Anderson Hsien-Cheng Huang

Putative Dental Pulp-Derived Stem/Stromal Cells Promote Proliferation and Differentiation of Endogenous Neural Cells in the Hippocampus of Mice

Until now, interest in dental pulp stem/stromal cell (DPSC) research has centered on mineralization and tooth repair. Beginning a new paradigm in DPSC research, we grafted undifferentiated, untreated DPSCs into the hippocampus of immune‐suppressed mice. The rhesus DPSC (rDPSC) line used was established from the dental pulp of rhesus macaques and found to be similar to human bone marrow/mesenchymal stem cells, which express Nanog, Rex‐1, Oct‐4, and various cell surface antigens, and have multipotent differentiation capability. Implantation of rDPSCs into the hippocampus of mice stimulated proliferation of endogenous neural cells and resulted in the recruitment of pre‐existing Nestin+ neural progenitor cells (NPCs) and β‐tubulin‐III+ mature neurons to the site of the graft. Additionally, many cells born during the first 7 days after implantation proliferated, forming NPCs and neurons, and, to a lesser extent, underwent astrogliosis, forming astrocytes and microglia, by 30 days after implantation. Although the DPSC graft itself was short term, it had long‐term effects by promoting growth factor signaling. Implantation of DPSCs enhanced the expression of ciliary neurotrophic factor, vascular endothelial growth factor, and fibroblast growth factor for up to 30 days after implantation. In conclusion, grafting rDPSCs promotes proliferation, cell recruitment, and maturation of endogenous stem/progenitor cells by modulating the local microenvironment. Our results suggest that DPSCs have a valuable, unique therapeutic potential, specifically as a stimulator and modulator of the local repair response in the central nervous system. DPSCs would be a preferable cell source for therapy due to the possibility of a “personalized” stem cell, avoiding the problems associated with host immune rejection.

Isolation and characterization of dental pulp stem cells from a supernumerary tooth

BACKGROUND Dental pulp stem cells (DPSCs) were primarily derived from the pulp tissues of primary incisors and permanent third molar teeth, whereas no report to our knowledge has yet been documented on deriving DPSCs from the other tooth types. The aim of this study is to present a novel approach of harvesting stem cells from a supernumerary tooth (a mesiodens). MATERIALS AND METHODS The pulp tissues from a mesiodens of a 20-year-old healthy male patient and the left lower deciduous canine of a healthy 10-year-old boy (the positive control) were extracted and cultured for DPSCs, which were examined with stem cells (Oct-4, Nanog and Rex-1) and differentiation (Osteonectin and Nestin) markers. Furthermore, DPSCs were directionally differentiated to osteogenic and adipogenic cell lineages. RESULTS Dental pulp stem cells derived from the mesiodens were capable of differentiating into adipogenic and osteogenic lineages. The mesiodens DPSCs also expressed stem cell and differentiation markers, which suggested their stem cell origin and differentiation capability. All the aforementioned results for the mesiodens were consistent with those of the DPSCs derived from the positive control. CONCLUSION We have demonstrated the feasibility of deriving DPSCs from a usually discarded tissue such as a supernumerary tooth.

Isolation and characterization of human dental pulp stem/stromal cells from nonextracted crown-fractured teeth requiring root canal therapy.

INTRODUCTION Human dental pulp stem/stromal cells (hDPSCs) in adults are primarily derived from the pulp tissues of permanent third molar teeth in existing literatures, whereas no reports exist, to our knowledge, on deriving hDPSCs from a tooth without the need for surgical procedure. The aim of this study was to raise a novel idea to source hDPSCs from complicated crown-fractured teeth requiring root canal therapy. METHODS hDPSCs were harvested from the pulp tissues for two complicated crown-fractured teeth requiring root canal therapy, retaining the teeth for subsequent prosthodontic rehabilitation, in a 41-year-old woman who had suffered a motorcycle accident. Pulp tissue from the left lower deciduous canine of a healthy 10-year-old boy (the positive control) was also removed because of high mobility and cultured for hDPSCs. RESULTS The hDPSCs derived from the two complicated crown-fractured teeth and the deciduous tooth were able to differentiate into adipogenic, chondrogenic, and osteogenic lineages and also expressed stem cells markers and differentiation markers, which indicated their stem cell origin and differentiation capability. In addition, hDPSCs from both the complicated crown-fractured teeth and the deciduous tooth showed high expression for bone marrow stem cell markers including CD29, CD90, and CD105 and exhibited very low expression of markers specific for hematopoietic cells such as CD14, CD34, and CD45. CONCLUSIONS This report describes the successful isolation and characterization of hDPSCs from the pulp tissue of complicated crown-fractured teeth without tooth extraction. Therefore, pulp exposed in complicated crown-fractured teeth might represent a valuable source of personal hDPSCs.

