察爾斯大夫 美麗殿堂 Dr. Charles Meridien Palace

神經榮養藥物（Neurotrophic）有助修復受損認知

作者健康醫療網／記者曾楷婷報導 | 健康醫療網 – 2014年9月12日

（健康醫療網／記者曾楷婷報導）

頭部外傷嚴重者恐導致後遺症出現，包括長期記憶受損、認知功能下降、注意力不集中等等；將造成患者無法回到工作崗位上繼續工作，影響家庭經濟收入，生活品質可能也因此受到影響。

中國醫藥大學附設醫院神經外科部醫師陳春忠於第七屆亞洲神經再生與神經重塑大會提出的研究成果顯示，使用某種神經藥物，將會引導神經再生、重塑，有助於神經繁榮滋養，簡稱「榮養」，可幫助傷後認知功能的恢復，對患者有實質助益。

陳春忠醫師針對頭部外傷且24小時內就醫、伴隨顱內出血，但意識仍清醒的患者32人，持續使用神經榮養藥物5天；給予神經榮養藥物的患者，在傷後三個月的認知功能，包括長程記憶改善及繪圖功能均明顯優於對照組，顯示在頭部外傷患者受傷後即時給予神經榮養藥物，有助於受損神經修復及再生。另外，若家中有阿茲海默症與中風患者，榮養藥物對於頭部外傷患者的復原也有幫助。

神經外科醫師陳春忠表示，意外事故導致傷殘、失能仍是影響國人健康的重要議題，尤其頭部外傷患者就算治癒，經常留下記憶力減退、注意力不集中，情緒不穩、性格改變等後遺症，過去對於頭部外傷患者認知失能現象並無有效治療方法。這項研究顯示，及時給予傷患神經修復所需的胜肽和胺基酸藥物，有助於受損腦神經修復，減少傷患日後認知失能的現象，協助傷患改善生活品質，重回工作崗位。

第七屆亞洲神經再生與神經重塑大會今天於台北舉行；大會邀集台灣、中國、奧地利、越南等國神經專家擔任專題講者，針對其研究領域與專長與各方相關領域先進共同交流，探討神經再生（Neurogenesis）與神經重塑（Neuroplasticity）等相關議題。

傷筋動骨不須一百天！「再生療法」有望解決退化關節炎

優活健康網 – 2014年7月30日

（優活健康網記陳承璋／綜合報導）

傷筋動骨一百天，以直白的說法，就是當民眾進行有關於脊椎與骨頭的手術，通常都要花上好一陣時間來復健養傷，也因此讓眾多人對於與骨頭有牽連的手術，幾乎退避三舍，能拖就拖，但事實上，針對於許多的關節疼痛或腰部的慢性疼痛等，其實還有多種療法來代替開刀，也因此民眾無須對肩頸疼痛、腰痛、退化關節炎等如此絕望。

男子退化關節炎　痠痛難忍想開刀

台中就有一位退休男子，過去因創傷致雙肩疼痛無力。超音波檢查顯示，他一側旋轉肌部分撕裂，另一側則為沾粘性與退化性肩關節炎，以致痠痛難忍。同樣求治過民俗療法，甚至想開刀圖個一勞永逸，但在經過再生與增生療法雙管齊下，輔以玻尿酸注射之後，雙肩力量已逐漸恢復，手術治療已無必要考慮。

中國附醫復健科賴宇亮醫師說，疼痛是復健病患普遍的主訴。而治療頭痛，顳顎關節痛，頸部、下背、腰部的慢性疼痛，肩部旋轉肌肉疼痛，頑固性網球肘，髖部及大腿上外側疼痛，膝關節周圍疼痛，腳踝扭傷合併韌帶鬆弛及慢性疼痛，最常使用的當屬葡萄糖增生療法，以及PRP再生療法。

難纏關節炎　可用再生療法解決

賴宇亮醫師指出，增生療法對關節周邊損傷，效果特別明顯，像是五十肩、網球肘、媽媽手、退化性膝關節炎、脊椎周邊韌帶損傷等。其中高濃度的葡萄糖較其他注射液安全，最常被用來引起輕微的發炎反應，以啟動身體的修補機制。人體本來就有受傷後自行修補的反應，借助些許外力製造，可以增強肌腱和韌帶的強度，進而減緩慢性疼痛。

至於近期發展出的PRP再生療法，則是注射自體血清，來刺激血管增生，加速軟組織修復。也就是自身的血小板經過純化之後，分泌出的細胞激素，在短時間內幫助組織修復，遠離疼痛。適用範圍包括退化性關節炎、運動傷害、慢性肌腱炎、網球肘等。

超商店長網球肘增生療法打針解疼痛

中廣新聞網 – 2014年7月31日

寇世菁報導

台中一名超商店長，每天忙進忙出搬貨上架，操勞過頭，得了網球肘，手肘疼痛兩年多，試過民俗療法，也接受針灸，毫無起色。輾轉到中國附醫求診，復健部醫師採取增生療法，前後十次，疼痛逐漸緩解。

中國附醫復健部醫師賴宇亮表示，疼痛是復健病患常見的問題，治療頭痛，顳顎關節痛，頸部、下背、腰部的慢性疼痛，肩部旋轉肌肉疼痛，頑固性網球肘，髖部及大腿上外側疼痛，膝關節周圍疼痛，腳踝扭傷合併韌帶鬆弛及慢性疼痛，最常使用的就是葡萄糖增生療法，以及PRP再生療法。醫師指出，增生療法對關節周邊損傷，效果特別明顯，像是五十肩、網球肘、媽媽手、退化性膝關節炎、脊椎周邊韌帶損傷等。一名超商店長，每天忙進忙出搬貨上架，操勞過頭，得了網球肘，兩年來，試過民俗療法，也接受針灸，毫無起色。輾轉到中國附醫求診，復健部醫師採取增生療法，前後十次，疼痛逐漸緩解。簡單的說，他是靠打針解決了問題。

另外一名退休男子，過去因創傷導致雙肩疼痛無力。超音波檢查發現，一側旋轉肌部分撕裂，另一側則是沾粘性與退化性肩關節炎，才會痠痛難忍。經過再生與增生療法雙管齊下，加上玻尿酸注射後，雙肩力量逐漸恢復，不必手術治療。

賴宇亮醫師說，高濃度的葡萄糖比其他注射液安全，最常被用來引起輕微的發炎反應，以啟動身體的修補機制。人體本來就有受傷後自行修補的反應，借助些許外力製造，可以增強肌腱和韌帶的強度，進而減緩慢性疼痛。

至於最新發展出的PRP再生療法，則是注射自體血清，刺激血管增生，加速軟組織修復。也就是自身的血小板經過純化之後，分泌出的細胞激素，在短時間內幫助組織修復，遠離疼痛。適用範圍包括退化性關節炎、運動傷害、慢性肌腱炎、網球肘等。

移植幹細胞治療脊椎癱瘓背部長出鼻子

Stem cell patient ACCIDENTALLY grows a NOSE on her back eight years after surgeons injected tissue there to try to cure her paralysis

2014年07月10日

潘勛

女性病患移植幹細胞治癱瘓不成，背部反而長出鼻狀物。Nose-like：A 3cm growth formed from nasal tissue, bone and nerve branches was discovered.（翻攝自每日郵報）

A new nose, was grown by surgeons on Xiaolian's forehead, pictured, before being transplanted to replace the original nose, which is infected and deformed, after a car crash.（翻攝自每日郵報）

一名美國女子8年前到葡萄牙首都里斯本，接受鼻部摘取幹細胞移植到脊椎，盼能治好癱瘓，結果手術沒成功，去年背部開始疼痛，就醫後發現手術部位竟然長出3公分的鼻子，目前經手術已經摘除，但傳為罕見案例。

