During neural development caudalization and dorsoventral patterning of the neural tube is directed by several inductive factors including retinoic acid, sonic hedgehog (Shh), bone morphogenetic proteins (BMPs), and Wnt signaling. The purpose of the current study was to investigate whether dorsal interneurons specific for the spinal cord can be generated from mouse embryonic stem (ES) cells using known inductive signals. Here we show that specific combination of developmental signaling molecules including all trans-retinoic acid, Shh, bone morphogenetic protein 2 (BMP2), and Wnt3A can direct differentiation of ES cells into dorsal interneurons possessing appropriate neuronal markers, synaptic proteins and functional neurotransmitter machineries. We introduce a concept that Wnt3A morphogenic action relies on crosstalk with both Shh and BMP2 signaling pathways.
Embryonic stem (ES) cells have been investigated in repair of the CNS following neuronal injury and disease; however, the efficacy of these cells in treatment of postinjury pain is far from clear. In this study, we evaluated the therapeutic potential of predifferentiated mouse ES cells to restore sensory deficits following spinal cord injury (SCI) in mice. The pain model used unilateral intraspinal injection of quisqualic acid (QUIS) into the dorsal horn between vertebral levels T13 and L1. Seven days later, 60,000 predifferentiated ES cells or media were transplanted into the site of the lesion. Histological analysis at 7, 14, and 60 days posttransplantation revealed that animals receiving ES cell transplants suffered significantly less tissue damage than animals receiving media alone. Transplanted cells provided immediate effects on both spontaneous and evoked pain behaviors. Treatment with ES cells resulted in 0% (n = 28) excessive grooming behavior versus 60% (18 of 30) in media-treated animals. In the acetone test (to assess thermal allodynia), mice recovered to preinjury levels by 12 days after ES cell transplant, whereas control animals injected with media after SCI did not show any improvement up to 60 days. Similarly, the von Frey test (to assess mechanical allodynia) and the formalin test (to assess nociceptive hyperalgesia) showed that transplantation of predifferentiated ES cells significantly reduced these pain behaviors following injury. Here we show that predifferentiated ES cells act in a neuroprotective manner and provide antinociceptive and therapeutic effects following excitotoxic SCI.
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