Rigid matrix supports osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHED)

Stem cell fate can be induced by the grade of stiffness of the extracellular matrix, depending on the developed tissue or complex tissues. For example, a rigid extracellular matrix induces the osteogenic differentiation in bone marrow derived mesenchymal stem cells (MSCs), while a softer surface induces the osteogenic differentiation in dental follicle cells (DFCs). To determine whether differentiation of ectomesenchymal dental precursor cells is supported by similar grades of extracellular matrices (ECMs) stiffness, we examined the influence of the surface stiffness on the proliferation and osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHED). Cell proliferation of SHED was significantly decreased on cell culture surfaces with a muscle-like stiffness. A dexamethasone-based differentiation medium induced the osteogenic differentiation of SHED on substrates of varying mechanical stiffness. Here, the hardest surface improved the induction of osteogenic differentiation in comparison to that with the softest stiffness. In conclusion, our study showed that the osteogenic differentiation of ectomesenchymal dental precursor cells SHED and DFCs are not supported by similar grades of ECM stiffness.     

Stem cells from human exfoliated deciduous teeth ameliorate concanavalin A-induced autoimmune hepatitis by protecting hepatocytes from apoptosis

BACKGROUNDAutoimmune hepatitis is a serious autoimmune liver disease that threatens human health worldwide, which emphasizes the urgent need to identify novel treatments. Stem cells from human exfoliated deciduous teeth (SHED), which are easy to obtain in a non-invasive manner, show pronounced proliferative and immunomodulatory capacities.AIMTo investigate the protective effects of SHED on concanavalin A (ConA)-induced hepatitis in mice, and to elucidate the associated regulatory mechanisms.METHODSWe used a ConA-induced acute hepatitis mouse model and an in vitro co-culture system to study the protective effects of SHED on ConA-induced autoimmune hepatitis, as well as the associated underlying mechanisms.RESULTSSHED infusion could prevent aberrant histopathological liver architecture caused by ConA-induced infiltration of CD3⁺, CD4⁺, tumor necrosis-alpha⁺, and interferon-gamma⁺ inflammatory cells. Alanine aminotransferase and aspartate aminotransferase were significantly elevated in hepatitis mice. SHED infusion could therefore block ConA-induced alanine aminotransferase and aspartate aminotransferase elevations. Mechanistically, ConA upregulated tumor necrosis-alpha and interferon-gamma expression, which was activated by the nuclear factor-kappa B pathway to induce hepatocyte apoptosis, resulting in acute liver injury. SHED administration protected hepatocytes from ConA-induced apoptosis. CONCLUSIONSHED alleviates ConA-induced acute liver injury via inhibition of hepatocyte apoptosis mediated by the nuclear factor-kappa B pathway. Our findings could provide a potential treatment strategy for hepatitis.     

Effect of lithium chloride on cell proliferation and osteogenic differentiation in stem cells from human exfoliated deciduous teeth

Lithium Chloride (LiCl) has been used as a canonical Wnt pathway activator due to its ability to inhibit a glycogen synthase kinase-3. The aim of the present study was to investigate the effect of LiCl on cell proliferation and osteogenic differentiation in stem cells isolated from human exfoliated deciduous teeth (SHEDs). SHEDs were isolated and cultured in media supplemented with LiCl at 5, 10, or 20 mM. The results demonstrated that LiCl significantly decreased SHEDs colony forming unit ability in a dose dependent manner. LiCl significantly enhanced the percentage of cells in the sub G0 phase, accompanied by a reduction of the percentage of cells in the G1 phase at day 3 and 7 after treatment. Further, LiCl markedly decreased OSX and DMP1 mRNA expression after treating SHEDs in an osteogenic induction medium for 7 days. In addition, no significant difference in alkaline phosphatase enzymatic activity or mineral deposition was found. Together, these results imply that LiCl influences SHEDs behavior.     