《每日郵報》9日報導，葡萄牙「埃斯加莫尼茲醫院」（Hospital de Egas Moniz）醫生摘取該女生鼻部幹細胞，移植到她脊椎，原因是希望能發展成神經細胞，從而修補該女脊椎受損的神經。可惜治療未能奏效。

只是在2013年，也就是該幹細胞手術8年後，當時28歲的女病患抱怨手術部位疼痛日增。就醫之後，醫生們發現她背部長出3公分的東西，主要是鼻部組織，還有一些骨骼及神經分叉，但沒連上脊椎神經。

愛荷華大學附屬醫院暨診療中心神經外科專家德勞希Brian Dlouhy已動手術摘除該「背部鼻子」，表示它並非惡性孳生物，但會分泌痰狀黏液物質，可能因此造成病人脊椎疼痛。

密西根州底特律市「韋恩州立大學」幹細胞學者培杜齊－尼爾森Jean Peduzzi-Nelson表示，大多數病人接受鼻組織移植復健，若是施術得當，癱瘓程度都能大為改善，像那位女性病患的案例不到1%。

PRF富含血小板纖維蛋白益於重建齒槽骨

作者：【記者何弘斌／高雄報導】 | 台灣新生報 – 2014年6月23日

血小板含有許多與傷口癒合有關的生物活性蛋白質，包括數種生長因子能促進幹細胞與修復細胞的移動、增生、分化、新血管生成及膠原蛋白合成，甚至再生新的組織；血小板的濃度越高，生長因子越多。

自體血小板濃縮產物開始由單純的濃縮血小板，進入需藉由牛的凝血酉每與氯化鈣引發血小板活化的PRP富含血小板的血漿，再進入完全天然的PRF富含血小板的纖維蛋白。自體血小板濃縮產物具有多種正常傷口癒合所需的生長因子、細胞激素與活性蛋白質，放進傷口內或加入植入物中，根據越來越多的文獻顯示，原本難纏的傷口，因有PRF的參與可讓傷口加速癒合。

相較於PRP，PRF的優點是：不必添加抗凝血劑與牛的凝血酉每，產製的時間較少，且完全是自體的產物，沒有免疫抗原抗體的問題；血液離心時，逐漸地自然形成緻密的纖維蛋白基質，較有耐受力，不像PRP所形成的纖維蛋白很快地就溶解掉。

高密集的纖維蛋白基質將血小板、白血球、與細胞激素拌在其間，血小板可慢慢地釋出生長因子及活性蛋白質，作用的時間加長；緻密的纖維蛋白基質有如生物材料，可支撐與癒合有關的細胞移動與細胞激素的釋放；被拌在其間的白血球，有對抗感染及免疫調節方面的作用。

目前在牙科領域，富含血小板的纖維蛋白大多應用在齒槽骨的重建。另有新的研究果顯示，在上顎竇提高術、牙根旁的牙周病手術治療、牙根覆蓋手術、拔牙傷口修復及引導骨頭再生術中，合併骨移植材料或自體牙齦，均有理想的治療成果。

高醫牙科部亦將此一技術應用在牙周病的治療。嚴重的牙周病破壞後，齒槽骨常呈現明顯缺損，傳統的引導骨頭再生術雖可重建齒槽骨，但對於傷口癒合的速度及再生膜材料的選取，仍是一項須要加強的項目。富含血小板的纖維蛋白則可針對以上特點加以補強，同時因為是自體的產物，所以沒有免疫抗原抗體及感染的問題。

退化性關節炎臨床注射高濃度血小板血漿（PRP）

作者：【記者蔡清欽／台南報導】 | 台灣新生報 – 2014年6月10日

骨科門診中退化性關節炎是病人最多困擾的問題之一，其中又以膝蓋疼痛的患者人數最多，台南市立醫院骨科醫師顏大千指出，當藥物治療效果不佳，但非到開刀手術不可的地步，以高濃度血小板血漿（PRP）注射來治療退化性關節炎，可獲得不錯的治療效果，是退化性關節炎及運動傷害治療上的新選擇。

顏大千醫師說，根據統計，65歲以上者，有6成人口正在忍受膝蓋退化之苦，75歲以上的銀髮族，更有高達9成有退化性關節炎。過去醫療技術中，退化性問題僅能延後或舒緩的方式治療，幾乎無法促進軟骨生長，常見的治療方式有復健物理治療、消炎藥物或葡萄糖胺補充、玻尿酸注射、關節鏡灌洗手術等，甚至退化進展至末期，不得不採用人工關節置換手術。

顏大千表示，PRP技術成功地由實驗室階段進入臨床使用。自2010年開始於美國骨科醫學會臨床經驗報告；2012年也開始於國內骨科醫學會報告國人使用臨床經驗，此項技術成為退化性關節炎的最新療法。

PRP注射治療是自患者本身抽取適量的血液，經由離心處理後，僅抽取上層富含生長因子的高濃度血小板血漿（25cc血液約可分離3.5cc），再由醫師注射進患者退化或需要治療的關節內；藉由生長因子的作用，開始促進關節內軟骨等組織生長及修補功能，達到減輕關節疼痛、增加膝蓋功能的效果。

台南市立醫院自今年初也特別引進全自動離心設備，讓每一位接受治療的患者，都有自己一套專屬的抽血離心拋棄式耗材；透過全自動離心，可以得到比傳統人工抽取更精確的濃度（約3.5倍），以達到最高品質的治療效果。

顏大千指出，以該院骨科的PRP注射治療為例，自今年2月起至今完成38例，，膝關節有35例，肩關節則有3例。PRP主要是由醫師注射進關節處，讓注射部位與軟骨細胞充分結合後，逐漸產生療效，當日即可返家，但注射部位須保護二周，建議避免從事劇烈運動，以利修補進行，一般效果可持續一年，比玻尿酸治療療效長一倍，並減少注射療程。

顏醫師也說，PRP也廣泛應用於運動傷害及手術後加速癒合的治療上，不僅限於膝關節，凡背、腰、肩、肘、腕，髖、踝等各種容易受傷的身體部位皆可應用；美國NBA明星Kobe·Bryant應用於阿基里斯腱縫合手術、大聯盟洋基隊A·Rod及王建民也都曾接受注射治療；國內大聯盟級的義大犀牛隊長胡金龍用於肩部傷勢治療、LAMIGO、桃猿隊的中華隊主炮、大師兄林智勝用於膝關節的韌帶修補手術回到運動場上拚戰。

自體血液純化注射退化性關節炎治療首選

作者：台北訊 | 中時電子報 – 2014年6月5日

中國時報【台北訊】

（中時健康╱王宇仁報導）

旅日多年的職棒選手林威助，今年決定回國投效中華職棒，加盟中信兄弟隊，但3月底卻因滑壘不幸造成左膝半月軟骨受傷，注射玻尿酸及消炎針治療3周後仍不見好轉，經醫療團隊及球團的評估後，決定開刀治療。

林威助接受核磁共振檢查時，左膝傷勢並不明朗，十分擔心可能嚴重到必須縫合，要休息半年，等於球季提前報銷。所幸開刀時發現傷勢比想像中來得輕微，只須整理破損部分，預估3至4個月後即能重返球場與球迷相見歡。

退化性關節炎是職棒、職籃選手常見的運動傷害，不少人甚至因此被迫提前結束職業運動生涯。然而，退化性關節炎並非運動選手的專利，一般民眾隨著年紀增長，長期運動傷害，也容易患有退化性關節炎。近年來，路跑及騎單車運動盛行，民眾發生退化性關節炎的機會也明顯增加。

新光醫院骨科主任暨微創中心主任釋高上醫師表示，退化性關節炎是目前全世界最常見的關節疾病，台灣65歲以上民眾，大約每3人就有1人罹患，佔全國人口的 5%到6%。由於患病初期沒有明顯疼痛，容易受輕忽，直到關節痠痛，經影像學檢查發現關節邊緣長了骨刺，治療就變得相當棘手。

