Human exfoliated deciduous teeth conditioned medium (SHED-CM)
6-12 years
Superior laryngeal nerve (SLN)-PNS
Rat model: the SLN was exposed bilaterally and injured with a vascular clip (60 g/mm2) over a period of 30 min in male Wistar/ST rats weighing 300–330 g (9-10 weeks old). Systemic administration in rats with 1 ml SHED-CM injected into the tail vein for 10 s simultaneously.
Unspecified passage number. Conditioned media were collected after 48 hours cultured in serum-free DMEM
SHED-CM promotes axon regeneration after SLN injury. Many nerve myelinated fibers were identified in the SHED-CM group. The SHED-CM group showed higher fiber densities when compared with the DMEM (-) group. G-ratio showed that the degree of myelination in the SHED-CM group was significantly higher compared to the DMEM (-) group
Administration of SHED-CM improved functional recovery in SLN model.
DMSC from human third molar age range 12-25 years: (1) Apical papilla stem cells (SCAP) (2) Dental pulp stem cells (DPSC) (3) Periodontal ligament stem cells (PDLSC)
12-25 years
Sciatic nerve-PNS
In vitro model (neurite outgrowth assay): SHSY-5Y neuronal cells were retinoic acid-differentiated (48 h) prior to exposure to conditioned media (CM) derived from all dental MSCs. In vivo rat model: 10 mm gap was excised (5 mm below sciatic nerve, 5 mm length of sciatic nerve). Fibrin conduits were inserted to the nerve and sutures at each ends. DMSC populations were stimulated with the following: (1) 200 ng/ml neuregulin1-beta1 (R&D systems) (2) 10 ng/ml basic fibroblast growth factor (Millipore) (3) 5 ng/ml platelet-derived growth factor (Millipore) (4) 14 μM forskolin (Sigma), for two weeks
Conditioned medium were collected from 2-week stimulated DMSCs.
In vitro: total neurite outgrowth increased in length when cultured in CM compared to medium-only control. In vivo: immunohistochemistry analysis demonstrated that DSCs enhance axon regeneration.
Human SCAP, DPSC, and PDLSC provide an alternative to Schwann cells to support regeneration after peripheral nerve injury and repair.
3
Kano et al. 2016
(i) Human exfoliated deciduous teeth conditioned medium (SHED-CM) (ii) sSinglec-9 or MCP-1 depleted-SHED-CM
6-12 years
Rat facial nerves-PNS
Facial nerve injury was created by removing 5 mm segments of the buccal and marginal branches at sites that were 10 mm distal to the stylomastoid foramen. Atelocollagen sponge impregnated with 20 μl (i) CM, (ii) DMEM, (iii) recombinant human MCP-1, and (iv) recombinant sSiglec-9 was placed in the nerve gap.
Passages 3 to 9 after cultured for 48 hours in serum-free DMEM
SHED-CM depleted both MCP-1 and sSiglec-9 showed lost ability to restore neurological function.
MCP-1 and sSiglec-9 in SHED-CM play important role to regenerate peripheral nerves.
11-25 years (donor undergoing tooth extraction for orthodontic reasons)
In vitro neuronal differentiation of IMR-32 preneuroblastic cell line using the secretome
All stem cells were maintained with a-MEM for 48 h prior to secretome collection. Secretome were used for neuronal differentiation of IMR-32 preneuroblastic cell line
Passages 3 to 7 after cultured for 48 hours in serum-free αMEM
Higher colony forming efficiency of DMSC secretome as compared to BMSC secretome. Neurite extension assay: neurite length was highest for IMR-32 cells treated with DPSC secretome. Neural gene profiling: IMR-32 induced DMSC secretome showed significant regulation of B-tubulin III and sox-1 expression as compared to IMR-32 induced BMSC secretome. Neural regulatory molecules present in secretome: (1) NGF: no significant difference between DPSC and BMSC secretomes (2) BDNF: higher in DMSC compared to BMSC (3) NTF: higher in DMSC compared to BMSC
DPSC secretome molecules can enhance neural differentiation by increasing the expression of neural genes, preventing apoptosis of neurons, or by maintaining a neuronal fate during differentiation.
