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Comparative Study
, 25 (19), 4694-705

Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitor Cell Transplants Remyelinate and Restore Locomotion After Spinal Cord Injury

Affiliations
Comparative Study

Human Embryonic Stem Cell-Derived Oligodendrocyte Progenitor Cell Transplants Remyelinate and Restore Locomotion After Spinal Cord Injury

Hans S Keirstead et al. J Neurosci.

Abstract

Demyelination contributes to loss of function after spinal cord injury, and thus a potential therapeutic strategy involves replacing myelin-forming cells. Here, we show that transplantation of human embryonic stem cell (hESC)-derived oligodendrocyte progenitor cells (OPCs) into adult rat spinal cord injuries enhances remyelination and promotes improvement of motor function. OPCs were injected 7 d or 10 months after injury. In both cases, transplanted cells survived, redistributed over short distances, and differentiated into oligodendrocytes. Animals that received OPCs 7 d after injury exhibited enhanced remyelination and substantially improved locomotor ability. In contrast, when OPCs were transplanted 10 months after injury, there was no enhanced remyelination or locomotor recovery. These studies document the feasibility of predifferentiating hESCs into functional OPCs and demonstrate their therapeutic potential at early time points after spinal cord injury.

Figures

Figure 1.
Figure 1.
Commitment of hESCs to oligodendrocyte progenitors. a, Undifferentiated hESCs readily expand in colonies, separated by stromal cells. b, Yellow spheres appeared within 5 d of exposure to RA and grew rapidly in the presence of GRM, evidenced by an increase in their size and proportion relative to other culture components. c, A total of 83 ± 7% of cells expressed the transcription factor Olig1 (red) associated with oligodendrocyte and motoneuron specification. d, A total of 72 ± 12% of cells expressed the DNA binding protein SOX10 (red) expressed within oligodendrocyte precursors. e, More than 95% of cells labeled with the mature oligodendroglial marker RIP (red). Cells that did not label with oligodendroglial markers were primarily GFAP positive or Tuj1 positive. f, g, Plated cells adopted a typical oligodendroglial morphology characterized by multiple branches. h, Quantitation of immunolabeling. Error bars illustrate SD. Magnification: a, 50×; c, d, 100×; e, f, 200×; b, g, 400×.
Figure 2.
Figure 2.
Acute transplantation of hESC-derived OPCs resulted in cell survival, limited redistribution from the site of implantation, and differentiation to mature oligodendrocytes. a, Anti-human nuclei-positive OPCs (arrows) double labeled with the mature oligodendrocyte marker APC-CC1 (arrows; b); a composite is shown in c. d, Anti-human nuclei-positive, APC-CC1-positive double labeling was confirmed using 3-D reconstruction of confocally scanned thin-plane images. e, Distribution of total numbers of BrdU-prelabeled cells within spinal cord transverse sections 2 months after transplantation at 7 d after SCI. Error bars illustrate SD. f, Anti-human nuclei-immunostained transverse section 1 mm caudal to the site of implantation showing transplanted cells (black dots) located primarily within the gray matter but also redistributed throughout the white matter. g, Anti-human nuclei-immunostained transverse section 6 mm cranial to the site of implantation, showing transplanted cells (black dots) located primarily within the dorsal column. Magnification: a-c, 400×; d, 2000×; f, g, 20×.
Figure 3.
Figure 3.
Oligodendrocyte remyelination can be distinguished from Schwann cell remyelination. a, b, Toluidine blue-stained transverse sections of hESC-derived OPC-transplanted animals at the magnification used for quantification, illustrating measurements of the myelin sheath thickness of oligodendrocyte-remyelinated axons (a) and Schwann cell-remyelinated axons (b). Magnification: a, b, 2000×. c, Myelin sheath thickness against axon diameter in oligodendrocyte-remyelinated and Schwann cell-remyelinated axons. The G-ratio was 10 ± 4 (53)* for oligodendrocyte-myelinated axons and 41 ± 16 (48) for Schwann cell-remyelinated axons. Myelin sheath thickness in axons remyelinated by oligodendrocytes never reached the thickness observed in Schwann cell-remyelinated axons. *, Data are expressed as mean ± SD; the number in parentheses shows the number of axons scored.
Figure 4.
Figure 4.