目前治療初期退化性關節炎，常見的方式是注射玻尿酸來降低關節摩擦，但效果只能維持數月，若疼痛復發就必須反覆施打。如今在歐美風行的PRP高濃度血小板生長因子血漿療法引進國內，只要從自己身上抽出約10cc血液，純化後注射到膝關節中，就能達到改善疼痛及初期退化性關節炎的目標。

釋高上主任解釋，患者抽出10cc的血液並非直接注射至關節，而是必須利用離心機加以純化、去除紅血球，取得能釋放出生長因子，具有實際療效的高濃度的血小板血漿，才能注射到關節發炎處。

PRP治療已被證實能加速骨骼肌肉細胞的復原，促進膠原增生，以及微血管周邊循環及軟骨組織的再生。由於不需要手術，沒有傷口，也不需要修復期，結果優於其他療法，如今已成為職業運動員在關節受傷時的治療首選。

PRP療法除了用於改善膝關節疼痛，運用範圍也延伸到肩關節、跟腱及韌帶損傷的修復上。骨折開刀的病人也可藉由PRP注射緩解疼痛，縮短癒合時間。許多外科手術的止痛修復，甚至皮膚再生抗老化，都有人應用PRP的技術來輔助。

必須提醒的是，PRP療法並非骨科治療萬靈丹。釋高上主任指出，嚴重關節退化，還是得靠手術修復，或置換人工膝關節才能改善，PRP療法反而無顯著成果。民眾若有關節疼痛問題，千萬不要隱忍輕忽，應把握治療時機，接受專科醫師檢查，找出病因，尋求安全有用、適合自己的治療方式，才能真正擺脫退化性關節炎的威脅。

高BMI者幹細胞移植卡安全

作者：【記者陳敬哲／綜合外電報導】 | 台灣新生報 – 2014年5月20日

High BMI stem-cell donors “safe”. Stem cell donations are used to treat blood cancers and disorders. People with a high Body Mass Index can make equally good stem-cell donors as those who are a healthy weight, according to new research.

High BMI stem-cell donors 'have no side effects'.

高身體質量指數（BMI）者，幹細胞移植越安全。英國骨髓移植中心與安東尼諾蘭慈善機構發現，相較於健康體重民眾，肥胖者幹細胞捐贈副作用較少，術後不容易產生疼痛；德國、美國、加拿大，最近放寬幹細胞捐贈規定，減少體重限制，都沒有重大副作用病患出現。

白血病患者必須透過骨髓移植才能痊癒，但3分之2患者在家族中找不到適合對象，必須仰賴非血緣配對找到骨髓捐贈者才能存活，然而過往醫學界認為BMI質超標民眾捐贈骨髓後，發生骨頭疼痛與副作用機率較高，因此部分國家規定，BMI超過一定標準就不在配對名單中，以免捐贈者健康受到威脅。

但研究團隊追蹤19位骨髓捐贈者一年，BMI值多在40上下，發現副作用影響不會比BMI值35民眾更明顯，反而更少，研究學者Annelies Billen表示，這項研究提出證據，高BMI值民眾，雖然身體健康危害比較大，但幹細胞捐贈影響小，部分國家未來若能放寬配對標準，有助於提高成功配對機會。

研究團隊將擴大追蹤對象，找到更多潛在骨髓捐贈者，讓白血病患者成功接受骨髓捐贈機會增加，Annelies Billen強調，會持續追蹤19位研究對象，確認骨髓捐贈有無長期性影響，藉此提高幹細胞知識基礎，部分放寬肥胖者捐贈骨髓的國家，目前都沒有顯著副作用案例出現，提高白血病治癒率。

明年登興櫃未演先轟動賽宇細胞獲日本疫苗廠大單

2014年05月08日

工商時報記者杜蕙蓉／台北報導

賽宇細胞小檔案

基亞疫苗大股東之一賽宇細胞進軍國際市場大報佳音，總經理陳宏飛證實已接獲日本大疫苗廠採用全套設備，包括培養基、製程開發等；在四川爆發全球首例人感染H5N6致死病例下，預期將帶動疫苗產業再掀風潮，並讓賽宇預計明年登錄興櫃也未演先轟動。

近年營運呈現三級跳的賽宇，2011年每股淨損0.67元，但2012年起則大逆轉展現獲利實力，尤其是2013年來自於日本四大疫苗廠之一採購其設備，每年獲利幾乎都以倍數成長。

陳宏飛表示，該日本疫苗廠正在積極興建疫苗廠，因此，採購的全套設備是按進度進行，今、明兩年都持續有業績貢獻，而且因客戶端目前反應頗佳，預計另一家日本大疫苗廠也來洽談合作中。

賽宇目前資本額1.2億元，原本是由工研院化工所張景明領軍生醫中心組織工程部成立，2002年引進前鑽全創辦人陳宏飛後，至今未曾辦理過減資和增資，且在陳宏飛經營後，2年前也開始獲利。

賽宇是亞洲唯一能提供細胞量產全套設備的廠商，產品包括提供細胞生長環境的生物反應器、提供細胞養分培養的培養基、提供細胞生長空間的載體和細胞培養製程開發等，除日本前四大疫苗廠，國衛院在H1N1新流感疫苗的開發也是採用其培養基。

陳宏飛表示，賽宇細胞產品線可應用包括蛋白質藥（Protein Drugs）、單株抗體（Monoclonal Antibody）、疫苗（Vaccine）等產品製造，皆屬於細胞培養（cell culture）範疇，甚至當紅的幹細胞、免疫細胞療法，只要進入量產階段，同樣都必須使用到細胞培養方式；而賽宇細胞在長達十年努力下，開發出自動化的細胞培養設備。日本四大疫苗為北里、化學暨血清、電氣暨化學、大阪微生物研究所，此四大疫苗廠共占日本80%疫苗市占率。

注入嫩血「老」鼠回春「吸血鬼療法」（Vampire therapy）有助治療阿茲海默症、心臟病

自由時報 – 2014年5月6日

〔編譯魏國金／綜合報導〕

美國兩個科學家團隊研究發現，年輕老鼠的血液扭轉了高齡鼠的老化，使其大腦、肌肉返老還童，形成新血管、提升記憶力與學習力。研究人員表示，這種「吸血鬼療法」（Vampire therapy）或許聽來殘酷陰森，卻可能對諸如阿茲海默症、心臟病等帶來革命性方案。

紐約時報報導，二○○○年代初期，史丹福大學醫學院神經學教授蘭杜（Thomas Rando）與其同僚探究，若將老化幹細胞「沉浸於」青春的血液中，可接收什麼樣的訊號。○五年的研究報告指出，老者肌肉復原的速度快如年輕者，老者也以青春速度生成新的肝細胞。研究顯示，年輕鼠血液中的成分可喚醒老幹細胞，並使老化細胞回春，高齡鼠血液中的成分則會損害年輕鼠的恢復能力。

該團隊成員、哈佛大學副教授瓦格絲（Amy Wagers）繼續相關研究，去年發現年輕老鼠的血液中含有豐富的蛋白質GDF11，老者則鮮少GDF11，研究人員將該蛋白質注入高齡鼠內，結果發現GDF11使其心臟恢復活力。

至於GDF11是否對其他組織也具回春功效，刊登於四日出刊的「科學」（Science）期刊的研究報告顯示，在老鼠的骨骼肌實驗中，GDF11恢復高齡鼠的幹細胞，使其肌力與耐力皆有所提升。該團隊發表於「科學」的研究也發現，年輕鼠的血液使高齡鼠有關嗅覺的神經元生長，使其嗅覺更敏銳。

另一方面，史丹福大學研究團隊也試圖了解年輕鼠的血液，是否能改善老者的大腦功能。四日刊登於「自然醫學」（Nature Medicine）期刊的研究指出，三週內持續將三個月大老鼠的血液，注射到十八個月大的高齡鼠內八次，結果高齡鼠的記憶表現改善。研究員維列達（Saul Villeda）表示，相關回春之效或許對阿茲海默症等病症也有裨益。

