Elsevier

Journal of Neuroscience Methods

Volume 312, 15 January 2019, Pages 126-138
Journal of Neuroscience Methods

Direct transdifferentiation of human Wharton’s jelly mesenchymal stromal cells into cholinergic-like neurons

https://doi.org/10.1016/j.jneumeth.2018.11.019Get rights and content

Highlights

  • Cholinergic-like neurons (ChLNs) were obtained in 4 days transdifferentiation.

  • Cholinergic-N-Run medium induced ∼76% ChAT/ VChAT positive cells by day 4.

  • ChLNs expressed neuronal markers NeuN, TUC-4, NF-L, MAP2 and β-Tubulin III.

  • ChLNs expressed cholinergic markers ChAT, VChAT and AChE.

  • ChLNs expressed neither TH, GABA nor GFAP.

Abstract

Barckground Alzheimer’s disease (AD) is mainly caused by cellular loss and dysfunction of the basal forebrain cholinergic neurons and cholinergic axons in the cortex leading to slowly progressive decline in learning and memory performance. Unfortunately, no definitive treatment to halt neural cell loss exists to date. Therefore, it is necessary to obtain an unlimited source of cholinergic neurons for future pharmacological applications in AD. Human mesenchymal stromal cells (hMSCs) represent a unique source of cholinergic-like neurons (ChLNs).

New method hWJ-MSCs were incubated with Cholinergic-N-Run medium for 4 and 7 days.

Results hWJ-MSCs cultured with Cholinergic-N-Run medium differentiated into ChLNs in 4 days as evidenced by high levels of protein expression of the neuronal markers ChAT, VAChT, AChE, MAP2, β-Tubulin III, NeuN, TUC-4, NF-L and no expression of the immature marker SOX2, the dopaminergic marker TH, GABAergic marker GAD67 and glial marker GFAP.

Comparison with existing method(s) The hWJ-MSCs form ChLNs (e.g., ∼26% IF+) within 20 days by using complex conditioned mediums that are expensive and time-consuming. We report for the first time, to our best knowledge, a direct method of hWJ-MSCs transdifferentiation into ChLNs (∼76% ChAT /VAChT assessed by immunofluorescence microscopy and flow cytometry) in an economic, efficient and timely fashion.

Conclusions The fastest method to obtain ChLNs from hWJ-MSCs takes only four days using the one-step incubation medium Cholinergic-N-Run.

Introduction

Alzheimer's disease is a progressive neurodegenerative disorder that predominantly involves memory and cognitive disorder due mainly to severe cellular loss and dysfunction of the basal forebrain cholinergic neurons and cholinergic axons in the cortex (Douchamps and Mathis, 2017). Despite several advances in the understanding the physiopathology of AD (Kumar et al., 2018), there is no definitive treatment to halt the neural cell loss in AD patients to date. Therefore, it is urgent to obtain an unlimited source of cholinergic neurons for future large-scale drug screening and/ or precision pharmacology (Frozza et al., 2018; Hampel et al., 2018). Cholinergic-like neurons (ChLNs) have been obtained from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) (Hu et al., 2016), and human umbilical cord matrix (hUCM) (Liang et al., 2013). Although the hESCs and hiPSCs sources are appealing, their isolation and purification procedures are technically challenging (e.g., Goparaju et al., 2017), expensive, and ethically complex (e.g., Ghosh et al., 2016). The hUCM (also known as Wharton’s jelly (WJ) tissue) is considered a promising source of mesenchymal stromal cells (MSCs) not only because of their unique characteristics of having prenatal and postnatal stem cell features (Arutyunyan et al., 2016) but also because are considered a robust and non-controversial source for current stem cell therapy and research (Ding et al., 2015; Van Pham et al., 2016). Most importantly, WJ-MSCs might be equivalent to hESCs/ hiPSCs (Choi et al., 2015; Song et al., 2018). Indeed, the WJ-MSCs are multipotent cells that differentiate into osteogenic, chondrogenic, and adipogenic cells (Dominici et al., 2006), and transdifferentiated into neural and glial cell types (Bonilla-Porras et al., 2017; Zhang et al., 2010). Current methods of MSCs transdifferentiation into cholinergic neurons from human sources (e.g., UC, adipocyte) uses a complex conditioned medium that is expensive and time-consuming, and the result of cellular transdifferentiation is not fully cholinergic-specific (Liang et al., 2013; Liu et al., 2013a; Marei et al., 2017,2018). Therefore, the aim of this study was to obtain cholinergic neurons from hUC-MSCs in an efficient and timely fashion. Here, we report for the first time a direct transdifferentiation method to induce MSCs into ChLNs by the incubation of MSCs in Dulbecco’s Modified Eagle`s medium F12 (DMEM/F12) 1:1 Nutrient Mixture supplemented with fetal bovine serum (FBS, 1%), basic fibroblast growth factor (bFGF, 10 ng/ mL), sonic hedgehog peptide (SHH, 50 ng/ mL), all-trans retinoic acid (RA, 0.5 μM) and sodium heparin (Hep, 50 μg/ mL), thereafter denominated Cholinergic-N-Run medium. Using this medium, ChLNs (∼76%) were formed in 4-days. This is a new fast and time/ low cost-effective direct protocol to obtain ChLNs for potential future applications in AD research.

