Custom-engineered hydrogels for delivery of human iPSC-derived neurons into the injured cervical spinal cord

Abstract

Cervical damage is the most prevalent type of spinal cord injury clinically, although few preclinical research studies focus on this anatomical region of injury. Here we present a combinatorial therapy composed of a custom-engineered, injectable hydrogel and human induced pluripotent stem cell (iPSC)-derived deep cortical neurons. The biomimetic hydrogel has a modular design that includes a protein-engineered component to allow customization of the cell-adhesive peptide sequence and a synthetic polymer component to allow customization of the gel mechanical properties. In vitro studies with encapsulated iPSC-neurons were used to select a bespoke hydrogel formulation that maintains cell viability and promotes neurite extension. Following injection into the injured cervical spinal cord in a rat contusion model, the hydrogel biodegraded over six weeks without causing any adverse reaction. Compared to cell delivery using saline, the hydrogel significantly improved the reproducibility of cell transplantation and integration into the host tissue. Across three metrics of animal behavior, this combinatorial therapy significantly improved sensorimotor function by six weeks post transplantation. Taken together, these findings demonstrate that design of a combinatorial therapy that includes a gel customized for a specific fate-restricted cell type can induce regeneration in the injured cervical spinal cord.

Keywords: Biomaterials; Cell transplantation; Hydrogel; Induced pluripotent stem cell; Spinal cord injury.

Figure 1.. The SHIELD family of hydrogels is designed to address cell loss during transplantation and provide bioactive cues that promote cell survival and neurite extension.

A. Schematic of the SHIELD system, which is composed of a C7 engineered protein and a PEG-P1 peptide copolymer with varying amounts of PNIPAM copolymer. B. The shear storage modulus of SHIELD formulations at 37°C is controlled by tuning the percentage of thermosensitive PNIPAM. Data are mean ± SEM. ****P < 0.0001; Tukey post hoc test; n = 3 to 8. C. Representative frequency sweeps of storage (G’) and loss (G”) moduli were performed at 24°C to characterize the viscoelastic behavior of SHIELD (0 wt% PNIPAM) and a negative control sample that includes only the C7 engineered protein without the PEG-P1 binding partner.

Figure 2.. SHIELD can be customized to promote viability and neurite extension of hiPSC-DCNs in vitro.

A. Schematic of the directed differentiation protocol to drive hiPSCs toward a deep cortical neuron (DCN) commitment. B. Representative brightfield images of hiPSC cultures at different points of the directed differentiation protocol. At day 0, the culture has a “cobblestone” morphology, consistent with pluripotent stem cells. At day 17, the culture has begun to radially organize into “rosette” structures, consistent with neuroepithelium. By day 32, the culture is largely interconnected by long processes, consistent with maturing neurons. C. Percentage of live hiPSC-DCNs (Calcein-AM⁺) after 7 days following encapsulation in SHIELD variants with fibronectin- (RGDS) or laminin- (YIGSR, IKVAV) derived binding domains, or a scrambled non-adhesive control sequence (RDGS). Data are mean ± SD. *P = 0.0150 (RGDS vs. RDGS), *P = 0.0306 (YIGSR vs. RDGS), F = 5.466; one-way analysis of variance (ANOVA) with Dunnet’s multiple comparisons post hoc test; N = 2 independent experiments, each with ≥2 technical replicates. D. Quantification of hiPSC-DCN neurite length in SHIELD gels with varying amount of PNIPAM after 3 days in culture. Data are mean ± SD. **P = 0.0020, ***P = 0.0006, ****P < 0.0001, F = 26.40; one-way ANOVA with Dunnet’s multiple comparisons post hoc test; N = 2 independent experiments, each with ≥ 2 technical replicates. E. Percentage of dead hiPSC-DCNs (ethidium homodimer-1⁺) following exposure to pipetting in saline (i.e. no injection), injection through a syringe needle (33-G) in saline at 500 nL/min, or injection through a syringe needle (33-G) in SHIELD (RGD binding domain and 0% PNIPAM) at 500 nL/min. Data are mean ± SD. **P = 0.0011, F = 7.648; one-way ANOVA with Dunnet’s multiple comparisons post hoc test; N = 2 independent experiments, each with ≥2 technical replicates.