Mobilized dental pulp stem cells (MDPSCs) (human third molar)
18-29 years
MDPSC secretomes were used to clarify its effect on Schwann cell in terms of migration, proliferation, and antiapoptotic analyses-PNS
DPSCs were treated with G-CSF to induce stem cell mobilization by culture in Transwell (upper and lower chambers) for 48 h. The medium was changed into serum-free DMEM at 70% confluence and CMs were collected after 24 hours and concentrated using centrifugal filter
Passage 3 after cultured for 24 hours in serum-free DMEM
MDPSC secretome: (1) Enhanced proliferation of RT4-D6P2T cells (rat Schwann cells) (2) Enhanced the migratory activity of RT4-D6P2T cells (3) Decreased apoptosis of RT4-D6P2T cell
MDPSCs contribute to peripheral nerve regeneration by secretion of neurogenic/angiogenic factors in the close proximity of newly migrated Schwann cells while regulating their apoptosis and proliferation.
SCAPs were seeded at 20,000 cells/cm2. When reached 90%, cells were washed and cultured in serum-free medium for 24 hours. Collected secretomes were concentrated.
Passages 3 to 5 after cultured for 24 hours in serum-free αMEM
SCAP secretomes contain higher secretion of chemokines and neutrophins than BMSCs.
SCAPs were found to secrete angiogenic, immunomodulatory, antiapoptotic, chemokine, and neuroprotective factors.
M2-like bone marrow macrophage induction assay—BMMs were subjected to IHC after supplemented with each CM ELISA—measurement of cytokines in CMs and protein depletion assays Cytokine antibody arrays using laser scanning, cytokines in SHED-CM, BMSC-CM, and serum-free DMEM were detected by 274-human-cytokine array plates Western blot, lectin blot, and coimmunoprecipitation—to detect ED Siglec-9 in CMs, the CMs were concentrated up to 50 times
Passages 3 to 9 after cultured for 48 hours in serum-free DMEM
Treatment with SHEDs or SHED-CM promotes functional recovery after SCI. Rats treated with SHEDs or SHED-CM exhibited less tissue loss & more 5-HT-positive descending raphe spinal axon fibers compared with the control. Effect of CMs on the SCI inflammatory response Treatment with SHED-CM or BMSC-CM similarly suppressed these proinflammatory mediators for 1 week after the injury. Factors in SHED-CM that induce macrophage differentiation 79 proteins expressed in SHED-CM, 28 were known to be involved in neuroregenerative processes. SHED-CM lacking of MCP-1 & ED Siglec-9 fails to induce M2 or to restore locomotor function after SCI. The depletion of IL-6 from SHED-CM (IL-6) had little or no effect.
MSC-derived secreted factors directly convert the proinflammatory conditions prevalent in the damaged CNS to tissue-repairing ones by modulating the microglia/macrophage phenotype.
(1) Human exfoliated deciduous teeth conditioned medium (SHED-CM) (2) Human bone marrow mesenchymal stem cells (BMSCs-CM) (3) Human skin fibroblast (Fibros-CM)
Unspecified
Alzheimer’s disease/primary neurons-CNS
All three cells were cultured in SF DMEM. CM collected after 48 h of culture. 50 μl of each CM administered to the ICR mice (9 weeks old) intranasally with microsyringe over the course of 10 min at a 2 min interval. Performed 2x a day for 4 days. Primary neuronal cultures prepared from the cortices of C57BL/6 mice embryos with supplementation of SHED-CM for 24 h. Cell viability determined by WST assay
Passages 3 to 5 after cultured for 48 hours in serum-free DMEM
SHED-CM ameliorates neurological dysfunction in a mouse AD-like model. Mice with SHED-CM exhibited significantly improved RI (recognition index), while rats with BMSC-CM or Fibro-CM exhibited only modestly improved RI. SHED-CM inhibits the generation of 3-NT. Treatment with SHED-CM, BMSC-CM, or Fibro-CM significantly inhibited the generation of both 3-NT & iNOS in AD mice. SHED-CM converts the proinflammatory brain environment of the mouse AD-like model to an anti-inflammatory one & increases neurotrophic factor expression. SHED-CM shifted the M1-type proinflammatory microenvironment associated with mouse AD toward the M2-type anti-inflammatory/neuroprotective one.
