Examining the fundamental biology of a novel population of directly reprogrammed human neural precursor cells

Abstract

Background Cell reprogramming is a promising avenue for cell-based therapies as it allows for the generation of multipotent, unipotent, or mature somatic cells without going through a pluripotent state. While the use of autologous cells is considered ideal, key challenges for their clinical translation include the ability to reproducibly generate sufficient quantities of cells within a therapeutically relevant time window. Methods We performed transfection of three distinct human somatic starting populations of cells with a non-integrating synthetic plasmid expressing Musashi 1 ( MSI1 ), Neurogenin 2 ( NGN2 ), and Methyl-CpG-Binding Domain 2 ( MBD2 ). The resulting directly reprogrammed neural precursor cells (drNPCs) were examined in vitro using RT-qPCR, karyotype analysis, immunohistochemistry, and FACS at early and late time post-transfection. Electrophysiology (patch clamp) was performed on drNPC-derived neurons to determine their capacity to generate action potentials. In vivo characterization was performed following transplantation of drNPCs into two animal models (Shiverer and SCID/Beige mice), and the numbers, location, and differentiation profile of the transplanted cells were examined using immunohistochemistry. Results Human somatic cells can be directly reprogrammed within two weeks to neural precursor cells (drNPCs) by transient exposure to Msi1, Ngn2, and MBD2 using non-viral constructs. The drNPCs generate all three neural cell types (astrocytes, oligodendrocytes, and neurons) and can be passaged in vitro to generate large numbers of cells within four weeks. drNPCs can respond to in vivo differentiation and migration cues as demonstrated by their migration to the olfactory bulb and contribution to neurogenesis in vivo. Differentiation profiles of transplanted cells onto the corpus callosum of myelin-deficient mice reveal the production of oligodendrocytes and astrocytes. Conclusions Human drNPCs can be efficiently and rapidly produced from donor somatic cells and possess all the important characteristics of native neural multipotent cells including differentiation into neurons, astrocytes, and oligodendrocytes, and in vivo neurogenesis and myelination. Electronic supplementary material The online version of this article (10.1186/s13287-019-1255-4) contains supplementary material, which is available to authorized users.

Intra-Arterial Stem Cell Transplantation in Experimental Stroke in Rats: Real-Time MR Visualization of Transplanted Cells Starting With Their First Pass Through the Brain With Regard to the Therapeutic Action

“…Directly reprogrammed neural precursor cells (drNPC) were kindly provided by New World Laboratories, Inc. (Laval, Quebec, Canada). They were prepared from cultured adult bone marrow MSC by direct reprogramming, skipping the pluripotency stage in the programming of human bone marrow MSC using transient transfection with the following 3 factors: Musashi−1 (Msi1), Neurogenin−2 (Ngn2), and methyl−CpG binding domain protein 2 (MBD2) ( Ahlfors et al, 2019 ). Cells were cultured in Petri dishes (SPL Life Sciences, South Korea) coated with human laminin (L2020, Sigma-Aldrich), at 37°C in the atmosphere of 5% CO 2 and 5% O 2 in complete NeuroCult TM -XF Proliferation medium (StemCell Technologies) supplemented with B27 (1×, Gibco), 20 ng/ml of epidermal growth factor (EGF, Biolegend), 40 ng/ml of fibroblast growth factor-2 (FGF-2, Biolegend) and 100 ng/ml heparin (Stem cells technology).…”

“…Direct reprogramming is likely to deliver less risky cell products. In this study we used directly reprogrammed NPC (drNPC) derived from human bone marrow mesenchymal cells ( Ahlfors et al, 2019 ). These cells or their oligodendrogenic derivatives proved to be therapeutically effective in rodent ( Nagoshi et al, 2008 ; Nori et al, 2018 ) and non-human primate ( Baklaushev et al, 2019 ) models of spinal cord injury and in a mouse focal ischemic stroke model with formation of small cortical infarctions ( Vonderwalde et al, 2020 ).…”

Substrate-Dependent Galvanotaxis of Directly Reprogrammed Human Neural Precursor Cells

“…Herein, we examine the galvanotactic response of clinically relevant human directly reprogrammed NPCs (drNPCs). 9 In a 2D in vitro model, we show that drNPCs undergo rapid and directed migration in the presence of a 250 mV/mm EF, and that the direction of migration (anodal vs. cathodal) can be modulated by the substrate stiffness. We have found that the migration of drNPCs under an applied EF is independent of secreted factors from neighboring cells and extracellular pH.…”

“…drNPCs were generated from mature human bone marrow cells as previously described. 9 The drNPCs were cultured in low oxygen conditions in basal medium (NeuroCultÔ NS-A Basal Medium Human -Catalog #05750) containing 10% proliferation supplement (NeuroCult Proliferation Supplement Human -Catalog #05753), fibroblast growth factor-2 (FGF-2; 30 ng/mL, Peprotech), epidermal growth factor (EGF, 20ng/mL; Peprotech), and heparin (100 lg/mL; Scientific Protein Laboratories), termed EFH. drNPCs were cultured as free-floating colonies (neurospheres) for 3-7 days on an ultra-low attachment 24-well plate (CLS3473 Sigma-Aldrich).…”

“…To better mimic the physiological conditions in vivo, we plated drNPCs onto a cell monolayer comprising neurons, astrocytes, and oligodendrocytes from differentiated drNPCS, as previously described. 9 First, we examined the migratory behavior of differentiated drNPCs in the presence of an EF. Briefly, drNPCs were differentiated on a galvanotaxis chamber coated with matrigel for 10-15 days, in the presence of 10% FBS, and an EF was applied across the chamber.…”