Tissue Engineered Neural Constructs Composed of Neural Precursor Cells, Recombinant Spidroin and PRP for Neural Tissue Regeneration

V G Bogush,V P Baklaushev,A V Troitskiy,N N Sovetnikov,E M Samoilova,J.-E Ahlfors,V A Kalsin,S L Kotova,M A Konoplyannikov,P S Timashev,K B Yushkov,A V Averyanov,V A Revkova,K V Sidoruk

doi:10.1038/s41598-019-39341-9

Abstract

We have designed a novel two-component matrix (SPRPix) for the encapsulation of directly reprogrammed human neural precursor cells (drNPC). The matrix is comprised of 1) a solid anisotropic complex scaffold prepared by electrospinning a mixture of recombinant analogues of the spider dragline silk proteins – spidroin 1 (rS1/9) and spidroin 2 (rS2/12) - and polycaprolactone (PCL) (rSS-PCL), and 2) a “liquid matrix” based on platelet-rich plasma (PRP). The combination of PRP and spidroin promoted drNPC proliferation with the formation of neural tissue organoids and dramatically activated neurogenesis. Differentiation of drNPCs generated large numbers of βIII-tubulin and MAP2 positive neurons as well as some GFAP-positive astrocytes, which likely had a neuronal supporting function. Interestingly the SPRPix microfibrils appeared to provide strong guidance cues as the differentiating neurons oriented their processes parallel to them. Implantation of the SPRPix matrix containing human drNPC into the brain and spinal cord of two healthy Rhesus macaque monkeys showed good biocompatibility: no astroglial and microglial reaction was present around the implanted construct. Importantly, the human drNPCs survived for the 3 month study period and differentiated into MAP2 positive neurons. Tissue engineered constructs based on SPRPix exhibits important attributes that warrant further examination in spinal cord injury treatment.

Highlights

(without viral transduction, pluripotency factors or any other gene engineering procedures), demonstrates the greatest potential from a clinical viewpoint. drNPC[13,14,15] are non-immunogenic, have a stable genome and pose a minimal risk of malignant transformation, especially when compared to iPS and embryonic stem cells; they exhibit the expected characteristics of neural stem cells such as self-renewal and multipotency
A number of studies have shown that platelet-rich plasma (PRP) promotes neurogenesis and axonal growth[28], as well as enhancement of proliferation and migration of Schwann cells[29] due to the presence of different growth factors (PDGF-AB, TGF-β1, IGF-1, VEGF, NGF and GDNF) and platelet-released exosomes containing microRNA and other signaling molecules
Using flow cytometry and immunocytochemical analysis, we demonstrated high level expression of the neural stem cell marker SOX2 and neural markers βIII-tubulin and MAP2 in the drNPC culture (Fig. 1A). drNPC cultured on laminin-coated adhesive plastic in complete medium exhibited a characteristic “honeycomb” pattern and co-expressed βIII-tubulin and GFAP (Fig. 1B,C), SOX2 and Nestin (Fig. 1D)

Summary

Introduction

(without viral transduction, pluripotency factors or any other gene engineering procedures), demonstrates the greatest potential from a clinical viewpoint. drNPC (directly reprogrammed Neural Precursor Cells)[13,14,15] are non-immunogenic, have a stable genome and pose a minimal risk of malignant transformation, especially when compared to iPS and embryonic stem cells; they exhibit the expected characteristics of neural stem cells such as self-renewal and multipotency. A number of studies have shown that PRP promotes neurogenesis and axonal growth[28], as well as enhancement of proliferation and migration of Schwann cells[29] due to the presence of different growth factors (PDGF-AB, TGF-β1, IGF-1, VEGF, NGF and GDNF) and platelet-released exosomes containing microRNA and other signaling molecules. These properties make PRP a prospective source of autologous growth factors providing the necessary biomimetic microenvironment for neuroregeneration[30]. The aims of this study were to (1) develop a two-component matrix SPRPix, based on PRP and an anisotropic complex scaffold of recombinant spidroins and polycaprolactone (rSS-PCL) for the creation of 3D tissue engineered constructs with drNPC (2) to test the biocompatibility of the drNPC-SPRPix construct in the brain and spinal cord of the Rhesus macaque and 3) to examine the survival and behaviour of the implanted human drNPC cells

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Conclusion