Stem cells from human exfoliated deciduous teeth affect mitochondria and reverse cognitive decline in a senescence-accelerated mouse prone 8 model

Background aimsStem cell therapy is a novel therapy being explored for AD. The molecular mechanism of its effect is still unclear. The authors investigated the effects and mechanism by injection of SHEDs into an AD mouse model.MethodsSHEDs were cultured in vitro and injected into AD SAMP8 mice by caudal vein, and SHEDs labeled via synthetic dye showed in vivo migration to the head. The cognitive ability of SAMP8 mice was evaluated via Barnes maze and new object recognition. The pathological indicators of AD, including Tau, amyloid plaques and inflammatory factors, were examined at the protein or RNA level. Next, macro-proteomics analysis and weighted gene co-expression network analysis (WGCNA) based on protein groups and behavioral data were applied to discover the important gene cluster involved in the improvement of AD by SHEDs, which was further confirmed in an AD model in both mouse and cell lines.ResultsSHED treatment improved the cognitive ability and pathological symptoms of SAMP8 mice. Proteomics analysis indicated that these improvements were tightly related to the mitochondria, which was proved through examination of the shape and function of mitochondria both in vivo (SAMP8 brain) and in vitro (SH-SY5Y cells). Finally, the core targets of SHEDs in the mitochondrial pathway, Hook3, Mic13 and MIF, were screened out and confirmed in vivo.ConclusionsSHED treatment significantly relieved AD symptoms, improved cognitive ability and reversed memory loss in an AD mouse model, possibly through the recovery of dysfunctional mitochondria. These results raise the possibility that SHED may ease the symptoms of AD by targeting the mitochondria.     

Osteogenic differentiation of stem cells derived from human periodontal ligaments and pulp of human exfoliated deciduous teeth

Multipotent stem cells derived from periodontal ligaments (PDLSC) and pulp of human exfoliated deciduous teeth (SHED) represent promising cell sources for bone regeneration. Recent studies have demonstrated that retinoic acid (RA) and dexamethasone (Dex) induce osteogenesis of postnatal stem cells. The objective of this study was to examine the effects of RA and Dex on the proliferation and osteogenic differentiation of SHED and PDLSC and to compare the osteogenic characteristics of SHED and PDLSC under RA treatment. SHED and PDLSC were treated with serum-free medium either alone or supplemented with RA or Dex for 21 days. The proliferation of SHED and PDLSC was significantly inhibited by both RA and Dex. RA significantly upregulated gene expression and the activity of alkaline phosphatase in SHED and PDLSC. Positive Alizarin red and von Kossa staining of calcium deposition was seen on the RA-treated SHED and PDLSC after 21 days of culture. The influences of RA on the osteogenic differentiation of SHED and PDLSC were significantly stronger than with Dex. Supplemention with insulin enhanced RA-induced osteogenic differentiation of SHED. Thus, RA is an effective inducer of osteogenic differentiation of SHED and PDLSC, whereas RA treatment in combination with insulin supplementation might be a better option for inducing osteogenic differentiation. Significantly higher cell proliferation of PDLSC results in greater calcium deposition after 3-week culture, suggesting that PDLSC is a better osteogenic stem cell source. This study provides valuable information for efficiently producing osteogenically differentiated SHED or PDLSC for in vivo bone regeneration.     

Transplantation of stem cells from human exfoliated deciduous teeth for bone regeneration in the dog mandibular defect

AIM: To investigate the effect of stem cells from human exfoliated deciduous teeth(SHED) transplanted for bone regeneration in the dog mandibular defect.METHODS: In this prospective comparative study, SHEDs had been isolated 5 years ago from human exfoliated deciduous teeth. The undifferentiated stem cells were seeded into mandibular bone through-andthrough defects of 4 dogs. Similar defects in control group were filled with cell-free collagen scaffold. After 12 wk, biopsies were taken and morphometric analysis was performed. The percentage of new bone formation and foreign body reaction were measured in each case. The data were subject to statistical analysis using the Mann-Whitney U and Kruskalwalis statistical tests. Differences at P 0.05 was considered as significant level.RESULTS: There were no significant differences between control and SHED-seeded groups in connective tissue(P = 0.248), woven bone(P = 0.248) and compact bone(P = 0.082). There were not any side effects in transplanted SHED group such as teratoma or malignancy and abnormalities in this period.CONCLUSION: SHEDs which had been isolated and characterized 5 years ago and stored with cryopreservation banking were capable of proliferation and osteogenesis after 5 years, and no immune response was observed after three months of seeded SHEDs.     

Intrastriatal transplantation of stem cells from human exfoliated deciduous teeth reduces motor defects in Parkinsonian rats

Background

This study explored the neural differentiation and therapeutic effects of stem cells from human exfoliated deciduous teeth (SHED) in a rat model of Parkinson's disease (PD).

