Local Delivery of Chondroitinase ABC Promotes Neuronal Differentiation of Human Induced Pluripotent Stem Cell-Derived Neural Grafts in a Spinal Cord Injury Model

Führmann, Tobias (Institute of Biomaterials and Biomedical Engineering, University of Toronto)
Anandakumaran, Priya N (Institute of Biomaterials and Biomedical Engineering, University of Toronto)
Payne Samantha L (Institute of Biomaterials and Biomedical Engineering, University of Toronto)
Pakulska, Malgosia (Institute of Biomaterials and Biomedical Engineering, University of Toronto)
Varga, Balazs (Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital,)
Nagy, Andras (Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital,)
Tator, Charles (Krembil Neuroscience Centre, University Health Network; Toronto, Canada)
Shoichet, Molly S (Institute of Biomaterials and Biomedical Engineering, University of Toronto)

Introduction

Functional recovery following spinal cord injury (SCI) is limited due to a cascade of cellular and biomolecular events, termed secondary injury, that lead to cell loss and the expression of growth inhibitory molecules. Because of the diversity of these events, it is likely that any regenerative treatment strategy has to target multiple aspects of the injury including neuroprotective and neuroregenerative molecules, endogenous cell stimulation and/or exogenous cell transplants, and enzymes to break down inhibitory molecules.
This project focuses on replacing lost cells, and degrading chondroitin sulfate proteoglycans (CSPGs), a group of mostly growth inhibitory proteins that are highly expressed at the lesion site. The main aim was to replace lost neurons with pre-differentiated human induced pluripotent stem cell-derived neuroepithelial cells (hNECs). To aid in cell survival they were delivered in an injectable hydrogel. The hNECs were combined with the local delivery of chondroitinase ABC (ChABC), which degrades CSPGs. ChABC can also degrade perineuronal nets, potentially promoting synapse formation between grafted and endogenous neurons. Importantly, both systems are injectable and in situ gelling for minimally invasive delivery to the injured spinal cord.

Materials and Methods

hNECs were differentiated into immature neuronal precursor cells and sorted for PSA-NCAM using magnetic assisted cell sorting (MACS) prior to grafting. Cells were transplanted in a physical hydrogel comprised of hyaluronan (HA) and methyl cellulose (MC). HA is shear thinning and can be delivered through a fine needle, whereas MC is inverse thermal gelling, enabling it to form a gel at 37°C to provide localized cell delivery.
Recombinant ChABC with an N-terminal His tag and a C-terminal FLAG tag was expressed as a fusion protein with Src homology domain 3 (SH3) in E. coli (ChABC-SH3)(1). MC-thiol and MC-peptide were formed by chemical modification of methylcellulose (MC), as previously described (2). ChABC-SH3 was combined with MC-peptide, and MC-thiol in artificial cerebrospinal fluid (aCSF). MC-thiol was crosslinked with poly(ethylene glycol)-bismaleimide (PEGMI2, 3000 Da, 0.75 maleimide: 1 thiol mol ratio).
One week following a moderate clip compression injury (26g) at level T2, female rats received cell transplants in HAMC (0.75% / 0.75% w/w) at 4 sites rostral and caudal to the lesion (20,000 cells/µl, 8µl in total injected). ChABC was injected intrathecally (5µl) at the time of injury and at one week post injury. Animals were tested for motor (BBB, ladder walk) and sensory function (tail flick) for up to 9 weeks. All animal procedures were performed in accordance with the Guide to the Care and Use of Experimental Animals (Canadian Council on Animal Care) and protocols were approved by the Animal Care Committee of the Research Institute of the University Health Network. Transplanted cells were indentified with antibodies against human nuclei (hNUC) and human cytoplasm (STEM121). Cell fate was determined using markers for neural stem cells (nestin), neurons (bIII-tubulin (TUBB3)), astrocytes (GFAP), and oligodendrocytes (SOX10). Data was subjected to an analysis of variance (ANOVA) followed by a Bonferroni’s post-hoc test for comparisons between means of multiple groups, a t-test was performed to assess differences between the cell groups only.

Results

hNECs were successfully differentiated into immature neurons, as indicated by the down-regulation of neural stem cell marker (SOX2, Nestin) and the up-regulation of neuronal marker (TUBB3, doublecortin). Sorting further enriched the population of neuronal cells, leading an increase of TUBB3-positive cells from 36 ± 3% to 81±5 %.
ChABC-SH3 was successfully expressed and purified from E. coli. Active ChABC-SH3 was released from MC-peptide for a period of at least 7 days. This release was tunable, either by choosing peptides with different dissociation constants or by varying the ratio of protein to peptide within the gel(1). ChABC decreased CSPG levels at 2, but not at 9, weeks following injury.
Surviving cells were found with and without co-delivery of ChABC 9 weeks after injury, and some cells retained the ability to proliferate (Ki67) (Figure 1). At 2 weeks following injury, most of the human cells expressed the neuronal marker TUBB3 (65-75%). At 9 weeks post-injury, the fate of the transplanted NESCs changed to having more nestin+ progenitors, more SOX10+ oligodendrocytes, similar GFAP+ astrocytes and fewer TUBB3+ neurons. While the percentages of hNESCs positive for nestin, GFAP, and SOX10 were similar between animals transplanted with cells alone and cells co-delivered with chABC at 9 weeks post-injury, the percentage of TUBB3+ neurons was significantly different: 32±3.02 % of the transplanted hNESCs were TUBB3-positive when transplanted with chABC and only 16±2.5 % were positive when transplanted alone (Figure 2). This effect seems to be partially mediated by the EGF-receptor (EGF-R), as treating cells in culture, in the presence of chondroitin sulfate A, with EGF-R antagonists promoted neuronal differentiation.

Discussion and Conclusion

The combined therapy did not have any deleterious effects on motor or sensory function, demonstrating that the neuronal cells, ChABC and delivery vehicles are safe. hNECs survived and integrated into the host spinal cord. ChABC reduced early CSPG levels and promoted neuronal differentiation of transplanted cells.


Figure 1: Quantification of surviving (a) and proliferating cells (b). (c) Surviving cells (hNUC, green) at 9 weeks following injury. Some cells were still proliferating (Ki67, red).

Figure 2: (a) Transplanted human cells at 9 weeks post injury stained against human cytoplasm (STEM121, green), and bIII-tubulin (TUBB3, red). (b) Histogram showing the percentage of hNESCs positive for the markers identifying progenitor cells, astrocytes, oligodendrocytes, and neurons.

Acknowledgements

We are grateful to the Craig H Neilsen Foundation for funding this research.

References

1.             Pakulska MM, Vulic K, Shoichet MS. Affinity-based release of chondroitinase ABC from a modified methylcellulose hydrogel. J Control Release Off J Control Release Soc. 2013 Oct 10;171(1):11–6.

2.             Vulic K, Shoichet MS. Tunable growth factor delivery from injectable hydrogels for tissue engineering. J Am Chem Soc. 2012 Jan 18;134(2):882–5.

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