Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Direct Reprogramming of Fibroblasts into Neural Stem Cells by Defined Factors

ABSTRACT: Recent advances in the stem cell biology have revealed that cell type-specific transcription factors could reset the somatic memory and induce direct reprogramming into specific cellular identities. The induction of pluripotency in terminally differentiated cells has been a major achievement in the field of direct reprogramming. Recent studies have shown that fibroblasts could be directly converted into specific cell types, such as neurons, cardiomyocytes, blood progenitor cells, and epiblast stem cells, without first passing through an induced pluripotent stem cell state3-7. However, direct reprogramming of differentiated cells into somatic stem cell types has not been described yet4. Here we show that a combination of neural-specific transcription factors (Sox2, Klf4, c-Myc, Brn4/Pou3f4 or Sox2, Klf4, c-Myc, Brn4, E47/Tcf3) can induce a neural stem cell (NSC) fate on the fibroblasts. Induced neural stem cells (iNSCs) showed morphology, gene expression, epigenetic features, differentiation potential, and functionality similar to wild-type NSCs. Therefore, our data suggest that cell type-specific defined factors can induce specific stem cell identities on somatic cells. Fibroblasts (5 x 104 cells) were infected with retroviruses for two days, and cells were maintained in NSC media: DMEM/F-12 supplemented with N2 or B27 supplements (Gibco-BRL), 10 ng/ml EGF, 10 ng/ml bFGF (both from Invitrogen), 50 ug/ml BSA (Fraction V Gibco-BRL), and 1x penicillin/streptomycin/glutamine (Gibco-BRL). In order to establish stable iNSC lines, were either manually picked mature iNSC clumps or passaged and seeded the whole dishes of cells onto either gelatin- or laminin-coated dishes. RNA samples to be analysed on microarrays were prepared using QIAGEN RNeasy columns with on-column DNA digestion. 500 ng of total RNA per sample was used as input RNA into a linear amplification protocol (Ambion) involving synthesis of T7-linked double-stranded cDNA and 12 h of in-vitro transcription incorporating biotin-labelled nucleotides. Purified and labelled cRNA was hybridised onto MouseRef-8 v2 expression BeadChips (Illumina) for 18 h according to the manufacturer's instructions. After washing, as recommended, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using iScan reader (Illumina) and accompanying software. Samples were hybridised as biological replicates. 9 samples were analyzed. Fibroblast: CF1 Mouse embryonic fibroblasts, 1 biological rep Control NSC: OG2-Rosa Neural Stem Cells, 2 biological rep 4F iNSC (late): 4 factors induced Neural Stem Cells (late passage), 2 biological rep 4F iNSC (early): 4 factors induced Neural Stem Cells (early passage), 2 biological rep 5F iNSC: 5 factors induced Neural Stem Cells, 2 biological rep

ORGANISM(S): Mus musculus

SUBMITTER: Marcos Araúzo-Bravo

PROVIDER: E-GEOD-30500 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Direct reprogramming of fibroblasts into neural stem cells by defined factors.

Han Dong Wook DW Tapia Natalia N Hermann Andreas A Hemmer Kathrin K Höing Susanne S Araúzo-Bravo Marcos J MJ Zaehres Holm H Wu Guangming G Frank Stefan S Moritz Sören S Greber Boris B Yang Ji Hun JH Lee Hoon Taek HT Schwamborn Jens C JC Storch Alexander A Schöler Hans R HR

Cell stem cell 20120322 4

Recent studies have shown that defined sets of transcription factors can directly reprogram differentiated somatic cells to a different differentiated cell type without passing through a pluripotent state, but the restricted proliferative and lineage potential of the resulting cells limits the scope of their potential applications. Here we show that a combination of transcription factors (Brn4/Pou3f4, Sox2, Klf4, c-Myc, plus E47/Tcf3) induces mouse fibroblasts to directly acquire a neural stem c ...[more]

PMID: 22445517

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Project description:Recent advances in the stem cell biology have revealed that cell type-specific transcription factors could reset the somatic memory and induce direct reprogramming into specific cellular identities. The induction of pluripotency in terminally differentiated cells has been a major achievement in the field of direct reprogramming. Recent studies have shown that fibroblasts could be directly converted into specific cell types, such as neurons, cardiomyocytes, blood progenitor cells, and epiblast stem cells, without first passing through an induced pluripotent stem cell state3-7. However, direct reprogramming of differentiated cells into somatic stem cell types has not been described yet4. Here we show that a combination of neural-specific transcription factors (Sox2, Klf4, c-Myc, Brn4/Pou3f4 or Sox2, Klf4, c-Myc, Brn4, E47/Tcf3) can induce a neural stem cell (NSC) fate on the fibroblasts. Induced neural stem cells (iNSCs) showed morphology, gene expression, epigenetic features, differentiation potential, and functionality similar to wild-type NSCs. Therefore, our data suggest that cell type-specific defined factors can induce specific stem cell identities on somatic cells. Fibroblasts (5 x 104 cells) were infected with retroviruses for two days, and cells were maintained in NSC media: DMEM/F-12 supplemented with N2 or B27 supplements (Gibco-BRL), 10 ng/ml EGF, 10 ng/ml bFGF (both from Invitrogen), 50 ug/ml BSA (Fraction V Gibco-BRL), and 1x penicillin/streptomycin/glutamine (Gibco-BRL). In order to establish stable iNSC lines, were either manually picked mature iNSC clumps or passaged and seeded the whole dishes of cells onto either gelatin- or laminin-coated dishes. RNA samples to be analysed on microarrays were prepared using QIAGEN RNeasy columns with on-column DNA digestion. 500 ng of total RNA per sample was used as input RNA into a linear amplification protocol (Ambion) involving synthesis of T7-linked double-stranded cDNA and 12 h of in-vitro transcription incorporating biotin-labelled nucleotides. Purified and labelled cRNA was hybridised onto MouseRef-8 v2 expression BeadChips (Illumina) for 18 h according to the manufacturer's instructions. After washing, as recommended, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using iScan reader (Illumina) and accompanying software. Samples were hybridised as biological replicates.