Stem cell recruitment factors secreted from cord blood-derived stem cells that are not secreted from mature endothelial cells enhance wound healing

Wounds are one of the most frequently occurring medical complication. Stem cells were recently highlighted as a novel therapeutic approach to treating wounds, although some negative aspects of allogenic stem cell transplantation were observed, such as cellular source limitations and unknown side effects in vivo. To address and eliminate these side effects, we examined the wound healing effect of secretory factors released from human cord blood-derived stem cells (hCB-SCs) and human umbilical vascular endothelial cells (HUVECs) on cutaneous excisional wound models. The hCB-SCs retained endothelial progenitor cell characteristics and expressed MSC markers such as CD73, CD105, and CD44. Analysis of hCB-SC-conditioned medium (CM) indicated that hCB-SCs secrete distinctly unique cytokines and chemokines such as TGF-β, PDGF, bFGF, EGF, KGF, and VEGF, which are well known to be important in normal angiogenesis and wound healing. Furthermore, hCB-SCs also secreted stem cell-recruiting factors such as G-CSF and GM-CSF, whereas HUVECs did not. When CB-SC-CM was applied to wound sites, hCB-SC-CM accelerated the wound healing rate compared with HUVEC-CM- and control medium-treated groups. In addition, hCB-SC-CM treatment caused a more rapid re-formation of granulation tissue and re-epithelialization of wounds, which indicates that the therapeutic effect of hCB-SC-CM is due to secreted stem cell-recruiting factors from stem cells, not just from endothelial lineage cells. Taken together, these results suggest that secretory factors released from stem cells, not just from endothelial cells, could be an important mediator of stem cell therapy in ischemic tissue diseases.     

A simple method for the quantitation of the stem cells derived from human exfoliated deciduous teeth using a luminescent cell viability assay

Stem cells from human exfoliated deciduous teeth are a population of highly proliferative postnatal stem cells and have been characterized as multipotent stem cells. In this study we developed a fast and sensitive method for stem cells derived from human exfoliated deciduous teeth count, using a luminescent viability assay. We isolated stem cells from normal exfoliated deciduous teeth using collagenase/dispase digestions solutions. Separated stem cells were placed in opaque-walled multiwall plates in culture alpha Modified Eagle’s Medium. For dental pulp stem cells quantitation we used a simple method for determining the number of viable cells based on ATP concentration. Cells attached to the bottom of the multiwall plates were counted with the luminescent assay and were cultured for mesenchymal markers expression. Moreover cells attached to the bottom of the multiwall plates were directed toward the osteogenic, adipogenic, lineages at the respective passages. Flow cytometry was used for immunophenotyping of cultured dental stem cells from exfoliated deciduous teeth. Cells that were counted with the luminescent assay, after culture formed fibroblastic morphology and were expressed the mesenchymal stem cell markers CD29, CD105, CD146, CD44. There was a correlation between the number of cells plated for culture and the number of mesenchymal stem cells after culture. Osteogenic and adipogenic differentiation of the cells counted with the luminescent assay was performed. The luminescent signal of viable mesenchymal dental stem cells isolated from dental pulp of exfoliated teeth represents an ideal method for mesenchymal stem cells count before culturing.     

Induced in vitro differentiation of neural-like cells from human exfoliated deciduous teeth-derived stem cells

Stem cells from human exfoliated deciduous teeth (SHED) are highly proliferative, clonogenic and multipotent stem cells with a neural crest cell origin. Additionally, they can be collected with minimal invasiveness in comparison with other sources of mesenchymal stem cells (MSCs). Therefore, SHED could be a desirable option for potential therapeutic applications. In this study, SHEDs were established from enzyme-disaggregated deciduous dental pulp obtained from 6 to 9 year-old children. The cells had typical fibroblastoid morphology and expressed antigens characteristic of MSCs, STRO1, CD146, CD45, CD90, CD106 and CD166, but not the hematopoietic and endothelial markers, CD34 and CD31, as assessed by FACS analysis. Differentiation assessment revealed a strong osteogenic and adipogenic potential of SHEDs. In order to further evaluate the in vitro differentiation potential of SHED into neural cells, a simple short time growth factor-mediated induction was used. Immunofluorescence staining and flow cytometric analysis revealed that SHED rapidly expressed nestin and b-III tubulin, and later expressed intermediate neural markers. In addition, the intensity and percentages of nestin and b-III tubulin and mature neural markers (PSA-NCAM, NeuN, Tau, TH, or GFAP) increased significantly following treatment. Moreover, RT-PCR and Western blot analyses showed that the neural markers were strongly up-regulated after induction. In conclusion, these results provide evidence that SHED can differentiate into neural cells by the expression of a comprehensive set of genes and proteins that define neural-like cells in vitro. SHED cells might be considered as new candidates for the autologous transplantation of a wide variety of neurological diseases and neurotraumatic injuries.     