Postnatal stem/progenitor cells derived from the dental pulp of adult chimpanzee

BackgroundBackgroundChimpanzee dental pulp stem/stromal cells (ChDPSCs) are very similar to human bone marrow derived mesenchymal stem/stromal cells (hBMSCs) as demonstrated by the expression pattern of cell surface markers and their multipotent differentiation capability.ResultsChDPSCs were isolated from an incisor and a canine of a forty-seven year old female chimpanzee. A homogenous population of ChDPSCs was established in early culture at a high proliferation rate and verified by the expression pattern of thirteen cell surface markers. The ChDPSCs are multipotent and were capable of differentiating into osteogenic, adipogenic and chondrogenic lineages under appropriate in vitro culture conditions. ChDPSCs also express stem cell (Sox-2, Nanog, Rex-1, Oct-4) and osteogenic (Osteonectin, osteocalcin, osteopontin) markers, which is comparable to reported results of rhesus monkey BMSCs (rBMSCs), hBMSCs and hDPSCs. Although ChDPSCs vigorously proliferated during the initial phase and gradually decreased in subsequent passages, the telomere length indicated that telomerase activity was not significantly reduced.ConclusionThese results demonstrate that ChDPSCs can be efficiently isolated from post-mortem teeth of adult chimpanzees and are multipotent. Due to the almost identical genome composition of humans and chimpanzees, there is an emergent need for defining the new role of chimpanzee modeling in comparative medicine. Teeth are easy to recover at necropsy and easy to preserve prior to the retrieval of dental pulp for stem/stromal cells isolation. Therefore, the establishment of ChDPSCs would preserve and maximize the applications of such a unique and invaluable animal model, and could advance the understanding of cellular functions and differentiation control of adult stem cells in higher primates.

Human dental pulp stem cells derived from different cryopreservation methods of human dental pulp tissues of diseased teeth

BACKGROUND Successful isolation of human dental pulp stem cells (hDPSCs) has been documented at least 120h after tooth extraction. Viable hDPSCs have been isolated chiefly from cryopreserved healthy molar teeth and their undigested dental pulp tissue. Isolation of hDPSCs from diseased but vital teeth after cryopreservation has not been reported. This study aimed to isolate hDPSCs from cryopreserved diseased but vital teeth of various tooth types. MATERIALS Fifty tooth samples were divided into group A (n = 20) - freshly derived dental pulp tissues, group B (n = 20) - liquid nitrogen (liq N(2) )-stored dental pulp tissues and group C (n = 10) - liq N(2) -stored intact teeth. METHODS AND RESULTS The success rate for hDPSCs isolation was 100% for groups A and B and only 20% for group C. hDPSCs from all groups demonstrated self-renewal properties and similar multipotent potential characteristics of adipogenic, chondrogenic and osteogenic differentiation. In addition, hDPSCs showed high expression of bone-marrow mesenchymal stem-cell markers (CD29, CD90 and CD105) and very low expression of specific hematopoietic cells markers (CD14, CD34 and CD45). CONCLUSION Our results indicate that hDPSCs isolated from diseased but vital teeth of various tooth types can be stored in liq N(2) for future usage.

