Issei Saitoh - Academia.edu (original) (raw)

Papers by Issei Saitoh

Research paper thumbnail of Alkaline phosphatase and OCT-3/4 as useful markers for predicting susceptibility of human deciduous teeth-derived dental pulp cells to reprogramming factor-induced iPS cells

Journal of investigative and clinical dentistry, Jan 18, 2016

The aim of the present study was to prove that primary cells enriched with stem cells are more ea... more The aim of the present study was to prove that primary cells enriched with stem cells are more easily reprogrammed to generate induced pluripotent stem (iPS) cells than those with scarce numbers of stem cells. We surveyed the alkaline phosphatase (ALP) activity in five primarily-isolated human deciduous teeth-derived dental pulp cells (HDDPC) with cytochemical staining to examine the possible presence of stem cells. Next, the expression of stemness-specific factors, such as OCT(Octumer-binding transcription factor)3/4, NANOG, SOX2(SRY (sex determining region Y)-box 2), CD90, muscle segment homeodomain homeobox (MSX) 1, and MSX2, was assessed with a reverse transcription polymerase chain reaction method. Finally, these isolated HDDPC were transfected with plasmids carrying genes coding Yamanaka factors to determine whether these cells could be reprogrammed to generate iPS cells. Of the five primarily-isolated HDDPC, two (HDDPC-1 and -5) exhibited higher degrees of ALP activity. OCT-3...

Research paper thumbnail of Efficient CRISPR/Cas9-based gene correction in induced pluripotent stem cells established from fibroblasts of patients with sickle cell disease

Stem cell investigation, 2016

Research paper thumbnail of Relationships among nasal resistance, adenoids, tonsils, and tongue posture and maxillofacial form in Class II and Class III children

American journal of orthodontics and dentofacial orthopedics : official publication of the American Association of Orthodontists, its constituent societies, and the American Board of Orthodontics, 2017

The purpose of this study was to clarify the relationships between upper airway factors (nasal re... more The purpose of this study was to clarify the relationships between upper airway factors (nasal resistance, adenoids, tonsils, and tongue posture) and maxillofacial forms in Class II and III children. Sixty-four subjects (mean age, 9.3 years) with malocclusion were divided into Class II and Class III groups by ANB angles. Nasal resistance was calculated using computational fluid dynamics from cone-beam computed tomography data. Adenoids, tonsils, and tongue posture were evaluated in the cone-beam computed tomography images. The groups were compared using Mann-Whitney U tests and Student t tests. The Spearman rank correlations test assessed the relationships between the upper airway factors and maxillofacial form. Nasal resistance of the Class II group was significantly larger than that of the Class III group (P = 0.005). Nasal resistance of the Class II group was significantly correlated with inferior tongue posture (P <0.001) and negatively correlated with intermolar width (P = 0...

Research paper thumbnail of RCAN-11R peptide provides immunosuppression for fully mismatched islet allografts in mice

Scientific reports, Jan 8, 2017

Calcineurin inhibitors have been used for transplant therapy. However, the inhibition of calcineu... more Calcineurin inhibitors have been used for transplant therapy. However, the inhibition of calcineurin outside the immune system has a number of side effects. We previously developed a cell-permeable inhibitor of NFAT (nuclear factor of activated T cells) using the polyarginine peptide delivery system. This peptide (11R-VIVIT) selectively interferes with calcineurin-NFAT interaction without affecting the activity of calcineurin phosphatase and provides immunosuppression for fully mismatched islet allografts in mice. However, our recent study showed that 11R-VIVIT affected cell viability in vitro when it was used at higher concentration because of the VIVIT sequence. The aim of this study is to develop a safer NFAT inhibitor (RCAN-11R) that does not affect cell viability, and which is less toxic than calcineurin inhibitors. The minimal sequence of the protein family of regulators of calcineurin (RCAN) that is responsible for the inhibition of calcineurin-NFAT signaling was recently cha...

Research paper thumbnail of Isolation and characterization of lymphoid enhancer factor-1-positive deciduous dental pulp stem-like cells after transfection with a piggyBac vector containing LEF1 promoter-driven selection markers

Archives of oral biology, Jan 7, 2017

Lymphoid enhancer-binding factor-1 (LEF1) is a 48-kD nuclear protein that is expressed in pre-B a... more Lymphoid enhancer-binding factor-1 (LEF1) is a 48-kD nuclear protein that is expressed in pre-B and T cells. LEF1 is also an important member of the Wnt/β-catenin signaling pathway that plays important roles in the self-renewal and differentiation of embryonic stem cells. We speculated that LEF1 might function in the stem cells from human exfoliated deciduous teeth (SHED). In this study, we attempted to isolate such LEF1-positive cells from human deciduous dental pulp cells (HDDPCs) by genetic engineering technology, using the human LEF1 promoter. A piggyBac transposon plasmid (pTA-LEN) was introduced into HDDPCs, using the Neon(®) transfection system. After G418 selection, the emerging colonies were assessed for EGFP-derived fluorescence by fluorescence microscopy. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed using RNA isolated from these colonies to examine stem cell-specific transcript expression. Osteoblastic or neuronal differentiation was ind...

