Schwann cells (SCs) are neural crest (NC) derived cells able to produce the myelin sheaths, wrapping neuronal axons in the peripheral nervous system (PNS). Transplantations of SCs might become an interesting therapeutic opportunity for the treatment of spinal cord and peripheral nerves injuries and demyelinating diseases of the PNS. However, these therapeutic approaches are strongly limited by the current lack of a renewable source of SCs. Cell reprogramming strategies have proven to be effective in providing a variety of tissue-specific cells for disease modelling, and cell transplantation procedure by over expression of cardinal developmental transcription factors of the interest cell type. I have identified the two transcription factors Sox10 and Egr2 able to generate induced Schwann Cells (iSCs) when co-expressed in murine fibroblasts with high efficiency. iSCs resembled primary SCs in global gene expression profiling and expressed cardinal markers of SCs including S100ß, O4 and MPZ. When co-cultured with mouse dorsal root ganglion (DRG) explants, iSCs generated compact myelin sheaths organized in Mbp+ internodes spaced by Caspr+ paranodal and Na+ channel nodal domains. Conversely, iSCs from Twitcher mice showed a severe loss in the myelinogenic potential, indicating iSCs as an attractive system for in vitro modeling of PNS diseases. Then, I derived iSCs from rats that were subjected to median nerve axotomy followed by transplantation of chitosan conduits previously seeded with autologous iSCs. These iSC-seeded conduits supported accelerated nerve regeneration with improved myelin content. Similarly, Sox10 and Egr2 are sufficient to convert human fibroblasts into iSCs. Moreover, their expression strongly facilitate the SC differentiation of human induced pluripotent stem cells (iPSCs), including in the reprogramming strategy few intermediate steps that provide different trophic stimuli to the differentiating cells. In particular, after the lentiviral transduction with the Sox10 and Egr2 expressing lentiviruses, I added neuralizing small molecules (SB431542 and LDN193189 in hiPS medium), together with a neural crest differentiation medium (B27, Ascorbic Acid and FGF2 in neurobasal medium), and finally a specific medium for Schwann cell growth (Forskoline, NRG1, FGF2 in DMEM 10% FBS), providing a simple procedure for obtaining a large number of homogeneous and well-differentiated SCs. Altogether, Sox10 and Egr2 is a unique combination of factors for the effective generation of myelinogenic iSCs from rodent as well as human fibroblasts and iPSCs. The fast and straightforward process to generate iSCs will facilitate in vitro disease modeling and autologous cell transplantation approaches for PNS diseases.

Le cellule di Schwann (SC) sono cellule derivate dalla cresta neurale (NC) in grado di produrre la guaina mielinica avvolgendo gli assoni neuronali nel sistema nervoso periferico (PNS). I trapianti di SC potrebbero diventare un'opportunità terapeutica interessante per il trattamento delle lesioni del midollo spinale, dei nervi periferici e delle malattie demielinizzanti del PNS. Tuttavia, questi approcci terapeutici sono fortemente limitati dall'attuale mancanza di una fonte rinnovabile di SC. Le strategie di riprogrammazione cellulare si sono rivelate efficaci nel fornire una varietà di cellule specifiche per la modellizzazione delle malattie e per le procedure di trapianto di cellule, ottenute mediante sovraespressione di fattori di trascrizione cardine nello sviluppo embrionale del tipo cellulare di interesse. Ho identificato i due fattori di trascrizione Sox10 ed Egr2 in grado di generare cellule di Schwann indotte (iSCs) ad alta efficienza quando co-espresse in fibroblasti murini. Le iSC assomigliano a SC primarie nel profilo globale di espressione genica ed esprimono marcatori specifici di SC, tra cui S100ß, O4 e MPZ. Quando co-coltivati con espianti di gangli della radice dorsale di topo (DRG), le iSCs hanno generato guaine di mielina compatte organizzate in internodi Mbp+ intervallati da domini paranodali caspr + e domini nodali con canali del sodio. Al contrario, le iSC derivate da topi Twitcher hanno mostrato una grave perdita del potenziale mielinogenico, indicando le iSC come un sistema attraente per la modellazione in vitro delle malattie del PNS. Quindi, ho derivato iSCs da ratti sottoposti ad assotomia del nervo mediano seguito da trapianto di condotti di chitosano precedentemente seminati con iSC autologhe. Questi condotti con le iSC supportano e accelerano la rigenerazione dei nervi con un migliorato contenuto di mielina. Allo stesso modo, Sox10 ed Egr2 sono sufficienti per convertire i fibroblasti umani in iSC. Inoltre, la loro espressione facilita fortemente la differenziazione in SC delle cellule staminali pluripotenti indotte umane (iPSC), includendo nella strategia di riprogrammazione pochi passaggi intermedi che forniscono diversi stimoli trofici alle cellule differenzianti. In particolare, dopo la trasduzione lentivirale con i lentivirus esprimenti Sox10 ed Egr2, ho aggiunto piccole molecole neuralizzanti (SB431542 e LDN193189 in terreno iPSC), insieme a un terreno di differenziazione della cresta neurale (B27, acido ascorbico e FGF2 in terreno neurobasico) e infine un medium specifico per la crescita delle cellule di Schwann (Forskoline, NRG1, FGF2 in DMEM 10% FBS), fornendo una semplice procedura per ottenere un gran numero di SC omogenee e ben differenziate. Complessivamente, Sox10 ed Egr2 sono una combinazione unica di fattori per la generazione efficace di iSC mielinogeniche da fibroblasti di roditori e umani e da iPSC. Il processo rapido e diretto per generare iSC faciliterà la modellazione in vitro e gli approcci di trapianto di cellule autologhe per le malattie del PNS.