逆轉老化吸血鬼療法是真的！？”'Vampire therapy” could reverse ageing, scientists find

A transfusion of youthful blood may halt or even reverse the ageing process as two studies find that the chemical make-up of younger blood has surprising health benefits

2014年05月06日

中時電子報郭匡超／綜合報導

老鼠換血實驗發現可改善身體器官功能。吸血鬼電影「換血保青春」情節可望成真！圖摘自英國電訊報

根據英國電訊報報導，科學家最近通過老鼠實驗發現，向年齡大的老鼠輸入年輕老鼠血液，能夠產生大腦「充電」現象，可以增生血管，改善記憶和認知能力。哈佛大學另一研究也發現，老鼠血液中的「GDF11」蛋白質能保持大腦和肌肉的年輕和強壯，只要提高「GDF11」含量水平可改善身體器官功能。我們在吸血鬼電影所看到的「換血保青春」情節可望成真！

進行這項實驗的科學家指出，輸入年輕者的血液可以逆轉衰老過程，甚至俗稱老年痴呆的阿兹海默症也找到治療新方法。並希望兩到三年內開始人體實驗。

（新聞來源：http://www.telegraph.co.uk/science/science-news/10807478/Vampire-therapy-could-reverse-ageing-scientists-find.html）

成人細胞首度「治療性複製」（Somatic cell nuclear transfer）出幹細胞

自由時報 – 2014年4月19日

〔編譯張沛元／綜合17日外電報導〕

科學家17日宣布，他們首度透過複製桃莉羊技術，利用成人皮膚細胞培育出完全符合病患細胞系的幹細胞。此舉朝日後打造出完全符合病患DNA的幹細胞以協助治病的目標更邁進一步，但也再度引發道德爭議。

利用「治療性複製」（體細胞核轉植技術，Somatic cell nuclear transfer）技術

根據這份刊登於網路期刊《細胞幹細胞》的研究報告，這是研究人員首度利用成人的細胞進行「治療性複製」，即「體細胞核轉植技術」（Somatic cell nuclear transfer），意指製造出在基因上與捐贈者完全相同的胚胎幹細胞，其目的通常是用於治療疾病，但也是生殖複製或製造出某人基因複製品的第一步—該技術自1997年用來成功複製出桃莉羊後引發極大爭議，聯合國於2005年呼籲各國禁止，美國也禁止將聯邦資金用於生殖或治療性複製。

去年嬰兒細胞研究成功

17日發表的最新研究，由洛杉磯「CHA Health Systems」幹細胞研究所的研究員鍾永基（Young Gie Chung，譯音）領導，資金來自一家基金會與南韓政府；所使用的技術與當年複製桃莉羊的技術類似，也就是對卵子細胞進行重新編程（拿掉卵子細胞原有的DNA，以成年捐贈者的DNA取而代之），然後對該細胞進行電擊，使之分裂與呈倍數增加，其結果就是製造出與捐贈者DNA完全相同的細胞。此研究去年就在人類上獲得成功，但當時使用的捐贈細胞取自嬰兒，這次的捐贈細胞則來自兩名分別為35歲與75歲的成年男性。

治療重症出現生機

倘若此一研究結果獲得其他實驗室證實，其意義顯然相當重大，因為包括心臟衰竭與失明等許多影響成年人的疾病，終有一日將能以幹細胞治癒；而且這些為個別病患量身打造的幹細胞，可以由較老的細胞、而非嬰兒或胚胎細胞製造出來。本身也研究幹細胞的美國加州大學戴文斯分校副教授諾佛勒就稱此研究「令人興奮、重大與技術上令人信服」。不過，儘管看似可行，但此一技術並不容易；科學家們總共嘗試39次，其中只有兩次成功製造出胚胎。

1985年4月16日台灣第一個試管嬰兒在台北榮總醫院出生國內第一個試管嬰兒張先生現在29歲正在念動物學博士學位

2014年04月16日

莫忘來時路／中國時報黃如萍

29年前的今天，台灣第一個試管嬰兒在台北榮總醫院出生（張昇平提供），為不孕夫婦帶來希望。

29年前的今天，台灣第一個試管嬰兒在台北榮總醫院出生，為不孕夫婦帶來希望。

所謂試管嬰兒是由人工操作，將卵子和精子取出後體外受精，並培養成胚胎，2至6天後再將胚胎植回母體內，利用體外受精技術所生出來的嬰兒。

世界第一個試管嬰兒是1978年在英國誕生，台灣晚了7年，香港則是到1986年、大陸1988年才有本土試管嬰兒誕生。目前台灣有40多個不孕症診所或中心，每年約7000～8000個試管嬰兒出生，全球約有3百萬名試管嬰兒。

試管嬰兒成功機率大約35%，台灣和美國差不多；國際上最著名的就是一對以色列夫婦，前後做了8次試管嬰兒才順利懷孕。

為了增加成功機率，試管嬰兒常會植入多胞胎，考量母體與孩子教養，台灣限制不得超過4胞胎；多胞胎的成功機率也和歐美相同，約25%～30%。台灣的紀錄保持人是連續3次進行試管嬰兒，成功產下一胞胎、三胞胎及雙胞胎，3女3男共6個小孩。

世界第一個試管嬰兒布朗小姐，已結婚並育有兒子，是上班族；國內第一個試管嬰兒張先生，現在29歲，正在念動物學博士學位。醫學專家證實，試管嬰兒在健康上和一般人無異，人格與發展成就端視家庭教育與養成，以及個人的努力。

試管嬰兒的誕生證明了醫學技術可以彌補不孕夫婦的遺憾，但有人擔心，未來隨著基因診斷、重組等技術的純熟，試管嬰兒的體外授精技術將被更精進的利用，未來訂做一個有諾貝爾獎得主頭腦、名模漂亮臉蛋與身材或NBA球星壯碩體格的下一代，不是夢想，遺傳、種族、倫理等概念將被顛覆，甚至連自我價值都得重新審視。試管嬰兒將造成文化、倫理與道德的爭議。

Awakened by Cellular Stress：Isolation and Characterization of a Novel Population of Pluripotent Stem Cells Derived from Human Adipose Tissue（3）

（http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0064752）

Results

Muse-ATs Isolated from Lipoaspirated Human Adipose Tissue

Adipose tissue is composed of adipocytes (mature cells) and the stromal vascular fraction (SVF) containing a heterogeneous population of cells, including adipose tissue macrophages (ATMs), adipose stem cells (ASCs), mesenchymal stem cells, and fibroblasts.

We explored the possibility of both activating and isolating Muse-AT cells from their quiescent state by exposing them to cellular stress (Fig. 1A). Lipoaspirated material was first incubated in collagenase for 30 min at 37°C to release adipocytes (floating cells) and different cellular components present in the SVF as previously described. This material was then subjected to severe cellular stress, including long incubation with collagenase, low temperatures, low serum and hypoxia, to kill fragile adipose cells and release Muse-AT cells. Optimal conditions for the release of Muse-AT cells were determined to be 16 hours incubation with collagenase in DMEM medium without FCS at 4°C under very low O2, which subsequently gave way to a homogenous population of Muse-AT cells. Approximately 90% of isolated cells were both SSEA3 and CD105 positive, as determined by flow cytometry (Fig. 1B). This high purity is presumably due to the severity of the cellular stress conditions, responsible for the depletion of other cell types. As all other components of the adipose tissue lipoaspirate failed to survive, a population of highly purified Muse-AT cells was obtained, and therefore further purification processes were not necessary. Muse-AT cells were plated in both adherent and non-adherent cell culture dishes. We observed that Muse-AT cells can grow either in suspension or in adherence culture to form the characteristic cell clusters observed in ES cell-derived embryoid body, as described in bone marrow and dermal fibroblast-derived Muse cells in previous reports (Fig. 1C, D). Under both conditions, individual Muse-AT cells reached a diameter of around 10µm and cell clusters reached a diameter of up to 50µm by day 3 (Fig. 1C–D), which has been previously demonstrated to mark the limit of their proliferative capacity.