Research paper thumbnail of Masticatory movement during occlusal phase in children with primary dentition and women

The Journal of Japanese Society of Stomatognathic Function, 2002

Research paper thumbnail of Tissue-Specific Stem Cells Obtained by Reprogramming of Non-Obese Diabetic (NOD) Mouse-Derived Pancreatic Cells Confer Insulin Production in Response to Glucose

PloS one

Type 1 diabetes occurs due to the autoimmune destruction of pancreatic β-cells in islets. Transpl... more Type 1 diabetes occurs due to the autoimmune destruction of pancreatic β-cells in islets. Transplantation of islets is a promising option for the treatment of patients with type 1 diabetes that experience hypoglycemic unawareness despite maximal care, but the present shortage of donor islets hampers such transplantation. Transplantation of insulin-producing cells derived from the patients themselves would be one of the most promising approaches to cure type 1 diabetes. Previously, we demonstrated that insulin-producing cells could be produced by transfecting murine pancreatic cells with Yamanaka's reprogramming factors. Non-obese diabetic (NOD) mice are naturally occurring mutant mice defective in insulin production due to autoimmune ablation of pancreatic β-cells. In this study, we showed that glucose-sensitive insulin-producing cells are successfully generated by transfecting primary pancreatic cells from NOD mice (aged 6 months old) with a plasmid harboring the cDNAs for Oct-...

Research paper thumbnail of Comparison of Purification Solutions With Different Osmolality for Porcine Islet Purification

Cell Medicine, 2017

The osmolality of the purification solution is one of the most critical variables in human islet ... more The osmolality of the purification solution is one of the most critical variables in human islet purification during islet isolation. We previously reported the effectiveness of a combined continuous density/osmolality gradient for the supplemental purification of human islets. We herein applied a combined continuous density/osmolality gradient for regular purification. The islets were purified with a continuous density gradient without osmolality preparation [continuous density/normal osmolality (CD/NO)] or continuous density/osmolality solution with osmolality preparation by 10× Hank&amp;amp;amp;amp;#39;s balanced salt solution (HBSS) [continuous density/continuous osmolality (CD/CO)]. The osmolality of the low-density solution was 400 mOsm/kg in both groups and that of the high-density solution was 410 mOsm/kg in the CD/NO group and 500 mOsm/kg in the CD/CO group. Unexpectedly, we noted no significant differences between the two solutions in terms of the islet yield, rate of viability and purity, score, stimulation index, or the attainability and suitability of posttransplantation normoglycemia. Despite reports that the endocrine and exocrine tissues of pancreata have distinct osmotic sensitivities and that high-osmolality solutions result in greater purification efficiency, the isolation and transplant outcomes did not markedly differ between the two purification solutions with different osmolalities in this study.

Research paper thumbnail of The Evaluation of Islet Purification Methods That Use Large Bottles to Create a Continuous Density Gradient

Cell Medicine, 2017

Islet purification is one of the most important steps of islet isolation for pancreatic islet tra... more Islet purification is one of the most important steps of islet isolation for pancreatic islet transplantation. The most common method of islet purification is density gradient centrifugation using a COBE 2991 cell processor. However, this method can damage islets mechanically through its high shearing force. We recently reported that a new purification method using large plastic bottles effectively achieves a high yield of islets from the porcine pancreas. In the present study, we evaluated the methods of making a continuous density gradient. The gradient was produced with a gradient maker and two types of candy cane-shaped stainless steel pipes. One method was to use a &amp;amp;amp;amp;quot;bent-tipped&amp;amp;amp;amp;quot; stainless steel pipe and to load from a high-density solution to a low-density solution, uploading the stainless steel pipe. The other method was to use a regular stainless steel pipe and to load from a low-density solution to a high-density solution, leaving the stainless steel pipe in place. There were no significant differences between the two solutions in terms of the islet yield, rate of viability or purity, score, or the stimulation index after purification. Furthermore, there were no differences in the attainability or suitability of posttransplantation normoglycemia. Our study shows the equivalency of these two methods of islet purification.

Research paper thumbnail of A simplified protocol for the semi-large scale recovery of plasmids from <i>Escherichia coli</i> grown on agar plates

Journal of Biomedical Science and Engineering, 2012

Semi-large scale liquid cultivation of transformed Escherichia coli (E. coli) in medium (100 -200... more Semi-large scale liquid cultivation of transformed Escherichia coli (E. coli) in medium (100 -200 ml) has been widely used for the acquisition of relatively large amounts of plasmid DNA (50 -300 μg). However, this method requires an expensive high-speed centrifugation apparatus to precipitate E. coli before lysis, which is both laborious and time-consuming. Here, we demonstrate a method for agar plate-based cultivation of bacteria that does not employ a high-speed centrifugation apparatus. This procedure proves to be simple and reproducible, yielding an average of 82 μg of plasmid DNA per experiment. It may therefore be valuable for cloning/transfection experiments under limited financial backgrounds.