Figure 3.. Crosslinking SHIELD is required for long-term retention within the injured cervical spine.

A. Timeline for biodegradation and functional studies depicting surgical procedures (injury, injection; white), live imaging (IVIS; purple), behavioral functional assays (behavior; pink), and endpoints for tissue processing (explant; yellow). B. The fluorescence of intraspinally injected cyanine-7 labeled C7 and SHIELD was quantified up to 6 weeks post injection. Data are normalized to C7 at 1 week post injection. Data are mean ± SEM, n = 3–4 per condition. C. Representative IVIS images from two individual rats intraspinally injected with labeled C7 or SHIELD from 1–6 weeks post injection. In each image, skin was retracted to expose the underlying muscle to avoid masking of signal due to the pigmentation patterns in hooded rats.

Figure 4.. Encapsulation in SHIELD improves hiPSC-DCN transplantation metrics.

A. At 6 weeks post transplantation, graft volume was calculated by measuring the area occupied by SC101⁺ (human nuclear marker) hiPSC-DCNs in serially labeled spinal cord tissue sections. Delivery in saline resulted in a significantly higher graft volume average and larger variability in graft volume when compared to delivery in SHIELD. Data are violin plots; median = dashed line, quartiles = dotted lines; *P = 0.0469, F = 16.17; Welch’s t test; n = 4. B. Quantification of SC101⁺ transplanted cells demonstrated no significant difference in the average number of human cells present within the graft regardless of delivery vehicle. Rats transplanted with hiPSC-DCNs delivered in saline had higher variability in quantified SC101⁺ transplanted human cells. Data are violin plots; median = dashed line, quartiles = dotted lines; P = 0.254, F = 4.409; Welch’s t test; n = 4. C. Quantification of co-labeled Ki67⁺/SC101⁺ human transplanted cells demonstrates a significantly higher number of proliferating human cells in grafts delivered in saline versus SHIELD. Data are violin plots; median = dashed line, quartiles = dotted lines; ***P = 0.0005, F = 1.980; Welch’s t test; n = 4. D. At 6 weeks post hiPSC-DCN transplantation, SC101⁺ human cell bodies (top) and SC121⁺ human projections (bottom) were quantified in 1-mm increments in serial-labeled sections of spinal cord tissue to determine distribution along the continuum of the spinal cord. Distribution and quantity of SC101⁺ human cells were similar regardless of delivery vehicle. Delivery in SHIELD, however, resulted in a higher number of SC121⁺ projections and extension over a longer distance, as appropriate for this cellular phenotype. Data are mean ± SEM. Bottom schematic shows typical spinal cord cross-section at the location of the graft epicenter (C5) and 5 mm rostral (negative) and caudal (positive). E. Left: Schematic of the rat cranium and spinal cord vertebrae, depicting location of histological slices. Right top: Representative images of SC101⁺ human cells taken from the transverse plane of the graft epicenter in a rat transplanted with hiPSC-DCNs delivered in SHIELD. Area of inset shown with white box. Right bottom: Representative images of SC121⁺ human projections taken from the transverse plane at locations 1, 5, and 10 mm caudal to the graft epicenter in rats transplanted with hiPSC-DCNs delivered in SHIELD or saline.

Figure 5.. Transplantation of hiPSC-DCNs encapsulated in SHIELD promotes functional recovery.

A. Simplified schematic depicting the major muscles of the forelimb and the behavioral assays that target their primary function. B. Legend depicting the five different treatment conditions for data in panels C-E. C-E. All data are mean ± SEM, with one-way ANOVA with Tukey’s multiple comparisons post hoc test. Per experimental condition, n=6. C. The cylinder assay quantifies the percentage of right forelimb touches per total touches as a metric of forelimb preference, normalized to pre-injury levels. *P = 0.0243, **P = 0.0027, ****P < 0.0001. D. Grip strength of both forelimbs was quantified using a metered bar, normalized to pre-injury levels. *P = 0.0336, ****P < 0.0001. E. Forelimb coordination was assessed with the horizontal ladder walk test. A decrease in coordination presents as an increased percentage of step errors per total steps, normalized to pre-injury levels. *P = 0.0217, ****P < 0.0001.