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Stem Cell Res Ther. 2019 Jun 13;10(1):166. doi: 10.1186/s13287-019-1255-4.

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

Author information

1
New World Laboratories, Laval, Quebec, H7V 5B7, Canada.
2
Institute of Medical Sciences, University of Toronto, Toronto, Ontario, M5S 1A8, Canada.
3
Division of Anatomy, Department of Surgery, University of Toronto, Ontario, M5S 1A8, Canada.
4
Division of Neurosurgery, Department of Surgery, University of Toronto, Ontario, M5T 1P5, Canada.
5
Division of Genetics and Development, Krembil Research Institute, University Health Network, Toronto, Ontario, M5T 2S8, Canada.
6
Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, M5S 3G9, Canada.
7
Institute of Medical Sciences, University of Toronto, Toronto, Ontario, M5S 1A8, Canada. cindi.morshead@utoronto.ca.
8
Division of Anatomy, Department of Surgery, University of Toronto, Ontario, M5S 1A8, Canada. cindi.morshead@utoronto.ca.
9
Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, M5S 3G9, Canada. cindi.morshead@utoronto.ca.
10
Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, M5S3E1, Canada. cindi.morshead@utoronto.ca.

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.

KEYWORDS:

Direct reprogramming; In vivo neurogenesis; In vivo remyelination; Neural precursor cells; Neural stem cells; drNPC

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