SHED-CM provide many neuroreparative effects for the treatment of cognitive deficit; thus, it may provide a novel cell-free neuroreparative therapy for AD.
9
Song et al. 2015
(1) Human dental pulp from healthy permanent teeth (hDPSCs) (2) Human bone marrow mesenchymal stem cells (hMSCs)
14-22 years
Human astrocytes (hAs) Oxygen-glucose deprivation (OGD) model
hDPSCs & hMSCs cultured in serum-free DMEM. CM was collected after 48 h. Pretreatment: hAs were cultured in CM-hDPSCs or CM-hMSCs CM, exposed to oxygen-glucose deprivation (OGD) for 2 h followed by reoxygenation/reperfusion for 30 min. Posttreatment: hAs were cultured in CM-hDPSCs or CM-hMSCs CM, exposed to oxygen-glucose deprivation (OGD) for 2 h followed by reoxygenation/reperfusion for 2 h.
Passages 5 after cultured for 48 hours in serum-free DMEM
hDPSCs and CM-hDPSCs protect against cell death in OGD-induced hAs. Pre- or posttreatment with hDPSCs or CM-hDPSCs conferred a superior cytoprotective effect compared to hMSCs & CM-hMSCs. hDPSCs and CM-hDPSCs inhibit ROS production and IL-1β (proinflammatory cytokines) in ischemic hAs. Pre- or posttreatment with hDPSCs or CM-hDPSCs effectively blocked OGD/reperfusion-induced ROS production. Pre- or posttreatment with CM-hDPSCs or CM-hMSCs significantly reduced OGD-stimulated upregulation of IL-1β. CM-hDPSCs reduced astrogliosis of ischemic hAs. IF results revealed upregulation of musashi-1 (marker for reactive astrocytes) in OGD-treated hAs. VGF was neuroprotective for RGC. Significantly higher transcription of VGF detected in hDPSC compared to hBMSC/hAMSC.
Pre- & posttreatment with hDPSCs or CM-hDPSCs promote superior cytoprotective effects on hAs, due to reduced gliosis and suppressed free radicals & proinflammatory cytokines.
SHEDs cultured until 80% confluent & replenished with SF DMEM. CM was collected after 48 h. In vitro: migration-SCs cultured in FBS+DMEM on top chamber of Transwell with supplementation of SHED-CM at the lower chamber for 48 h. Cell removed with swab n filter stained with hematoxylin. Migrated cells were counted. Proliferation-SCs cultured in FBS+DMEM for 24 h. Medium removed & replenished with 100 μl SHED-CM for 48 h. MTT assay was performed. Tube formation assay-mixed HUVECs & HDF seeded under optimal tubule formation conditions. EM medium contains VEGF, HGF, or SHED-CM replaced on days 1, 4, 7, 9, and 11 & incubated with antibody. Total tube lengths & capillary were counted. Neurite length-DRG incubated with SHEM CM and subjected for IF. CCK-8 assay performed to measure cell viability. In vivo: male Wistar/ST rat (250-300 g) left sciatic nerve exposed & isolated. 12 mm nerve segment excised & nerve stumps were pulled 1 mm inside each end of the 12 mm SHEM CM-filled silicon conduit. Rats subjected to walking track analysis, electrophysiological testing, target muscle weight, Masson’s trichrome staining & histomorphological analysis.
Unspecified passage number. Conditioned media were collected after 48 hours cultured in serum-free DMEM
SHED-CM enhances SC migration & proliferation. SHED-CM increased SC migration rate by 7-fold & significantly increased proliferation compared to control. Various growth factors present in SHED-CM SHED-CM significantly regulates NTF, angiogenic, & ECM molecules compared to control. SHED-CM stimulates neurite outgrowth & increases DRG neuron viability. Neurite growth & cell viability significantly higher in DRG neuron supplemented SHED-CM compared to control. SHED-CM stimulates angiogenesis in vitro. HUVECS cultured in SHED-CM significantly increased tube length & joint number. SHED-CM enhances nerve regeneration. Nerve regenerated in SHED-CM thicker than control. SHED-CM enhances axon regeneration & remyelination. Number of myelinated nerve fiber & degree of myelination are significantly higher in SHED-CM than the other groups. SHED-CM improves motor function recovery. Walking track analysis (SFI) value is significantly higher in SHED-CM compared to control. SHED-CM prevents muscle atrophy & maintains muscle fiber. Gastrocnemius muscle wet weigh & collagen fiber percentage are significantly higher in SHED-CM compared to control.