Research paper thumbnail of The piggyBac-Based Gene Delivery System Can Confer Successful Production of Cloned Porcine Blastocysts with Multigene Constructs

International Journal of Molecular Sciences, 2016

The introduction of multigene constructs into single cells is important for improving the perform... more The introduction of multigene constructs into single cells is important for improving the performance of domestic animals, as well as understanding basic biological processes. In particular, multigene constructs allow the engineering and integration of multiple genes related to xenotransplantation into the porcine genome. The piggyBac (PB) transposon system allows multiple genes to be stably integrated into target genomes through a single transfection event. However, to our knowledge, no attempt to introduce multiple genes into a porcine genome has been made using this system. In this study, we simultaneously introduced seven transposons into a single porcine embryonic fibroblast (PEF). PEFs were transfected with seven transposons containing genes for five drug resistance proteins and two (red and green) fluorescent proteins, together with a PB transposase expression vector, pTrans (experimental group). The above seven transposons (without pTrans) were transfected concomitantly (control group). Selection of these transfected cells in the presence of multiple selection drugs resulted in the survival of several clones derived from the experimental group, but not from the control. PCR analysis demonstrated that approximately 90% (12/13 tested) of the surviving clones possessed all of the introduced transposons. Splinkerette PCR demonstrated that the transposons were inserted through the TTAA target sites of PB. Somatic cell nuclear transfer (SCNT) using a PEF clone with multigene constructs demonstrated successful production of cloned blastocysts expressing both red and green fluorescence. These results indicate the feasibility of this PB-mediated method for simultaneous transfer of multigene constructs into the porcine cell genome, which is useful for production of cloned transgenic pigs expressing multiple transgenes.

Research paper thumbnail of Evaluation of serum-free, xeno-free cryopreservation solutions for human adiposederived mesenchymal stem cells

Cell Medicine, 2016

Adipose-derived mesenchymal stem cells (ASCs) have the potential to differentiate into cells of m... more Adipose-derived mesenchymal stem cells (ASCs) have the potential to differentiate into cells of mesodermal origin, such as osteoblasts, adipocytes, myocytes, and chondrocytes, and cryopreservation is currently performed as a routine method for preserving ASCs to safely acquire large numbers of cells. For clinical application of ASCs, serum-free, xeno-free cryopreservation solutions should be used. This study determined the viability and adipo-osteogenic potential of cryopreserved ASCs using four cryopreservation solutions: 10% DMSO, Cell Banker 2 (serum free), Stem Cell Banker (=Cell Banker 3: serum free, xeno free), and TC protector (serum free, xeno free). The viability of the cryopreserved ASCs was over 80% with all cryopreservation solutions. No difference in the adipo-osteogenic potential was found between the cells that did or did not undergo cryopreservation in these cryopreservation solutions. These data suggest that Cell Banker 3 and TC protector are comparable with 10% DMSO and Cell Banker 2 for ASCs, and cryopreserved as well as noncryopreserved ASCs could be applied for regenerative medicine.

Research paper thumbnail of Mosaic expression of pluripotency-related proteins oct-3/4 and alkaline phosphatase in human pancreatic carcinoma cell PANC-1

Advanced Studies in Biology, 2013

Research paper thumbnail of A reproducibility method to test lip-closing strength in preschool children

Cranio the Journal of Craniomandibular Practice, Oct 1, 2010

This cross-sectional study tested the reproducibility of a simple button-pulling system for measu... more This cross-sectional study tested the reproducibility of a simple button-pulling system for measuring lip-closing strength in normal preschool children and compared their strength to that of normal adults. The sample consisted of 348 preschool children and 123 adults. Lip-closing strength was measured by inserting a button, fastened to a piece of string, into the vestibule between the incisors and lips with minimal mouth opening. The string was attached to a digital tension gauge and was pulled parallel to the floor. Maximum tension, with three repetitions, was recorded at the instant that the button was pulled out of the mouth. Multilevel statistical models were used to evaluate any differences in contractive muscle strength between age groups and between the genders. The strength in children increased significantly from three years to five years (p<0.01). Gender-related differences were found in adults but not in preschool children. Inter-individual variation at each age was larger than intra-individual variation. Measurement of lip-closing strength by button pulling is highly reproducible in children and has potential clinical and research applications.

Research paper thumbnail of Overlap of the primary dentition in children

The Journal of Clinical Pediatric Dentistry, Feb 1, 2009

OBJECTIVE: The aim of the present study was to estimate the amount of overlap in children with th... more OBJECTIVE: The aim of the present study was to estimate the amount of overlap in children with the primary dentition.STUDY DESIGN: The sample consisted of 20 Japanese boys between 3 and 5 years of age (mean age: 4 years 10 months). Landmarks on their maxillary and mandibular dental models were digitized using a three-dimensional mechanical digitizer in a single coordinate system. Multilevel statistical models created best-fit polynomial curves to determine overbite, overjet and buccal height of all primary teeth and describe the dental arch forms.RESULTS: No significant side differences were detected. The primary canine showed the largest overbite (1.87 mm) and buccal height (9.07 mm). The primary second molar exhibited the largest overjet (2.76 mm). Buccal height was the least variable measurement.CONCLUSION: Occlusal relationships of the primary dentition were evaluated in 3-dimensions, establishing overlap variables for clinical diagnosis and treatment planning.

Research paper thumbnail of Normalization of masticatory function of a scissors-bite child with primary dentition: a case report

Cranio the Journal of Craniomandibular Practice, Apr 1, 2008

Normalization of mandibular function in children is important for normal orofacial development be... more Normalization of mandibular function in children is important for normal orofacial development because their function is not still matured. This case report examined jaw movement during chewing in a young patient with unilateral scissors-bite. He could hardly chew on the affected side, preferring to chew only on the unaffected side, and his minimum opening position was initially unstable, i.e., he had two positions before active treatment. Retention did not stabilize his minimal opening position and his dual-bite was not corrected. His minimal opening position was stabilized after equipping his upper canines with a resin cap. Although orthodontic treatment morphologically improved the patient's malocclusion, his function did not improve. Normal jaw movement on both sides was achieved after interfering with his old chewing pattern. Because normalization is needed for acquisition of normal function in children, long-term observations of their growth and functional changes are necessary after orthodontic treatment.