Project description:The differentiation capability of induced pluripotent stem cells (iPSCs) towards certain cell types for disease modelling and drug screening assays might be influenced by their somatic cell of origin. Here, we have compared the neural induction of human iPSCs generated from either fetal neural stem cells (fNSCs), dermal fibroblasts or CD34+ hematopoietic stem cells. Neural progenitor cells (NSCs) and neurons could be generated at similar efficiencies from all iPSCs. Whole genome transcriptome analysis and an expression analysis of neural genes in iPSC-derived NSCs revealed a separation of neuroectoderm- from mesoderm-derived cells. Furthermore, we found genes, which were similarly expressed between fNSCs and neuroectoderm- but not mesoderm-derived NSCs. Notably, these neural signatures were retained after transplantation into the cortex of mice and paralleled with increased survival and integration of neuroectoderm-derived cells in vivo. These results indicated distinct origin- dependent neural cell identities in differentiated human iPSCs both in vitro and in vivo. RNA samples for microarray analysis were prepared using RNeasy columns (Qiagen, Germany) with on-column DNA digestion. 300 ng of total RNA per sample were used as the input in the linear amplification protocol (Ambion), which involved the synthesis of T7-linked double-stranded cDNAs and 12hrs of in vitro transcription incorporating biotin-labeled nucleotides. Purified and labeled cRNA was then hybridized for 18 hrs onto HumanHT-12 v4 expression BeadChips (Illumina, USA) following the manufacturer's instructions. After the recommended washing, the chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and the accompanying software. The samples were exclusively hybridized as biological replicates. The RNA samples from in vivo transplanted and FACS sorted cells were amplified using the TargetAmp 2-Round Biotin-aRNA Amplification Kit 3.0 (Epicentre, USA) from 100 pg to 50 microg. 43 samples were analyzed Fib, Human fibroblast F134, 2 replicates CD34+, Human Cord Blood CD34+ Hematopoyetic Stem Cell (HSC), 1 replicate fNSC, Human fetal Neural Stem Cells (NSC), 1 replicate fNSC-1FiPSCa-NSC, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone a, in vitro differentiated to NSC, 2 replicates fNSC-1FiPSCb-NSC, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone b, in vitro differentiated to NSC, 2 replicates fNSC-2FiPSCa-NSC, Human two factors (POU5F1, KLF4) fetal Neural Stems Cell (NSC) induced reprogrammed cell (iPSC) clone a, in vitro differentiated to NSC, 2 replicates CD34-2FiPSCa-NSC, Human two factors (POU5F1, KLF4) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone a, in vitro differentiated to NSC, 2 replicates CD34-4FiPSCa-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone a, in vitro differentiated to NSC, 2 replicates CD34-4FiPSCb-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone b, in vitro differentiated to NSC, 2 replicates Fib-4FiPSCa-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone a, in vitro differentiated to NSC, 2 replicates Fib-4FiPSCb-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone b, in vitro differentiated to NSC, 2 replicates Fib-4FiPSCc-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone c, in vitro differentiated to NSC, 1 replicate H1-ESC-NSC, Human H1 embryonic stem cell (ESC), in vitro differentiated to NSC, 2 replicates HUES6-ESC-NSC, Human HUES6 embryonic stem cell (ESC), in vitro differentiated to NSC, 1 replicate fNSC-1FiPSCa, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone a, 1 replicate fNSC-1FiPSCb, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone b, 1 replicate fNSC-2FiPSCa, Human two factors (POU5F1, KLF4) fetal Neural Stems Cell (NSC) induced reprogrammed cell (iPSC) clone a, 1 replicate CD34-2FiPSCa, Human two factors (POU5F1, KLF4) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone a, 1 replicate CD34-4FiPSCa, Human four factors (POU5F1, KLF4, SOX2, CMYC) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone a, 1 replicate CD34-4FiPSCb, Human four factors (POU5F1, KLF4, SOX2, CMYC) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone b, 1 replicate Fib-4FiPSCa, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone a, 1 replicate Fib-4FiPSCb, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone b, 1 replicate H1-ESC, Human H1 embryonic stem cell (ESC), 3 replicates HUESC6-ESC, Human HUESC6 embryonic stem cell (ESC) , 2 replicates Fib-4FiPSCa-NSC-In Vivo, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone a, in vivo differentiated to NSC, 2 replicates fNSC-1FiPSCa-NSC-In Vivo, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone a, in vivo differentiated to NSC, 4 replicates

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

Dataset Information

Direct Reprogramming of Fibroblasts into Neural Stem Cells by Defined Factors

Publications

Direct reprogramming of fibroblasts into neural stem cells by defined factors.

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