Implantation of 3D Constructs Embedded with Oral Mucosa-Derived Cells Induces Functional Recovery in Rats with Complete Spinal Cord Transection.

Javier Ganz,Izhak Michaelevski,Erez Shor,Anton Sheinin,Ina Arie,Daniel Offen,Sandu Pitaru,Shulamit Levenberg,Shaowei Guo

doi:10.3389/fnins.2017.00589

Abstract

Spinal cord injury (SCI), involving damaged axons and glial scar tissue, often culminates in irreversible impairments. Achieving substantial recovery following complete spinal cord transection remains an unmet challenge. Here, we report of implantation of an engineered 3D construct embedded with human oral mucosa stem cells (hOMSC) induced to secrete neuroprotective, immunomodulatory, and axonal elongation-associated factors, in a complete spinal cord transection rat model. Rats implanted with induced tissue engineering constructs regained fine motor control, coordination and walking pattern in sharp contrast to the untreated group that remained paralyzed (42 vs. 0%). Immunofluorescence, CLARITY, MRI, and electrophysiological assessments demonstrated a reconnection bridging the injured area, as well as presence of increased number of myelinated axons, neural precursors, and reduced glial scar tissue in recovered animals treated with the induced cell-embedded constructs. Finally, this construct is made of bio-compatible, clinically approved materials and utilizes a safe and easily extractable cell population. The results warrant further research with regards to the effectiveness of this treatment in addressing spinal cord injury.

Highlights

Spinal cord injury (SCI) results in structural and functional damage to neural circuitry, arising from axon loss, local inflammation, glial scarring, and progressive tissue cavitation extending beyond the boundaries of the primary lesion (Cregg et al, 2014)
Cells were passaged at 70–80% confluence. human oral mucosa stem cells (hOMSC) were cultured in expansion medium consisting of low-glucose Dulbecco’s modified Eagle’s medium, supplemented with 100 μg/ml streptomycin, 100 U/ml penicillin (Biological Industries, Beit-Haemek, Israel), 2 mM glutamine (Invitrogen, Carlsbad, CA, USA) and 10% fetal calf serum (FCS) (Gibco), as described by Marynka-Kalmani et al (2010)
We first analyzed whether 3D cultures support the induction of trophic factors and astrocyte-like phenotype by mixing hOMSCs with fibrin, seeding those on PLLA/polylactic-glycolic acid (PLGA) scaffolds (Supplementary Figure 1) and culturing the constructs under our previously reported astrocyte induction method originally designed for monolayer induction (Ganz et al, 2014) (Figure 1A)

Summary

Introduction

Spinal cord injury (SCI) results in structural and functional damage to neural circuitry, arising from axon loss, local inflammation, glial scarring, and progressive tissue cavitation extending beyond the boundaries of the primary lesion (Cregg et al, 2014). Mesenchymal stromal cells from various sources and olfactory ensheathing cells are the most studied cell types for autologous transplantation, showing efficacy to ameliorate SCI in animal models and in early human clinical trials (Tabakow et al, 2013; Jarocha et al, 2014; Kakabadze et al, 2016; Assinck et al, 2017; Melo et al, 2017) These can be used in combination with scaffolds, genetic engineering, medium-based induction or co-transplanted with other cell types (Assinck et al, 2017). We recently reported that hOMSCs can be induced into astrocyte-like cells, which exhibit elevated secretion of neurotophic factors (NTFs), provide neuroprotection to motor neurons in vitro and enhance neural repair in rats with sciatic nerve injury (Ganz et al, 2014)

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