Research paper thumbnail of Pharyngeal airway in children with prognathism and normal occlusion

Http Dx Doi Org 10 2319 013010 65 1, Oct 11, 2010

Objective: To test the hypothesis that there is no difference in the pharyngeal airway width and ... more Objective: To test the hypothesis that there is no difference in the pharyngeal airway width and the position of the maxillofacial skeleton between prognathic and normal children. Materials and Methods: Twenty-five girls with prognathism (mean, 7.9 6 0.9 years old) and 15 girls with normal occlusion (mean, 8.4 6 1.5 years) participated in this study. On each girl's lateral cephalogram, the coordinates of all points were marked and systematically digitized using a mechanical three-dimensional digitizing system. An independent-groups t-test was used to detect significant upper and lower pharyngeal width differences between the two groups. Correlations between the horizontal positions of each point and upper and lower pharyngeal widths were examined. Results: Prognathic girls had a significantly wider lower pharyngeal airway compared with those with normal occlusion (P 5 .01). Furthermore, the horizontal coordinate of Ar was significantly positively correlated with lower pharyngeal airway width in both groups of girls. Conclusions: The hypothesis is rejected. The mandible in prognathic girls tends to be positioned more anteriorly, resulting in a wider lower pharyngeal airway. (Angle Orthod. 2011;81:75-80.)

Research paper thumbnail of Choice of Feeders Is Important When First Establishing iPSCs Derived From Primarily Cultured Human Deciduous Tooth Dental Pulp Cells

Cell Medicine, 2015

Feeder cells are generally required to maintain embryonic stem cells (ESCs)/induced pluripotent s... more Feeder cells are generally required to maintain embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs). Mouse embryonic fibroblasts (MEFs) isolated from fetuses and STO mouse stromal cell line are the most widely used feeder cells. The aim of this study was to determine which cells are suitable for establishing iPSCs from human deciduous tooth dental pulp cells (HDDPCs). Primary cultures of HDDPCs were cotransfected with three plasmids containing human OCT3/4, SOX2/KLF4, or LMYC/LIN28 and pmaxGFP by using a novel electroporation method, and then cultured in an ESC qualified medium for 15 days. Emerging colonies were reseeded onto mitomycin C-treated MEFs or STO cells. The colonies were serially passaged for up to 26 passages. During this period, colony morphology was assessed to determine whether cells exhibited ESC-like morphology and alkaline phosphatase activity to evaluate the state of cellular reprogramming. HDDPCs maintained on MEFs were successfully reprogrammed into iPSCs, whereas those maintained on STO cells were not. Once established, the iPSCs were maintained on STO cells without loss of pluripotency. Our results indicate that MEFs are better feeder cells than STO cells for establishing iPSCs. Feeder choice is a key factor enabling efficient generation of iPSCs.

Research paper thumbnail of Occlusal Contacts during Chewing in Children with Primary Dentition

ABSTRACT Objectives: Occlusal contacts are most important during the occlusal phase, when food pa... more ABSTRACT Objectives: Occlusal contacts are most important during the occlusal phase, when food particles are being pulverized. However, measument of occlusal contacts during chewning is difficult, and clear differences between children and adults have not been established. The purpose of this study was to estimate differences in occusal contacts between children and adults during chewing. Methods: Thirteen healthy children (4 to 6 years of age; mean age 5 years, 7 months) and thirteen adult females (18 to 26 years of age; mean age 20 years, 7 months) with normal occlusion participated in this study. Occlusal contact area was estimated with a measurement system combining 3-D digitization of tooth shape with 3-D tracking of mandibular movements during 1) the closing stroke, 2) at maximum closing position (MCP) and 3) during the opening stroke. Occlusal contact area at static maximum intercuspation (ICP) was also estimated. Results: At the MCP, the children&#39;s contact area was less than 82% compared to that at ICP. The timing of maximum occlusal contact area in children was shifted more towards the opening stroke compared with adults, and the occlusal contact area remained larger during opening in children than adults. The occurrence of the MCP was less stable in children than in adults, both between subjecst and within subjects. Conclusion: We conclude that both the amount of occusal contact area and the pattern of occlusal contact changes during the occlusal phase of chewing differ between children and adults.

Research paper thumbnail of Relationship between nasal and skeletal landmarks on lateral cephalograms of adults

Http Dx Doi Org 10 1080 00450618 2013 877079, Jul 1, 2014

ABSTRACT The purpose of this study was to investigate the relationship between cephalometric nasa... more ABSTRACT The purpose of this study was to investigate the relationship between cephalometric nasal soft tissue and skeletal landmarks in adults. Lateral cephalograms from Japanese adults (30 men: mean age, 24.5 ± 4.9 years; 30 women: mean age, 20.3 ± 3.3 years; overall mean age, 22.4 ± 2.4 years) were used in this study. Twenty-two skeletal points and three soft tissue nasal points were marked on each subject’s lateral cephalogram, and the coordinates of all the points were systematically digitised and transformed to a standardised plane. A forward stepwise regression analysis determined how combinations of the skeletal landmarks predict the location of the nasal soft tissue landmarks. Based on the result of our research, the location of the nasal soft tissue cephalometric landmarks in our adult subjects may be predicted based on skeletal landmarks, and different skeletal landmarks predicted the position of each soft tissue landmark in the adult males and females in this study.

