Acute reduction in oxygen tension enhances the induction of neurons from human fibroblasts

Abstract

We and others have reported the successful conversion of human fibroblasts into functional induced neuronal (iN) cells; however the reprogramming efficiencies were very low. Robust reprogramming methods must be developed before iN cells can be used for translational applications such as disease modeling or transplantation-based therapies. Here, we describe a novel approach in which we significantly enhance iN cell conversion efficiency of human fibroblast cells by reprogramming under hypoxic conditions (5% O2). Fibroblasts were derived under high (21%) or low (5%) oxygen conditions and reprogrammed into iN cells using a combination of the four transcription factors BRN2, ASCL1, MYT1L and NEUROD1. An increase in Map2 immunostaining was only observed when fibroblasts experienced an acute drop in the O2 tension upon infection. Interestingly, cells derived and reprogrammed under hypoxic conditions did not produce more iN cells. Approximately 100% of patched cells fire action potentials in low O2 relative to 50% under high O2 growth conditions, confirming the beneficial aspect of reprogramming under low O2. Further characterization showed no significant difference in the intrinsic properties of iN cells reprogrammed in either condition. Surprisingly, the acute drop in oxygen tension did not affect cell proliferation or cell survival and is not synergistic with blockade of GSK3 beta and Smad-mediated pathways. Our results show that lowering the O2 tension at initiation of reprogramming is a simple and efficient manner to enhance the production of iN cells which will facilitate their use for basic discovery and regenerative medicine.

Induced Neural Lineage Cells as Repair Kits: So Close, Yet So Far Away

“…The data show that these induced NSC‐like cells express high level of Sox2 and NSC markers such as Pax6 , can be expanded for more than five passages, are capable to differentiate into neurons and glia both in vitro and in vivo (Su et al, ). Interestingly, simple change in oxygen tension (from 21% to 5% O 2 ) during human fibroblasts neural conversion could boost the efficiency of functional iNs generation (Davila et al, ). Developing a system‐level set up that can clarify the regulatory interactions beyond extrinsic and intrinsic factor interplays may help us to provide more powerful transdifferentiation schemes for future cell‐based therapies.…”

Mash1 efficiently reprograms rat astrocytes into neurons

“…Hypoxic conditions during the initial reprogramming stages significantly enhance the efficiency of inducing human fibroblasts into neuronal cells[19]. Mouse fibroblast cells were transdifferentiated into an intermediate state characterized by spherical cell aggregates, somewhere between mouse embryonic stem cells and mouse fibroblasts, using specialized medium containing lipid-rich albumin.…”

Direct Neural Conversion from Human Fibroblasts Using Self-Regulating and Nonintegrating Viral Vectors

“…These data demonstrate that miR-124.T-regulated vectors stably convert human fibroblasts to induced neurons at the same efficiency and purity as nonregulated vectors, without significant effects on subtype specificity. It is worth noting that the conversion efficiency is remarkably high in these experiments compared to most other published studies (Ambasudhan et al, 2011;Caiazzo et al, 2011;Davila et al, 2013;Pang et al, 2011;Pfisterer et al, 2011a;Wang et al, 2014;Yoo et al, 2011), and on par with the highest conversion efficiencies that have been reported using doxycycline-regulated vectors (Ladewig et al, 2012;Liu et al, 2013;Pereira et al, 2014). One likely explanation for this is the use of phosphoglycerate kinase (PGK) promoter in these studies.…”