Transcription factorbased modulation of neural st

Authors: Kristin Stock Lars Nolden Frank Edenhofer Tamara Quandel Oliver Brüstle

Publish Date: 2010/03/30

Volume: 67, Issue: 14, Pages: 2439-2449

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Abstract

In contrast to conventional gene transfer strategies the direct introduction of recombinant proteins into cells bypasses the risk of insertional mutagenesis and offers an alternative to genetic intervention Here we explore whether protein transduction of the gliogenic transcription factor Nkx22 can be used to promote oligodendroglial differentiation of mouse embryonic stem cell ESCderived neural stem cells NSC To that end a recombinant cellpermeant form of Nkx22 protein was generated Exposure of ESCderived NSC to the recombinant protein and initiation of differentiation resulted in a twofold increase in the number of oligodendrocytes Furthermore Nkx22transduced cells exhibited a more mature oligodendroglial phenotype Comparative viral gene transfer studies showed that the biological effect of Nkx22 protein transduction is comparable to that obtained by lentiviral transduction The results of this proofofconcept study depict direct intracellular delivery of transcription factors as alternative modality to control lineage differentiation in NSC cultures without genetic modificationControlled differentiation into neuronal and glial lineages is a key prerequisite for the biomedical application of neural stem cells NSC In addition to exposure to extrinsic growth and differentiation factors expression of instructive transcription factors has been employed to modulate cell fate decisions Viral transduction of the transcription factors Nurr1 and Lmx1a has recently been used to promote the differentiation of midbrain dopamine neurons from mouse embryonic stem cells ESC 1 2 3 A central problem in most viral and nonviral gene transduction systems is random integration of the vectors in the host genome which can result in insertional mutagenesis potentially leading to aberrant differentiation and tumor formation On the other hand direct intracellular delivery of bioactive proteins and nucleic acids is limited by low permeability of the eukaryotic cell membrane Recently the generation of cellpermeant proteins has emerged as a promising new avenue for intracellular protein transfer 4 5 6 Transduction is enabled by linking the protein of interest to protein transduction domains PTD also designated cellpenetrating peptides CPP The short PTD from the transactivator protein TAT of HIV1 7 8 9 the viral protein 22 VP22 of Herpes simplex virus 10 and the antennapedia homeoprotein of Drosophila 11 12 have been fused to a variety of proteins including cell cycle factors 13 14 DNA recombinases 15 16 and transcription factors 17 18 19 20 21 Highly efficient delivery of biologically active proteins was reported for a wide variety of cultured cells including mouse 22 and human ESC 23 as well as neural precursor cells and postmitotic neurons 24 Although cellpermeant proteins have mainly been shown to work in vitro their functionality was also demonstrated in vivo 25 26 27 28 29 The mechanism of cellular uptake is not fully understood but appears to involve at least two cellular processes endocytosis and direct membrane penetration 30 31There is strong evidence that the molecular control of oligodendrocyte specification and differentiation from neural stem or progenitor cells is regulated by the transcription factors Olig1/2 Nkx22 Sox9 and Sox10 32 Fig 2a Several studies have demonstrated that expression of the basic helixloophelix transcription factors Olig1 and Olig2 is required for oligodendrocyte lineage determination in vivo 33 34 35 In addition the highmobility transcriptional regulator Sox10 and the homeodomain transcription factor Nkx22 have been proposed to directly regulate oligodendrocyte differentiation and myelin gene expression Mutations of both genes result in a decreased number of oligodendrocytes in the CNS 36 37 These findings suggest that Sox10 and Nkx22 cooperatively mediate the function of Olig2 to control oligodendrocyte differentiation and maturation Conversely expression of Nkx22 in combination with Olig2 can induce ectopic formation of mature myelin basic protein MBPpositive oligodendrocytes in embryonic chicken spinal cord 38 Along this line inducible expression of Olig2 has been used to promote the generation of oligodendrocytes from murine ESC 39 and transduction of human fetal neural precursors with an Olig2encoding lentivirus induces their commitment towards an oligodendroglial fate in vitro and in vivo 40 In human adultderived NSC cotransfection with Olig2 and Nkx22 or Sox10 and Nkx22 induces oligodendrocyte differentiation and maturation 41 Thus overexpression of transcription factors that play essential roles in oligodendrocyte development facilitate oligodendrocyte specification and differentiation in neural stem and progenitor cellsClonally derived mouse ESCderived NSC line NS5 were cultured according to Conti et al 42 Oligodendroglial differentiation was performed as described 43 Briefly cells were plated on polyornithin/laminincoated dishes and proliferated in NSC expansion medium which is composed of NSA medium Euroclone Pero Italy plus N2 supplement Invitrogen Karlsruhe Germany 10 ng/ml fibroblast growth factor 2 FGF2 Invitrogen and 10 ng/ml epidermal growth factor EGF Invitrogen For differentiation this medium was replaced by DMEM/F12 Invitrogen supplemented with N2 differentiation medium and the cells were propagated for 4 days without FGF2 and EGF but in the presence of 335triiodothyronine T3 30 ng/µl Sigma Steinheim Germany and ascorbic acid AA 200 µM SigmaRecombinant Nkx22 fusion protein was expressed from a pTriEx1based plasmid in E coli Briefly Nkx22specific primers were used to amplify the coding sequence from human nkx22 cDNA clones Open Biosystems Huntsville AL by PCR reaction The sequence was cloned between the AvrII and NheI sites of the pSESAMEC vector 17 to construct a fusion protein comprising the protein transduction sequence at the Cterminus The fusion protein was purified from the E coli lysates using Ni2+ affinity chromatography eluted and concentrated in a glycerol stock For protein transduction the protein was diluted in cell culture medium For most experiments the protein was used at a final concentration of 5 µg/ml Fresh protein was added every day NSC were treated with protein during the last day of proliferation and throughout the subsequent 4day growth factor withdrawalinduced differentiation in the presence of T3 and AAFor tracking of transducible Nkx22 the protein was labeled with Nhydroxysuccinimiderhodamine NHSrhodamine Pierce Rockford IL To that end 25 ml of the recombinant protein 200 µg/ml were mixed with 250 µl NHSrhodamine 37 µg/ml and incubated for 2 h in darkness The labeled protein was transferred onto a desalting column and eluted with PBS/DMEM high glucose 11 Protein concentration was quantified by Bradford staining BioRad Cambridge MA The eluate was used immediately or divided into aliquots and frozen in a dry ice/ethanol bath and stored at −80°C For application of the labeled protein on NSC the cells were washed twice with PBS and incubated with NSC expansion medium containing the labeled protein 50 µg/ml After 30 min the cells were washed three times with heparin 05 mg/ml Sigma to detach protein bound to the cell surface Nuclei were visualized by Hoechst staining 11000 Sigma for 15 min at 37°C and the labeled protein was tracked using fluorescence microscopy

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