Research paper thumbnail of Alkaline phosphatase and OCT-3/4 as useful markers for predicting susceptibility of human deciduous teeth-derived dental pulp cells to reprogramming factor-induced iPS cells

Journal of investigative and clinical dentistry, Jan 18, 2016

The aim of the present study was to prove that primary cells enriched with stem cells are more ea... more The aim of the present study was to prove that primary cells enriched with stem cells are more easily reprogrammed to generate induced pluripotent stem (iPS) cells than those with scarce numbers of stem cells. We surveyed the alkaline phosphatase (ALP) activity in five primarily-isolated human deciduous teeth-derived dental pulp cells (HDDPC) with cytochemical staining to examine the possible presence of stem cells. Next, the expression of stemness-specific factors, such as OCT(Octumer-binding transcription factor)3/4, NANOG, SOX2(SRY (sex determining region Y)-box 2), CD90, muscle segment homeodomain homeobox (MSX) 1, and MSX2, was assessed with a reverse transcription polymerase chain reaction method. Finally, these isolated HDDPC were transfected with plasmids carrying genes coding Yamanaka factors to determine whether these cells could be reprogrammed to generate iPS cells. Of the five primarily-isolated HDDPC, two (HDDPC-1 and -5) exhibited higher degrees of ALP activity. OCT-3...

Research paper thumbnail of Efficient CRISPR/Cas9-based gene correction in induced pluripotent stem cells established from fibroblasts of patients with sickle cell disease

Stem cell investigation, 2016

Research paper thumbnail of Relationships among nasal resistance, adenoids, tonsils, and tongue posture and maxillofacial form in Class II and Class III children

American journal of orthodontics and dentofacial orthopedics : official publication of the American Association of Orthodontists, its constituent societies, and the American Board of Orthodontics, 2017

The purpose of this study was to clarify the relationships between upper airway factors (nasal re... more The purpose of this study was to clarify the relationships between upper airway factors (nasal resistance, adenoids, tonsils, and tongue posture) and maxillofacial forms in Class II and III children. Sixty-four subjects (mean age, 9.3 years) with malocclusion were divided into Class II and Class III groups by ANB angles. Nasal resistance was calculated using computational fluid dynamics from cone-beam computed tomography data. Adenoids, tonsils, and tongue posture were evaluated in the cone-beam computed tomography images. The groups were compared using Mann-Whitney U tests and Student t tests. The Spearman rank correlations test assessed the relationships between the upper airway factors and maxillofacial form. Nasal resistance of the Class II group was significantly larger than that of the Class III group (P = 0.005). Nasal resistance of the Class II group was significantly correlated with inferior tongue posture (P <0.001) and negatively correlated with intermolar width (P = 0...

Research paper thumbnail of RCAN-11R peptide provides immunosuppression for fully mismatched islet allografts in mice

Scientific reports, Jan 8, 2017

Calcineurin inhibitors have been used for transplant therapy. However, the inhibition of calcineu... more Calcineurin inhibitors have been used for transplant therapy. However, the inhibition of calcineurin outside the immune system has a number of side effects. We previously developed a cell-permeable inhibitor of NFAT (nuclear factor of activated T cells) using the polyarginine peptide delivery system. This peptide (11R-VIVIT) selectively interferes with calcineurin-NFAT interaction without affecting the activity of calcineurin phosphatase and provides immunosuppression for fully mismatched islet allografts in mice. However, our recent study showed that 11R-VIVIT affected cell viability in vitro when it was used at higher concentration because of the VIVIT sequence. The aim of this study is to develop a safer NFAT inhibitor (RCAN-11R) that does not affect cell viability, and which is less toxic than calcineurin inhibitors. The minimal sequence of the protein family of regulators of calcineurin (RCAN) that is responsible for the inhibition of calcineurin-NFAT signaling was recently cha...

Research paper thumbnail of Isolation and characterization of lymphoid enhancer factor-1-positive deciduous dental pulp stem-like cells after transfection with a piggyBac vector containing LEF1 promoter-driven selection markers

Archives of oral biology, Jan 7, 2017

Lymphoid enhancer-binding factor-1 (LEF1) is a 48-kD nuclear protein that is expressed in pre-B a... more Lymphoid enhancer-binding factor-1 (LEF1) is a 48-kD nuclear protein that is expressed in pre-B and T cells. LEF1 is also an important member of the Wnt/β-catenin signaling pathway that plays important roles in the self-renewal and differentiation of embryonic stem cells. We speculated that LEF1 might function in the stem cells from human exfoliated deciduous teeth (SHED). In this study, we attempted to isolate such LEF1-positive cells from human deciduous dental pulp cells (HDDPCs) by genetic engineering technology, using the human LEF1 promoter. A piggyBac transposon plasmid (pTA-LEN) was introduced into HDDPCs, using the Neon(®) transfection system. After G418 selection, the emerging colonies were assessed for EGFP-derived fluorescence by fluorescence microscopy. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed using RNA isolated from these colonies to examine stem cell-specific transcript expression. Osteoblastic or neuronal differentiation was ind...

