Si-Jung Jang

Multilineage potential and proteomic profiling of human dental stem cells derived from a single donor.

Dental tissues provide an alternative autologous source of mesenchymal stem cells (MSCs) for regenerative medicine. In this study, we isolated human dental MSCs of follicle, pulp and papilla tissue from a single donor tooth after impacted third molar extraction by excluding the individual differences. We then compared the morphology, proliferation rate, expression of MSC-specific and pluripotency markers, and in vitro differentiation ability into osteoblasts, adipocytes, chondrocytes and functional hepatocyte-like cells (HLCs). Finally, we analyzed the protein expression profiles of undifferentiated dental MSCs using 2DE coupled with MALDI-TOF-MS. Three types of dental MSCs largely shared similar morphology, proliferation potential, expression of surface markers and pluripotent transcription factors, and differentiation ability into osteoblasts, adipocytes, and chondrocytes. Upon hepatogenic induction, all MSCs were transdifferentiated into functional HLCs, and acquired hepatocyte functions by showing their ability for glycogen storage and urea production. Based on the proteome profiling results, we identified nineteen proteins either found commonly or differentially expressed among the three types of dental MSCs. In conclusion, three kinds of dental MSCs from a single donor tooth possessed largely similar cellular properties and multilineage potential. Further, these dental MSCs had similar proteomic profiles, suggesting their interchangeable applications for basic research and call therapy.

Cryopreservation of human dental follicle tissue for use as a resource of autologous mesenchymal stem cells.

The main purpose of this study was to develop a cryopreservation method for human dental follicle tissue to maintain autologous stem cells as a resource. A modified cryoprotectant, consisting of 0.05 m glucose, 0.05 m sucrose and 1.5 m ethylene glycol in phosphate‐buffered saline (PBS) was employed, with a slow‐ramp freezing rate. We observed > 70% of cell survival rate after 3 months of tissue storage. Isolated and cultured human dental stem cells (hDSCs) from cryopreserved dental follicles expressed mesenchymal stem cell markers at a level similar to that of hDSCs from fresh tissue. They also successfully differentiated in vitro into the mesenchymal lineage, osteocytes, adipocytes and chondrocytes under specific inductions. Using immunohistochemistry, the early transcription factors OCT4, NANOG and SOX2 were moderately or weakly detected in the nucleus of both fresh and cryopreserved dental follicles. In addition, p63, CCND1, BCL2 and BAX protein expression levels were the same in both fresh and cryopreserved tissues. However, the positive‐cell ratio and intensity of p53 protein was higher in cryopreserved tissues than in fresh tissues, indicating direct damage of the freeze–thawing process. Real‐time PCR analysis of hDSCs at passage 2 from both fresh and cryopreserved dental follicles showed similar levels of mRNA for apoptosis‐ and transcription‐related genes. Based on these results, a newly developed cryoprotectant, along with a slow ramp rate freezing procedure allows for long‐term dental tissue preservation for later use as an autologous stem cell resource in regenerative cell therapy. Copyright

Positive expression of NANOG, mutant p53, and CD44 is directly associated with clinicopathological features and poor prognosis of oral squamous cell carcinoma

BackgroundIn order to predict long-term prognosis and define individual treatment modalities for patients with oral squamous cell carcinoma (OSCC), more reliable tumor biomarkers are needed during the pretreatment workup period. The present study aimed to identify more reliable immunohistochemical tumor prognostic markers in the pretreatment biopsy specimens of patients with OSCC.MethodsWe selected 57 patients who were diagnosed with primary OSCC through histopathological analysis. Pretreatment biopsy specimens were immunohistochemically analyzed for the transcription factor NANOG, cancer stem cell marker CD44, and mutant tumor protein 53 (mutant p53). The immunostaining patterns were assessed for their association with the clinicopathological features of OSCC and overall survival rates.ResultsLate tumor stage, positive neck node metastasis, and high-grade differentiation were associated with significantly poorer survival rates. Enhanced expression of NANOG and mutant p53 positivity were significantly associated with clinically late-stage tumors, positive neck node metastasis, histologically high-grade tumors, and poor overall survival rates. OSCCs with strong co-detection of NANOG and mutant p53 were linked to significantly lower survival rates than those with both weak NANOG expression and p53 negativity. Increased expression of CD44 had a limited correlation with unfavorable clinicopathological features.ConclusionHigh expression of NANOG and positive expression of mutant p53 in the pretreatment biopsy specimens of patients with OSCC were associated with poor survival rates and unfavorable clinicopathological features. These results demonstrate that NANOG, mutant p53, and CD44 could be used as immunohistochemical markers in the pretreatment specimens of OSCC. In particular, analysis for co-expression of NANOG and mutant p53 should be made highly available as a tool for prognosis and selecting individual treatment modalities.

