Characterization of Stem Cells in the Orofacial Region

牙科生物科由石松涛博士指导，目前包括三浦雅子博士、文武锡博士和竹尾刚雄博士。由于该单位独立仅有几个月，与S博士的密切合作是显而易见的。我们的研究重点是牙科/颅面相关干细胞的分离和鉴定，包括牙髓干细胞(DPSCs)和人类脱落乳牙(SHEAD)。 我们以前的研究发现，成人牙髓含有一种克隆形成并快速增殖的细胞群，称为牙髓干细胞(DPSCS)。然后将DPSCs与人骨髓基质干细胞(BMSSCs)进行比较，BMSSCs是成骨细胞的已知前体。尽管它们在体外具有相似的免疫表型，但功能研究表明，DPSCs只产生零星但密集的钙化结节。当DPSCs被移植到免疫低下的小鼠体内时，它们产生了一种牙本质样结构，里面排列着人成牙本质样细胞，环绕着牙髓样的间质组织。相反，BMSSCs形成包含骨细胞和表面衬里的成骨细胞的板层骨，包围着活跃的造血细胞和脂肪细胞的纤维血管组织。这项研究是首次分离出具有形成牙本质/牙髓样复合体能力的出生后DPSCs。此外，我们还对其自我更新能力、多系分化能力和克隆形成效率进行了研究。从原代DPSC移植的培养中重建基质样细胞，并再次移植到免疫低下的小鼠体内，生成牙本质牙髓样组织，显示其自我更新的能力。DPSCs也被发现能够分化为脂肪细胞和神经样细胞。对12株单个菌落来源的DPSC菌株的成牙潜能进行了测定。在单个菌落来源的DPSC菌株中，三分之二的菌株在体内产生了丰富的异位牙本质，而在其余三分之一的菌株中只检测到有限数量的牙本质。这些结果表明，单个菌落来源的DPSC株之间的牙齿形成率不同。 从可获得的资源中分离出高质量的人类出生后干细胞是干细胞研究的重要目标。我们发现人类脱落的乳牙含有多能干细胞(来自人类脱落的乳牙的干细胞，Share)。SHEEP被鉴定为一群高度增殖的克隆细胞，能够分化为各种类型的细胞，包括神经细胞、脂肪细胞和成牙本质细胞。体内移植后，SHEAD能够诱导大量的骨形成，产生牙本质，并在小鼠脑内存活，并表达神经标志物。我们的研究表明，自然剥离的人体器官包含一组与先前已鉴定的干细胞完全不同的干细胞。Share不仅来源于非常容易获得的组织资源，而且能够为潜在的临床应用提供足够的细胞。因此，剥离的牙齿可能是包括自体干细胞移植和组织工程在内的干细胞治疗的意想不到的独特来源。 最近，CSDB已经做了大量的工作来确定间充质干细胞的潜在生态位，这将是了解其功能特征的关键。我们发现DPSCs存在于牙髓的微血管系统中，它们是通过免疫选择的方式分离出来的，这种抗体可以识别牙髓组织中间质成分和血管周围细胞上的抗原。新分离的Stro-1阳性的DPSCs缺乏血管内皮细胞标志物von Willebrand因子的表达，但α-平滑肌肌动蛋白(平滑肌细胞标志物)和MUC-18(内皮细胞标志物)阳性。此外，大多数DPSCs表达周细胞标志物3G5。DPSCs表现出与不同血管周围细胞群一致的表型，这一发现可能对理解调节矿化基质和其他相关结缔组织形成的因素有进一步的意义。

The Dental Biology Unit is directed by Dr. Songtao Shi and currently includes Drs. Masako Miura, Byoung-Moo Seo and Takeo Tsutsui. Because the unit as been independent for only several months, the close collaboration with Dr. Shi!'s previous Section (Skeletal Biology Section at CSDB led by Dr. Pamela G. Robey) is obvious. Our research is focused on isolation and characterization of dental/craniofacial associated stem cells including stem cells from Dental Pulp Stem Cells (DPSCs) and Human Exfoliated Deciduous teeth (SHED). Our previous studies identified that adult human dental pulp contained a clonogenic and rapidly proliferative population of cells, named dental pulp stem cells (DPSCs). DPSCs were then compared to human bone marrow stromal stem cells (BMSSCs), known precursors of osteoblasts. Although they share a similar immunophenotype in vitro, functional studies showed that DPSCs produced only sporadic, but densely calcified nodules. When DPSCs were transplanted into immunocompromised mice, they generated a dentin-like structure lined with human odontoblast-like cells that surrounded a pulp-like interstitial tissue. In contrast, BMSSCs formed lamellar bone containing osteocytes and surface lining osteoblasts, surrounding a fibrous vascular tissue with active hematopoiesis and adipocytes. This study was the first to isolate post-natal DPSCs that have the ability to form a dentin/pulp-like complex. Furthermore, we characterized the self-renewal capability, multi-lineage differentiation capacity, and clonogenic efficiency of DSPCs. Stromal-like cells were re-established in culture from primary DPSC transplants and re-transplanted into immunocompromised mice to generate a dentin-pulp-like tissue, demonstrating their self-renewal capability. DPSCs were also found to be capable of differentiating into adipocytes and neural-like cells. The odontogenic potential of twelve individual single-colony derived DPSC strains was determined. Two thirds of the single-colony derived DPSC strains generated abundant ectopic dentin in vivo, while only a limited amount of dentin was detected in the remaining one third. These results indicate that single-colony derived DPSC strains differ from each other with respect to their rate of odontogenesis. To isolate high quality human postnatal stem cells from accessible resources is an important goal for stem cell research. We found that exfoliated human deciduous teeth contain multipotent stem cells (Stem cells from Human Exfoliated Deciduous tooth, SHED). SHED were identified to be a population of highly proliferative clonogenic cells capable of differentiating into a variety of cell types including neural cells, adipocytes, as well as odontoblasts. Upon in vivo transplantation, SHED were found to be able to induce a significant amount of bone formation, generate dentin, and survive in mouse brain along with expression of neural markers. Our studies demonstrated that a naturally exfoliated human organ contains a novel population of stem cells that are completely different from previously identified stem cells. SHED are not only derived from a very accessible tissue resource, but are also capable of providing enough cells for potential clinical application. Thus, exfoliated teeth may be an unexpected unique resource for stem cell therapies including autologous stem cell transplantation and tissue engineering. Recently, a great deal of effort has been done in CSDB to identify the potential niche of mesenchymal stem cells, which will be critical for understanding their functional characteristics. We found that DPSCs reside in the microvasculature of dental pulp and they were isolated by immunoselection using the antibody, STRO-1, that recognizes an antigen on stromal elements and perivascular cells in dental pulp tissue. Freshly isolated STRO-1 positive DPSCs were found to lack expression of von Willebrand Factor (an endothelial cell marker) but were positive for alpha-smooth muscle actin (smooth muscle cell marker) and MUC-18 (endothelial cell marker). Furthermore, the majority of DPSCs expressed the pericyte marker, 3G5. The finding that DPSCs display phenotypes consistent with different perivascular cell populations may have further implications in understanding the factors that regulate the formation of mineralized matrices and other associated connective tissues.