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: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

Project description:Neural stem cell (NSC) transplantation induces recovery in animal models of central nervous system (CNS) diseases. Although the replacement of lost endogenous cells was originally proposed as the primary healing mechanism of NSC grafts, it is now clear that transplanted NSCs operate via multiple mechanisms, including the horizontal exchange of therapeutic cargoes to host cells via extracellular vesicles (EVs). EVs are membrane particles trafficking nucleic acids, proteins, metabolites and metabolic enzymes, lipids, and entire organelles. However, the function and the contribution of these cargoes to the broad therapeutic effects of NSCs is yet to be fully understood. Mitochondrial dysfunction is an established feature of several inflammatory and degenerative CNS disorders, most of which are potentially treatable with exogenous stem cell therapeutics. Herein we investigated the hypothesis that NSCs release and traffic functional mitochondria via EVs to restore mitochondrial function in target cells. Untargeted proteomics revealed a significant enrichment of mitochondrial proteins spontaneously released by NSCs in EVs. Morphological and functional analyses confirmed the presence of ultrastructurally intact mitochondria within EVs with conserved membrane potential and respiration. We found that the transfer of these mitochondria from EVs to mtDNA-deficient L929 Rho 0 cells rescued mitochondrial function and increased Rho 0 cell survival. Furthermore, the incorporation of mitochondria from EVs into inflammatory mononuclear phagocytes restored normal mitochondrial dynamics and cellular metabolism and reduced the expression of proinflammatory markers in target cells. When transplanted in an animal model of multiple sclerosis, exogenous NSCs actively transferred mitochondria to mononuclear phagocytes and induced a significant amelioration of clinical deficits. Our data provide the first evidence that NSCs deliver functional mitochondria to target cells via EVs, paving the way for the development of novel (a)cellular approaches aimed at restoring mitochondrial dysfunction not only in multiple sclerosis, but also in degenerative neurological diseases.

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Prospective identification and characterization of multipotent peripheral neural stem cells out of mammalian central nervous system

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