The Effect of iPS-Derived Neural Progenitors Seeded on Laminin-Coated pHEMA-MOETACl Hydrogel with Dual Porosity in a Rat Model of Chronic Spinal Cord Injury

Abstract

Spinal cord injury (SCI), is a devastating condition leading to the loss of locomotor and sensory function below the injured segment. Despite some progress in acute SCI treatment using stem cells and biomaterials, chronic SCI remains to be addressed. We have assessed the use of laminin-coated hydrogel with dual porosity, seeded with induced pluripotent stem cell-derived neural progenitors (iPSC-NPs), in a rat model of chronic SCI. iPSC-NPs cultured for 3 weeks in hydrogel in vitro were positive for nestin, glial fibrillary acidic protein (GFAP) and microtubule-associated protein 2 (MAP2). These cell-polymer constructs were implanted into a balloon compression lesion, 5 weeks after lesion induction. Animals were behaviorally tested, and spinal cord tissue was immunohistochemically analyzed 28 weeks after SCI. The implanted iPSC-NPs survived in the scaffold for the entire experimental period. Host axons, astrocytes and blood vessels grew into the implant and an increased sprouting of host TH⁺ fibers was observed in the lesion vicinity. The implantation of iPSC-NP-LHM cell-polymer construct into the chronic SCI led to the integration of material into the injured spinal cord, reduced cavitation and supported the iPSC-NPs survival, but did not result in a statistically significant improvement of locomotor recovery.

Keywords: Chronic spinal cord injury; HEMA hydrogel; human induced pluripotent stem cells; laminin; neural progenitors; surface charge.

Fig. 1.

Experimental set-up and representative TMA⁺ diffusion curves in native gel, and 3 (3-d) and 21 days (3-w) after cell seeding. To stabilize the inter-tip distance of the electrode array, an iontophoretic micropipette and TMA⁺-selective microelectrode (ISM) were glued together with dental cement (A). Note the increased curve amplitude reflecting the reduction of the ECS volume fraction α in 3-w hydrogel, already populated with cells (B). Development of the changes in the ECS volume fraction α and tortuosity λ, calculated as a percentage of their values in the native hydrogel, which were set to 100% (C). Note that an increase in tortuosity, indicating the production of diffusion barriers created, for example by cell processes, was already observed in 3-d hydrogel, while a significant decrease in the ECS volume fraction was present only in 3-w gel, after a robust stem cell proliferation. ECS: extracellular space; ISM: ion-selective microelectrode; TMA: tetramethylammonium.

Fig. 2.

(A) MRI scan of spinal cord lesion 5 weeks after SCI in rat (before hydrogel implantation). Lesion cavity is indicated by hyper-intensive signal (circle). The insertion of LHM hydrogel (with or without cells) was performed according to individual MRI scans. (B) LHM hydrogel seeded with cells (circle) adhered well to the host tissue; only a small cavity is visible at the rostral part of the implant (arrow). MRI scan was taken 28 weeks after SCI. Scale bar is 500 μm. LHM: laminin-coated pHEMA-MOETACl; MRI: magnetic resonance imaging; SCI: spinal cord injury.

Fig. 3.

Immunocytochemistry of iPSC-NPs in gel samples. IPSC-NPs were cultivated for 3 weeks in gels and stained for various markers. Phalloidin (A), GFAP (B), olig2 (C), nestin (D), MAP2 (E), NF160 (F) (red). Nuclei were stained with DAPI (blue). Scale bar is 50 μm. DAPI: 4’, 6-2, dihydrochloride; GFAP: glial fibrillary acidic protein; iPSC-NP: induced pluripotent stem cell-derived neural progenitor; MAP2: microtubule-associated protein 2.

Fig. 4.

Immunohistochemistry showing the chronic lesion injected with saline 28 weeks after SCI. Tissue in the lesion center was atrophic with glial scar (GFAP, red, A, detail A1). Axons were present in the remaining tissue (NF200, red B, detail B1). Nuclei were stained with DAPI (blue). Scale bars are 500 μm (A, B), 20 μm (A1, B1). DAPI: 4′,6-diamidino-2-phenylindole; GFAP: glial fibrillary acidic protein; SCI: spinal cord injury.

Fig. 5.

Immunohistochemistry showing the tissue element incorporation into an implanted LHM hydrogel without (A, C, E) or with iPSC-NPs (B, D, F), 28 weeks after SCI. Almost no ingrowth of axons (NF200, red, A, detail A1) was observed into the implanted LHM gel without cells. On the contrary, an implanted cell-polymer construct was infiltrated with cells that survived and endogenous axons (NF200, red B, detail B1, 2). Blood vessels infiltrated gels with or without cells (RECA, red, C, D, detail C1, D1, D2). The implanted hydrogel was always surrounded with a GFAP-positive glial scar (GFAP, red, E, F). GFAP-positive cells were found within the hydrogel in close vicinity with implanted stem cells (detail F1, 2). Human iPSC-NPs were stained with MTC02 (green). Nuclei were stained with DAPI (blue). Scale bars are 500 μm (A, B, E, F), 400 μm (C, D), 40 μm (C1, D1, 2) and 20 μm (A1, B1, 2, E1, F1, 2). DAPI: 4’, 6-2, dihydrochloride; GFAP: glial fibrillary acidic protein; iPSC-NP: induced pluripotent stem cell-derived neural progenitor; LHM: laminin-coated pHEMA-MOETACl; SCI: spinal cord injury.

Fig. 6.

Survival and differentiation of iPSC-NPs outside the hydrogel 28 weeks after SCI. iPSC-NPs that migrated out of the LHM hydrogel, differentiated into NF200 (A–C) and MAP2 (D–F) positive cells. Most of the cells with neuron-like morphology were not positive for GFAP (G–I). Images were taken from three different animals. All the scale bars are 20 μm. GFAP: glial fibrillary acidic protein; iPSC-NP: induced pluripotent stem cell-derived neural progenitor; LHM: laminin-coated pHEMA-MOETACl; MAP2: microtubule-associated protein 2; SCI: spinal cord injury.

Fig. 7.

Density and distribution of TH-positive fibers in the injured spinal cords of control rats (n=4), and rats transplanted with gel (n=3) and gel seeded with iPSC-NPs (n=4). From each animal at least 8–10 slides were analyzed. (A) Quantitative analysis of the number of TH-positive fibers in the spinal cord tissue of control and transplanted animals. (B) Distribution of TH-positive fibers in the spinal cord tissue of control and transplanted animals. (C) Histochemical staining demonstrates the different density of TH-positive fibers in the injured spinal cord. (D) Injured spinal cord transplanted with gel only and (E) transplanted with iPSC-NP-seeded gel (detail of TH⁺fiberE1). All the scale bars are 200 μm. iPSC-NP: induced pluripotent stem cell-derived neural progenitor; TH: tyrosine hydroxylase.

Fig. 8.

Functional recovery after LHM + iPSC-NP combined treatment of chronic SCI. No significant differences were found between all tested groups, neither in motoric recovery (BBB test, A), nor in thermal nociceptive stimulus (plantar test, B). BBB: Basso, Beattie, and Bresnahan; iPSC-NP: induced pluripotent stem cell-derived neural progenitor; LHM: laminin-coated pHEMA-MOETACl; SCI: spinal cord injury.