinguishable from other iPSCs or embryonic stem cells. Cell culture conditions: iNdiPSCs were grown on feeder cells in 2i medium (Knockout DMEM supplemented with 20% Knockout serum replacement, 0.2x -Mercaptoethanol (all GIBCO), 1x nonessential amino acids and 1x penicillin/streptomycin/glutamine (PSG) (both PAA), 1nM PD0325901, 3nM CHIR99021 (both Axon Medchem), and 1000 U/ml leukemia inhibitory factor [in house preparation]). Virus production and infection: Commercially available pMX retroviruses expressing mouse Oct4 (Addgene # 13366) and Klf4 (# 13370) were transfected in 293T cells in iNSC medium: DMEM/F-12 supplemented with B27 (both GIBCO), 10ng/ml EGF, 10ng/ml bFGF (both Peprotech) and 1x PSG. Supernatant was collected after 48 h, filtered and used for infection of 5 x 104 iNSCs. After 48 h cells were cultured in 2i medium. Alkaline phosphatase staining Cells were fixed for 5 min in 4% paraformaldehyde (PFA) at room temperature (RT), washed twice with phosphate buffered saline (PBS) (PAA) and stained with the SIGMA FAST Fast Red TR/Napthol AS-MX kit (product no. F4648) for 20 min in the dark at RT. Chromosome spread: Cells were subjected to 100ng/ml KaryoMax Colcemid (GIBCO) for 3 h at 37C. Cells were harvested and resuspended by slowly adding 4 ml 0.56% prewarmed KCl, incubated for 15 min at 37C and spun down at 900 rpm for 5 min. Fresh fixative (3 parts methanol plus 1 part acetic acid) was very slowly added to the pellet to a final volume of 3 ml and incubated for 30 min at RT. Cells were subsequently washed 3 times in fixative and resuspened in fixative. Approximately 20 microl were dropped from a height of about 2 m on a dry slide. Drops were air dried for 30 min and mounted with DAPI containing mounting solution. RNA extraction, cDNA synthesis and quantitative real time PCR (qRTPCR) RNA was extracted using the QIAGEN RNeas Mini Kit (cat no. 74106) and cDNA synthesis performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRTPCR was carried out using iTaq SYBR Green Supermix With ROX (Bio-Rad). Oct4 promoter methylation analysis: Bisulfite conversion was performed using the QIAGEN EpiTectﾮ Bisulfite Kit (Cat no. 59104). Followed by nested PCR using a set of target region specific primers. The first PCR was performed using 1micro-l of bisulfite converted and purified DNA, SuperTaq polymerase (Promega) according to the manufacturers protocol and a total of 35 cycles with denaturation at 94C for 30 s, followed by 30 s of annealing at 50C and 72C elongation for 1 min with an initial denaturation at 94C and a final elongation step at 72C for 3 min each. For the second PCR, we used Taq polymerase according to the manufacturers protocol (New England Biolabs) and 1 microl of the first PCR product with denaturation at 95C for 30 s, followed by 40 cycles of denaturation for 30 s at 95C, annealing for 30 s at 55C and elongation for 1 min at 68C, and finally an additional elongation step for 5 min at 68C. The amplified fragments were resolved on a 1.5% agarose gel, purified using the QIAGEN QIAquick Gel Extraction Kit (Cat no. 28706) and subsequently cloned into the pCRII-TOPO-vector using the TOPO TA Cloning Kit (Invitrogen). The cloned products were transformed into One Shot TOP10 competent bacteria (Invitrogen) and plated on agar plates. The picked colonies were sent to GATC for sequencing. The results were then analyzed with the QUMA software (online available at www.quma.cdb.riken.jp). Immunocytochemistry: Cells were fixed for 5 min in 4% PFA at RT, washed twice in PBS, permeabilized with 0.1% Triton X-100 for 10 min, and washed again twice in PBS. Followed by blocking with 5% bovine serum albumin (BSA) (GIBCO) for 1 h shaking at RT. Subsequently the first antibody was added in 1% BSA and incubated for 1 h shaking at RT. The cells were again washed twice in PBS and subjected to the second antibody in 1% BSA for 1 h shaking at RT and then washed three times with PBS, including DAPI in the second wash. The following primary antibodies were used: anti-Nanog (REC-RCAB0002P-F, Cosmo Bio Co.) 1:1000 anti SSEA1, (MC-480, Millipore), 1:160, anti-3-Tubulin (T8660, Sigma), 1:2000, anti-Smooth muscle actin (M0851, DAKO), 1:200, anti-Sox17 (AF1924, R&D Systems), 1:100. The following secondary antibodies were used: Alexa Fluor 568 goat anti-rabbit, rabbit anti-mouse, and rabbit anti-goat (Invitrogen), all 1:2000. DNA extraction and PCR: DNA extraction was performed using the QIAGEN QIAampﾮ DNA Mini Kit (cat no. 51306). PCR was carried out using Taq polymerase including buffers from New England Biolabs, following their recommended protocol. The following primers were used for genotyping: pMX-Oct4, -Klf4, -Sox2, -c-Myc and -Brn4. In vitro differentiation: First, hanging drops were formed placing 20ﾵl drops containing 600 cells each on the lid of a 10cm cell culture dish, which was then