Human oral mucosa-derived neural crest-like stem cells differentiate into functional osteoprogenitors that contribute to regeneration of critical size calvaria defects.

BACKGROUND AND OBJECTIVE: Regeneration of large bony defects is an unmet medical need. The therapeutic effect of fully developed bony constructs engineered in vitro from mineralized scaffold and adult stem cells is hampered by deficient long-term graft integration. The purpose of the present study was to investigate the regenerative capacity of a bony primordial construct consisting of human oral mucosa stem cells (hOMSC)-derived osteoprogenitors and absorbable Gelfoam® sponges. METHODS: Gingiva and alveolar mucosa-derived hOMSC were differentiated into osteoprogenitors (Runx2 and osterix positive) and loaded into Gelfoam® sponges to generate primordial hOMSC constructs. These were implanted into critical size calvaria defects in the rat. Defects treated with human dermal fibroblasts (HDF) constructs; Gelfoam® sponges and untreated defects served as controls. RESULTS: After 120-day post-implantation defects treated with hOMSC constructs, HDF constructs and gelatin and untreated defects exhibited 86%, 30%, 21%, and 9% of new bone formation, respectively. Immunofluorescence analysis for human nuclear antigen (HNA), bone sialoprotein (BSP), and osteocalcin (OCN) revealed viable hOMSC-derived osteoblasts and osteocytes that formed most of the cell population of the newly formed bone at 30 and 120 days post surgery. Few HNA-positive HDF that were negative for BSP and OCN were identified together with inflammatory cells in the soft tissue adjacent to new bone formation only at 30 days post implantation. CONCLUSION: Collectively, the results demonstrate that primordial in vitro engineered constructs consisting of hOMSC-derived osteoprogenitors and absorbable gelatin almost completely regenerate critical size defects in an immunocompetent xenogeneic animal by differentiating into functional osteoblasts that retain the immunomodulatory ability of naïve hOMSC.

Examination of the Therapeutic Potential of Mouse Oral Mucosa Stem Cells in a Wound-Healing Diabetic Mice Model.

Diabetic wounds' delayed healing response is still considered a major therapeutic challenge. Stem cells and derived cellular products have been an active field of research for novel therapies referred to as regenerative medicine. It has recently been shown that human oral mucosa stem cells (hOMSCs) are a readily accessible source for obtaining large quantities of stem cells. This study evaluates the potential of mouse oral mucosa stem cells (mOMSCs) to enhance wound healing in a diabetic (db/db) mouse model by morphological and histological analysis. We show that mOMSCs-treated wounds displayed a significantly faster wound-healing response (p ≤ 0.0001), featuring faster re-epithelialization and a larger area of granulation tissue (p ≤ 0.05). Taken together, these results suggest that oral mucosa stem cells might have therapeutic potential in diabetic wound healing.

Implantation of 3D Constructs Embedded with Oral Mucosa-Derived Cells Induces Functional Recovery in Rats with Complete Spinal Cord Transection.

Spinal cord injury (SCI), involving damaged axons and glial scar tissue, often culminates in irreversible impairments. Achieving substantial recovery following complete spinal cord transection remains an unmet challenge. Here, we report of implantation of an engineered 3D construct embedded with human oral mucosa stem cells (hOMSC) induced to secrete neuroprotective, immunomodulatory, and axonal elongation-associated factors, in a complete spinal cord transection rat model. Rats implanted with induced tissue engineering constructs regained fine motor control, coordination and walking pattern in sharp contrast to the untreated group that remained paralyzed (42 vs. 0%). Immunofluorescence, CLARITY, MRI, and electrophysiological assessments demonstrated a reconnection bridging the injured area, as well as presence of increased number of myelinated axons, neural precursors, and reduced glial scar tissue in recovered animals treated with the induced cell-embedded constructs. Finally, this construct is made of bio-compatible, clinically approved materials and utilizes a safe and easily extractable cell population. The results warrant further research with regards to the effectiveness of this treatment in addressing spinal cord injury.

Dopaminergic-like neurons derived from oral mucosa stem cells by developmental cues improve symptoms in the hemi-parkinsonian rat model.

Achieving safe and readily accessible sources for cell replacement therapy in Parkinson's disease (PD) is still a challenging unresolved issue. Recently, a primitive neural crest stem cell population (hOMSC) was isolated from the adult human oral mucosa and characterized in vitro and in vivo. In this study we assessed hOMSC ability to differentiate into dopamine-secreting cells with a neuronal-dopaminergic phenotype in vitro in response to dopaminergic developmental cues and tested their therapeutic potential in the hemi-Parkinsonian rat model. We found that hOMSC express constitutively a repertoire of neuronal and dopaminergic markers and pivotal transcription factors. Soluble developmental factors induced a reproducible neuronal-like morphology in the majority of hOMSC, downregulated stem cells markers, upregulated the expression of the neuronal and dopaminergic markers that resulted in dopamine release capabilities. Transplantation of these dopaminergic-induced hOMSC into the striatum of hemi-Parkinsonian rats improved their behavioral deficits as determined by amphetamine-induced rotational behavior, motor asymmetry and motor coordination tests. Human TH expressing cells and increased levels of dopamine in the transplanted hemispheres were observed 10 weeks after transplantation. These results demonstrate for the first time that soluble factors involved in the development of DA neurons, induced a DA phenotype in hOMSC in vitro that significantly improved the motor function of hemiparkinsonian rats. Based on their neural-related origin, their niche accessibility by minimal-invasive procedures and their propensity for DA differentiation, hOMSC emerge as an attractive tool for autologous cell replacement therapy in PD.

Astrocyte-like cells derived from human oral mucosa stem cells provide neuroprotection in vitro and in vivo.

Human oral mucosa stem cells (hOMSC) are a recently described neural crest-derived stem cell population. Therapeutic quantities of potent hOMSC can be generated from small biopsies obtained by minimally invasive procedures. Our objective was to evaluate the potential of hOMSC to differentiate into astrocyte-like cells and provide peripheral neuroprotection. We induced hOMSC differentiation into cells showing an astrocyte-like morphology that expressed characteristic astrocyte markers as glial fibrillary acidic protein, S100ß, and the excitatory amino acid transporter 1 and secreted neurotrophic factors (NTF) such as brain-derived neurotrophic factor, vascular endothelial growth factor, glial cell line-derived neurotrophic factor, and insulin-like growth factor 1. Conditioned medium of the induced cells rescued motor neurons from hypoxia or oxidative stress in vitro, suggesting a neuroprotective effect mediated by soluble factors. Given the neuronal support (NS) ability of the cells, the differentiated cells were termed hOMSC-NS. Rats subjected to sciatic nerve injury and transplanted with hOMSC-NS showed improved motor function after transplantation. At the graft site we found the transplanted cells, increased levels of NTF, and a significant preservation of functional neuromuscular junctions, as evidenced by colocalization of α-bungarotoxin and synaptophysin. Our findings show for the first time that hOMSC-NS generated from oral mucosa exhibit neuroprotective effects in vitro and in vivo and point to their future therapeutic use in neural disorders.