Hyun Nam

The effects of platelet-rich plasma derived from human umbilical cord blood on the osteogenic differentiation of human dental stem cells

Platelet-rich plasma (PRP) is an emerging therapeutic application because PRP contains various growth factors that have beneficial effects on tissue regeneration and engineering. Mesenchymal stem cells and PRP derived from peripheral blood have been well studied. In this study, we investigated the effects of PRP derived from human umbilical cord blood (UCB-PRP) on proliferation, alkaline phosphatase (ALP) activity, and osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHEDs), dental pulp stem cells (DPSCs), and periodontal ligament stem cells (PDLSCs). Three types of dental stem cells were primarily isolated and characterized by flow cytometric analysis. Dental stem cells were exposed to various concentrations of UCB-PRP, which resulted in the proliferation of dental stem cells. Treatment with 2% UCB-PRP resulted in the highest level of proliferation. The ALP activity of DPSCs and PDLSCs increased following treatment with UCB-PRP in a dose-dependent manner up to a concentration of 2%. ALP activity decreased with higher concentration of UCB-PRP. The effects of UCB-PRP on calcium deposition were similar to those on proliferation and ALP activity. Treatment with 2% UCB-PRP resulted in the highest calcium depositions in DPSCs and PDLSCs; however, treatment with 1% UCB-PRP resulted in the highest calcium deposition in SHEDs. The concentrations of platelet-derived growth factor-AB and transforming growth factor-β1 in UCB-PRP were investigated and found to be comparable to the amounts in peripheral blood. Overall, UCB-PRP had beneficial effects on the proliferation and osteogenic differentiation of dental stem cells. Determination of the optimal concentration of UCB-PRP requires further investigation for clinical applications.

Identification of novel epithelial stem cell-like cells in human deciduous dental pulp.

It is well known that interactions between epithelial components and mesenchymal components are essential for tooth development. Therefore, it has been postulated that both types of stem cells might be involved in the regeneration of dental hard tissues. Recently, mesenchymal dental pulp stem cells that have odontogenic potential were identified from human dental pulp. However, the existence of epithelial cells has never been reported in human dental pulp. In the present study, we isolated and characterized epithelial cell-like cells from human deciduous dental pulp. They had characteristic epithelial morphology and expressed epithelial markers. Moreover, they expressed epithelial stem cell-related genes such as ABCG2, Bmi-1, DeltaNp63, and p75. Taken together, our findings suggest that epithelial stem cell-like cells might exist in human deciduous dental pulp and might play a role as an epithelial component for the repair or regeneration of teeth.

Expression profile of the stem cell markers in human hertwig's epithelial root sheath/epithelial rests of malassez cells.

Hertwig’s epithelial root sheath/Epithelial rests of Malassez (HERS/ERM) cells are unique epithelial cells in the periodontal ligament. They remain in periodontal tissues through-out the adult life, and it is expected that their functional role is to maintain the homeostasis of the periodontium through reciprocal interactions with other periodontal cells. In this study, we investigated whether HERS/ERM cells have primitive stem cell characteristics: those of embryonic stem cells as well as of epithelial stem cells. Primary HERS/ERM cells had typical epithelial cell morphology and characteristics and they maintained for more than five passages. They expressed epithelial stem cell-related genes: ABCG2, ANp63, p75, EpCAM, and Bmi-1. Moreover, the expression of embryonic stem cell markers such as Oct-4, Nanog, and SSEA-4 were detected. Next, we investigated whether the expression of these stem cell markers was maintained during the sub-culture process. HERS/ERM cells showed different expression levels of these stemness genes at each passage, but their expression was maintained throughout the passages. Taken together, our data suggest that a primary culture of HERS/ERM cells contains a population of primitive stem cells that express epithelial stem cell markers and embryonic stem cell markers. Furthermore, these cell populations were maintained during the sub-culturing process in our culture conditions. Therefore, our findings suggest that there is a strong possibility of accomplishing cementum tissue engineering with HERS/ERM cells.

Known-plaintext cryptanalysis of the Domingo-Ferrer algebraic privacy homomorphism scheme

We propose cryptanalysis of the First Domingo-Ferrers algebraic privacy homomorphism e: Zn → (Zp × Zq)d where n = pq. We show that the scheme can be broken by (d + 1) known plaintexts in O(d3 log2 n) time. Even when the modulus n is kept secret, it can be broken by 2(d + 1) known plaintexts in O(d4 log dn + d3 log2 n + e(m)) time with overwhelming probability.