In vitro comparative analysis of human dental stem cells from a single donor and its neuronal differentiation potential evaluated by electrophysiology.

AIMS The aim of this study was to find out a mesenchymal stem cells (MSCs) source from human dental tissues of the same donor (follicle, papilla and pulp), which exhibits higher neurogenic differentiation potential in vitro. MAIN METHODS MSCs were isolated from dental tissues (follicle, papilla and pulp) by digestion method. All MSCs were analyzed for pluripotent makers by western blot, cell surface markers by flow cytometry, adipo- and osteocytes markers by RT-qPCR. The neuronal differentiated MSCs were characterized for neuronal specific markers by RT-qPCR and immunofluorescence. Functional neuronal properties were analyzed by electrophysiology and synaptic markers expression. KEY FINDINGS All MSCs expressed pluripotent markers (Oct4, Sox2 and Nanog) and were found positive for mesenymal markers (CD44, CD90, CD105) while negative for hematopoietic markers (CD34 and CD45). Furthermore, MSCs were successfully differentiated into adipocytes, osteocytes and trans-differentiated into neuronal cells. Among them, dental pulp derived MSCs exhibits higher neurogenic differentiation potential, in term of expression of neuronal specific markers at both gene and protein level, and having higher Na(+) and K(+) current with the expression of synaptic markers. SIGNIFICANCE The three types of dental MSCs from a single donor broadly possessed similar cellular properties and can differentiate into neuronal cells; however, pulp derived MSCs showed higher neurogenic potential than the follicle and papilla, suggesting their use in future stem cells therapy for the treatment of neurodegenerative disorders.

Characterization and Evaluation of Neuronal Trans-Differentiation with Electrophysiological Properties of Mesenchymal Stem Cells Isolated from Porcine Endometrium

Endometrial stromal cells (EMSCs) obtained from porcine uterus (n = 6) were positive for mesenchymal stem cell markers (CD29, CD44 and CD90), and negative for epithelial marker CD9 and hematopoietic markers CD34, CD45 analyzed by flow cytometry. Further the cells were positive for expression of mesenchymal markers, CD105, CD140b, and CD144 by PCR. Pluripotent markers OCT4, SOX2, and NANOG were positively expressed in EMSCs analyzed by Western blotting and PCR. Further, differentiation into adipocytes and osteocytes was confirmed by cytochemical staining and lineage specific gene expression by quantitative realtime-PCR. Adipocyte (FABP, LPL, AP2) and osteocyte specific genes (ON, BG, RUNX2) in differentiated EMSCs showed significant (p < 0.05) increase in expression compared to undifferentiated control cells. Neurogenic transdifferentiation of EMSCs exhibited distinctive dendritic morphology with axon projections and neuronal specific genes, NFM, NGF, MBP, NES, B3T and MAP2 and proteins, B3T, NFM, NGF, and TRKA were positively expressed in neuronal differentiated cells. Functional analysis of neuronal differentiated EMSCs displayed voltage-dependence and kinetics for transient outward K+ currents (Ito), at holding potential of −80 mV, Na+ currents and during current clamp, neuronal differentiated EMSCs was more negative than that of control EMSCs. Porcine EMSCs is a suitable model for studying molecular mechanism of transdifferentiation, assessment of electrophysiological properties and their efficiency during in vivo transplantation.

Overexpression of Oct4 in porcine ovarian stem/stromal cells enhances differentiation of oocyte-like cells in vitro and ovarian follicular formation in vivo.

