Poppy O Smith: Endothelial Cells Differentiated from Human Induced Pluripotent Stem Cells Form Aligned Network Structures in Engineered Neural Tissue

Fortunately, the peripheral nervous system has an intrinsic ability to regenerate following injury when provided with a supportive environment where a connection is formed between the ends of the damaged nerve [2]. Endothelial cells play multiple support roles in nerve regeneration and repair. As in all tissues, endothelial cells are required to form the blood vessels necessary to vascularise nerve tissue. Vascularisation is required for long-term tissue survival and provides a transport system for the supply of oxygen and nutrients, and the removal of waste [3]. 

Pre-vascularised engineered nerve tissues have been shown to integrate with the host vasculature and provide a conducive environment for axon re-growth and functional recovery in long sciatic nerve defects in rabbits and rodents [4,5,6]. Beyond their vascular function, endothelial cells secrete various factors beneficial to regeneration [7], such as brain-derived neurotrophic factor (BDNF) which when secreted by human umbilical vein endothelial cells (HUVECs) enhanced chicken and rat dorsal root ganglion neuron outgrowth in vitro [8]. Exosomes secreted from HUVECs have also been shown to promote the repair phenotype in cultures of RSC96 Schwann cells and improve functional recovery and remyelination in a rat sciatic nerve crush model when injected following injury [9].

Following a nerve transection injury in rodents, endothelial cells in both nerve stumps undergo angiogenesis and can migrate through the tissue bridge that forms across the nerve gap, forming multiple thin blood vessels which link the two nerve stumps. This polarised structure guides the subsequent migration of Schwann cells and provides a path for axonal regeneration [10]. Disruption of organised vascular growth through genetic manipulation of Plexin-D1 in mice resulted in the abnormal regrowth of nerve fibres across the nerve bridge, delaying regeneration [11]. The maximal distance between nerve stumps that can support spontaneous formation of this endothelial cell bridge has not been determined, but the length is typically a few millimetres in animal models. 

The contribution of organised vascular structures to the support and guidance of nerve regeneration across small gaps following transection raises the possibility that mimicking such features could be a useful tool in nerve tissue engineering for bridging larger gaps in humans, which are generally treated using a nerve autograft [2]. Our previous work indicated that collagen gels containing aligned structures made from HUVECs supported nerve regeneration in a rat nerve gap model to a greater extent than equivalent materials containing Schwann cells [5]. To progress nerve tissue engineering research based on this concept, the present study aimed to develop EngNT containing organised and stable tube-like structures formed from endothelial cells suitable for translational development as an advanced therapy medicinal product (ATMP).

Human endothelial cells can be isolated from patients, with endogenous sources of mature and progenitor endothelial cells in adults found in the peripheral blood, blood vessels, bone marrow, and adipose tissue [12,13,14]. While autologous cells would reduce the likelihood of graft rejection, the number of autologous cells suitable for isolation is limited and endothelial cells are subject to inter-organ and inter-patient variability [15,16]. Furthermore, in nerve tissue engineering to treat trauma, rapid repair is desirable, increasing the possibility of a full recovery [17]. 

Therefore, allogeneic human induced pluripotent stem cell (hiPSC) derived cells, which have recently gained much attention in Parkinson’s disease clinical trials [18], could be suitable for an ‘off-the-shelf’ application, overcoming the time-consuming harvesting and processing of patient-derived autologous cells [19,20,21]. Endothelial cells differentiated from hiPSCs [22,23] could potentially be used as part of an allogenic engineered tissue approach. Issues around immunogenicity and graft rejection could be addressed through the use of human leukocyte antigen (HLA) haplotype matching, HLA cloaking, or immunosuppression, and risks of tumorigenicity mitigated by stringent purification and the use of inducible suicide genes [20,24]. In this study, endothelial cells were differentiated from hiPSCs and characterised at the molecular, protein, and functional level. Subsequently, collagen hydrogel-based engineered neural tissue (EngNT) containing the derived endothelial cells (EngNT-ECs) was produced and the formation of stable aligned tube-like structures within the constructs was investigated. The potential of the EngNT-EC constructs to support neuronal regeneration was then assessed in vitro.This work advanced engineered nerve tissue by incorporating endothelial luminal structures that mimic key features of the vascular structures seen early in the natural nerve regeneration process. This provides a platform suitable for investigating the role of aligned vascular structures in peripheral nerve injury treatment and translational development.

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