Spinal cord injury (SCI) often leads to persistent functional deficits due to loss of neurons and glia and to limited axonal regeneration after injury. Here we report that transplantation of human dental pulp stem cells into the completely transected adult rat spinal cord resulted in marked recovery of hind limb locomotor functions. Transplantation of human bone marrow stromal cells or skin-derived fibroblasts led to substantially less recovery of locomotor function. The human dental pulp stem cells exhibited three major neuroregenerative activities. First, they inhibited the SCI-induced apoptosis of neurons, astrocytes, and oligodendrocytes, which improved the preservation of neuronal filaments and myelin sheaths. Second, they promoted the regeneration of transected axons by directly inhibiting multiple axon growth inhibitors, including chondroitin sulfate proteoglycan and myelin-associated glycoprotein, via paracrine mechanisms. Last, they replaced lost cells by differentiating into mature oligodendrocytes under the extreme conditions of SCI. Our data demonstrate that tooth-derived stem cells may provide therapeutic benefits for treating SCI through both cell-autonomous and paracrine neuroregenerative activities.

Head formation requires simultaneous inhibition of multiple caudalizing signals during early vertebrate embryogenesis. We identified a novel antagonist against Wnt and FGF signaling for head formation, Shisa, which functions cell autonomously in the endoplasmic reticulum (ER). Shisa is specifically expressed in the prospective head ectoderm and the Spemann organizer of Xenopus gastrulae. Overexpression of Shisa inhibited both Wnt and FGF signaling in Xenopus embryos and in a cell line. Loss of Shisa function sensitized the neuroectoderm to Wnt signaling and suppressed head formation during gastrulation. Shisa physically interacted with immature forms of the Wnt receptor Frizzled and the FGF receptor within the ER and inhibited their posttranslational maturation and trafficking to the cell surface. Taken together, these findings indicate that Shisa is a novel molecule that controls head formation by regulating the establishment of the receptors for caudalizing factors.

Engrafted mesenchymal stem cells from human deciduous dental pulp (SHEDs) support recovery from neural insults via paracrine mechanisms that are poorly understood. Here we show that the conditioned serum-free medium (CM) from SHEDs, administered intrathecally into rat injured spinal cord during the acute postinjury period, caused remarkable functional recovery. The ability of SHED-CM to induce recovery was associated with an immunoregulatory activity that induced anti-inflammatory M2-like macrophages. Secretome analysis of the SHED-CM revealed a previously unrecognized set of inducers for anti-inflammatory M2-like macrophages: monocyte chemoattractant protein-1 (MCP-1) and the secreted ectodomain of sialic acid-binding Ig-like lectin-9 (ED-Siglec-9). Depleting MCP-1 and ED-Siglec-9 from the SHED-CM prominently reduced its ability to induce M2-like macrophages and to promote functional recovery after spinal cord injury (SCI). The combination of MCP-1 and ED-Siglec-9 synergistically promoted the M2-like differentiation of bone marrow-derived macrophages in vitro, and this effect was abolished by a selective antagonist for CC chemokine receptor 2 (CCR2) or by the genetic knock-out of CCR2. Furthermore, MCP-1 and ED-Siglec-9 administration into the injured spinal cord induced M2-like macrophages and led to a marked recovery of hindlimb locomotor function after SCI. The inhibition of this M2 induction through the inactivation of CCR2 function abolished the therapeutic effects of both SHED-CM and MCP-1/ED-Siglec-9. Macrophages activated by MCP-1 and ED-Siglec-9 extended neurite and suppressed apoptosis of primary cerebellar granule neurons against the neurotoxic effects of chondroitin sulfate proteoglycans. Our data suggest that the unique combination of MCP-1 and ED-Siglec-9 repairs the SCI through anti-inflammatory M2-like macrophage induction.

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Stem cells from human exfoliated deciduous teeth (SHED) reside within the perivascular niche of the dental pulp. They are thought to originate from the cranial neural crest, and express early markers for both mesenchymal and neuroectodermal stem cells. 1,2 We previously showed that SHED transplantation into the completely transected rat spinal cord results in remarkable functional recovery of hindlimb locomotion.2 However, whether engrafted SHED or the paracrine factors derived from them can offer therapeutic benefits in other neurological disease settings is still largely unknown. In this study, we investigated the therapeutic benefits of SHED on mouse neonatal hypoxia-ischemia (HI). Materials and MethodsAn expanded version of the Methods section is available in the online-only Data Supplement. SHED, human skin fibroblasts, and their serum-free conditioned medium (CM) were prepared as described. 2The SHED's multi-differentiation potential and their expression of both mesenchymal stem cell and neural lineage markers were similar to those reported previously.2 HI brain injury was induced in postnatal day 5 (P5) mice as described. Cells (2×10 5 ) in 2 μL phosphate buffered saline or phosphate buffered saline alone (as a control) were transplanted into the ipsilateral hemisphere at 2.0 mm anterior and 2.0 mm lateral to bregma, and 2.0 mm deep to the dural surface, using a glass needle and a Kopf microstereotaxic injection system, 24 hours after HI ( Figure 1A). These animals were given daily administration of cyclosporin A (Novartis, Nurnberg, Germany, 10 mg/kg, IP) throughout the experimental period, except when they were used for cytokine expression analysis. For the experiments using CM, mice were given a 2-μL injection of CM or Dulbecco's modification of Eagle's medium (as a control) without cyclosporin A treatment. The animals' neurological recovery was examined by a foot-fault test in 4-, 6-, and 8-week-old HI mice.3 Tissue loss was examined by staining with hematoxylin and eosin, and brain injury was evaluated using a neuropathological scoring system, 4,5 by an observer blinded to the identity of the animal group. The level of apoptosis was analyzed by staining with anticaspase-3 (Cell Signaling). Real-time reverse transcription PCR was carried out as described.2 GAPDH cDNA was amplified as an internal control. Primer sequences are shown in the online-only Supplemental Table 1.Data are expressed as means±SEM. Survival data were analyzed by applying the Kaplan-Meier curve, followed by the Mental-Cox Background and Purpose-Perinatal hypoxia-ischemia (HI) has high rates of neurological deficits and mortality. So far, no effective treatment for HI brain injury has been developed. In this study, we investigated the therapeutic effects of stem cells from human exfoliated deciduous teeth (SHED) for the treatment of neonatal HI brain injury. Methods-Unilateral HI was induced in postnatal day 5 (P5) mice. Twenty-four hours later, SHED, human skin fibroblasts, or serum-free conditioned medium derived from these ...

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