BackgroundRecent findings have revealed that the female gonad may have regenerative activity with having germ line stem cells in juveniles and adults. Application of these germ line stem cells could be an alternative therapy for reproductive disorders in regenerative medicine.MethodsTo enhance the potency of differentiation into oocyte-like cells (OLCs) and folliculogenesis, we overexpressed Oct4 in ovarian stem/stromal cell (OvSCs) and examined the cellular properties related to stemness and self-renewal ability and finally demonstrated the ability of in vitro differentiation and folliculogenesis.ResultsOvarian cortex included putative stem cells in terms of AP activity, cell cycle status, cell proliferation, expression of mesenchymal lineage surface markers and pluripotent transcriptional markers. Further, Oct4 transfected OvSCs (Oct4-OvSCs) were enhanced their AP activity and cell proliferation compared to OvSCs. The potential on in vitro differentiation into OLCs and in vivo folliculogenesis was also evaluated in OvSCs and Oct4-OvSCs, respectively. Oct4-OvSCs possessed higher oogenesis potential in vitro than OvSCs, in terms of expression of germ cell markers by RT-PCR and the number of OLCs. When OvSCs and Oct4-OvSCs were xeno-transplanted into infertile mice ovaries, the OvSCs transplantation induced new primary follicle formation and hormonal levels of estradiol and FSH remained similar to that of normal mice. However, Oct4-OvSCs possessed higher ability for folliculogenesis based on inducing developing follicles with thecal layer and granulosa cells and more similar estradiol level to normal mice.ConclusionsThese findings demonstrated that putative stem cells were present in ovarian cortex and exhibited differentiation ability into OLCs and folliculogenesis in vivo, and Oct4-overexpression enhanced these ability, suggesting their cellular models based on gene therapy in understanding the mechanisms of oogenesis and folliculogenesis, and finally in view of reproductive cell therapy.

Cryopreservation of Human Wharton’s Jelly-derived Mesenchymal Stem Cells Following Controlled Rate Freezing Protocol Using Different Cryoprotectants; A Comparative Study

Objectives To compare the effect of three different cryoprotectants on basic stem cell characteristics for the possibility of using well defined, dimethyl sulfoxide (DMSO) and serum free freezing solutions to cryopreserve human Wharton’s jelly-derived mesenchymal stem cells (WJMSCs) following controlled rate freezing protocol. Methods The mesenchymal stem cells isolated from human Wharton’s jelly were cryopreserved using 10% DMSO, 10% polyvinylpyrrolidone (PVP) and a cocktail solution comprising of 0.05 M glucose, 0.05 M sucrose and 1.5 M ethylene glycol following controlled rate freezing protocol. We investigated the post-thaw cell viability, morphology, proliferation capacity, basic stem cell characteristics, in vitro differentiation potential and apoptosis-related gene expression profile before and after cryopreservation. Results The cryoprotectant 10% DMSO has shown higher post-thaw cell viability of 81.2±0.58% whereas 10% PVP and cocktail solution have shown 62.87±0.35% and 72.2±0.23%, respectively at 0 h immediately thawing. The cell viability was further reduced in all the cryopreserved groups at 24 h later post-thaw culture. Further, the complete elimination of FBS in cryoprotectants has resulted in drastic reduction in cell viability. Cryopreservation did not alter the basic stem cell characteristics, plasticity and multipotency except proliferation rate. The expression of pro-apoptotic BAX and p53 genes were higher whilst p21 was lower in all the cryopreserved groups when compare to the control group of WJMSCs. Conclusion Although 10% DMSO has shown higher post-thaw cell viability compare to 10% PVP and cocktail solution, the present study indicates the feasibility of developing a well-defined DMSO free cryosolution which can improve storage and future broad range applications of WJMSCs in regenerative medicine without losing their basic stem cell characteristics.

Immunomodulatory properties and in vivo osteogenesis of human dental stem cells from fresh and cryopreserved dental follicles.

