Embryonic stem cells overexpressing high molecular weight FGF2 isoform enhance recovery of pre-ganglionic spinal root lesion in combination with fibrin biopolymer mediated root repair

Abstract

Background Spinal ventral root avulsion results in massive motoneuron degeneration with poor prognosis and high costs. In this study, we compared different isoforms of basic fibroblast growth factor 2 (FGF2), overexpressed in stably transfected Human embryonic stem cells (hESCs), following motor root avulsion and repair with a heterologous fibrin biopolymer (HFB). Methods In the present work, hESCs bioengineered to overexpress 18, 23, and 31 kD isoforms of FGF2, were used in combination with reimplantation of the avulsed roots using HFB. Statistical analysis was conducted using GraphPad Prism software with one-way or two-way ANOVA, followed by Tukey’s or Dunnett’s multiple comparison tests. Significance was set at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Results For the first set of experiments, rats underwent avulsion of the ventral roots with local administration of HFB and engraftment of hESCs expressing the above-mentioned FGF2 isoforms. Analysis of motoneuron survival, glial reaction, and synaptic coverage, two weeks after the lesion, indicated that therapy with hESCs overexpressing 31 kD FGF2 was the most effective. Consequently, the second set of experiments was performed with that isoform, so that ventral root avulsion was followed by direct spinal cord reimplantation. Motoneuron survival, glial reaction, synaptic coverage, and gene expression were analyzed 2 weeks post-lesion; while the functional recovery was evaluated by the walking track test and von Frey test for 12 weeks. We showed that engraftment of hESCs led to significant neuroprotection, coupled with immunomodulation, attenuation of astrogliosis, and preservation of inputs to the rescued motoneurons. Behaviorally, the 31 kD FGF2 - hESC therapy enhanced both motor and sensory recovery. Conclusion Transgenic hESCs were an effective delivery platform for neurotrophic factors, rescuing axotomized motoneurons and modulating glial response after proximal spinal cord root injury, while the 31 kD isoform of FGF2 showed superior regenerative properties over other isoforms in addition to the significant functional recovery.

1. Introduction

Preganglionic nerve root injuries have been considered untreatable for the most part due to the challenging surgical approach necessary to reattach the ruptured rootlets to the surface of the spinal cord, together with the long distance that regrowing axons need to travel toward the target. Nerve root injuries have been documented since the 19th century, predominantly affecting the brachial plexus [1], including high-energy traumatic events such as automobile accidents, particularly involving motorcycles [2]; and iatrogenic factors associated with surgical procedures [3]. These injuries often result in significant and permanent loss of function, together with muscle atrophy and severe pain. Overall, such outcome leads to poor quality of life, depression, and elevated treatment costs [4, 5].

The first step towards developing efficient treatment approaches to plexus injuries is the establishment of animal models. In this regard, models of neonatal peripheral nerve injuries have been considered, since during the first week of postnatal life, axotomy at the mid-tight level results in loss of up to 70% of the motoneurons in the spinal cord [6–10]. However, these models face important limitations regarding regeneration and glial reaction due to the immaturity of the nervous system. Thus, the use of adult rats has been proposed [11] together with surgical reconstruction of the ruptured roots [12–16].

Although pial suture was proposed to reattach avulsed roots to the surface of the spinal cord, the use of a biological glue, the heterologous fibrin biopolymer (HFB), has been considered efficient, allowing the reimplantation of multiple rootlets [15, 17]. In fact, a recent review by Buchaim et al., (2019) [18] on HFB, emphasized its promising hemostatic, adhesive, drug delivery, and scaffold (support structure) properties. The scaffold property, in particular, facilitates advanced therapies including stem cells (SC). Cell therapy represents a recent and promising approach that can be combined with such scaffolds to enhance regeneration.