Figure 1. Isolation and morphologic characterization of Muse-ATs.

(A) Schematic of Muse-AT isolation and activation from their quiescent state by exposure to cellular stress. Muse-AT cells were obtained after 16 hours, with incubation with collagenase in DMEM medium without FCS at 4°C under very low O2 (See Methods). (B) FACS analysis demonstrates that 90% of isolated cells are both SSEA3 and CD105 positive. (C) Muse-AT cells can grow in suspension, forming spheres or cell clusters as well as individual cells (see red arrows) or (D) Muse-AT cells can adhere to the dish and form cell aggregates. Under both conditions, individual Muse-AT cells reached a diameter of approximately 10µm and cell clusters reached a diameter of up to 50µm, correlating to stem cell proliferative size capacity.

doi:10.1371/journal.pone.0064752.g001

Muse-ATs Spontaneously Express Pluripotent Stem Cell Markers

Upon transfer and adherence to chamber slides for immunofluorescent staining, both the Muse-AT cell clusters and individual Muse-AT cells strongly expressed all of the characteristic pluripotent stem cell markers that were examined. These included SSEA3, a cell-surface glycosphingolipid frequently used to detect human ES cells and to purify Muse cells from bone marrow and dermis; Oct3/4 a protein involved in the self-renewal of human ES cells; Nanog, a transcription factor involved in the self-renewal of human ES cells; Sox2, a transcription factor that controls genes involved in embryonic development; and TRA-1-60, which reacts with the antigen TRA-1-60 on the surface of embryonic germ cells and ES cells (Fig. 2). Comparatively, ASCs derived from the same lipoaspirated tissue were either negative or weakly positive for these pluripotent stem cell markers (Fig. 2).

Figure 2. Muse-ATs express pluripotent stem cell markers.

Immunofluorescence microscopy demonstrates that Muse-AT aggregates, along with individual Muse-AT cells, express characteristic pluripotent stem cell markers, including SSEA3, Oct3/4, Nanog, Sox2, and TRA1-60. Comparatively, ASCs (right panel) derived from the same lipoaspirate under standard conditions (see above, were negative for these pluripotent stem cell markers. Nuclei were stained with DAPI (blue). Original magnification, 600 X.

doi:10.1371/journal.pone.0064752.g002

Awakened by Cellular Stress：Isolation and Characterization of a Novel Population of Pluripotent Stem Cells Derived from Human Adipose Tissue（2）

Methods

Isolation of Muse-AT cells from Lipoaspirated Fat

Lipoaspirates (100–200 g per aspirate) were obtained from subcutaneous abdominal adipose of women undergoing elective liposuction. None of the investigators of this study had any contact with, nor any knowledge of any personal information relating to, these patients. Furthermore, human subjects were unidentifiable as well as all their characteristics and clinical records. Therefore, this study did not meet the criteria of human subjects research and HHS regulations did not apply (45 CFR 46.102(f)).

Lipoaspirate was repeatedly Washed with PBS until blood was completely removed from the tissue, and then incubated with equal volume of DMEM containing collagenase (0.1%, Sigma Aldrich) for 30 min at 37°C in a shaking incubator at 110 rpm, followed by incubation in 4°C, while still in collagenase and nutritionally deficient medium (no FCS), for 16 hours under severe hypoxia conditions. Digested material was then centrifuged at 1500 rpm for 10 minutes at 4°C. Supernatant containing adipose cell debris (dead adipocytes, macrophages, red blood cells, adipose stem cells among other cell components) was removed by aspiration and the remaining cell Pellets were washed several times with PBS. Pellets were re-suspended in PBS and incubated with a red blood cell lysis buffer (eBiosciences, San Diego, CA) for 10 min at R/T (2×). Remaining cell pellets containing cells highly resistant to severe cellular stress, were re-suspended in Dulbecco’s Modified Eagle Medium 1× (DMEM; CellGro, MediatechInc, Manassas, VA) comprised of 10% fetal bovine serum (FBS; Thermo Scientific Hyclone, Logan, UT) and 5% antibiotic-antimyocotic solution (CellGro, Mediatech Inc, Manassas, VA), and plated as cells in suspension as well as adherent cells. For ASC isolation, lipoaspirate material was subjected to collagenase digestion (0.1%, Sigma Aldrich) for 30 min at 37°C in a shaking incubator at 110 rpm, and ASCs were isolated and cultured as previously described.

Flow Cytometry Analysis

Floating Muse-AT cells were cultured in DMEM/10% FCS for 2 days followed by FACS analysis. Cells were washed in 2% inactivate FCS/0.05% sodium Azide/PBS and were re-suspended in 100 µl of the same buffer and incubated at 4°C for 1 hour in the presence or absence of primary unconjugated rat anti-human SSEA3 (EMD Millipore; Billerica, Massachusetts). Cells were then washed twice with the same buffer and incubated with the corresponding secondary FITC-conjugated anti-rat IgM (BD Biosciences; San Diego, CA) for 45 minutes at 4°C. After two consecutive washes, cells were incubated with PE-mouse anti-human CD105 (BD Biosciences, San Diego, CA) at 4°C for 1 hour. Cells were then washed and re-suspended in 200 µl of the same buffer. Analysis of count and cell type was performed using a FACS Calibur flow cytometer and cEllQuest Pro software.

Immunocytochemistry

Cells were fixed in 4% paraformaldehyde (20 min at R/T), washed in PBS, then incubated in 0.2% Triton for 20 min. After 2 successive washes in PBS, cells were blocked with 10% normal goat serum in 1% BSA solution for 60 min at R/T. Cells were then incubated with the primary antibodies overnight at 4°C. The following pluripotent stem cell markers were used: rat anti-human stage-specific embryonic antigen (SSEA3, Millipore, Billerica, MA), mouse anti-human octamer-binding transcription factor 3 and 4 (Oct3/4, Santa Cruz Biotech, Santa Cruz, CA), rabbit anti-human Nanog (Millipore, Billerica, MA), rabbit anti-human SRY-box 2 (Sox2, Millipore, Billerica, MA), and mouse anti-human TRA-1-60 (Abcam, Cambridge, MA); for mesenchymal cell lineages: rabbit anti-human preadipocyte factor 1 (Pref-1, [a.k.a. delta-like 1 homolog (drosophila), DLK1] preadipocyte marker, Santa Cruz Biotech, Santa Cruz, CA); mouse anti-human myosin D (MyoD, myocyte marker, R&D Systems, Minneapolis, MN), and mouse anti-human smooth muscle actin (SMA, myocyte marker, Thermo Scientific, Waltham MA); for endodermal cell lineages: mouse anti-human pan keratin (Santa Cruz, CA); rabbit anti-human α-fetoprotein (Dako, Santa Clara, CA); and mouse anti-human cytokeratin 7 (Millipore, Billerica, MA); and for ectodermal cell lineages: mouse anti-human neuron specific enolase (NSE, Millipore, Billerica, MA); rabbit anti-human glutamate receptor (Abcam, Cambridge, MA); rabbit anti-human NeuroD (Chemicon, Temecula CA); mouse anti-human nestin (Chemicon, Temecula CA); and rabbit anti-human microtubule-associated protein 2 (MAP2, AbDSerotech, Raleigh, NC). All primary antibodies were diluted 1:200 in PBS/0.1% BSA solution. Following treatment with primary antibodies, cells were washed 3 times with PBS and incubated for 1 hour at R/T with PBS/0.1% BSA containing secondary immunofluorescent antibodies (1:1000) Alexa Fluor 488 conjugated dye (mouse or rat, Invitrogen, Carlsbad, CA) or Texas Red conjugated dye (rabbit, Invitrogen, Carlsbad, CA). Cells were washed 4X with PBS and treated with PBS/0.2% DAPI for 10 minutes. Cells were then washed 3X with PBS. Images were acquired with an Evos immunofluorescence inverted microscope (Advanced Microscopy, Mill Creek, WA).