In our previous study, dental follicle tissues from extracted wisdom teeth were successfully cryopreserved for use as a source of stem cells. The goals of the present study were to investigate the immunomodulatory properties of stem cells from fresh and cryopreserved dental follicles (fDFCs and cDFCs, respectively) and to analyze in vivo osteogenesis after transplantation of these DFCs into experimental animals. Third passage fDFCs and cDFCs showed similar expression levels of interferon-γ receptor (CD119) and major histocompatibility complex class I and II (MHC I and MHC II, respectively), with high levels of CD119 and MHC I and nearly no expression of MHC II. Both fresh and cryopreserved human DFCs (hDFCs) were in vivo transplanted along with a demineralized bone matrix scaffold into mandibular defects in miniature pigs and subcutaneous tissues of mice. Radiological and histological evaluations of in vivo osteogenesis in hDFC-transplanted sites revealed significantly enhanced new bone formation activities compared with those in scaffold-only implanted control sites. Interestingly, at 8 weeks post-hDFC transplantation, the newly generated bones were overgrown compared to the original size of the mandibular defects, and strong expression of osteocalcin and vascular endothelial growth factor were detected in the hDFCs-transplanted tissues of both animals. Immunohistochemical analysis of CD3, CD4, and CD8 in the ectopic bone formation sites of mice showed significantly decreased CD4 expression in DFCs-implanted tissues compared with those in control sites. These findings indicate that hDFCs possess immunomodulatory properties that involved inhibition of the adaptive immune response mediated by CD4 and MHC II, which highlights the usefulness of hDFCs in tissue engineering. In particular, long-term preserved dental follicles could serve as an excellent autologous or allogenic stem cell source for bone tissue regeneration as well as a valuable therapeutic agent for immune diseases.

Evaluation of phenotypic, functional and molecular characteristics of porcine mesenchymal stromal/stem cells depending on donor age, gender and tissue source.

The biological properties of mesenchymal stem cells (MSCs) are influenced by donor age, gender and/or tissue sources. The present study investigated the cellular and molecular properties of porcine mesenchymal stromal/stem cells (MSCs) isolated from different tissues (adipose & dermal skin) and sex at different ages (1 week & 8 months after birth) with similar genetic and environmental backgrounds. MSCs were analyzed for alkaline phosphatase (AP) activity, CD90 and Oct3/4 expression, in vitro differentiation ability, senescence-associated β-galactosidase (SA-β-Gal) activity, telomeric properties, cell cycle status and expression of senescence (IL6, c-myc, TGFβ, p53 and p21)- and apoptosis (Bak and Bcl2)-related proteins. An age-dependent decline in AP activity and adipogenesis was observed in all MSCs, except for male A-MSCs. CD90 expression did not change, but SA-β-Gal activity increased with advancement in age, except in A-MSCs. Telomeric properties were similar in all MSCs, whereas expression levels of Oct3/4 protein declined with the advancement in age. p21 expression was increased with increase in donor age. Male derived cells have shown higher IL6 expression. The expression of p53 was slightly lower in MSCs of dermal tissue than in adipose tissue. Bak was expressed in all MSCs regardless of age, but up regulation of Bcl2 was observed in DS-MSCs derived at 1 week after birth. In conclusion, adipose tissue-derived MSCs from young female individuals were found to be more resistant to senescence under in vitro culture conditions.

Development of Porous Beads to Provide Regulated BMP-2 Stimulation for Varying Durations: In Vitro and In Vivo Studies for Bone Regeneration

It is commonly accepted that the sustained release of bone morphogenetic protein-2 (BMP-2) can enhance bone regeneration and minimize its safety issues. However, little is known regarding the appropriate duration of BMP-2 stimulation for sufficient osteogenic differentiation and new bone formation because of the short half-life of BMP-2 in the physiological environment and the lack of a well-defined delivery matrix that can regulate the release period of BMP-2. In this study, we prepared porous poly(lactic-co-glycolic acid) (PLGA) beads with different surface pore sizes that can regulate the release period of BMP-2 (i.e., 7, 17, and 30 days) while providing the BMP-2 concentration required for bone regeneration. Our findings in both in vitro cell culture and in vivo animal studies using these BMP-2-loaded beads demonstrate that release of BMP-2 within 7 days affects only the initial differentiation of human periosteum-derived cells (hPDCs) and does not significantly enhance their subsequent differentiation into mature functional cells. However, extending the duration of BMP-2 stimulation over 17 days can provide a suitable environment for osteogenic differentiation of hPDCs and new bone formation.