Sequentially induced motor neurons from human fibroblasts facilitate locomotor recovery in a rodent spinal cord injury model

Abstract

Generation of autologous human motor neurons holds great promise for cell replacement therapy to treat spinal cord injury (SCI). Direct conversion allows generation of target cells from somatic cells, however, current protocols are not practicable for therapeutic purposes since converted cells are post-mitotic that are not scalable. Therefore, therapeutic effects of directly converted neurons have not been elucidated yet. Here, we show that human fibroblasts can be converted into induced motor neurons (iMNs) by sequentially inducing POU5F1(OCT4) and LHX3. Our strategy enables scalable production of pure iMNs because of the transient acquisition of proliferative iMN-intermediate cell stage which is distinct from neural progenitors. iMNs exhibited hallmarks of spinal motor neurons including transcriptional profiles, electrophysiological property, synaptic activity, and neuromuscular junction formation. Remarkably, transplantation of iMNs showed therapeutic effects, promoting locomotor functional recovery in rodent SCI model. Together, our advanced strategy will provide tools to acquire sufficient human iMNs that may represent a promising cell source for personalized cell therapy.

A Monolayer System for the Efficient Generation of Motor Neuron Progenitors and Functional Motor Neurons from Human Pluripotent Stem Cells

“…To date, MNs have been successfully differentiated from human embryonic stem cells (hESCs) and human induced PSCs (hiPSCs) by direct differentiation, or from somatic cells (i.e., fibroblasts) by an induction process based on the overexpression of specific transcription factors for the conversion of somatic cells towards MNs. Most of the xeno-free chemically defined available protocols for the generation of hiPSC-derived neurons are based on floating EB formation, a step that makes the process longer and that, due to the complex EB structure, does not allow proper monitoring of the differentiation [ 15 , 16 , 17 , 18 , 19 , 20 , 21 ]. To bypass these limitations of the EB-based procedure, some protocols have been developed for differentiating neural progenitor cells (NPCs) in monolayer conditions, yet most of them are based on dual-SMAD inhibition during the early stage of neuralization [ 22 , 23 , 24 , 25 ].…”

Stem Cell Neurodevelopmental Solutions for Restorative Treatments of the Human Trunk and Spine

“…Mesenchymal stem cells (MSCs) have a long history in transplantation (reviewed in Cofano et al, 2019 ), as well as other primary candidate cell types such as olfactory ensheathing cells and Schwann cells. More recently OPCs, NSPCs, lineage-restricted progenitors, and in some instances, postmitotic neurons have seen increased use (Assinck et al, 2017 ; Lee et al, 2020 ). In 2006, the advent of reprogramming to induce pluripotency (Takahashi and Yamanaka, 2006 ) and improved differentiation protocols accelerated human neural stem cell endeavors, as it became possible to establish patient-specific, renewable cell resources for grafting that have tailored neural identities and reduced immunogenicity concerns (Trawczynski et al, 2019 ).…”

Direct Reprogramming of Somatic Cells to Neurons: Pros and Cons of Chemical Approach

“…The importance of age preservation for disease modeling was recently illustrated also in Hungtington's disease where aggregation of the disease-causing mutant Huntingtin protein can be recapitulated in directly converted striatal neurons but not in neurons derived-iPSC, probably linked to the erasure of age signatures [9]. Among the various strategies to obtain direct reprogramming, ectopic expression of TFs in non-neuronal cells has generated neurons and neural progenitors both in vitro and in vivo [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26]. Direct conversion by TFs stands on their ability to bind to inaccessible neuronal genes in differentiated non-neuronal cell types which are generally called as pioneer TFs (Fig.…”

“…Among the various strategies to obtain direct reprogramming, ectopic expression of TFs in non-neuronal cells has generated neurons and neural progenitors both in vitro and in vivo [ 10 – 26 ] . Direct conversion by TFs stands on their ability to bind to inaccessible neuronal genes in differentiated non‐neuronal cell types which are generally called as pioneer TFs (Fig.…”