Induced Differentiation of Muse-ATs

Various differentiation media were used to induce differentiation of Muse cells-AT to the three germline cell lineages. For adipocyte formation, adherent Muse-AT cells were treated with adipogenic differentiation medium containing DMEM with 0.5 mM isobutylmethylxanthine, 1 µM dexamethasone, 10 µM insulin, 200 µM indomethacin and PPAR-γ (ZenBio, Inc, Research Triangle Park, NC) over 3 or 6 days at 37°C and 5% CO2. Adipocytes were detected using fluorescence lipid drop marker BODIPY-C16 (1:1000, Invitrogen, Carslbad, CA) following manufacturer specification.

For myocyte formation, adherent Muse-AT cells were incubated in DMEM containing with 10% FBS, 5% NHS, 50µM hydrocortisone, and 1% antibiotic-antimycotic solution over 3 or 6 days at 37°C and 5% CO2. Smooth muscle cells were identified by expression of smooth muscle actin (SMA) and skeletal muscle cells myosin D.

For hepatocyte and biliary cell induction, adherent Muse-AT cells were incubated in hepatocyte differentiation medium for 3 or 6 days, as previously described adherent Muse-AT cells were incubated in DMEM supplemented with 10% FBS, 10 µg/ml insulin, 5.5 µg/ml transferring, 6.7 ng/ml sodium selenite (ITS; Gibco, Life Technologies, Grand Island, NY), 10 nM dexamethasone (Sigma-Aldrich, St. Louis, MO), 100 ng/ml hepatocyte growth factor (HGF, Peprotech, Rocky Hill, NJ) and 50 ng/ml and fibroblast growth factor- 4 (FGF-4, R & D Systems, Minneapolis, MN) for 3 or 6 days. Hepatocytes were identified by immunohistochemistry using cytokeratin 7 and α-fetoprotein expression.

For neural cell formation, Muse cells-AT were incubated as non-adherent cells in ultra-low attachment plates (Corning Incorporated, Life Sciences, Manassas, VA) in the presence of neural differentiation medium 1 containing Neurobasal medium (Gibco, Life Technology, Grand Island, NY) supplemented with B-27 supplement serum free (Gibco, Life Technology, Grand Island, NY), 100 µg/ml kanamycin (Gibco, Life Technology, Grand Island, NY), 2 mM glutamine (Sigma-Aldrich, St. Louis, MO), 30 ng/ml bFGF (Peprotech, Rocky Hill, NJ) and 30 ng/ml EGF (Peprotech, Rocky Hill, NJ) for 7 days. Cells were then transferred to polystyrene culture slides (BD Biosciences, San Jose, CA) and cultured for another 7 days as adherent cells in the presence of neural differentiation medium 2 containing 1 DMEM supplemented with 2% FCS, 25 ng/ml bFGF and 25 ng/ml BDNF (Peprotech, Rocky Hill, NJ). Neural cells were identified by immunohistochemistry using nestin and MAP2 as indicated above.

Microarray Analysis

Muse-AT cells and ASCs were isolated from lipoaspirate material of three different patients. RNA was extracted using an RNeasy Mini Kit (Qiagen) and analyzed by Hokkaido System Science Co. Ltd. Array signals were processed and normalized using the GeneSpring GX version 12.1.0 (Agilent Technologies). Data has been deposited into the Gene Expression Omnibus databank with the access number GSE46353. The criteria for selecting differentially-expressed genes were preset as at least 2-fold difference in either direction plus statistical significance (P<0.05, unpaired t test). Microarray analysis was performed using the software program IPA via a license to Ingenuity (https://analysis.ingenuity.com/pa/login/​login.jsp) to identify (1) functional pathways (cell function, physiological function, diseases), (2) canonical signaling pathways (3) networks of related genes derived from genes changed in the analyzed comparisons and (4) upstream regulators. Further information regarding gene function was obtained from the program GeneDecks V3 at www.genecards.org. Statistical analyses were carried out by Fischer’s exact test (as performed automatically by the software). In determining which genes are only expressed in either Muse-ATs or ASCs, all samples, having been performed in triplicate, had to display uniform detection (indicated with at least 100 standard units) or absence (at most 30 standard units) along with a P-value <0.05.

Awakened by Cellular Stress：Isolation and Characterization of a Novel Population of Pluripotent Stem Cells Derived from Human Adipose Tissue（1）

Saleh Heneidi, Ariel A. Simerman, Erica Keller, Prapti Singh, Xinmin Li, Daniel A. Dumesic, Gregorio Chazenbalk  

Published：June 05, 2013

DOI：10.1371/journal.pone.0064752

Abstract

Advances in stem cell therapy face major clinical limitations, particularly challenged by low rates of post-transplant cell survival. Hostile host factors of the engraftment microenvironment such as hypoxia, nutrition deprivation, pro-inflammatory cytokines, and reactive oxygen species can each contribute to unwanted differentiation or apoptosis. In this report, we describe the isolation and characterization of a new population of adipose tissue (AT) derived pluripotent stem cells, termed Multilineage Differentiating Stress-Enduring (Muse) Cells, which are isolated using severe cellular stress conditions, including long-term exposure to the proteolytic enzyme collagenase, serum deprivation, low temperatures and hypoxia. Under these conditions, a highly purified population of Muse-AT cells is isolated without the utilization of cell sorting methods. Muse-AT cells grow in suspension as cell spheres reminiscent of embryonic stem cell clusters. Muse-AT cells are positive for the Pluripotency markers SSEA3, TR-1-60, Oct3/4, Nanog and Sox2, and can spontaneously differentiate into Mesenchymal, endodermal and ectodermal cell lineages with an efficiency of 23%, 20% and 22%, respectively. When using specific differentiation media, differentiation efficiency is greatly enhanced in Muse-AT cells (82% for mesenchymal, 75% for endodermal and 78% for ectodermal). When compared to adipose stem cells (ASCs), microarray data indicate a substantial up-regulation of Sox2, Oct3/4, and Rex1. Muse-ATs also exhibit gene expression patterns associated with the down-regulation of genes involved in cell death and survival, embryonic development, DNA replication and repair, cell cycle and potential factors related to oncogenecity. Gene expression analysis indicates that Muse-ATs and ASCs are mesenchymal in origin; however, Muse-ATs also express numerous Lymphocytic and Hematopoietic genes, such as CCR1 and CXCL2, encoding chemokine receptors and ligands involved in stem cell homing. Being highly resistant to severe cellular stress, Muse-AT cells have the potential to make a critical impact on the field of regenerative medicine and cell-based therapy.

