Direct Conversion of Adult Human Fibroblasts into Induced Neural Precursor Cells by Non-Viral Transfection.

Abstract

Direct reprogramming offers a unique approach by which to generate mature neural lineages for the study and treatment of neurological diseases and neurodevelopmental disorders. However, few studies have directly generated neural stem/precursor cells (iNPs) from adult human fibroblasts that are capable of producing a wide range of mature neuronal phenotypes. Further, current reprogramming protocols require the use of viral-mediated gene delivery to induce the generation of iNPs from human fibroblasts. Here we describe a robust and efficient protocol to directly reprogram adult human fibroblasts to expandable iNPs using transient non-viral gene delivery in a feeder-free cell culture system. The two transfection factors required for direct conversion to iNP cells, SOX2 and PAX6, do not generate a pluripotent cell state, reducing the potential risk of tumor formation following transplantation. By following the current protocol, iNPs are observed 6-9 weeks after transfection. Upon differentiation, iNPs generate both astrocytes and a range of phenotype-specific neurons.

Conversion of adult human fibroblasts into neural precursor cells using chemically modified mRNA

“…Regardless, the aim of the current study was to identify and confirm an efficient, non-integrative gene delivery approach for direct-to-iNP reprogramming to prevent the risk of genomic recombination or insertional mutagenesis, and provide a neural cell source suitable for neurological disease modelling and therapeutic application. We have previously demonstrated the use of plasmid DNA and recombinant protein delivery methods by which to generate human iNPs [ 18 , 23 ]. While these strategies allowed for transgene-free and vector-free reprogramming, the major limitation associated with the use of plasmid DNA was a low level of transfection efficiency.…”

“…The objective is to provide a direct source of non-pluripotent, expandable iNPs with the capability of generating multiple neural lineages. Two main approaches have been used to generate iNPs; either transient expression of the four pluripotent factors OCT4, KLF4, SOX2 , and cMYC [ 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 ], or forced expression of either neural-specific [ 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 ] or non-neural transcription factors [24] . Regardless of the strategy or transcription factor combination employed, each protocol results in the production of either bi- or tri-potent iNPs.…”

“…Our protocol utilises the neural promoting transcription factors SOX2 and PAX6 to directly reprogram adult human fibroblasts to a neural precursor cell-like state [ 18 , 23 ]. SOX2 and PAX6 were identified based on their prominent roles in human neural development [ 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 ], and forced expression in adult human fibroblasts results in the generation of bipotent iNPs with the capability to differentiate into GFAP-positive astrocytes and mature region-specific neurons [ 18 , 23 ]. A key feature of this protocol for clinical translation is the utilization of non-viral plasmid DNA transfection to transiently over-express SOX2 and PAX6 in adult human dermal fibroblasts, without requirement of oncogenic-promoting transcription factors [ 18 , 23 ].…”

“…SOX2 and PAX6 were identified based on their prominent roles in human neural development [ 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 ], and forced expression in adult human fibroblasts results in the generation of bipotent iNPs with the capability to differentiate into GFAP-positive astrocytes and mature region-specific neurons [ 18 , 23 ]. A key feature of this protocol for clinical translation is the utilization of non-viral plasmid DNA transfection to transiently over-express SOX2 and PAX6 in adult human dermal fibroblasts, without requirement of oncogenic-promoting transcription factors [ 18 , 23 ]. Although DNA transfection-based methodologies significantly reduce the risk of genomic recombination or insertional mutagenesis, considerable limitations accompany non-integrative cell reprogramming strategies [ 33 , 34 , 35 , 36 , 37 , 38 ].…”

“…Although DNA transfection-based methodologies significantly reduce the risk of genomic recombination or insertional mutagenesis, considerable limitations accompany non-integrative cell reprogramming strategies [ 33 , 34 , 35 , 36 , 37 , 38 ]. In particular, while the use of plasmid DNA provides a robust and transient mechanism by which to generate human iNPs [ 18 , 23 ], the major limitation is a low level of transfection efficiency. We propose the use of chemically modified mRNA as an alternative strategy for the generation of clinically acceptable cells.…”