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DR.WU粉餅 驗出有毒重金屬鋇(Barium

20140416

蘋果日報

(方翊倩/綜合報導)

DR.WU粉餅,驗出有毒重金屬鋇。翻攝《壹週刊》

DR.WU粉餅,驗出有毒重金屬鋇。翻攝《壹週刊》

DR.WU登記的土城工廠地址,未見門牌號碼。翻攝《壹週刊》

DR.WU粉餅,驗出有毒重金屬鋇。翻攝《壹週刊》

DR.WU粉餅,驗出有毒重金屬鋇。翻攝《壹週刊》

 

《壹週刊》抽驗美妝龍頭DR.WU旗下產品,意外發現其中一項粉餅產品檢驗出有毒重金屬鋇(Barium),其數值還比環保署公告的污水排放容許值高出6倍。

今日出刊的《壹週刊》報導,《壹週刊》在北市新光三越A8館購買DR.WU旗下產品,包括「礦質無暇雙效粉餅(自然色)」和「角鯊潤澤修護精華」,送交SGS(台灣檢驗科技股份有限公司)進行常見的八大重金屬檢驗。結果礦質無暇雙效粉餅(自然色)驗出鋇。

327日再度購買DR.WU礦質無暇雙效粉餅(自然色)和礦質無暇雙效粉餅(白皙色)送驗。結果礦質無暇雙效粉餅(自然色)鋇含量高達6.76ppm,白皙色粉餅鋇含量是5.12ppm

醫師表示,重金屬鋇具有心臟、神經毒性,吸入或誤食後,會產生心律不整和血壓升高的症狀,嚴重會造成呼吸衰竭,以及傷害肝臟、腎臟功能,如果皮膚接觸到鋇,也會有刺激性,輕則發癢,重則潰爛。

DR.WU天昱生物科技公司田姓公關表示,礦質無暇雙效粉餅是委託韓國製造,他們沒有添加這個成分,為何會有鋇?可能是原料天然礦物晶石有鋇所致,公司事先有把關送驗。

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B15A00_P_04_01  

1985416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

20140416

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,26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70008000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,33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。

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20140416000017  

英國東倫敦大學21歲男子毀容手術重現俊臉成模特兒參加時裝秀

20140416

旺報即時吳貴奉

羅賓斯整容前後對比照片。(摘自環球網)

 

模特兒一般對相貌要求很高,但英國一名曾經毀容的男子最後圓了自己的模特兒夢。據香港《東方日報》415日報導,英國東倫敦大學21歲男生羅賓斯樣貌俊朗,且獲模特兒公司相中,準備向模特兒工作發展。但沒想到後來因事故遭到暴打被毀容。

據環球網報導說,在20131221日晚,羅賓斯在科爾賈斯特一間酒吧外,見到一名女子遭6人圍住。他上前為女子解圍時,反遭圍毆,導致鼻骨、頰骨和眼窩被打傷。

羅賓斯在醫院看見自己樣貌被毀,以為模特兒夢已粉碎。但經過4個多月的治療,醫生為他進行臉部重整手術,包括固定鼻骨和在頰骨植入金屬片,羅賓斯最後重現俊臉。雖然他的左臉還有少許腫,和鼻子有疤痕,但他於2周前終得償宿願,首次踏上「天橋」,參加了時裝秀。

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B07A00_P_02_02B07A00_T_02_03  

國健署:肥胖、少運動、飲食不健康三大新興致癌因子

20140416

中國時報洪欣慈/台北報導

國健署指出,不健康的飲食、肥胖率高及運動量少是台灣人癌症的3項新型殺手。圖為民眾在休閒中心運動的畫面。(本報資料照片)

3大新興致癌因子

 

台灣癌症人數居高不下,致癌因子除了已知的菸酒、檳榔外,不健康的飲食、肥胖及運動量少,更是癌症的3項新型殺手。

國健署指出,肥胖者罹患乳癌、子宮內膜癌、結直腸癌等疾病的危險性皆較常人高12倍;身體運動量不足,也與乳癌、大腸癌息息相關。

國健署長邱淑媞表示,根據FAO(世界農糧組織)統計,台灣在肉類及油脂性食物的可獲性都高過日本、韓國等鄰近亞洲國家,顯示肉類和油脂性食物在國人飲食結構中相當重要。

愛吃肉卻少吃菜、少運動,國健署統計顯示,國內有150019歲以上成年人每日蔬果攝取量未達建議標準;男女缺乏運動的比率分別達64.4%73.1%,與OECD(經濟合作暨發展組織)其他30個國家相比,各排名第二及居冠。