Research paper thumbnail of Masticatory movement during occlusal phase in children with primary dentition and women

The Journal of Japanese Society of Stomatognathic Function, 2002

Research paper thumbnail of Tissue-Specific Stem Cells Obtained by Reprogramming of Non-Obese Diabetic (NOD) Mouse-Derived Pancreatic Cells Confer Insulin Production in Response to Glucose

PloS one

Type 1 diabetes occurs due to the autoimmune destruction of pancreatic β-cells in islets. Transpl... more Type 1 diabetes occurs due to the autoimmune destruction of pancreatic β-cells in islets. Transplantation of islets is a promising option for the treatment of patients with type 1 diabetes that experience hypoglycemic unawareness despite maximal care, but the present shortage of donor islets hampers such transplantation. Transplantation of insulin-producing cells derived from the patients themselves would be one of the most promising approaches to cure type 1 diabetes. Previously, we demonstrated that insulin-producing cells could be produced by transfecting murine pancreatic cells with Yamanaka's reprogramming factors. Non-obese diabetic (NOD) mice are naturally occurring mutant mice defective in insulin production due to autoimmune ablation of pancreatic β-cells. In this study, we showed that glucose-sensitive insulin-producing cells are successfully generated by transfecting primary pancreatic cells from NOD mice (aged 6 months old) with a plasmid harboring the cDNAs for Oct-...

Research paper thumbnail of Comparison of Purification Solutions With Different Osmolality for Porcine Islet Purification

Cell Medicine, 2017

The osmolality of the purification solution is one of the most critical variables in human islet ... more The osmolality of the purification solution is one of the most critical variables in human islet purification during islet isolation. We previously reported the effectiveness of a combined continuous density/osmolality gradient for the supplemental purification of human islets. We herein applied a combined continuous density/osmolality gradient for regular purification. The islets were purified with a continuous density gradient without osmolality preparation [continuous density/normal osmolality (CD/NO)] or continuous density/osmolality solution with osmolality preparation by 10× Hank&amp;amp;amp;amp;#39;s balanced salt solution (HBSS) [continuous density/continuous osmolality (CD/CO)]. The osmolality of the low-density solution was 400 mOsm/kg in both groups and that of the high-density solution was 410 mOsm/kg in the CD/NO group and 500 mOsm/kg in the CD/CO group. Unexpectedly, we noted no significant differences between the two solutions in terms of the islet yield, rate of viability and purity, score, stimulation index, or the attainability and suitability of posttransplantation normoglycemia. Despite reports that the endocrine and exocrine tissues of pancreata have distinct osmotic sensitivities and that high-osmolality solutions result in greater purification efficiency, the isolation and transplant outcomes did not markedly differ between the two purification solutions with different osmolalities in this study.

Research paper thumbnail of The Evaluation of Islet Purification Methods That Use Large Bottles to Create a Continuous Density Gradient

Cell Medicine, 2017

Islet purification is one of the most important steps of islet isolation for pancreatic islet tra... more Islet purification is one of the most important steps of islet isolation for pancreatic islet transplantation. The most common method of islet purification is density gradient centrifugation using a COBE 2991 cell processor. However, this method can damage islets mechanically through its high shearing force. We recently reported that a new purification method using large plastic bottles effectively achieves a high yield of islets from the porcine pancreas. In the present study, we evaluated the methods of making a continuous density gradient. The gradient was produced with a gradient maker and two types of candy cane-shaped stainless steel pipes. One method was to use a &amp;amp;amp;amp;quot;bent-tipped&amp;amp;amp;amp;quot; stainless steel pipe and to load from a high-density solution to a low-density solution, uploading the stainless steel pipe. The other method was to use a regular stainless steel pipe and to load from a low-density solution to a high-density solution, leaving the stainless steel pipe in place. There were no significant differences between the two solutions in terms of the islet yield, rate of viability or purity, score, or the stimulation index after purification. Furthermore, there were no differences in the attainability or suitability of posttransplantation normoglycemia. Our study shows the equivalency of these two methods of islet purification.

Research paper thumbnail of A simplified protocol for the semi-large scale recovery of plasmids from <i>Escherichia coli</i> grown on agar plates

Journal of Biomedical Science and Engineering, 2012

Semi-large scale liquid cultivation of transformed Escherichia coli (E. coli) in medium (100 -200... more Semi-large scale liquid cultivation of transformed Escherichia coli (E. coli) in medium (100 -200 ml) has been widely used for the acquisition of relatively large amounts of plasmid DNA (50 -300 μg). However, this method requires an expensive high-speed centrifugation apparatus to precipitate E. coli before lysis, which is both laborious and time-consuming. Here, we demonstrate a method for agar plate-based cultivation of bacteria that does not employ a high-speed centrifugation apparatus. This procedure proves to be simple and reproducible, yielding an average of 82 μg of plasmid DNA per experiment. It may therefore be valuable for cloning/transfection experiments under limited financial backgrounds.