Section snippets

Mesenchymal stromal cells (MSCs) derived from human umbilical cord Wharton’s jelly tissue (hUC-WJ)

The MSCs were obtained from the Neuroscience tissue bank (NTB) code number 8330, University of Antioquia. The isolated MSCs showed the following characteristics: a colony formation capacity (i.e., colony displayed typical adherent growth, colony-forming unit fibroblast activity, and spindle-shaped and fibroblast-like morphology); positive for the mesenchymal-associated surface markers CD90, CD9 and CD73 (99% positive); and karyotype with normal shape, number and distribution of MSC chromosomes

Characterization of WJ-MSCs

Previously, we demonstrated that isolated Wharton’s jelly MSCs from hUC (e.g., NTB code #8330) displayed the typical minimal set of standard criteria for defining MSCs, according to the International Society for Cellular Therapy (Mendivil-Perez et al., 2016). Therefore, we first wanted to assess whether the freeze-thawed sample code #8330, cryo-conserved for ∼2 years, still preserved stromal cell features. As shown in Fig. 1A, WJ-MSCs reached more than 90% confluency and displayed adherent

Discussion

We report for the first time that a modified medium, named Cholinergic-N-Run promotes the transdifferentiation of hWJ-MSCs into ChLNs (∼76%) in 4 days. Several observations support this finding: (i) ChLNs expressed high levels of specific cholinergic lineage markers ChAT, VAChT and AChE, as well as expressed high levels of neuronal specific protein markers NeuN, TUC-4, NF-L, MAP2 and β-Tubulin III; (ii) ChLNs expressed neither TH (DAergic), GFAP (glial) nor GAD67 (GABAergic) cellular lineage

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported by Colciencias grant #1115-657-40786 to MJ-Del-Rio and CV-P. MM-P is enrolled as a post-Doctoral fellowship grant #784-2017 from Colciencias. We greatly acknowledge Fundación San Vicente Hospital Universitario, Fabian Alberto Jaimes Barragan, MD, PhD, director of the Research Unit; and Sandra Maria Velez Cuervo, Department of Obstetrics & Gynecology, for human umbilical cord tissue.

References (48)

  • W.L. Strauss et al.

    Human choline acetyltransferase gene maps to region 10q11-q22.2 by in situ hybridization

    Genomics

    (1991)
  • P. Taylor et al.

    Acetylcholinesterase. In Squire LR, editor. Encyclopedia of Neuroscience

    (2009)
  • W. Thangnipon et al.

    Potential role of N-benzylcinnamide in inducing neuronal differentiation from human amniotic fluid mesenchymal stem cells

    Neurosci. Lett.

    (2016)
  • H.T. Zhang et al.

    Human Wharton’s jelly cells can be induced to differentiate into growth factor-secreting oligodendrocyte progenitor-like cells

    Differentiation

    (2010)
  • E. Zieger et al.

    New insights into the roles of retinoic acid signaling in nervous system development and the establishment of neurotransmitter systems

    Int. Rev. Cell Mol. Biol.

    (2017)
  • I. Arutyunyan et al.

    Umbilical cord as prospective source for mesenchymal stem cell-based therapy

    Stem Cells Int.

    (2016)
  • U. Arvidsson et al.

    Vesicular acetylcholine transporter (VAChT) protein: a novel and unique marker for cholinergic neurons in the central and peripheral nervous systems

    J. Comp. Neurol.

    (1997)
  • Y. Belgacem et al.

    The many hats of sonic hedgehog signaling in nervous system development and disease

    J. Dev. Biol.

    (2016)
  • J. Briscoe et al.

    Morphogen rules: design principles of gradient-mediated embryo patterning

    Development

    (2015)
  • J. Choi et al.

    A comparison of genetically matched cell lines reveals the equivalence of human iPSCs and ESCs

    Nat. Biotechnol.

    (2015)
  • D.C. Ding et al.

    Human umbilical cord mesenchymal stem cells: a new era for stem cell therapy

    Cell Transplant.

    (2015)
  • V. Douchamps et al.

    A second wind for the cholinergic system in Alzheimer’s therapy

    Behav. Pharmacol.

    (2017)
  • N.A. Franken et al.

    Clonogenic assay of cells in vitro

    Nat. Protoc.

    (2006)
  • R.L. Frozza et al.

    Challenges for Alzheimer’s disease therapy: insights from novel mechanisms beyond memory defects

    Front. Neurosci.

    (2018)
  • View full text