Overexpression of Smad proteins, especially Smad7, in oral epithelial dysplasias

ObjectiveTransforming growth factor β, via membrane-bound receptors and downstream Smad2–4, 7, can modulate tumorigenesis. Smad2 and Smad3 heterodimerize with Smad4, and the complex migrates to the nucleus to regulate the expression of target genes. Smad7 is a key negative regulator of this signaling pathway. This study aimed to examine Smad2–4, 7 expression and phosphorylated Smad2–3 (p-Smad2–3) in oral epithelial dysplasia and compared it with normal oral mucosa, hyperkeratosis/epithelial hyperplasia and squamous cell carcinoma (SCC).Materials and methodsImmunohistochemical staining of Smad2–4, 7 and p-Smad2–3, was performed for 75 samples of human oral mucosa, including hyperkeratosis/epithelial hyperplasia (n = 20), mild epithelial dysplasia (n = 11), moderate to severe epithelial dysplasia (n = 11), and SCC (n = 43). Normal buccal mucosa samples (n = 9) were also included.ResultsA significant increase in Smad7 expression was observed in the ascending order of samples of normal oral mucosa, hyperkeratosis/epithelial hyperplasia/mild oral epithelial dysplasia, moderate to severe oral epithelial dysplasia, and well-differentiated oral SCC/moderately to poorly differentiated oral SCC. Additionally, significant increases in Smad7 expression were noted as compared with expression of Smad2–4 and p-Smad2–3 in lesions of hyperkeratosis/epithelial hyperplasia, mild oral epithelial dysplasia, moderate to severe oral epithelial dysplasia, well-differentiated oral SCC, and moderately to poorly differentiated oral SCC.ConclusionsOur results indicate that Smad proteins, particularly Smad7, in oral epithelial dysplasia and SCC could contribute to the attenuation of Smads anti-proliferative signaling in cancer development.Clinical relevanceSmad7 could be a marker for risk of malignant transformation of oral epithelial dysplasia.

Reversal of Cellular Phenotypes in Neural Cells Derived from Huntington's Disease Monkey-Induced Pluripotent Stem Cells

Summary Huntington’s disease (HD) is a dominant neurodegenerative disorder caused by the expansion of glutamine residues in the N-terminal region of the huntingtin (HTT) protein. The disease results in progressive neuronal loss, leading to motor, cognitive, and psychiatric impairment. Here, we report the establishment of neural progenitor cell (NPC) lines derived from induced pluripotent stem cells (iPSCs) of transgenic HD monkeys. Upon differentiation to neurons, HD neural cells develop cellular features of HD, including the formation of nuclear inclusions and oligomeric mutant HTT (mHTT) aggregates, as well as increased apoptosis. These phenotypes are rescued by genetic suppression of HTT and pharmacological treatment, demonstrating the ability of our HD cell model to respond to therapeutic treatment. The development and reversal of HD-associated phenotypes in neural cells from HD monkeys provides a unique nonhuman primate (NHP) model for exploring HD pathogenesis and evaluating therapeutics that could be assessed further in HD monkeys.

Aberrant expression in multiple components of the transforming growth factor-β1-induced Smad signaling pathway during 7,12-dimethylbenz[a]anthracene-induced hamster buccal-pouch squamous-cell carcinogenesis.

UNLABELLED Transforming growth factor (TGF)-β1 signaling controls a plethora of cellular processes including tumorigenesis. The TGF-β1 ligand initiates signaling by binding to TGF-βreceptor II (TβRII) and allowing heterodimerization with TGF-βreceptor I (TβRI); thus, TβRI is phosphorylated by TβRII. After phosphorylation, Smad2 and Smad3 heterodimerize with Smad4, and this complex migrates to the nucleus to regulate the expression of specific target genes. However, Smad7 interrupts above signal transduction by preventing phosphorylation of Smad2 or Smad3. The objective of this study was to examine the TGF-β1-induced Smad signaling pathway during 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal-pouch squamous-cell carcinogenesis. Fifty 6-week-old male Syrian golden hamsters were divided into three experimental and two control groups (10 animals in each). Both pouches of each animal in the experimental groups were painted with 0.5% DMBA solution, and both pouches of each animal of one of the control groups were similarly treated with mineral oil; the other control group remained untreated throughout the experiment. Animals from three experimental groups were sacrificed at the end of 3rd, 9th, and 14th-weeks after DMBA treatment, respectively, and animals from two control groups were all sacrificed at 14th-weeks after the treatment. Immunohistochemical staining for TGF-β1, TβRI, TβRII, Smad2-4 and Smad7 were performed. RESULTS A significant increase in the expression of Smad7 and significant decreases in the expression of TβRII, Smad 2, Smad3 and Smad4 were noted during hamster buccal-pouch carcinogenesis induced by DMBA. Our findings indicate that a disruption in TGF-β1-induced Smad signaling occurs as a result of aberrant expression of multiple components in the TGF-β1 signaling pathway during DMBA-induced hamster buccal-pouch carcinogenesis, leading to loss of TGF-β1 growth-suppressive effects on transformed pouch keratinocytes.