Paracrine factors of mesenchymal stem cells recruit macrophages and endothelial lineage cells and enhance wound healing

Bone marrow derived mesenchymal stem cells (BM-MSCs) have been shown to enhance wound healing; however, the mechanisms involved are barely understood. In this study, we examined paracrine factors released by BM-MSCs and their effects on the cells participating in wound healing compared to those released by dermal fibroblasts. Analyses of BM-MSCs with Real-Time PCR and of BM-MSC-conditioned medium by antibody-based protein array and ELISA indicated that BM-MSCs secreted distinctively different cytokines and chemokines, such as greater amounts of VEGF-alpha, IGF-1, EGF, keratinocyte growth factor, angiopoietin-1, stromal derived factor-1, macrophage inflammatory protein-1alpha and beta and erythropoietin, compared to dermal fibroblasts. These molecules are known to be important in normal wound healing. BM-MSC-conditioned medium significantly enhanced migration of macrophages, keratinocytes and endothelial cells and proliferation of keratinocytes and endothelial cells compared to fibroblast-conditioned medium. Moreover, in a mouse model of excisional wound healing, where concentrated BM-MSC-conditioned medium was applied, accelerated wound healing occurred compared to administration of pre-conditioned or fibroblast-conditioned medium. Analysis of cell suspensions derived from the wound by FACS showed that wounds treated with BM-MSC-conditioned medium had increased proportions of CD4/80-positive macrophages and Flk-1-, CD34- or c-kit-positive endothelial (progenitor) cells compared to wounds treated with pre-conditioned medium or fibroblast-conditioned medium. Consistent with the above findings, immunohistochemical analysis of wound sections showed that wounds treated with BM-MSC-conditioned medium had increased abundance of macrophages. Our results suggest that factors released by BM-MSCs recruit macrophages and endothelial lineage cells into the wound thus enhancing wound healing.     

Simulated hypogravity stimulates cell spreading and wound healing in cultured human vascular endothelial cells

It is well known that endothelial cells (EC) are highly sensitive to mechanical influences such as hemodynamic conditions or pulsatile stretch. However, it is still unknown, how endothelium responds to the changed gravity. The results of some studies suggest that cellular elements of vascular wall and, particularly, endothelium, may directly participate in development of physiological responces to microgravity. On our suggestion, this is extremely attractive since vascular endothelium is one of the main regulators of vascular tone (via its interaction with vascular smooth muscle cells) and, consequently, can play not last role in maintaining of normal cardiovascular system operation in microgravity. On the other hand, the endothelium itself may be regarded as a widely dispersed organ of approximately 1.5 kg in weight (in the adult human organism). Finally, endothelium is not just a passive barrier between vascular wall and circulating blood but synthesizes, metabolizes, and releases a substances which act on adjacent cell systems or distant cell structures. The main aims of this study were: 1) the development of experimental model, allowing to study functional parameters of human endothelial cells in hypogravity conditions in vitro; 2) the verification of endothelial sensitivity to gravitational micro-environment.     

Mesenchymal stem cell-conditioned media: A novel alternative of stem cell therapy for quality wound healing

Mesenchymal stem cells-conditioned media (MSCs-CM) contains several growth factors and cytokines, thus may be used as a better alternative to stem cell therapy, which needs to be elucidated. The present study was conducted to evaluate the therapeutic potential of caprine, canine, and guinea pig bone marrow-derived MSCs-CM in excision wound healing in a guinea pig model. MSCs were obtained from bone marrow, expanded ex vivo and characterized as per ISCT criteria. CM was collected assayed by western blot to ascertain the presence of important secretory biomolecules. Quantitative estimation by enzyme-linked immunosorbent assay was done for a vascular epidermal growth factor (VEGF) and interleukin-6 (IL-6) in caprine MSCs-CM and optimum time for collection of CM was decided as 72 hr. CM from all the species was lyophilized by freeze-drying method. Full-thickness (2 × 2 cm²) excision skin wounds were created in guinea pigs (six animals in each group) and respective lyophilized CM mixed with laminin gel was applied topically at weekly interval. On Day 28, histopathological examinations of healed skin were done by hemotoxylin and eosin staining. MSCs were found to secrete important growth factors and cytokines (i.e., VEGF, transforming growth factor-β1, fibroblast growth factor-2, insulin-like growth factor-1, stem cell factor, and IL-6) as demonstrated by immunohistochemistry and western blot assay. It was found that allogenic and xenogenic application of CM significantly improved quality wound healing with minimal scar formation. Thus, MSCs-CM can be used allogenically as well as xenogenically for quality wound healing.     