SHED-CM promotes axonal regeneration & functional recovery in a sciatic nerve defect rat, enhances axon growth, angiogenesis, migration, proliferation, & neuron survival, and thus could be a potential for PNI treatment.
11
Mead et al. 2014
(1) Human dental pulp from healthy permanent teeth (hDPSCs) (2) Human bone marrow mesenchymal stem cells (hMSCs) (3) Human adipose-derived mesenchymal stem cells (hAMSC)
Unspecified age of donor: (1) hDPSCs were purchased from AllCell LLC (Berkeley, CA) (2) hBMSC and hAMSC from Lonza (Slough, UK) (3) Each represented pooled samples from 3 donors
Rat retinal ganglion cells (RGCs)/optic nerve-PNS
DPSCs or BMSCs were cocultured with retinal cells in Transwell chamber system and particular wells were treated 5 μg/ml of Fc-TrKA, Fc-TrKB and/or Fc-TrKC, Fc-VEGFr, Fc-GDNr, Fc-PDGFAr, & Fc-PDGFBr fusion protein inhibitors. Combination of 60 ng/ml NGF, BDNF, & NT-3 added to retinal cells as control. Particular wells of retinal cells treated with 0.1 μm, 1 μm, & 10 μm of VGF.
Passages 2 and 5 after 48 h in serum-free DMEM
hDPSC promoted significantly greater paracrine-mediated neuroprotection and neuritogenesis than hBMSC/hAMSC. hDPSC-treated retinal cultures showed significant RGC survival than that in hAMSC but not significantly than hBMSC. NTFR Fc-receptor blockers for multiple NTFR attenuated the neuroprotective and neuritogenic effect of hDPSC/hBMSC/hAMSC. NTFR blockers significantly attenuated hDPSC-mediated neuroprotection or neuritogenesis of cocultured RGC compared to uninhibited hDPSC/retinal cell cocultures. hDPSC, hBMSC, and hAMSC have distinct NTF expression profiles. Both NTF genes showed distinct expression in each stem cells. hDPSC secrete multiple NTF at higher levels than hBMSC/hAMSC. hDPSC secreted significantly greater titers of NTF factors than hBMSC/hAMSC.
hDPSC mediates neuroprotection & neuritogenesis through paracrine effects of secreted neutrophic factors; thus, hDPSCs may represent an effective cellular therapy for nerve repair.
12
Ishizaka et al. 2013
Porcine premolar teeth (CD31)-side population (SP) cells: (1) Porcine dental pulp (2) Porcine bone marrow (3) Porcine adipose
Unspecified
(1) NIH3T3 mouse embryonic fibroblast (2) Human peripheral blood mononuclear cells (PBMCs) (3) Human neuroblastoma cell line TGW
CM from DP, BM, & AD collected after 48 h of culture & concentrated using filter unit. In vitro: MEF: MEF cultured in DMEM+FBS for 24 h, then changed into DMEM+each CM (final conc. of 5 μg/ml). Cells were subjected to cell count & migration Apoptosis-MEF grown in DMEM (3 days) & incubated with staurosporine (100 nm) supplemented 5 μg/ml of each CM. After 8 h, MEF was analyzed by flow cytometry. Endothelial cell differentiation-MEF cultured in & 5 μg/ml of each CM & performed ICC. MEF seeded on Matrigel in , 5 mg/ml heparin, 5 mg/ml ascorbic acid, 5 mg/ml hydrocortisone, & 5 mg/ml of each CM. Network formation observed after 4 h. Lengths of networks of cords & tube-like structures was measured. PBMCs: immunomodulatory effect-cells were purified & treated with mitomycin C (3 h). Autologous PBMCs & allogenic stimulator PBMCs were cocultured & supplemented with 5 μg/ml of CM. Cell counted at 0, 12, 24, & 36 h. Human neuroblastoma cell line TGW: neurite outgrowth-TGW were serum starved & stimulated with each CM (48 h). Neurite length was measured. 100 cells/sample were counted.
