Functional assessment of cell transplants

Figure 1
Figure 1: Light and electron microscopy. Sagittal sections corresponding to the center of injury. H&E stained paraffin sections of spinal cords from animals subjected to contusive SCI and treated with human fibroblasts (A) or hES-derived OPCs (B) Arrows indicate areas of neuron like tracts transversing the center of injury in OPC treated rats. This is consistent with LFB (blue) and cresyl violet (purple) staining which showed significantly more cavitation (dotted circles) in the human fibroblast-treated group (C) compared to the hES-derived OPC group (D). Specifically the fibroblast treated groups shows overall loss of tissue integrity with reduced LFB and cresyl violet staining, while the OPC treated group shows the presence of neurons, stained with cresyl violet surrounded by LFB staining. Transmission electron microscopy of sagittal sections of spinal cords also showed disrupted myelin for the fibroblast-treated group (E), whereas remyelination with thin, compact sheaths was observed for the hES-OPC group (F, arrowheads). Magnification: (A-D) 40x and (E, F) 10000x.

The litmus test for any cell therapy is the behavior of the generated cells in an in vivo disease model. For acute SCI, a rat model of contusive SCI at the eigth thoracic vertebra-level was employed. hES- and iPS-derived OPCs were transplanted twenty four hours after this injury, directly into the spinal parenchyma. Functional assessments included histology (to study survival, integration, and maturation of cells in vivo), somatosensory evoked potential (to study repair of sensory pathways in the spinal cord), and electron microscopy (to study ultrastructure of de novo myelin formation). Cells were also tracked using bioluminescence imaging (BLI).

Figure 2
Figure 2: Bioluminescence of hES derived OPC-treated groups. Bioluminescence was followed at six different time points over a period of 4 weeks. A) Images taken at each time point that depict the bioluminescent activity of the transplanted cells. B) Total flux (photons/second) measured at various time points of the OPC-treated injury and laminectomy-only groups. Error bars represent standard deviations. C) An image of an animal showing bioluminescent activity at the site of OPC transplantation. *p<0.05.

hES-OPCs survived and integrated into the spinal cord parenchyma, as evidenced by expression of human nuclear antigen (HNA) in spinal cord sections six weeks after cell transplantation. Several of the HNA+ cells also expressed the myelin basic protein (MBP), indicating that the myelination machinery is active in these cells. Survival was also confirmed by the persistence of a BLI signal.

Somatosensory evoked potential (SSEP) measurements can be used to measure the integrity of ascending sensory pathways. These involve electrical stimulation at the hindlimb; the transmitted signal is recorded from the sensory cortex and subjected to shape analysis. In the case of hES-OPC transplantation after moderate injury, treated groups showed an increase in the signal amplitude and decrease in signal latency (indicative of better sensory pathway integrity) over the course of six weeks when compared to the negative control.

Histology and electron microscopy also showed better anatomical integrity of the hES-OPC treated group. In the case of iPS-OP treated animals, H&E staining revealed improved anatomy of treated groups compared to heat-killed controls. This was mainly seen in terms of the difference in manifestation of injury-associated cavity. The cavity of treated groups showed more tissue, resembling transplanted cells.

Luxol Fast Blue (LFB) staining revealed more myelination in the spinal cord tracts for iPS-OPC groups. This was further corroborated by electron microscopy which showed more myelinated axons compared to control.