中研院生物醫學研究所研究員潘文涵表示,肥胖、運動不足、不健康飲食三者息息相關,吃不對卻又不運動,等同雪上加霜;肥胖之所以成為新興致癌因子,原因在於肥胖細胞會分泌賀爾蒙、促進癌症發展,也會讓身體發炎指數升高、降低免疫力

她進一步說,飲食不當與各種癌症也都有關係,像食道癌與飲食過燙有關,大腸直腸癌則可能是攝取過量油脂,蔬果攝取量不足也與許多癌症密切相關。

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ihXd7Zuw4ybQ  

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Awakened by Cellular StressIsolation and Characterization of a Novel Population of Pluripotent Stem Cells Derived from Human Adipose Tissue3

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.

 journal.pone.0064752.g001

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).

journal.pone.0064752.g002 

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

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64960-XXLA26A00_T_04_02  

台積電法說陸行之提四問

鉅亨網新聞中心 來源:聯合報系/udndata.com 2014-04-14

 

台積電法說會本周四登場,挑起敲響台股2014年首季法說行情重任,成為法人觀察半導體產業景氣、甚至台股後續走勢的重要指標。外資圈包括巴克萊、麥格理率先上調台積電目標價,對營運展望投下肯定票。

巴克萊亞太區半導體產業首席分析師陸行之延續慣例,在法說會前端出「四問台積電」。分別是:

一、至明年下半年前,台積電如何規畫20奈米與16奈米的產能?

二、20奈米、16奈米量產除可推升營收,台積電認為對整體毛利率影響為何?

三、台積電未來二年如何看待指紋感測器、心跳感測、智慧手表、智慧眼鏡等產品的營收貢獻?

四、台積電對2014年營收成長預估值是否有上調至二成的機會?

 

陸行之說,台積電不僅首季可繳出每股純益上看1.75元的成績單,展望第2季,受惠蘋果、高通、聯發科等大廠需求,台積電的28奈米、20奈米金屬閘極(HKMG)製程將放量,第2季營收季增率可達14%18%,產能利用率也會來到100%水準。

就個別公司競爭力分析,德意志證券半導體產業分析師周立中認為,因二線晶圓廠的良率相對較低,會驅使客戶積極向台積電下單,預料一直到年底前,台積電2820奈米等製程產能都將十分吃緊。半導體一線大廠獨霸局面,儼然成形。

麥格理證券亞太科技產業研究部主管蘇志凱對台積電第2季營運展望則更加樂觀。麥格理估計,台積電第2季將交出營收季增二成成績單,更引人注目的是,從本季起至2015年底,台積電每一季的每股純益都將突破2元。換言之,半導體龍頭的營運榮景,現在還只是開端,未來會更亮麗。

【記者簡威瑟、魏興中/台北報導】

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704_565930_860329704_565937_538035  

保有適量脂肪才能維持身體健康不要過分在意體重反而維持適量活動、規律又均衡的飲食方式才是保持健康的最好方法

作者:【記者陳敬哲/綜合外電報導】 | 台灣新生報 – 2014415

 

健康是否與瘦直接劃上等號,可能要重新思考。學術期刊Journal of Epidemiology中提出,不到平均年紀死亡的民眾,70%體重過輕,人數遠大於身體肥胖者;然而社會風氣不斷將瘦與健康劃上等號,讓許多人不斷節食害怕體重上升,但身體保有適量脂肪,才能維持健康

美國心臟科醫師Carl Lavie提出,健康身體絕對不是瘦,而是保持良好的脂肪與肌肉比例,體重絕對不是魔鬼,某些方面,身體需要脂肪才能打敗疾病,但現在激進的社揮風氣,認為體脂肪是萬惡來源,不斷壓低身體質量指數,盡可能讓身體脂肪量趨近於零,可是這絕對不是健康之道,反而造成傷害。

民眾絕對不要過於擔心體脂肪,美國一名61歲男性,20年前曾經心臟病發,目前體重約86公斤,雖然體重超標,但醫生建議不要過分在意體重,反而維持適量活動、規律又均衡的飲食方式,才是保持健康的最好方法Carl Lavie提醒,許多美式足球運動員,雖然體重超出標準,但不代表不健康

身體質量指數能夠計算脂肪比率,超過25達到超重,超過30達到肥胖,但Carl Lavie提醒,體脂肪絕對不是惡夢,體脂肪比率超重的民眾,其實不需要過份擔心,只要每天保持活動,營養均衡的飲食方式,不會因多出標準體重10公斤導致傷害,仍然可以很健康維持生活。

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Awakened by Cellular StressIsolation and Characterization of a Novel Population of Pluripotent Stem Cells Derived from Human Adipose Tissue2

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.

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Awakened by Cellular StressIsolation and Characterization of a Novel Population of Pluripotent Stem Cells Derived from Human Adipose Tissue1

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

PublishedJune 05, 2013

DOI10.1371/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.

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