Citation: Heneidi S, Simerman AA, Keller E, Singh P, Li X, et al. (2013) Awakened by Cellular Stress: Isolation and Characterization of a Novel Population of Pluripotent Stem Cells Derived from Human Adipose Tissue. PLoS ONE 8(6): e64752. doi:10.1371/journal.pone.0064752

Editor: Alexander V. Ljubimov, Cedars-Sinai Medical Center, United States of America

Received: February 7, 2013; Accepted: April 17, 2013; Published: June 5, 2013

Copyright: © 2013 Heneidi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: Part of these studies were supported by the Department of Obstetrics/Gynecology at University of California Los Angeles and by the Eunice Kennedy Shriver National Institute of Child Health & Human Development, National Institutes of Health through cooperative agreement U54 HD071836. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Cellular stress is induced by abrupt disruption of the physiological niche: the optimal home most conducive to cell survival. Although adult stem cells have been considered an attractive source for cell therapy, their effectiveness and efficiency is hindered by a frequently low survival rate due to their exposure to a high cellular stress environment upon transplantation. This key limitation is observed when utilizing adult stem cells for regenerative purposes, as typical cell engraftment yields are extremely low (<3%). Multiple factors contribute to this low rate of cell survival, including the harsh environment of the recipient site, harboring pro-apoptotic factors including hypoxia, malnutrition, pro-inflammatory cytokines and reactive oxygen and nitrogen species. The severity of cellular stress is heightened when stem cells are administered to an acutely injured area, such as a myocardial infarction, stroke, or a peripheral ischemic injury, as are the chances of unwanted activation or differentiation of surviving cells. It is extremely difficult to alter the environment of the damaged tissue, which necessitates a viable alternative: to improve post-transplant stem cell survival rates through the administration of a stem cell population with the adaptations necessary for survival in the hostile host environment.

One potential solution to this problem is to gradually adapt stem cells to cellular stress prior to cell delivery. It has been shown that introducing stem cells to hypoxic conditions in vitro for a duration of 24–48 hours, also known as Hypoxia preconditioning (HPC), provides the opportunity for these cells to adapt to low oxygen concentrations, thus increasing chances for survival upon reintroduction to hypoxic conditions in vivo. HPC is a promising solution to the severe apoptosis that accompanies transplantation as it induces an adaptive mechanism that increases the likelihood of cell survival in a pro-apoptotic microenvironment in vivo. Adult human Mesenchymal stem cells (MSCs) and adult Hematopoietic stem cells (HSCs) have similarly been shown to increase expansion, survival, and self-renewal under hypoxia conditions while maintaining the capability for multi-lineage differentiation.

Another potential solution to the problem of successful delivery of stem cells to a hostile host environment is to utilize a purified population of stem cells, isolated during exposure to severe cellular stress conditions (e.g. long time incubation to proteolytic enzymes, hypoxic conditions, serum deprivation, low temperatures), for engraftment. Recently, a new stem cell population has been isolated from mesenchymal tissues such as human skin fibroblasts and bone marrow stromal cells under cellular stress conditions. These cells, termed Multilineage Differentiating Stress-Enduring (Muse) Cells, are of Mesenchymal stem cell origin and comprise 1–3% of the entire cell population. Muse cells exhibit characteristics of both Mesenchymal and Pluripotent stem cells. They are double positive for CD105, a mesenchymal stem cell marker, and Stage specific embryonic antigen-3 (SSEA3), well known for the characterization of undifferentiated human embryonic stem cells (ES) from bone marrow aspirates or from cultured mesenchymal cells such as bone marrow stromal cells and dermal fibroblasts. They express Pluripotency markers including Oct3/4, Nanog and Sox2, differentiate into cells of ectodermal, endodermal, and mesodermal lineages both in vitro and in vivo, and have the ability to self-renew. Advantageously, Muse cells do not appear to undergo tumorigenic proliferation, and therefore would not be prone to produce teratomas in vivo, nor do they induce immuno-rejection in the host upon autologous transplantation. In addition, Muse cells are shown to home into the damage site in vivo and spontaneously differentiate into Tissue specific cells according to the Microenvironment to contribute to Tissue regeneration when infused into the blood stream. Therefore, they exhibit the potential to make critical contributions to tissue regeneration in the absence of restrictions attributed to the difficult extraction of bone marrow stromal cells and human skin fibroblasts, and time-consuming purification methods such as cell sorting. In order to increase the viability of Muse cells as a source of tissue regeneration, a more accessible supply must be utilized.

Harvesting human adipose tissue by Lipoaspiration is a safe and non-invasive procedure, and hundreds of millions of cells can be isolated from 1–2 liters of lipoaspirate material. Therefore, adipose tissue could prove the ideal source for Muse cell isolation as opposed to bone marrow or dermis. Using lipoaspirate material, we developed a novel methodology for the isolation of a population of human Muse cells under Severe cellular stress conditions (long term incubation with Proteolytic enzyme, 4°C, serum deprivation, and Hypoxia). Purification of human Muse cells derived from adipose tissue (Muse-ATs) does not require the use of cell sorting, magnetic beads or special devices. Muse-ATs can grow either in suspension, forming cell spheres, or as adherent cells forming cell aggregates similar to human ES cell-derived embryoid bodies as previously reported. Furthermore, Muse-AT cells express Pluripotent stem cell markers and a variety of markers indicative of all three germlines. Upon the introduction to specific culture conditions, Muse-AT cells can differentiate to mesenchymal (adipocytes, skeletal and smooth muscle cells), endodermal (hepatocytes and biliary ducts) and ectodermal (neural cells) cell lineages both spontaneously and by differentiation induction. Immunocytochemistry and microarray data demonstrate Up-regulation of the Pluripotent stem cell markers Sox2, Oct3/4, and Rex1 in Muse-AT cells, as compared to previously studied multipotent adipose stem cells (ASCs). Microarray analysis reveals that Muse-AT cells highly express genes involved in Cellular protection against Oxidative stress. Additionally, these cells also exhibit up regulation of CXCL2 gene expression, a critical Chemokine involved in stem cell Homing. Muse-AT cells display down regulation of genes involved in cell death and survival, embryonic development, organism survival, cellular assembly and organization, mitosis, DNA replication, recombination and repair. Because lipoaspiration is a safe and non-invasive procedure and Muse-AT cell isolation requires a simple yet highly efficient purification technique, Muse-AT cells could provide an ideal source of pluripotent-like stem cells with the potential to have a critical impact on regenerative medicine and cell-based therapy.

Muse cell（Multi-lineage differentiating Stress Enduring cell）

Muse cell（Multi-lineage differentiating Stress Enduring cell）is a newly discovered non-tumorigenic pluripotent Stem cell.

Muse cells reside in mesenchymal tissues such as Bone marrow, Dermis and Adipose tissue as well as in commercially obtainable mesenchymal cells（human fibroblasts and bone marrow）. Muse cells can be obtained from：Bone marrow aspirate、Adipose tissue and liposuction、Dermis、Commercially available culture cells such as：Bone marrow-derived mesenchymal stem cells、Fibroblasts、Adipose-derived stem cells.

Muse cells are able to generate cells representative of all three germ layers from a single cell both spontaneously and under cytokine induction.

Muse cells do not undergo teratoma formation when transplanted into a host environment in vivo. This can be explained in part by their intrinsically low telomerase activity, eradicating the risk of tumorigenesis through unbridled cell proliferation.

Muse cell

• Pluripotent stem cells, which can generate various kinds of the cells representative of all three germ layers have the ability to self-renew.

• Non-tumorigenic.

• Exhibit Tissue repair effect when supplied to the blood stream.

• Can be collected from Bone marrow, Dermis, Adipose tissue and commercially available Fibroblasts.

• Comprise ~0.03% of bone marrow transplantation and several % of mesenchymal stem cell transplantation.

• Can be isolated as cells positive for SSEA-3, a well known human embryonic stem cell marker.

• Pluripotent stem cells can be directly obtained from normal human mesenchymal tissues without using artificial manipulations such as gene introduction.

Muse cells are identified as cells which

• are positive for SSEA-3+, a well-known marker for undifferentiated human ES cells. Cell isolation by SSEA-3 cell sorting can be done using SSEA-3 antibody.

• are positive for general mesenchymal stem cell markers such as CD105, CD90 and CD29.

• are double positive for Pluripotent and Mesenchymal stem cell markers.

• do not express CD34（hematopoietic and adipose stem cell markers）and CD117（hematopoietic stem cells markers）, Snai1 and Slug（skin-derived precursors markers）, CD271 and Sox10（neural crest-derived stem cells markers）, NG2 and CD146（perivascular cells）or CD31 and von Willebrand factor（endothelial progenitor markers）. This indicates that Muse cells do not belong to previously investigated stem cell types.