Acute transplantation of hESC-derived OPCs resulted in a significant increase in the density of oligodendrocyte remyelination compared with controls. a, Electron micrograph of the transplant environment at 7 d after injury, illustrating demyelinated axons (*) in an extracellular environment free of astrogliosis. b, Toluidine blue-stained transverse section and electron micrograph (c), illustrating robust oligodendrocyte remyelination (R; with characteristically thin myelin sheaths) among few normally myelinated axons (N). d, e, Anti-GFP and anti-neurofilament double immunostains illustrating highly branched GFP-positive OPCs (O) extending processes that ensheath nearby neurofilament-positive axons (arrows), confirming that remyelination was performed by eGFP-labeled transplanted cells. f, Quantification of normally myelinated, demyelinated, and oligodendrocyte or Schwann cell-remyelinated axons in hESC-derived OPC-transplanted, hFb-transplanted, and DMEM-injected animals. Error bars illustrate SD. The myelin sheath thickness for each class of axons is indicated in brackets. Magnification: a, 6000×; b, 400×; c, 3000×; d, 600×; e, 1000×.
Figure 5.
Figure 5.
Acute transplantation of hESC-derived OPCs resulted in a significant increase in locomotor recovery compared with controls. a, From ∼3 weeks after implantation for the duration of testing, animals that received 250,000 hESC-derived OPCs consistently demonstrated significantly greater locomotor capabilities (p < 0.01) compared with controls, as determined using the BBB locomotor rating scale. b, The degree of locomotor recovery in animals that received 1.5 million hESC-derived OPCs was not significantly (p > 0.1) different from those that received 250,000 hESC-derived OPCs, as determined using the BBB locomotor rating scale. c, Animals that received hESC-derived OPCs also exhibited significant increases in locomotor recovery as determined using four-parameter kinematic analyses.
Figure 6.
Figure 6.
Chronic transplantation of hESC-derived OPCs resulted in cell survival, limited redistribution from the site of implantation, and differentiation to mature oligodendrocytes. a, Anti-BrdU-positive OPCs (arrows) double labeled with the mature oligodendrocyte marker APC-CC1 (arrows; b); a composite is shown in c. d, Anti-BrdU-positive, APC-CC1-positive double labeling was confirmed using 3-D reconstruction of confocally scanned thin-plane images. e, Distribution of total numbers of BrdU-prelabeled cells within spinal cord transverse sections 2 months after transplantation and 10 months after SCI. Error bars illustrate SD. Magnification: a-c, 400×; d, 2000×.
Figure 7.
Figure 7.
Chronic transplantation of hESC-derived OPCs resulted in no change in the density of oligodendrocyte remyelination or locomotor recovery compared with controls. a, Toluidine blue-stained transverse section from a transplanted animal, illustrating very sparse remyelination among normally myelinated and demyelinated axons, and increased extracellular space. b, Electron micrograph of the transplant environment at 10 months after injury, illustrating that demyelinated axons (*) were present. c, Electron micrograph of the transplant environment at 10 months after injury, illustrating axons surrounded by enlarged intermediate filament-rich astrocytic processes (AP), which occupied virtually all of the extracellular space. d, Electron micrograph of the transplant environment at 10 months after injury, illustrating an astrocyte (a) with a large intermediate filament-rich process (AP) extending to demyelinated axons surrounded by intermediate filament-rich astrocytic processes (arrows) and myelinated axons surrounded by intermediate filament-rich astrocytic processes (arrowheads). e, Quantification of normally myelinated, demyelinated, and oligodendrocyte or Schwann cell-remyelinated axons in hESC-derived OPC transplanted, and DMEM-injected animals. Error bars illustrate SD. The myelin sheath thickness for each class of axons is indicated in brackets. f-h, The degree of locomotor recovery in animals that received 1.5 million cell transplants was not significantly (p > 0.1) different from those that received vehicle-only injections, regardless of the severity of injury. Magnification: a, 400×; b-d, 3000×.
Figure 8.
Figure 8.
Morphometric analysis indicated that transplantation of hESC-derived OPCs 1 week or 10 months after SCI decreased cavitation but did not alter the extent of tissue sparing or tissue loss. Animals receiving vehicle-only consistently demonstrated cavitation at 1 mm cranial to the injury epicenter (a), at the epicenter (b), and 1 mm caudal to the injury epicenter (c). Animals receiving either OPC transplants consistently demonstrated little or no cavitation at 1 mm cranial to the injury epicenter (d), at the epicenter (e), and 1 mm caudal to the injury epicenter (f). Morphometric analyses at 1 mm intervals through the spinal cord indicated that transplantation of OPCs 7 d (g) or 10 months (h) after SCI did not cause overt tissue sparing or tissue loss. Error bars illustrate SD. Magnification: a-f, 10×.

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