Research paper thumbnail of The piggyBac-Based Gene Delivery System Can Confer Successful Production of Cloned Porcine Blastocysts with Multigene Constructs

International Journal of Molecular Sciences, 2016

The introduction of multigene constructs into single cells is important for improving the perform... more The introduction of multigene constructs into single cells is important for improving the performance of domestic animals, as well as understanding basic biological processes. In particular, multigene constructs allow the engineering and integration of multiple genes related to xenotransplantation into the porcine genome. The piggyBac (PB) transposon system allows multiple genes to be stably integrated into target genomes through a single transfection event. However, to our knowledge, no attempt to introduce multiple genes into a porcine genome has been made using this system. In this study, we simultaneously introduced seven transposons into a single porcine embryonic fibroblast (PEF). PEFs were transfected with seven transposons containing genes for five drug resistance proteins and two (red and green) fluorescent proteins, together with a PB transposase expression vector, pTrans (experimental group). The above seven transposons (without pTrans) were transfected concomitantly (control group). Selection of these transfected cells in the presence of multiple selection drugs resulted in the survival of several clones derived from the experimental group, but not from the control. PCR analysis demonstrated that approximately 90% (12/13 tested) of the surviving clones possessed all of the introduced transposons. Splinkerette PCR demonstrated that the transposons were inserted through the TTAA target sites of PB. Somatic cell nuclear transfer (SCNT) using a PEF clone with multigene constructs demonstrated successful production of cloned blastocysts expressing both red and green fluorescence. These results indicate the feasibility of this PB-mediated method for simultaneous transfer of multigene constructs into the porcine cell genome, which is useful for production of cloned transgenic pigs expressing multiple transgenes.

Research paper thumbnail of Evaluation of serum-free, xeno-free cryopreservation solutions for human adiposederived mesenchymal stem cells

Cell Medicine, 2016

Adipose-derived mesenchymal stem cells (ASCs) have the potential to differentiate into cells of m... more Adipose-derived mesenchymal stem cells (ASCs) have the potential to differentiate into cells of mesodermal origin, such as osteoblasts, adipocytes, myocytes, and chondrocytes, and cryopreservation is currently performed as a routine method for preserving ASCs to safely acquire large numbers of cells. For clinical application of ASCs, serum-free, xeno-free cryopreservation solutions should be used. This study determined the viability and adipo-osteogenic potential of cryopreserved ASCs using four cryopreservation solutions: 10% DMSO, Cell Banker 2 (serum free), Stem Cell Banker (=Cell Banker 3: serum free, xeno free), and TC protector (serum free, xeno free). The viability of the cryopreserved ASCs was over 80% with all cryopreservation solutions. No difference in the adipo-osteogenic potential was found between the cells that did or did not undergo cryopreservation in these cryopreservation solutions. These data suggest that Cell Banker 3 and TC protector are comparable with 10% DMSO and Cell Banker 2 for ASCs, and cryopreserved as well as noncryopreserved ASCs could be applied for regenerative medicine.

Research paper thumbnail of Mosaic expression of pluripotency-related proteins oct-3/4 and alkaline phosphatase in human pancreatic carcinoma cell PANC-1

Advanced Studies in Biology, 2013

Research paper thumbnail of A reproducibility method to test lip-closing strength in preschool children

Cranio the Journal of Craniomandibular Practice, Oct 1, 2010

This cross-sectional study tested the reproducibility of a simple button-pulling system for measu... more This cross-sectional study tested the reproducibility of a simple button-pulling system for measuring lip-closing strength in normal preschool children and compared their strength to that of normal adults. The sample consisted of 348 preschool children and 123 adults. Lip-closing strength was measured by inserting a button, fastened to a piece of string, into the vestibule between the incisors and lips with minimal mouth opening. The string was attached to a digital tension gauge and was pulled parallel to the floor. Maximum tension, with three repetitions, was recorded at the instant that the button was pulled out of the mouth. Multilevel statistical models were used to evaluate any differences in contractive muscle strength between age groups and between the genders. The strength in children increased significantly from three years to five years (p<0.01). Gender-related differences were found in adults but not in preschool children. Inter-individual variation at each age was larger than intra-individual variation. Measurement of lip-closing strength by button pulling is highly reproducible in children and has potential clinical and research applications.

Research paper thumbnail of Overlap of the primary dentition in children

The Journal of Clinical Pediatric Dentistry, Feb 1, 2009

OBJECTIVE: The aim of the present study was to estimate the amount of overlap in children with th... more OBJECTIVE: The aim of the present study was to estimate the amount of overlap in children with the primary dentition.STUDY DESIGN: The sample consisted of 20 Japanese boys between 3 and 5 years of age (mean age: 4 years 10 months). Landmarks on their maxillary and mandibular dental models were digitized using a three-dimensional mechanical digitizer in a single coordinate system. Multilevel statistical models created best-fit polynomial curves to determine overbite, overjet and buccal height of all primary teeth and describe the dental arch forms.RESULTS: No significant side differences were detected. The primary canine showed the largest overbite (1.87 mm) and buccal height (9.07 mm). The primary second molar exhibited the largest overjet (2.76 mm). Buccal height was the least variable measurement.CONCLUSION: Occlusal relationships of the primary dentition were evaluated in 3-dimensions, establishing overlap variables for clinical diagnosis and treatment planning.

Research paper thumbnail of Normalization of masticatory function of a scissors-bite child with primary dentition: a case report

Cranio the Journal of Craniomandibular Practice, Apr 1, 2008

Normalization of mandibular function in children is important for normal orofacial development be... more Normalization of mandibular function in children is important for normal orofacial development because their function is not still matured. This case report examined jaw movement during chewing in a young patient with unilateral scissors-bite. He could hardly chew on the affected side, preferring to chew only on the unaffected side, and his minimum opening position was initially unstable, i.e., he had two positions before active treatment. Retention did not stabilize his minimal opening position and his dual-bite was not corrected. His minimal opening position was stabilized after equipping his upper canines with a resin cap. Although orthodontic treatment morphologically improved the patient's malocclusion, his function did not improve. Normal jaw movement on both sides was achieved after interfering with his old chewing pattern. Because normalization is needed for acquisition of normal function in children, long-term observations of their growth and functional changes are necessary after orthodontic treatment.