inverted. After 3 days, hanging drops were transferred to gelatin-coated dishes and cultured in standard MEF medium (DMEM containing 20% fetal calf serum (both GIBCO) and 1x PSG), which was also used for hanging drop formation. After approximately 10-14 days the cells were fixed and stained as described above. Teratoma formation: 1 x 106 iNdiPSCs were injected subcutaneously into SCID mice. Tumors were resected after 4 weeks and fixed in 4% PFA. Paraffin sections were cut, mounted on glass slides and stained with haematoxylin and eosin. Animal handling was performed in accordance with the German animal protection law and the guidelines of the Max Planck Institute. Microarray analysis: Purified cRNA samples were prepared with the linear TotalPrep RNA Amplification Kit (Ambion) from 500 ng original RNA, involving synthesis of double-stranded cDNA by T7-promoter-linked oligo(dT) primers and 14 h of in vitro transcription incorporating biotin-labeled nucleotides. Hybridization onto MouseRef 8 v2.0 Expression BeadChips (Illumina) was performed according to the manufacturerﾒs recommendations. Chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using an iScan reader (Illumina). The bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix robust multiarray analysis (RMA) background correction model. Variance stabilization was performed by log2 scaling, and gene expression normalization was calculated with the method implemented in the lumi package of R-Bioconductor. 8 samples were analyzed. MEF: Regular mouse embryonic fibroblasts with Tet-On background, 1 replicate NSC: Regular mouse neural stems cells with Tet-On background, 1 replicate iNSC: mouse induced neural stems cells, 1 replicate iNdiPSC-1: mouse induced pluripotent cells (IPSs) derived from iNSC (clon 1), 1 replicate iNdiPSC-4: mouse induced pluripotent cells (IPSs) derived from iNSC (clon 4), 1 replicate NSC2FiPSC: mouse induced pluripotent cells (IPSs) derived from regular NSC (using reprogramming 2 factors), 1 replicate MEF4FiPSC: mouse induced pluripotent cells (IPSs) derived from regular MEF (using reprogramming 4 factors), 1 replicate ESC: regular mouse embryonic stem cells with Tet-On background, 1 replicate

Project description:In the past years, transdifferentiation from one lineage to another has become an important research field. In that context, we recently reported the direct reprogramming of mouse embryonic fibroblasts (MEFs) into induced neural stem cells (iNSCs). In this study, we, for the first time, successfully reprogrammed these iNSCs into induced pluripotent stem cells (iPSCs), which we termed iNSC-derived iPSCs (iNdiPSCs). iNdiPSCs proved to be truly pluripotent, as shown by a pluripotent gene expression profile and the ability to differentiate into all three germ layers both, in vitro and in vivo. We could further show that iNdiPSCs do not retain an epigenetic memory neither from iNSCs nor MEFs. In conclusion, our data validate the stable somatic stem cell status of iNSCs and demonstrate their potential to give rise to bona fide iPSCs that are indistinguishable from other iPSCs or embryonic stem cells. Cell culture conditions: iNdiPSCs were grown on feeder cells in 2i medium (Knockout DMEM supplemented with 20% Knockout serum replacement, 0.2x -Mercaptoethanol (all GIBCO), 1x nonessential amino acids and 1x penicillin/streptomycin/glutamine (PSG) (both PAA), 1nM PD0325901, 3nM CHIR99021 (both Axon Medchem), and 1000 U/ml leukemia inhibitory factor [in house preparation]). Virus production and infection: Commercially available pMX retroviruses expressing mouse Oct4 (Addgene # 13366) and Klf4 (# 13370) were transfected in 293T cells in iNSC medium: DMEM/F-12 supplemented with B27 (both GIBCO), 10ng/ml EGF, 10ng/ml bFGF (both Peprotech) and 1x PSG. Supernatant was collected after 48 h, filtered and used for infection of 5 x 104 iNSCs. After 48 h cells were cultured in 2i medium. Alkaline phosphatase staining Cells were fixed for 5 min in 4% paraformaldehyde (PFA) at room temperature (RT), washed twice with phosphate buffered saline (PBS) (PAA) and stained with the SIGMA FAST Fast Red TR/Napthol AS-MX kit (product no. F4648) for 20 min in the dark at RT. Chromosome spread: Cells were subjected to 100ng/ml KaryoMax Colcemid (GIBCO) for 3 h at 37C. Cells were harvested and resuspended by slowly adding 4 ml 0.56% prewarmed KCl, incubated for 15 min at 37C and spun down at 900 rpm for 5 min. Fresh fixative (3 parts methanol plus 1 part acetic acid) was very slowly added to the pellet to a final volume of 3 ml and incubated for 30 min at RT. Cells were subsequently washed 3 times in fixative and resuspened in fixative. Approximately 20 microl were dropped from a height of about 2 m on a dry slide. Drops were air dried for 30 min and mounted with DAPI containing mounting solution. RNA extraction, cDNA synthesis and