Human feeder layer system derived from umbilical cord stromal cells for human embryonic stem cells

OBJECTIVE To evaluate the potential of human umbilical cord-derived stromal cells (hUCSCs) as a human feeder for human embryonic stem cells (ESCs). DESIGN Prospective study. SETTING Laboratory of Molecular Genetics and Stem Cell Differentiation, Dental Research Institute, School of Dentistry, Seoul National University. INTERVENTION(S) The hUCSCs were established, and human ESCs were cultured on established hUCSCs without serum. MAIN OUTCOME MEASURE(S) Cell-surface markers, karyotyping, and teratoma formation. RESULT(S) Primary cultures of hUCSCs from individual umbilical cords were maintained by an established protocol. Human ESCs on hUCSC layers were successfully maintained in serum-free culture medium past passage 30. Compared with hESCs on mouse feeder cells, the hESCs on hUCSCs showed similar levels of pluripotency-related cell-surface markers, self-renewal capacity, and teratoma formation in immune-deficient mice. These ESCs cultured on hUCSCs had a normal karyotype, even after long-term culture. CONCLUSION(S) The hUCSCs supported self-renewal of hESCs in serum-free conditions. This culture system has the potential to facilitate the development of clinical-grade hESCs for regenerative medicine.

Dental follicle cells and cementoblasts induce apoptosis of ameloblast-lineage and Hertwig's epithelial root sheath/epithelial rests of Malassez cells through the Fas-Fas ligand pathway.

Hertwigs epithelial root sheath (HERS), epithelial rests of Malassez (ERM) cells, and reduced ameloblasts undergo apoptosis during tooth development. This study examined the effects of dental follicle cells and cementoblasts on the apoptosis of ameloblast-lineage and HERS/ERM cells derived from the enamel organ. We also elucidated the induction pathways and identified the apoptotic pathway involved in this process. Here, we showed terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling (TUNEL)-positive HERS cells and reduced ameloblasts near dental follicle cells during tooth development. Co-culturing ameloblast-lineage cell line (ALC) ameloblasts and HERS/ERM cells with either dental follicle cells or OCCM-30 cementoblasts markedly enhanced the apoptosis of ameloblasts and HERS/ERM cells compared with cells cultured alone. However, dental follicle cells and cementoblasts did not modulate the apoptotic responses of co-cultured non-odontogenic MCF10A or KB cells. When ameloblasts + HERS and cementoblasts + dental follicle cells were co-cultured, the expression of Fas ligand (FasL) increased in cementoblasts + dental follicle cells, while the expression of Fas increased in ameloblasts + HERS. Interestingly, recombinant FasL induced ameloblast apoptosis while the cementoblast-induced ameloblast apoptosis was suppressed by the Fas/FasL antagonist Kp7-6. These results suggest that during tooth development, dental follicle cells and cementoblasts induce apoptosis of ameloblast-lineage and HERS/ERM cells through the Fas-FasL pathway, but do not induce the apoptosis of non-odontogenic epithelial cells.

A class of nonparametric DSSY nonconforming quadrilateral elements

A new class of nonparametric nonconforming quadrilateral finite elements is introduced which has the midpoint continuity and the mean value continuity at the interfaces of elements simul- taneously as the rectangular DSSY element (J. Douglas, Jr., J.E. Santos, D. Sheen and X. Ye, ESAIM: M2AN 33 (1999) 747-770.) The parametric DSSY element for general quadrilaterals requires five de- grees of freedom to have an optimal order of convergence (Z. Cai, J. Douglas, Jr., J.E. Santos, D. Sheen and X. Ye, Calcolo 37 (2000) 253-254.), while the new nonparametric DSSY elements require only four degrees of freedom. The design of new elements is based on the decomposition of a bilinear transform into a simple bilinear map followed by a suitable affine map. Numerical results are presented to compare the new elements with the parametric DSSY element. Mathematics Subject Classification. 65N30.