Muse cell in regenerative medicine

• Bone marrow transplantation：Muse cells are a subpopulation of bone marrow cells. They represent a small population of mono-nucleated bone marrow cells（～0.03%）. This means that they have already been supplied to patients many times all over the world in bone marrow transplantations; a well-known procedure that has been performed in clinics since 1958.

• Mesenchymal stem cell transplantation：Muse cells exist within cultured MSCs such as bone marrow mesenchymal stem cells and adipose-derived stem cells. MSC transplantation has been employed for repairing liver, heart, neural tissue, airway, skin, skeletal muscle, and intestine. Therefore, if Muse cells were purified or enriched, the effectiveness of currently performed MSC transplantation is expected to see vast improvements.

• Because Muse cells do not form teratomas in vivo, they could provide an ideal source of Pluripotent stem cells for Regenerative medicine and Cell-based therapy.

幹細胞治療效果有待檢驗

作者：洪欣慈╱台北報導 | 中時電子報 – 2014年4月13日

中國時報【洪欣慈╱台北報導】

幹細胞一直被脊髓損傷者、神經疾病患者視為治癒的一線曙光，數年前前總統陳水扁孫子出生時保留下來的臍帶血，也被寄予讓其夫人吳淑珍再站起來的厚望。醫師表示，幹細胞相關研究與臨床試驗一直在進行，但要證明療效，恐還有漫漫長路要走。

台大台成幹細胞治療中心主任唐季祿表示，周邊血液幹細胞是藉由施打白血球生長激素（G-CSF），將骨髓中的幹細胞驅動至血液中，再經由血液分離機收集取得，目前主要用於治療血癌等血液疾病，抽取技術已十分純熟。

唐季祿說，過去國內外有許多研究，欲試驗周邊血液幹細胞對於帕金森氏症、中風等神經疾病的療效，但都還有爭議，原因在於中風病人經過一段時間，也會自己慢慢恢復機能，是否由幹細胞促成不得而知，國內外報告結果也不一。

他表示，幹細胞要運用於治療中風，效果最好的應是神經幹細胞，但因其需在體外培養、培養成功後再放回身體裡，需嚴格的體外培養條件，國內外對這部分的法規也較嚴格。　

台大神經部腦中風加護病房主任鄭建興表示，很多國家都使用不同來源的幹細胞來進行腦中風試驗，但成效可能會依急性、慢性腦中風的不同，或患者病變狀況而有所差異。這次林欣榮的試驗看起來有得到一些效果，但要推廣到一般治療，恐怕還有很大一段距離。

馬偕醫院癌症中心主任謝瑞坤表示，人體免疫系統比想像中複雜，加上要操控生長因子並非易事，這也是多數幹細胞研究尚無法直接運用於臨床的原因，初步臨床結果還需更廣泛試驗，才能確認療效。

醫學新發現！移植自體周邊血液幹細胞改善腦中風失能

作者：華人健康網記者洪毓琪／台北報導 | 華人健康網 – 2014年4月12日

中風不僅會手腳發麻，還有可能出現眩暈、嘔吐、頭痛、步態不穩等 …

中風之所以令人害怕，除了它經常突然發生，事先沒有明顯的預兆、即易導致猝死外，就是即使將中風患者從鬼門關救回來，通常會留下行動不便的後遺症，嚴重影響日後生活。不過，針對這樣的情形未來也許有治療、改善的機會。在台灣醫師將於國際期刊「細胞移植Cell Transplantation」發表的臨床試驗成果指出，移植自體周邊血液幹細胞，可有效改善中風患者的身體失能。

中國醫藥大學北港附設醫院暨安南醫院院長林欣榮表示，國內每年有10萬以上的中風新增病例，使得腦中風成為導致國人殘疾的主因。一般來說，當中風沒有早期發現，在發病6個月以上就進入慢性期，復健較難再有起色，這是因為腦部神經受損，一直缺乏有效療法來改善。

該項臨床試驗，是透過收集患者本身的周邊血液幹細胞，再利用腦部定位技術，將幹細胞植入腦中，透過幹細胞的修復、再生功能，讓患者中風受損的腦神經再生，恢復原本的功能。這項試驗相當成功，術後患者的各項腦中風評估指數，都有明顯提升。影像醫學也顯示，腦部受傷部位的神經有再生現象。試驗過程未見嚴重副作用，證實其安全無虞。

2期臨床試驗結果成功　進行第3期臨床試驗確認可行性

林欣榮醫師表示，目前完成的二期臨床試驗結果，已被國際知名期刊《細胞移植》（Cell Transplantation）所接受，將於近期內刊出。這是全球首度進行的嘗試，希望能為再生醫學帶來新思維。接下來團隊將展開第三期臨床試驗，進一步確認該療法的可行性。除此之外，林欣榮醫師還將與全球脊椎治療權威，美國紐澤西Rutgers大學教授楊詠威（Dr. Wise Young）一同合作，進行臍帶血幹細胞治療脊髓損傷的試驗。

【醫學小知識—周邊血液幹細胞】

幹細胞只存在於骨隨之中？事實上，平常在骨髓內負責人體白血球，紅血球及血小板的製造幹細胞（Stem cell），在人類出生後，雖然主要分佈在人體的骨髓中，但其周邊血液中，仍有其少量的幹細胞存在，不過由於含量極低，在以往無法透過分離得到足夠幹細胞作為臨床移植用。

而周邊血液幹細胞則是指藉由施打白血球生長激素（G-CSF），將骨髓中的幹細胞驅動至血液中，再經由血液分離機收集取得之幹細胞的方式。由於與骨髓幹細胞極為相近，現已逐漸取代需要全身麻醉的骨髓抽取手術。

脂肪幹細胞療效好「肌萎縮性脊髓側索硬化症」漸動人家屬盼人體試驗

中廣新聞網 – 2014年4月12日

（張文祿報導）

一名四十歲的手機晶片硬體工程師，兩年前還是一個健康的中年人，現在卻已經無法抱起四歲女兒，言語表達也相當困難，醫師診斷他罹患「肌萎縮性脊髓側索硬化症」，也就是漸凍人症，由於國內醫藥大學跟生技公司合作，以自體脂肪幹細胞應用在漸凍鼠身上，有良好療效，八個月前申請人體試驗，遲遲沒有下文，由於漸凍人發病後，三到四年內會死亡，家屬今天懇求衛福部儘快審核通過，讓郭姓工程師可以延續生命。

郭姓工程師，曾到紐約攻讀電機碩士，平時健康愛運動，但是兩年前，卻開始沒有力氣，容易扭傷，喝水容易嗆到，最後被診斷出罹患漸凍人症，現在已經無法抱起四歲女兒，講話也很困難，一家人陷入愁雲慘霧中，郭先生自知來日無多，願意用僅存的千萬積蓄治療，換取生存的機會，而中國醫藥大學北港附設醫院院長林欣榮跟光麗生醫合作，進行漸凍鼠自體脂肪幹細胞治療，有很大的進展，八個月前申請衛福部人體試驗，但是還沒有核准，郭先生跟太太及媽媽站出來，自願成為試驗對象，郭媽媽說，服貿爭議問題都可以這麼快有進展，希望有關人命的問題，可以加速進行。

林欣榮院長說，自體脂肪幹細胞治療，不必再像以前需要大傷口手術，只需局部麻醉，進入腦部即可，而且可以體外大量培養，不會有免疫排斥。

另外，中國醫藥大學附設醫院與美商永生臍帶血公司也在台中金典酒店舉辦第7屆泛太平洋國際幹細胞及癌症研究研討會，林欣榮院長也在會中發表以幹細胞治療腦中風的臨床試驗成果，移植自體周邊血液幹細胞，可有效改善中風患者的身體失能狀況。