Single-Step Plasmid Based Reprogramming of Human Dermal Fibroblasts to Induced Neural Stem Cells

Abstract

Reprogramming of somatic cells to induced pluripotent stem cells (iPSC) and subsequent differentiation opened up the opportunity of deriving cell types in vitro which (like neurons) had a very restricted accessibility in the past. However, cell culture protocols for iPSC reprogramming, neural induction and differentiation tend to be labor and time intensive, costly and commonly depend on viral vector delivery. Single-step reprogramming to induced neural stem cells (iNSC) avoids many of the necessary intermediate steps of the aforementioned method but yields a cell type that proliferates over longer time spans and readily differentiates to mature neurons when required. Here we describe a plasmid based reprogramming protocol employing defined, commercially available components for induction and proliferation of iNSC, followed by a defined, small molecule based differentiation step toward mature neurons. The described method might be of particular interest for groups with limited resources and/or restricted access to higher biosafety level facilities required for viral transduction, but also for groups requiring a high throughput for dealing with large numbers of cell lines.

Visualizing the Synaptic and Cellular Ultrastructure in Neurons Differentiated from Human Induced Neural Stem Cells—An Optimized Protocol

“…A good compromise, in our opinion, is induced neural stem cells (iNSC) employed in this study: Derived from somatic cells by plasmid-based transfection, they are proliferative for at least 25 passages but cultivating them requires much less intervention and they are not prone to spontaneous differentiation [9,21]. Differentiation is simply initiated by a change in cell culture media and the addition of three recombinant growth factors and one small molecule.…”

“…Detailed protocols describing the reprogramming and culture of iNSC from human fibroblast cultures have been published before [9,21]. In short, fibroblast cultures from 2 healthy donors who gave informed consent following the requirements and positive votum (AZ12-219) of the ethics committee of the University of Lübeck, Germany, were transfected with three polycistronic plasmids overexpressing transcription factors associated with pluripotency (Oct3/4, Sox2, Klf4, L-myc, Lin28) as well as a small hairpin RNA directed against p53 [37].…”

Xeno- and transgene-free reprogramming of mesenchymal stem cells toward the cells expressing neural markers using exosome treatments

“…The iPS cells require the downregulation of ES cell genes, such as Nanog and Oct4, for their differentiation [ 32 ]. Recently, a cost-effective method to produce iNSCs directly from fibroblasts was reported, though it may have safety issues because of the use of transgenes [ 11 ]. This issue is also evident in the transdifferentiation of human umbilical cord blood cells to iNSCs with over expressions of Sox2 and HMGA2 [ 12 ].…”

“…Nevertheless, all these methods are still lengthy, unsafe, cumbersome, since the mechanism behind reprogramming is not yet well understood, limiting its improvement. Many researchers have been trying to directly convert somatic cells to induced neural stem-cell-like cells (iNSCs) with a process known as "direct reprogramming," bypassing the pluripotent state to avoid risks associated with iPS cells by a variety of ways [10][11][12][13][14][15]. The direct reprogramming strategy may be favorable for cell therapy and modeling of neurodegenerative diseases because it may directly produce NSCs from adult cells without issues associated with iPS cells.…”

How to reprogram human fibroblasts to neurons

“…2014 [ 110 ] CHIR99021 GSK3β inhibition Sodium butyrate HDAC inhibition Lysophosphatidic acid Unknown Rolipram PDE4 inhibition SP600125 JNK inhibition NPCs OCT4, SOX2,KLF4, C-MYC Unknown Unknown CHIR99021, SB431542 Unknown Unknown Meyer et al 2015 [ 111 ] NSCs ZFP521 Unknown Unknown CHIR99021, SB431542 Unknown Unknown Shahbazi et al 2016 [ 65 ] NSCs hOCT3/4-shp53-F Unknown Unknown Y-27632 Unknown Unknown Azmitia et.al. 2018 [ 112 ] NSCs BRN2, KLF4, SOX2, ZIC3 Unknown Have the ability of NSCs population maintenance and positive regulation of NPCs proliferation, induce neural crest identity of brain development and head development and the regional identity of a dorsal anterior hindbrain fate CHIR99021 GSK-3 inhibition Unknown Thier et al, 2019 [ 113 ] ALK5 inhibitor II ALK5 inhibition Purmorphamine Hedgehog-smoothened agonist Tranylcypromine Inhibitior of monoamine oxidase and CYP2 enzymes: A6; C19; and D6 <...>…”