Human iPS cell-derived astrocyte transplants preserve respiratory function after spinal cord injury

Abstract

Transplantation-based replacement of lost and/or dysfunctional astrocytes is a promising therapy for spinal cord injury (SCI) that has not been extensively explored, despite the integral roles played by astrocytes in the central nervous system (CNS). Induced pluripotent stem (iPS) cells are a clinically-relevant source of pluripotent cells that both avoid ethical issues of embryonic stem cells and allow for homogeneous derivation of mature cell types in large quantities, potentially in an autologous fashion. Despite their promise, the iPS cell field is in its infancy with respect to evaluating in vivo graft integration and therapeutic efficacy in SCI models. Astrocytes express the major glutamate transporter, GLT1, which is responsible for the vast majority of glutamate uptake in spinal cord. Following SCI, compromised GLT1 expression/function can increase susceptibility to excitotoxicity. We therefore evaluated intraspinal transplantation of human iPS cell-derived astrocytes (hIPSAs) following cervical contusion SCI as a novel strategy for reconstituting GLT1 expression and for protecting diaphragmatic respiratory neural circuitry. Transplant-derived cells showed robust long-term survival post-injection and efficiently differentiated into astrocytes in injured spinal cord of both immunesuppressed mice and rats. However, the majority of transplant-derived astrocytes did not express high levels of GLT1, particularly at early times post-injection. To enhance their ability to modulate extracellular glutamate levels, we engineered hIPSAs with lentivirus to constitutively express GLT1. Overexpression significantly increased GLT1 protein and functional GLT1-mediated glutamate uptake levels in hIPSAs both in vitro and in vivo post-transplantation. Compared to human fibroblast control and unmodified hIPSA transplantation, GLT1-overexpressing hIPSAs reduced (1) lesion size within the injured cervical spinal cord, (2) morphological denervation by respiratory phrenic motor neurons at the diaphragm neuromuscular junction, and (3) functional diaphragm denervation as measured by recording of spontaneous EMGs and evoked compound muscle action potentials. Our findings demonstrate that hiPSA transplantation is a therapeutically-powerful approach for SCI.

Affected astrocytes in the spinal cord of the leukodystrophy vanishing white matter

“…Remarkably, most transplantation studies have so far focused on the brain, while many leukodystrophies also show spinal cord involvement, such as Alexander disease (Liu et al, 2016; van der Knaap et al, 2006), PMD (Koeppen & Robitaille, 2002), MLD (Toldo, Carollo, Battistella, & Laverda, 2005) and Krabbe disease (Wang, Melberg, Weis, Mansson, & Raininko, 2007). In rodent models of spinal cord injury, glial transplantations in the spinal cord resulted in decreased pathology and functional recovery (Haidet‐Phillips et al, 2014; Li et al, 2015; Nicaise, Mitrecic, Falnikar, & Lepore, 2015). Also a clinical trial, where patients with spinal cord injury were transplanted with human neural stem cells, showed moderate clinical improvements (Shin et al, 2015).…”

Harnessing the power of cell transplantation to target respiratory dysfunction following spinal cord injury

“…These mechanisms include the obvious but challenging replacement of injured long-distance projecting neurons of the circuits controlling breathing. To date, transplantation-based interventions have been used to promote plasticity of axonal connections that are part of these respiratory circuits (Alilain et al, 2011; Decherchi and Gauthier, 2002; Decherchi et al, 1996; Li et al, 2003; Polentes et al, 2004; Stamegna et al, 2011), replacement of glial cell types (Li et al, 2015a; Li et al, 2015b) and local respiratory interneurons (Lee et al, 2014; White et al, 2010) of the cervical spinal cord, delivery of neurotrophic factor support (Gransee et al, 2015), and restoration of neurotransmitter signaling (Dougherty et al, 2016; Li et al, 2015a; Li et al, 2015b). While this list only represents a few of the possible benefits that transplantation can provide, the fact that only a limited number of studies have to date been conducted in this field means that many of the potential mechanistic benefits of transplanted cells have yet to be explored for targeting respiratory dysfunction in SCI.…”

“…This technology also allows for homogeneous derivation of mature cell types in large quantities, potentially in an autologous fashion from the eventual transplant recipient (Das and Pal, 2010; Kiskinis and Eggan, 2010). Despite the promise of this approach, the iPS cell field is in its infancy with respect to evaluating in vivo graft integration and therapeutic usefulness in relevant SCI models (Goulao and Lepore, 2016; Salewski et al, 2010), particularly in the context of respiratory dysfunction (Li et al, 2015b). …”

“…We derived pluripotent iPS cells from non-diseased human donors, generated glial progenitors and then differentiated these cells into astrocytes prior to transplantation into the unilateral C4 contusion model (Li et al, 2015b). Similar to rodent GRPs, human iPS cell-derived transplants survived for long periods of time in the injured cervical spinal, did not form tumors or show uncontrolled proliferation, differentiated into only GFAP-positive astrocytes, and did not localize to ectopic locations or differentiate into unexpected lineages.…”

Transplantation of human immature dental pulp stem cell in dogs with chronic spinal cord injury

“…A great number of studies have been performed with stem cells from different sources using animal models of spinal cord injury, and even therapeutic attempts on humans, presenting promising results [5][6][7] . However, there are some difficulties to perform a direct comparison of such results, due to the large variability of characterization grade of the transplanted cells, different animal models, and the time fluctuation between injury event and its treatment Commonly, dogs present clinical injuries of the thoracic and lumbar spinal medulla mainly due to herniation or protrusion of the intervertebral discs 9 .…”