VX2-induced rabbit buccal carcinoma: A potential cancer model for human buccal mucosa squamous cell carcinoma

The buccal mucosa is the site at highest risk of contracting malignancy in habitual betel-quid chewers who expose the buccal mucosa to high doses of carcinogens. Of all oral squamous cell carcinomas (SCCs), those of the buccal mucosa are most associated with the poorest prognoses. Therefore, an animal model would be helpful to evaluate new treatment modalities for buccal SCC. We evaluated whether the VX2 rabbit cancer model could be employed as a cancer model for human buccal SCC. Ten adult male, New Zealand White outbred rabbits were randomly divided into two groups A (n=2) and B (n=8). A 0.5 ml VX2 tumor cell suspension containing approximately 40 x 10(6) vital cells was injected intramuscularly into the right hind paw of the two rabbits of group A. Four weeks later, moderately to poorly differentiated hind paw SCCs were apparent in both rabbits of group A. No abdominal organ metastases, but multiple pulmonary metastases, were found in both animals. Fresh solid tumor pieces (about 5 x 5 mm) obtained from group A animals were subsequently inserted into the surgically created spaces of the left cheeks of the eight rabbits of group B. Ulcerated buccal tumors (moderately to poorly differentiated SCCs) were found in all eight animals 6 weeks later. No internal organ metastases were noted in any of the eight rabbits, but a total of 11 with an average of 2.75 cervical lymph node metastases were found in four of the eight animals. Mandibular bone and tooth pulp invasion by cancer cells was also noted in one animal. In conclusion, our findings indicated that VX2-induced rabbit buccal carcinomas could be a potential cancer model for human buccal mucosa squamous cell carcinoma.

Human dental pulp stem cells derived from cryopreserved dental pulp tissues of vital extracted teeth with disease demonstrate hepatic-like differentiation.

Reviewing the literature, hepatic differentiation of human dental pulp stem cells (hDPSCs) from cryopreserved dental pulp tissues of vital extracted teeth with disease has not been studied. This study is aimed to evaluate the hypothesis that hDPSCs from cryopreserved dental pulp tissues of vital extracted teeth with disease could possess potential hepatic differentiation. Forty vital extracted teeth with disease recruited for hDPSCs isolation, stem cell characterization and hepatic differentiation were randomly and equally divided into group A (liquid nitrogen‐stored dental pulp tissues) and group B (freshly derived dental pulp tissues). Samples of hDPSCs isolated from groups A and B but without hepatic growth factors formed negative controls. A well‐differentiated hepatocellular carcinoma cell line was employed as a positive control. All the isolated hDPSCs from groups A and B showed hepatic‐like differentiation with morphological change from a spindle‐shaped to a polygonal shape and normal karyotype. Differentiated hDPSCs and the positive control expressed hepatic metabolic function genes and liver‐specific genes. Glycogen storage of differentiated hDPSCs was noted from day 7 of differentiation‐medium culture. Positive immunofluorescence staining of low‐density lipoprotein and albumin was observed from day 14 of differentiation‐medium culture; urea production in the medium was noted from week 6. No hepatic differentiation was observed for any of the samples of the negative controls. We not only demonstrated the feasibility of hepatic‐like differentiation of hDPSCs from cryopreserved dental pulp tissues of vital extracted teeth with disease but also indicated that the differentiated cells possessed normal karyotype and were functionally close to normal hepatic‐like cells. Copyright