Studies in rats showed regenerative properties not seen with other types of stem cells.

Easily accessible stem cells taken from tooth pulp are capable of stimulating long-term regeneration of nerves in damaged spinal cords, scientists report. A team at Nagoya University Graduate School of Medicine transplanted tooth stem cells into rats with completely severed spinal cords.

The stem cells not only promoted the regeneration of transected axons by directly inhibiting multiple axon growth inhibitors, they also prevented damage-induced apoptosis of neurons, astrocytes, and oligodendrocytes as well as differentiated into mature oligodendrocytes to replace cells that were lost.

The onset of regeneration was evident during the acute phase of injury and enabled rats with severed spinal cords to coordinate hind limb joint movement and walk without weight support within five weeks.

Minoru Ueda, M.D., and colleagues, describe their findings in The Journal of Clinical Investigation in a paper titled “Human dental pulp-derived stem cells promote locomotor recovery after complete transection of the rat spinal cord by multiple neuro-regenerative mechanisms.” 

While results have demonstrated that stem cell transplantation can promote varying degrees of functional recovery in spinal cord injury (SCI), these types of cells have shown poor survival and/or differentiation capacity in vivo. Human adult dental pulp stem cells (DPSCs) and stem cells from human exfoliated deciduous teeth (SHEDs), meanwhile are easily obtained stem cell populations that display markers of both mesenchymal and neuroectodermal stem cells and can be obtained relatively easily. Like adult bone marrow stromal cells (BMSCs), they can also be prompted to differentiate into osteoblasts, chondrocytes, adipocytes, endothelial cells, and functionally active neurons in vitro, under defined conditions, the authors note.

To evaluate the neuoregenerative potential of DPSCs and SHEDs in axonal regeneration during the early phase of SCI, the authors devised a series of experiments in which the cells were transplanted into a completely transected rat SCI model.

Initial flow cytometric and immunohistochemical analyses indicated that SHEDS and DPSCs expressed mesenchymal stem cell (MSC) markers but not endothelial/hematopoietic markers. The majority of SHEDs and DPSCs also co-expressed several neural lineage markers. Real-time PCR analyses confirmed that both cell types expressed GDNF, BDNF, and CNTF at over 3–5 times the levels expressed by skin-derived fibroblasts of BMSCs.

Critically, when DPSCs and SHEDs were implanted into rats with a completely transected SC, the animals displayed much greater locomotor recovery than animals transplanted with either transplanted BMSCs or fibroblasts. Even more encouragingly, the authors note, was the observation that the improved recovery was evident soon after the operation, during the acute phase of SCI. Five weeks after the operation, rats that had received SHEDs were able to coordinate movement of three joints of the hind limb and walk without weight support.

Although levels of recovery were similar in the SHED- and DPSC-transplanted animals, the team focused subsequent evaluation on SHED-transplanted rats to see how the cells promoted regeneration of a transected SC. Immunohistochemical analyses indicated that after eight weeks, mice implanted with SHEDs demonstrated greater preservation of neurofilament-positive axons in the spinal cord than than PBS-treated control animals.

In the SHED-treated, but not control animals, corticospinal tract (CST) axons and serotonergic raphespinal axons (identified by antibody binding) both extended up to 3 mm caudal to the damage epicenter. The boutons of some of these axons could also been seen apposed to neurons in the caudal stump, “suggesting that the regenerated axons had established new neural connections,” the authors write. And while there weren’t many descending axons extending beyond the epicenter, many penetrated the scar tissue of the rostral stump.

Further analyses suggested that SHEDs also directly inhibited multiple axon growth inhibitor (AGI) signals generated by oligodendrocytes and reactive astrocytes that formed the glial scar. Conditioned medium from SHEDs (SHED-CM) or DPSCs (DPSC-CM) was similarly capable of promoting neurite extension of cerebral granular neurons CGNs grown on plates treated with an AGI.  

In a separate set of immunohistochemical staining experiments, SHED cell transplantation was shown to promote regeneration of myelin structures in the transected region of the spinal cord. By eight weeks after grafting, about 30% of the transplanted SHEDs still survived in the injured spinal cords, and the vast majority of these expressed two mature oligodendrocyte markers, APC and MBP, but no longer expressed markers of neural stem cells or astrocytes, “indicating that they specifically differentiated along the oligodendrocyte lineage in the injured SCs,” the team writes. Finally, TUNEL staining assays demonstrated that engrafted SHEDs inhibited the apoptosis of oigodendrocytes, neurons, and astrocytes, which would otherwise play a key role in secondary injury following SCI.

The tooth-derived stem cells thus demonstrate “remarkable neruoregenerative activity” following SCI, the authors state. These capabilities include inhibiting SCI-induced apoptosis to preserve neural fibers and myelin sheaths, regenerating the transected axon through direct inhibition of multiple AGI signals, and replacing damaged oligodendrocytes by differentiation into mature oligodendrocytes.

“To our knowledge, the latter two neuroregenerative activities are unique to tooth-derived stem cells and are not exhibited by any other previously described stem cells,” they state. “We propose that tooth-derived stem cells may be an excellent and practical cellular resource for the treatment of SCI.”

The authors admit that while the complete transaction model used in their experiments enabled them to precisely assess the axonal regeneration capacity of tooth-derived stem cells, contusion, and crush models would represent  forms of SCIs seen clinically in humans. Evaluating the stem cells in models similar to these is one of their aims for the future.  

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