幹細胞(stem cells)為一群具有不斷自我更新的能力(self-renewal)以及分化(differentiation)成各類下游成熟組織的細胞。2006年日本學者山中伸彌的團隊發現，將4個特定基因(cMyc, Klf4, Oct3/4, Sox-2)導入至老鼠纖維母細胞中，能使細胞重新獲得分化能力，並將之命名為誘導型多功能幹細胞(induced pluripotent stem cell, iPSC)。這個發現，讓原本胚胎幹細胞(embryonic stem cell, ESC)和成體幹細胞(adult stem cell)遭遇的問題，像是排斥反應、道德問題、細胞老化和細胞來源的限制，迎來了曙光。近年來，部分實驗致力於將誘導型多功能幹細胞分化成齒源性(odontogenic)細胞，包含造釉母細胞(ameloblast)、牙本質母細胞(odontoblast)、牙骨質母細胞(cementoblast)和牙周韌帶細胞(PDL)。然而這些體外實驗都只侷限在細胞層級且缺乏功能性測定；而在少數的老鼠體內實驗中，雖然iPSC在受體的牙胚幫助之下，有成功分化成類牙齒結構，但這些小型動物的研究，除了成功率偏低和仍須受體牙胚協助生長外，其結果是否能直接套用在人體上也依然存疑。此外，相關研究也顯示，比起其他種幹細胞，誘導型多功能幹細胞可能具有較高的致瘤性。故本實驗目的是一系列長期實驗的開端，先去觀察植入豬恆牙牙胚的誘導型多功能幹細胞和胚胎幹細胞，在恆牙發育過程中，齒源性組織的分化狀況，同時也去確認iPSC的安全性，最終希望以iPSC為來源，穩定一致地獲取齒源相關細胞。
在這個定性實驗中，我們先針對培養好的幹細胞進行免疫細胞化學染色(ICC)和免疫組織化學染色(IHC)來驗證其分化能力。接著，針對四隻蘭嶼豬的雙側下顎第四小臼齒恆牙以split-mouth設計，左側將帶有綠色螢光蛋白的豬幹細胞(其中兩隻iPSC，另兩隻ESC)植入其中，無植入細胞的右側則視為控制組，待其七個月萌發後犧牲，期間定期拍攝牙齒x光(第0, 3, 7個月)。在動物犧牲前，我們採集豬隻血液進行血球計數分析以及螢光顯微鏡鏡檢，觀察有無異常。動物犧牲後，除了記錄臨床照片外，我們取得下顎骨和牙齒檢體，經活體螢光影像系統(IVIS)和脫鈣後的IHC染色，去觀察具有綠色螢光的細胞分佈，並以齒源性相關抗體對目標牙齒進行IHC染色來驗證分化的牙齒組織。此外，我們也針對遠端器官的肺臟和軟組織進行IHC染色，檢查這些組織中，是否有綠色螢光的細胞存留。
結果發現，雙側牙齒皆有綠色螢光蛋白表現；而齒源性相關抗體的IHC染色中，也發現植入的幹細胞有成功分化成牙本質母細胞及牙骨質母細胞。臨床檢查則發現這些萌發的牙齒皆可行使正常咀嚼功能，並且沒有外型異常或畸胎瘤產生。另外，在定期拍攝的牙齒x光影像中，也無任何牙齒型態變異。動物犧牲前的血球計數檢驗整體落在正常範圍內，顯示實驗體處於健康狀況。然而，肺臟及軟組織在IHC染色下卻有觀察到綠色螢光蛋白的表現，而血液檢體中也有觀察到綠色螢光的影像，這些現象都說明了幹細胞能夠在血液中移動到遠端組織的能力。我們甚至發現其中一隻植入的iPSC的豬有瀰漫性肺出血的現象，這使得未來iPSC異體移植的應用有潛在的安全疑慮。
我們的實驗說明了iPSC能在豬恆牙牙胚中成功分化成齒源細胞。雖然沒有畸胎瘤產生，但瀰漫性肺出血以及幹細胞經血液移動到遠端組織的能力，讓iPSC的安全議題存在著風險。未來，我們除了重複檢驗iPSC的安全問題之外，也希望能以人源化動物模型(humanized animal model)作為平台，將人類iPSC植入其中，並期待能夠分化出作為臨床再生使用的牙齒相關細胞和組織。

Stem cells are undifferentiated cell with the ability of self-renewal and differentiate into various kinds of specialized cells. In 2006, Yamanaka’s team found 4 specific transcription factors (cMyc, Klf4, Oct3/4, Sox-2) were sufficient to convert mouse fibroblasts to pluripotent stem cells, which was named induced pluripotent stem cells (iPSC). This finding had break down many barriers lie in front of embryonic stem cells (ESC) and adult stem cells, such as immune rejection, ethical problems, cell aging, and inadequate cell source. Many studies had focused on the odontogenic differentiation from iPSC such as odontoblast-like, cementum-like and periodontal ligament (PDL) cells. However, results from these in vitro studies were limited in the transcription level and lacked functional assay for further confirmation. Few mouse studies show iPSC could differentiate into tooth-like structure with the aid of host’s own tooth germ, though the low successful rate and the need of recipient’s tooth germ still remained problems. And these results were not necessarily consistent with human. Furthermore, some studies had even shown that iPSC had higher tumorigenicity than ESC. Therefore, one of our aims in this study was to observe the odontogenic differentiation from the implanted iPSC & ESC in porcine tooth germ. Another aim here was to confirm the safety of iPSC.
In this qualitative experiment, we first examined stem cell pluripotency with immunocytochemistry (ICC) and immunohistochemistry (IHC). And we transplanted porcine stem cell in four Lanyu pigs. Under split mouth design, we implanted porcine stem cells with green florescent protein (GFP) gene expression to left mandibular 4th permanent premolar tooth germ and no cell as control group to right site. (iPSC in 2 pig, ESC in 2 pig). Then, we took dental periapical x-ray films at 0, 3, 7 month. Before animal sacrificed at the 7 month, we drew the blood for whole blood count and fluorescent microscope assay to examine the general health of pigs. After animals sacrificed, we took clinical photo as record. As to the distribution and differentiation of GFP-expressed cells, we obtained the mandible and teeth for In Vivo Imaging System (IVIS) assay and IHC stain. We also performed IHC to soft tissues and lungs for GFP-expressed cell tracking.
The result showed GFP-expressed odontoblasts and cementoblasts were fully developed in transplanted teeth. The dentition was in normal function without any teratoma formation. The x-ray films and whole blood count assay both showed no abnormality. However, we still found GFP-expressed cells in lung and soft tissues. The blood samples also showed green fluorescence under fluorescent microscope, which indicated the migration and homing ability of the stem cells. We even found the diffuse alveolar hemorrhage in one iPS-transplanted recipient.
Our study demonstrated iPSC could differentiate into odontogenic cells in the porcine tooth germ. Although there was no teratoma, the transplanted pig with diffuse alveolar hemorrhage posed a potential threat on clinical use in allografted iPSC. In the future, we hope to use humanized animal model to perform further human iPSC-transplanted study and test the feasibility of odontogenic differentiation from these animals as well as the safety issue. We expect to provide an alternative safety method for gaining regenerated odontogenic tissues from iPS cell in the surrogated animals.

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