Establishment of Hertwig’s Epithelial Root Sheath/Epithelial Rests of Malassez Cell Line from Human Periodontium

Human Hertwigs epithelial root sheath/epithelial rests of Malassez (HERS/ERM) cells are epithelial remnants of teeth residing in the periodontium. Although the functional roles of HERS/ERM cells have yet to be elucidated, they are a unique epithelial cell population in adult teeth and are reported to have stem cell characteristics. Therefore, HERS/ERM cells might play a role as an epithelial component for the repair or regeneration of dental hard tissues; however, they are very rare population in periodontium and the primary isolation of them is considered to be difficult. To overcome these problems, we immortalized primary HERS/ERM cells isolated from human periodontium using SV40 large T antigen (SV40 LT) and performed a characterization of the immortalized cell line. Primary HERS/ERM cells could not be maintained for more than 6 passages; however, immortalized HERS/ERM cells were maintained for more than 20 passages. There were no differences in the morphological and immunophenotypic characteristics of HERS/ERM cells and immortalized HERS/ERM cells. The expression of epithelial stem cell and embryonic stem cell markers was maintained in immortalized HERS/ERM cells. Moreover, immortalized HERS/ERM cells could acquire mesenchymal phenotypes through the epithelial-mesenchymal transition via TGF-β1. In conclusion, we established an immortalized human HERS/ERM cell line with SV40 LT and expect this cell line to contribute to the understanding of the functional roles of HERS/ERM cells and the tissue engineering of teeth.

Upregulation of GM-CSF by TGF-β1 in epithelial mesenchymal transition of human HERS/ERM cells.

Hertwig’s epithelial root sheath/epithelial rests of Malassez (HERS/ERM) have been suggested to play an important role in tooth root formation, particularly in periodontal development. Epithelial mesenchymal transition (EMT) has been suggested to contribute to root development in tooth. However, the mechanism of interaction between HERS/ERM cells and dental mesenchymal cells has not been fully understood. In this study, we investigated the effect of exogenous transforming growth factor beta 1 (TGF-β1) in human HERS/ERM cells in order to verify the role of granulocyte macrophage colony-stimulating factor (GM-CSF) in EMT process. Antibody array was used to screen secretion factors by exogenous TGF-β1. Secretion of GM-CSF was increased by exogenous TGF-β1. Expression levels of EMT markers, vimentin, ZEB1 (zinc finger E-box binding homeobox 1), and E-cadherin, were confirmed using reverse transcription polymerase chain reaction and immunocytochemistry. Treatment with GM-CSF increased the expression of vimentin and ZEB1, similar to TGF-β1 treatment, and decreased the expression of E-cadherin. Our results suggest that GM-CSF could induce EMT in human HERS/ERM cells.

The Significance of SDF-1α-CXCR4 Axis in in vivo Angiogenic Ability of Human Periodontal Ligament Stem Cells

Periodontal ligament stem cells (PDLSCs) are multipotent stem cells derived from periodontium and have mesenchymal stem cell (MSC)-like characteristics. Recently, the perivascular region was recognized as the developmental origin of MSCs, which suggests the in vivo angiogenic potential of PDLSCs. In this study, we investigated whether PDLSCs could be a potential source of perivascular cells, which could contribute to in vivo angiogenesis. PDLSCs exhibited typical MSC-like characteristics such as the expression pattern of surface markers (CD29, CD44, CD73, and CD105) and differentiation potentials (osteogenic and adipogenic differentiation). Moreover, PDLSCs expressed perivascular cell markers such as NG2, αsmooth muscle actin, platelet-derived growth factor receptor β, and CD146. We conducted an in vivo Matrigel plug assay to confirm the in vivo angiogenic potential of PDLSCs. We could not observe significant vessel-like structures with PDLSCs alone or human umbilical vein endothelial cells (HU-VECs) alone at day 7 after injection. However, when PDLSCs and HUVECs were co-injected, there were vessel-like structures containing red blood cells in the lumens, which suggested that anastomosis occurred between newly formed vessels and host circulatory system. To block the SDF-1α and CXCR4 axis between PDLSCs and HUVECs, AMD3100, a CXCR4 antagonist, was added into the Matrigel plug. After day 3 and day 7 after injection, there were no significant vessel-like structures. In conclusion, we demonstrated the peri-vascular characteristics of PDLSCs and their contribution to in vivo angiogenesis, which might imply potential application of PDLSCs into the neovascularization of tissue engineering and vascular diseases.