Research paper thumbnail of Pharyngeal airway in children with prognathism and normal occlusion

Http Dx Doi Org 10 2319 013010 65 1, Oct 11, 2010

Objective: To test the hypothesis that there is no difference in the pharyngeal airway width and ... more Objective: To test the hypothesis that there is no difference in the pharyngeal airway width and the position of the maxillofacial skeleton between prognathic and normal children. Materials and Methods: Twenty-five girls with prognathism (mean, 7.9 6 0.9 years old) and 15 girls with normal occlusion (mean, 8.4 6 1.5 years) participated in this study. On each girl's lateral cephalogram, the coordinates of all points were marked and systematically digitized using a mechanical three-dimensional digitizing system. An independent-groups t-test was used to detect significant upper and lower pharyngeal width differences between the two groups. Correlations between the horizontal positions of each point and upper and lower pharyngeal widths were examined. Results: Prognathic girls had a significantly wider lower pharyngeal airway compared with those with normal occlusion (P 5 .01). Furthermore, the horizontal coordinate of Ar was significantly positively correlated with lower pharyngeal airway width in both groups of girls. Conclusions: The hypothesis is rejected. The mandible in prognathic girls tends to be positioned more anteriorly, resulting in a wider lower pharyngeal airway. (Angle Orthod. 2011;81:75-80.)

Research paper thumbnail of Choice of Feeders Is Important When First Establishing iPSCs Derived From Primarily Cultured Human Deciduous Tooth Dental Pulp Cells

Cell Medicine, 2015

Feeder cells are generally required to maintain embryonic stem cells (ESCs)/induced pluripotent s... more Feeder cells are generally required to maintain embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs). Mouse embryonic fibroblasts (MEFs) isolated from fetuses and STO mouse stromal cell line are the most widely used feeder cells. The aim of this study was to determine which cells are suitable for establishing iPSCs from human deciduous tooth dental pulp cells (HDDPCs). Primary cultures of HDDPCs were cotransfected with three plasmids containing human OCT3/4, SOX2/KLF4, or LMYC/LIN28 and pmaxGFP by using a novel electroporation method, and then cultured in an ESC qualified medium for 15 days. Emerging colonies were reseeded onto mitomycin C-treated MEFs or STO cells. The colonies were serially passaged for up to 26 passages. During this period, colony morphology was assessed to determine whether cells exhibited ESC-like morphology and alkaline phosphatase activity to evaluate the state of cellular reprogramming. HDDPCs maintained on MEFs were successfully reprogrammed into iPSCs, whereas those maintained on STO cells were not. Once established, the iPSCs were maintained on STO cells without loss of pluripotency. Our results indicate that MEFs are better feeder cells than STO cells for establishing iPSCs. Feeder choice is a key factor enabling efficient generation of iPSCs.

Research paper thumbnail of Occlusal Contacts during Chewing in Children with Primary Dentition

ABSTRACT Objectives: Occlusal contacts are most important during the occlusal phase, when food pa... more ABSTRACT Objectives: Occlusal contacts are most important during the occlusal phase, when food particles are being pulverized. However, measument of occlusal contacts during chewning is difficult, and clear differences between children and adults have not been established. The purpose of this study was to estimate differences in occusal contacts between children and adults during chewing. Methods: Thirteen healthy children (4 to 6 years of age; mean age 5 years, 7 months) and thirteen adult females (18 to 26 years of age; mean age 20 years, 7 months) with normal occlusion participated in this study. Occlusal contact area was estimated with a measurement system combining 3-D digitization of tooth shape with 3-D tracking of mandibular movements during 1) the closing stroke, 2) at maximum closing position (MCP) and 3) during the opening stroke. Occlusal contact area at static maximum intercuspation (ICP) was also estimated. Results: At the MCP, the children&#39;s contact area was less than 82% compared to that at ICP. The timing of maximum occlusal contact area in children was shifted more towards the opening stroke compared with adults, and the occlusal contact area remained larger during opening in children than adults. The occurrence of the MCP was less stable in children than in adults, both between subjecst and within subjects. Conclusion: We conclude that both the amount of occusal contact area and the pattern of occlusal contact changes during the occlusal phase of chewing differ between children and adults.

Research paper thumbnail of Relationship between nasal and skeletal landmarks on lateral cephalograms of adults

Http Dx Doi Org 10 1080 00450618 2013 877079, Jul 1, 2014

ABSTRACT The purpose of this study was to investigate the relationship between cephalometric nasa... more ABSTRACT The purpose of this study was to investigate the relationship between cephalometric nasal soft tissue and skeletal landmarks in adults. Lateral cephalograms from Japanese adults (30 men: mean age, 24.5 ± 4.9 years; 30 women: mean age, 20.3 ± 3.3 years; overall mean age, 22.4 ± 2.4 years) were used in this study. Twenty-two skeletal points and three soft tissue nasal points were marked on each subject’s lateral cephalogram, and the coordinates of all the points were systematically digitised and transformed to a standardised plane. A forward stepwise regression analysis determined how combinations of the skeletal landmarks predict the location of the nasal soft tissue landmarks. Based on the result of our research, the location of the nasal soft tissue cephalometric landmarks in our adult subjects may be predicted based on skeletal landmarks, and different skeletal landmarks predicted the position of each soft tissue landmark in the adult males and females in this study.