Human neural stem cells differentiate and promote locomotor recovery in spinal cord-injured mice

Abstract

We report that prospectively isolated, human CNS stem cells grown as neurospheres (hCNS-SCns) survive, migrate, and express differentiation markers for neurons and oligodendrocytes after long-term engraftment in spinal cord-injured NOD-scid mice. hCNS-SCns engraftment was associated with locomotor recovery, an observation that was abolished by selective ablation of engrafted cells by diphtheria toxin. Remyelination by hCNS-SCns was found in both the spinal cord injury NOD-scid model and myelin-deficient shiverer mice. Moreover, electron microscopic evidence consistent with synapse formation between hCNS-SCns and mouse host neurons was observed. Glial fibrillary acidic protein-positive astrocytic differentiation was rare, and hCNS-SCns did not appear to contribute to the scar. These data suggest that hCNS-SCns may possess therapeutic potential for CNS injury and disease.

Fig. 1.

The progeny of hCNS-SCns migrate and are morphologically distinct in gray vs. white matter. Shown are human immunopositive cells (SC121, brown) 17 weeks postgrafting. (A) In white matter, many human cells have an elongated, oligodendrocyte-like morphology (arrowheads). (B) In gray matter, some human cells exhibit neuronal morphologies (arrowheads); lesion perimeter is above and left of the dashed line. Insets in A and B show higher magnification of the morphologies adopted by human cells within white and gray matter, respectively. (Scale bars: 50 μm; Insets, 10 μm.)

Fig. 2.

Surviving hCNS-SCns promote locomotor recovery. (A and B) BBB locomotor performance is significantly improved in 50-kd SCI hCNS-SCns-grafted mice compared with vehicle controls (repeated-measures ANOVA, P ≤ 0.01) (A) and in 60-kd SCI hCNS-SCns-grafted mice compared with vehicle or hfibroblast controls (B) in individual t tests of recovery times. Asterisks indicate t test (P ≤ 0.05). DT was administered after 16-week BBB scores were obtained. (C and D) Errors on a ladder beam task are significantly decreased in 50-kd SCI hCNS-SCns-grafted mice compared with vehicle controls (P ≤ 0.05) (C) and in 60-kd SCI hCNS-SCns-grafted mice compared with vehicle controls (P ≤ 0.05) (D). No significant differences were observed between either vehicle vs. hfibroblasts (P ≤ 0.87) or hfibroblasts vs. hCNS-SCns, although there was a trend toward the latter (P ≤ 0.17). Open-field BBB and ladder beam improvements in hCNS-SCns-transplanted mice were reversed 1 week after treatment with DT in A-D. (E-H) hCNS-SCns (E) or human hfibroblasts (F) 17 weeks posttransplantation after treatment with vehicle in comparison with DT (G and H), which ablated the majority of human cells. No evidence of toxicity to surrounding host tissues was apparent. See Fig. 7 for additional photos

Fig. 3.

Differentiation of hCNS-SCns into astrocytes, neurons, and oligodendrocytes at 17 weeks. (A) Few human cytoplasm-positive (SC121, red), GFAP-positive (green) astrocytes (arrowheads) could be detected. Most human cells were negative for GFAP (arrows). (B) Glutamate decarboxylase (GAD)-67-immunopositive (red) processes (arrows) were occasionally colocalized with human nuclei (SC101, green), indicating GABAergic neurons. (C) Human cytoplasm-positive cell (red) colocalized (arrowheads) with the neural marker NeuN (green). (D and E) Several APC-positive oligodendrocytes (red), one (arrow) colocalized with a human nuclei (SC101, green). Other APC-positive cells (arrowhead) were not colocalized. (F) Injection of hCNS-SCns into neonatal NOD-scid/shi mice demonstrated MBP (green, arrowheads) wrapping neurofilament-positive mouse axons (red) in the cerebellum. Other axons remained dysmyelinated (arrows). Blue nuclei are Hoechst-stained. (Scale bars: A, D, and E, 20 μm; B, C, and F, 10 μm.)

Fig. 4.

EM of differentiation of hCNS-SCns into putative neurons. Immuno-EM for the human cytoplasm marker SC121 reveals many engrafted cells within the spinal neuropil. (A) Nucleus of a human cytoplasm-labeled cell adjacent to the nucleus of an endogenous mouse cell (mN). Human positive perikaryal cytoplasm surrounds the nucleus (hN), which exhibits patchy chromatin aggregates (Cr) typical of neurons. A single apical process (arrowheads), together with the paucity of cytoplasm, suggests that this human cell could be an immature neuron. (B) Transverse section of two axons and a human cytoplasm-labeled process (Hp). The upper axon (Ax₁) is myelinated; the lower axon (Ax₂) is not. Ax₂ exhibits a bouton-like swelling containing synaptic vesicles (Sv) in apposition with structure Hp; the presence of mitochondria (Mt) and smooth endoplasmic reticulum (SR) within Hp suggests that this process may be a dendrite. (C) Human cytoplasm-labeled process (Hp) cradled by a mouse axon terminal (At). Synaptic vesicles (Sv) and a single mitochondrion (M) are visible in the mouse axon terminal in close apposition with structure Hp; there are no interposing membranes between Hp and At, but no cleft is visible. (D) Human cytoplasm-labeled process (Hp) forming a putative symmetric synapse with a mouse axon terminal. There is thickening of both the presynaptic (black arrowheads) and postsynaptic (white arrowheads) membranes, with widening of membrane apposition and greater electron density within the cleft. Synaptic vesicles (Sv) are present within the mouse axon terminal. Inset shows the “synapse region” without overlays

Fig. 5.

EM of myelination by hCNS-SCns in the spinal cord. Six-week-old NOD-scid/shi mice were grafted with hCNS-SCns after a contusion injury and examined at 10 weeks of age. (A) Normal myelination of axons (Ax) within the dorsal funiculus of heterozygous shiverer littermates. (B) Spinal cord from ungrafted NOD-scid/shi reveals hypomyelination (Inset shows eight lamellae), loops of myelin, and a lack of the major dense line but healthy mitochondria (mit). (C) Spinal cord from a hCNS-SCns-grafted NOD-scid/shi mouse demonstrating thicker, dense myelination. (D) Boxed area in C showing compact myelin, >20 lamellae, and presence of the major dense line. (E) In spinal cord of contusion-injured NOD-scid mice, immuno-EM for human cytoplasm (SC121) reveals grafted human stem cells 17 weeks after spinal cord injury. Two human oligodendrocytes (Oligo) appear associated with neighboring axons. (F) Cross section of a human immunopositive oligodendrocytic tongue process (Op) and residual immunopositive cytoplasm within the outermost wrap of myelin (arrowheads). (Scale bars: A-C and F, 200 nm; D, 100 nm; E, 1 μm.)