Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

Summary

Marrow stromal cells (MSCs) with neural potential exist within the bone marrow. Our protocol enriches this population of cells via hypoxic preconditioning and thereafter directs them to become mature Schwann cells.

Abstract

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the mature Schwann cell fate. We subjected rat and human MSCs to transient hypoxic conditions (1% oxygen for 16 h) followed by expansion as neurospheres upon low-attachment substratum with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) supplementation. Neurospheres were seeded onto poly-D-lysine/laminin-coated tissue culture plastic and cultured in a gliogenic cocktail containing β-Heregulin, bFGF, and platelet-derived growth factor (PDGF) to generate Schwann cell-like cells (SCLCs). SCLCs were directed to fate commitment via coculture for 2 weeks with purified dorsal root ganglia (DRG) neurons obtained from E14-15 pregnant Sprague Dawley rats. Mature Schwann cells demonstrate persistence in S100β/p75 expression and can form myelin segments. Cells generated in this manner have potential applications in autologous cell transplantation following spinal cord injury, as well as in disease modeling.

Introduction

The transplantation of neural progenitors and their derivatives demonstrates promise as a treatment strategy following traumatic nerve injury^1,2 and neurodegeneration^3,4. Prior to clinical application, it is essential to ensure: i) a method for accessing and expanding upon an autologous source of stem/progenitor cells and ii) a means to direct them to relevant, mature cell types³. Our interest in cell therapy for spinal cord injury led us to seek a robust, autologous cell source of neural progenitors from adult tissues.

Protocol

All procedures involving animals were performed in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Committee on Use of Live Animals for Teaching and Research, Li Ka Shing Faculty of Medicine, The University of Hong Kong. Human bone marrow samples were obtained from the iliac crest of healthy donors after obtaining informed consent. Protocols were approved by the Institutional Review Board, The University of Hong Kong.

1. Preparation of Rat MSC Cultures

1. Harvest of MSCs from the femur
   1. Autoclave all dissection tools (i.e., fine dissecting scis....

Results

An overview of the key stages in our protocol is illustrated in Figure 1. In summary, rat and human MSCs are selected for by adherence to tissue culture plastic. Expanded MSCs are preconditioned with hypoxia and are then subject to neurosphere-forming conditions. Neurospheres are plated and allowed to differentiate into SCLCs. SCLCs are cocultured with purified DRG neurons to generate fate-committed Schwann cells.

Discussion

It is essential to preserve the "stemness" of MSCs prior to the enrichment of neural progenitors via hypoxic preconditioning and neurosphere culture. From our experience, multipotent MSCs can be reliably identified by their elongated fibroblast-like morphology. In contrast, MSCs that have adopted a more flattened, quadrangular morphology, with prominent cytoskeletal stress fibers, do not readily adopt neural cell fates and should be discarded. In general, we do not utilize MSCs with passage numbers greater than e.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
αMEM	Sigmaaldrich	M4526
DMEM/F12	Thermofisher scientific	12400-024
Neurobasal medium	Thermofisher scientific	21103-049
FBS	Biosera	FB-1280/500
B27	Thermofisher scientific	17504-001
Epidermal growth factor (EGF)	Thermofisher scientific	PHG0313
Basic fibroblast growth factor (bFGF)	Peprotech	100-18B/100UG
Nerve growth factor (NGF)	Millipore	NC011
Platelet-derived growth factor-AA (PDGF-AA)	Peprotech	100-13A
Heregulin beta-3, EGF domain (β-Her)	Millipore	01-201
Uridine	Sigmaaldrich	U3003
5-Fluro-2' - deoxyuridine (FDU)	Sigmaaldrich	F0503
Poly-D-lysine (PDL)	Sigmaaldrich	P7886-1G
Laminin	Thermofisher scientific	23017015
GlutaMAX	Thermofisher scientific	35050061
Penicillin / streptomycin (P/S)	Thermofisher Scientific	15140-122
TrypLE Express	Thermofisher Scientific	12604-013
10 cm plate for adherent culture	TPP	93100	Used for selection of MSCs by tissue culture adherence
6-well plate for adherent culture	TPP	92006	Used for expansion of MSCs following passaging
UltraLow 6-well plate for non-adherent culture	Corning	3471	Used for neural progenitor enrichment
anti-human CD90(Thy-1)	BD Biosciences	555593
anti-human CD73	BD Biosciences	550256
anti-human/rat STRO-1	R&D Systems	MAB1038
anti-human nestin	R&D Systems	MAB1259
anti-human CD45	BD Biosciences	555480
anti-rat CD90(Thy-1)	BD Biosciences	554895
anti-rat CD73	BD Biosciences	551123
anti-rat nestin	BD Biosciences	MAB1259
anti-rat CD45	BD Biosciences	554875
Anti-S100β	Dako	Z031101
Anti-p75	Millipore	MAB5386
Anti-GFAP	Sigmaaldrich	G3893
Anti-Class III-beta tubulin (Tuj-1)	Covance	MMS-435P
Anti-Human nuclei	Millipore	MAB1281
Hypoxia chamber	Billups-Rothenberg	MIC-101
HEPES buffer	Sigmaaldrich	H4034-100G