Unspecified passage number. Conditioned media were collected after 48 hours cultured in serum-free DMEM
CM from DP produced higher migration activity, antiapoptotic activity, & immunomodulatory effects compared to BM & AD. Stimulatory effects of the CM from BM on migration & immunosuppression significantly higher than CM from AD. HUVECs differentiated into endothelial cells that were positive for VE-cadherin & formed extensive networks of cords and tube-like structures as early as 4 h with supplementation of each CM. CM from DPs had higher angiogenic potential than CM from AD. CM from DP had significantly higher stimulatory effects on neurite outgrowth than CM from BM & AD.
CM from DP had higher trophic effects on angiogenesis, neurite outgrowth, migration, antiapoptosis, & immunomodulation than BM & AD CM in vitro.
13
Mead et al. 2013
Upper and lower incisors of Sprague-Dawley rats weighing 170 to 200 g: (1) Rat dental pulp stem cells (DPSCs) (2) Rat bone marrow mesenchymal stem cells (BMSCs)
Unspecified
Rat retinal ganglion cells (RGCs)/optic nerve-PNS
Rats (weighing 170-200 g): In vitro: DPSCs or BMSCs-CM was collected after 48 h of culture & subjected for neurotrophins quantitation using ELISA. DPSCs or BMSCs were cocultured with retinal cells in Transwell chamber system and particular wells were treated with protein inhibitors. CM pooled from 3 samples for coculture & transplant. In vivo: intraorbital optic nerve crush (ONC) was exposed & crushed using forceps 1 mm posterior to the lamina cribrosa. ONC was created in rats’ eye and injected intravitreally (150,000 cells suspended in 5 ml PBS) either with DPSC, BMSC, or PBS (control). Every 7 days, optical coherence tomography (OCT) was performed to measure retinal nerve fiber layer (RNFL).
Passages 2 to 4 after cultured for 48 hours in serum-free DMEM
DPSCs secreted NGF, BDNF, & NT-3. These neurotrophic titers were higher in DPSCs-CM compared to BMSCs-CM. DPSCs promoted BIII-tubulin retinal cell survival & neurogenesis in a coculture assay. DPSCs promote significant increase in the survival & number of cocultured retinal cells compared with retinal cultured alone, cocultured with BMSCs or treated with recombinant factors. Fc-TrK receptor attenuates survival & neuritogenesis effects of DPSCs. DPSCs cocultured significantly decreased number of BIII-tubulin retinal cells after treated with Fc-TrKA, Fc-TrKB, & Fc-TrKC, whereas BMSCs cocultured reduced retinal cells survival in Fc-TrKA, Fc-TrKB, or combination of 3, but not after adding Fc-TrKC alone. DPSC transplants preserved RNFL thickness for 14 days after ONC injury. All animal survived with no adverse effects. There were no significant RNFL thinning at 7 pdl that injected with living DPSC compared to intact animals, indicating a neuroprotective effect of DPSC. Transplanted DPSCs survived for 21 days. Viable DPSCs detected in the transplanted site at 21 pdl with higher level of BDNF & NT-3 compared to eyes transplanted with dead DPSCs. DPSC transplant protects RGCs from death after ONC. DPSC transplant after ONC significantly increased RGC survival at 21 dl compared with animals received BMSCs, dead DPSCs, or ONC alone. DPSC transplant after ONC promotes RGC axon regeneration. At distances 100-1200 μm distal to crush site, number of regenerating GAP-43 RGC axon significantly increased in DPSCs compared with BMSCs, dead DPSCs, or untreated.
DPSCs secrete higher concentration of neurotrophins that are responsible for promoting axotomized RGC neuroprotection and neuritogenesis/axogenesis; hence, it may be a promising alternative for CNS cell therapy.