Exosomes derived from human amniotic epithelial cells accelerate wound healing and inhibit scar formation

Wound healing is a highly orchestrated physiological process consisting of a complex events, and scarless wound healing is highly desired for the development and application in clinical medicine. Recently, we have demonstrated that human amniotic epithelial cells (hAECs) promoted wound healing and inhibited scar formation through a paracrine mechanism. However, exosomes (Exo) are one of the most important paracrine factors. Whether exosomes derived from human amniotic epithelial cells (hAECs-Exo) have positive effects on scarless wound healing have not been reported yet. In this study, we examined the role of hAECs-Exo on wound healing in a rat model. We found that hAECs, which exhibit characteristics of both embryonic and mesenchymal stem cells, have the potential to differentiate into all three germ layers. hAECs-Exo ranged from 50 to 150 nm in diameter, and positive for exosomal markers CD9, CD63, CD81, Alix, TSG101 and HLA-G. Internalization of hAECs-Exo promoted the migration and proliferation of fibroblasts. Moreover, the deposition of extracellular matrix (ECM) were partly abolished by the treatment of high concentration of hAECs-Exo (100 μg/mL), which may be through stimulating the expression of matrix metalloproteinase-1 (MMP-1). In vivo animal experiments showed that hAECs-Exo improved the skin wound healing with well-organized collagen fibers. Taken together, These findings represent that hAECs-Exo can be used as a novel hope in cell-free therapy for scarless wound healing.     

Human umbilical cord perivascular cells (HUCPVC): A mesenchymal cell source for dermal wound healing

Human bone marrow mesenchymal stem cells (hBM-MSC) have recently been employed in the clinical treatment of challenging skin defects. We have described an MSC population that can be easily harvested from human umbilical cord perivascular tissue, human umbilical cord perivascular cells (HUCPVC), which exhibit a higher proliferative rate and frequency than hBM-MSC. Our objective was to establish whether HUCPVC could promote healing of full thickness murine skin defects, and thus find utility as a cell source for dermal repair. To this end, bilateral full thickness defects were created on the dorsum of Balb/c nude mice. Fibrin was used as a delivery vehicle for 1 x 106 PKH-67 labeled HUCPVC with contralateral controls receiving fibrin only. Epifluorescent and brightfield microscopic evaluation of the wound site was carried out at 3 and 7 days while mechanical testing of wounds was carried out at 3, 7, and 10 days. Our results show that by 3 days, marked contraction of the wound was observed in the fibrin controls whilst the HUCPVC samples exhibited neither collapse nor contraction of the defect, and the dermal repair tissue was considerably thicker and more organized. By 7 days, complete re-epithelialization of the HUCPVC wounds was observed whilst in the controls re-epithelialization was limited to the wound margins. Wound strength was significantly increased in the HUCPVC treatment group by 3 and 7 days but no statistical difference was seen at 10 days. We conclude that HUCPVCs accelerate early wound healing in full thickness skin defects and thus represent a putative source of human MSCs for use in dermal tissue engineering.     

The isolation and identification of exfoliated prostate cells from human semen

A prostatic acid phosphatase localization technique to identify exfoliated prostate cells in human semen is described. Three morphologically distinct types of exfoliated prostate cells were observed in the semen of 14 vasectomized and nonvasectomized men: (1) large, ovoid prostate cells with abundant cytoplasm; (2) small, ovoid prostate cells with little cytoplasm; and (3) columnar prostate cells. These three types of exfoliated prostate cells were also observed within the lumina of prostatic alveoli when stained by this method. Finally, this staining procedure was found to have certain advantages over the conventionally used staining techniques, for it allows the rapid identification of exfoliated prostate cells in human semen and provides a noninvasive technique for monitoring the functional state of the prostate gland.