Long-distance axonal growth from human induced pluripotent stem cells after spinal cord injury

Abstract

Human induced pluripotent stem cells (iPSCs) from a healthy 86-year-old male were differentiated into neural stem cells and grafted into adult immunodeficient rats after spinal cord injury. Three months after C5 lateral hemisections, iPSCs survived and differentiated into neurons and glia and extended tens of thousands of axons from the lesion site over virtually the entire length of the rat CNS. These iPSC-derived axons extended through adult white matter of the injured spinal cord, frequently penetrating gray matter and forming synapses with rat neurons. In turn, host supraspinal motor axons penetrated human iPSC grafts and formed synapses. These findings indicate that intrinsic neuronal mechanisms readily overcome the inhibitory milieu of the adult injured spinal cord to extend many axons over very long distances; these capabilities persist even in neurons reprogrammed from very aged human cells.

Figure 1. Survival, Differentiation and Growth of Human iPSC-Derived Neural Stem Cells in Sites of Spinal Cord Injury

(A) GFP-labeled human iPSC-derived neural stem cells were grafted into sites of C5 hemisection spinal cord injury. Horizontal section immunolabeled for GFP and GFAP indicates that implants survive well and distribute through the lesion cavity. Rostral is to left, caudal is to right. (B) The majority of cells within the graft immunolabel for mature neuronal markers NeuN, indicating neuronal differentiation. (C-E) Very large numbers of GFP-labeled axons extend caudally into the host spinal cord (D) white matter and (E) gray matter (region of NeuN labeling). Insets in panel C indicate that axons co-localize with Tuj1, but not neurofilament (NF). (F-G) GFP, MBP, and NeuN triple labeling of a coronal section 3 segments (C8) caudal to the graft shows dense distribution of human axons predominantly on right, lesioned side of the spinal cord. (G) Higher magnification of panel F from lateral white matter demonstrates remarkably high number of human axons interspersed in white matter. Scale bar: A, 350 μm; B, 10 μm; C, 600 μm; D-E, 32 μm; F, 250 μm; G, 20 μm.

Figure 2. Long-Distance Growth of iPSC-Derived NSCs

(A-D) Light-level GFP immunolabeling of human iPSC-derived axons in coronal sections shows very large numbers of axons extending into caudal host spinal cord. Insets in each panel show the sampled region from which higher magnification views were obtained: (A) C8, (B) T6, (C) T12 and (D) L4. (E) Fluorescent GFP labeled human iPSC-derived axons extend rostrally into brain in a sagittal section at a low magnification. (F-H) Higher magnification views from the boxed areas in panel E show that GFP labeled human axons extend into (F) midbrain and (G) gracile (Gr) and solitary (Sol) nuclei; (H) very large numbers of axons enter the medulla. NeuN labels host brain neurons. (I-K) Individual GFP-labeled human axons are present in (I) cortex, (J) olfactory bulb, and (K) cerebellum. Insets indicate region of sampling. Scale bar: A-C, 20 μm; D, 60 μm; E, 1.7 mm; F, H, 110 μm, G, 180 μm, I-K, 100 μm.

Figure 3. Association of Human Axons with Host Myelin, and Connectivity with Host

(A-B) GFP and MBP double labeling indicate close association of human axons with host myelin in white matter. (A) Horizontal and (B) coronal section (C8). Myelination of human axons is not evident. (C) Electron microscopy confirms that extending iPSC-derived graft (g) axons contact host myelin sheaths. (D) GFP-expressing human axon terminals are closely associated with MAP-2-expressing host neurons and dendrites caudal to the lesion site. (E) A z-stack image triple labeled for GFP, synaptophysin (Syn), and ChAT, indicating co-association of graft-derived human axon terminals with a synaptic marker in direct association with host motor neurons. (F) A z-stack image triple labeled for GFP, human-specific synaptophysin (hSyn), and MAP2, showing graft-derived human axon terminals with a synaptic marker in direct association with host dendrites. (G) Double labeling for GFP, vesicular glutamate transporter 1 (vGlut1) showing a graft-derived human axon terminal co-expression of vGlut1. (H) Electron microscopy confirms that DAB-labeled, GFP-expressing human axon terminals form synapses (arrowhead) with host dendrites (see inset). Scale bar: A, D, 4 μm; B, E, G, 2 μm; C, 500 nm; F, 1.8 μm; H 200 nm.

Figure 4. Lack of Cell Fusion; Host Axonal Ingrowth into Grafts

(A) Triple labeling for RFP, GFP, and NeuN reveals survival and neurite extension from RFP-expressing human iPSC-derived NSCs into GFP transgenic adult mice with C5 spinal cord lesions. (B) Higher magnification view from host white matter 2 mm caudal to the graft shows that RFP-labeled human axons do not co-localize with GFP-expressing host white matter processes. Individual RFP axons are distinct from host GFP axons in inset. (C) Host (h) raphespinal axons penetrate a control lesion (Les) site (injected at the time of grafting with a fibrin/thrombin matrix, but no cells); dashed lines indicate host/lesion interface. (D) Host raphespinal axons penetrate an iPSC graft (g) in the lesion site; the boxed region is shown in panel E. (F) Serotonergic axons penetrating grafts in the lesion site express the pre-synaptic protein synaptophysin. (G) Quantification shows a 6-fold increase in penetration of host serotonergic axons into the lesion site compared to controls, P<0.001. Scale bar: A, 100 μm; B, 22 μm; C-D, 120μm; E, 10μm; G, 5 μm.