quantitative real time PCR (qRTPCR) RNA was extracted using the QIAGEN RNeas Mini Kit (cat no. 74106) and cDNA synthesis performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRTPCR was carried out using iTaq SYBR Green Supermix With ROX (Bio-Rad). Oct4 promoter methylation analysis: Bisulfite conversion was performed using the QIAGEN EpiTectﾮ Bisulfite Kit (Cat no. 59104). Followed by nested PCR using a set of target region specific primers. The first PCR was performed using 1micro-l of bisulfite converted and purified DNA, SuperTaq polymerase (Promega) according to the manufacturers protocol and a total of 35 cycles with denaturation at 94C for 30 s, followed by 30 s of annealing at 50C and 72C elongation for 1 min with an initial denaturation at 94C and a final elongation step at 72C for 3 min each. For the second PCR, we used Taq polymerase according to the manufacturers protocol (New England Biolabs) and 1 microl of the first PCR product with denaturation at 95C for 30 s, followed by 40 cycles of denaturation for 30 s at 95C, annealing for 30 s at 55C and elongation for 1 min at 68C, and finally an additional elongation step for 5 min at 68C. The amplified fragments were resolved on a 1.5% agarose gel, purified using the QIAGEN QIAquick Gel Extraction Kit (Cat no. 28706) and subsequently cloned into the pCRII-TOPO-vector using the TOPO TA Cloning Kit (Invitrogen). The cloned products were transformed into One Shot TOP10 competent bacteria (Invitrogen) and plated on agar plates. The picked colonies were sent to GATC for sequencing. The results were then analyzed with the QUMA software (online available at www.quma.cdb.riken.jp). Immunocytochemistry: Cells were fixed for 5 min in 4% PFA at RT, washed twice in PBS, permeabilized with 0.1% Triton X-100 for 10 min, and washed again twice in PBS. Followed by blocking with 5% bovine serum albumin (BSA) (GIBCO) for 1 h shaking at RT. Subsequently the first antibody was added in 1% BSA and incubated for 1 h shaking at RT. The cells were again washed twice in PBS and subjected to the second antibody in 1% BSA for 1 h shaking at RT and then washed three times with PBS, including DAPI in the second wash. The following primary antibodies were used: anti-Nanog (REC-RCAB0002P-F, Cosmo Bio Co.) 1:1000 anti SSEA1, (MC-480, Millipore), 1:160, anti-3-Tubulin (T8660, Sigma), 1:2000, anti-Smooth muscle actin (M0851, DAKO), 1:200, anti-Sox17 (AF1924, R&D Systems), 1:100. The following secondary antibodies were used: Alexa Fluor 568 goat anti-rabbit, rabbit anti-mouse, and rabbit anti-goat (Invitrogen), all 1:2000. DNA extraction and PCR: DNA extraction was performed using the QIAGEN QIAampﾮ DNA Mini Kit (cat no. 51306). PCR was carried out using Taq polymerase including buffers from New England Biolabs, following their recommended protocol. The following primers were used for genotyping: pMX-Oct4, -Klf4, -Sox2, -c-Myc and -Brn4. In vitro differentiation: First, hanging drops were formed placing 20ﾵl drops containing 600 cells each on the lid of a 10cm cell culture dish, which was then inverted. After 3 days, hanging drops were transferred to gelatin-coated dishes and cultured in standard MEF medium (DMEM containing 20% fetal calf serum (both GIBCO) and 1x PSG), which was also used for hanging drop formation. After approximately 10-14 days the cells were fixed and stained as described above. Teratoma formation: 1 x 106 iNdiPSCs were injected subcutaneously into SCID mice. Tumors were resected after 4 weeks and fixed in 4% PFA. Paraffin sections were cut, mounted on glass slides and stained with haematoxylin and eosin. Animal handling was performed in accordance with the German animal protection law and the guidelines of the Max Planck Institute. Microarray analysis: Purified cRNA samples were prepared with the linear TotalPrep RNA Amplification Kit (Ambion) from 500 ng original RNA, involving synthesis of double-stranded cDNA by T7-promoter-linked oligo(dT) primers and 14 h of in vitro transcription incorporating biotin-labeled nucleotides. Hybridization onto MouseRef 8 v2.0 Expression BeadChips (Illumina) was performed according to the manufacturerﾒs recommendations. Chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using an iScan reader (Illumina). The bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix robust multiarray analysis (RMA) background correction model. Variance stabilization was performed by log2 scaling, and gene expression normalization was calculated with the method implemented in the lumi package of R-Bioconductor.

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Reprogramming to pluripotency through a somatic stem cell intermediate.

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Reprogramming to pluripotency through a somatic stem cell intermediate.

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