(2018). TWO FACTOR BASED REPROGRAMMING OF FIBROBLASTS AND INDUCED PLURIPOTENT STEM CELLS INTO MYELINOGENIC SCHWANN CELLS. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2018).

TWO FACTOR BASED REPROGRAMMING OF FIBROBLASTS AND INDUCED PLURIPOTENT STEM CELLS INTO MYELINOGENIC SCHWANN CELLS

MAZZARA, PIETRO GIUSEPPE
2018

Abstract

Schwann cells (SCs) are neural crest (NC) derived cells able to produce the myelin sheaths, wrapping neuronal axons in the peripheral nervous system (PNS). Transplantations of SCs might become an interesting therapeutic opportunity for the treatment of spinal cord and peripheral nerves injuries and demyelinating diseases of the PNS. However, these therapeutic approaches are strongly limited by the current lack of a renewable source of SCs. Cell reprogramming strategies have proven to be effective in providing a variety of tissue-specific cells for disease modelling, and cell transplantation procedure by over expression of cardinal developmental transcription factors of the interest cell type. I have identified the two transcription factors Sox10 and Egr2 able to generate induced Schwann Cells (iSCs) when co-expressed in murine fibroblasts with high efficiency. iSCs resembled primary SCs in global gene expression profiling and expressed cardinal markers of SCs including S100ß, O4 and MPZ. When co-cultured with mouse dorsal root ganglion (DRG) explants, iSCs generated compact myelin sheaths organized in Mbp+ internodes spaced by Caspr+ paranodal and Na+ channel nodal domains. Conversely, iSCs from Twitcher mice showed a severe loss in the myelinogenic potential, indicating iSCs as an attractive system for in vitro modeling of PNS diseases. Then, I derived iSCs from rats that were subjected to median nerve axotomy followed by transplantation of chitosan conduits previously seeded with autologous iSCs. These iSC-seeded conduits supported accelerated nerve regeneration with improved myelin content. Similarly, Sox10 and Egr2 are sufficient to convert human fibroblasts into iSCs. Moreover, their expression strongly facilitate the SC differentiation of human induced pluripotent stem cells (iPSCs), including in the reprogramming strategy few intermediate steps that provide different trophic stimuli to the differentiating cells. In particular, after the lentiviral transduction with the Sox10 and Egr2 expressing lentiviruses, I added neuralizing small molecules (SB431542 and LDN193189 in hiPS medium), together with a neural crest differentiation medium (B27, Ascorbic Acid and FGF2 in neurobasal medium), and finally a specific medium for Schwann cell growth (Forskoline, NRG1, FGF2 in DMEM 10% FBS), providing a simple procedure for obtaining a large number of homogeneous and well-differentiated SCs. Altogether, Sox10 and Egr2 is a unique combination of factors for the effective generation of myelinogenic iSCs from rodent as well as human fibroblasts and iPSCs. The fast and straightforward process to generate iSCs will facilitate in vitro disease modeling and autologous cell transplantation approaches for PNS diseases.
BROCCOLI, VANIA
Schwann,; reprogramming,; iPS,; nerve; regeneration
Schwann,; reprogramming,; iPS,; nerve; regeneration
BIO/13 - BIOLOGIA APPLICATA
English
20-mar-2018
MEDICINA TRASLAZIONALE E MOLECOLARE - DIMET - 76R
30
2016/2017
open
(2018). TWO FACTOR BASED REPROGRAMMING OF FIBROBLASTS AND INDUCED PLURIPOTENT STEM CELLS INTO MYELINOGENIC SCHWANN CELLS. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2018).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/199039
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