轉譯醫學 連結實驗室與診療室的天橋 以幹細胞及臍帶血應用為例 清大生科系莊永仁. Original image from:

Transcription

1 轉譯醫學 連結實驗室與診療室的天橋 以幹細胞及臍帶血應用為例 清大生科系莊永仁 Original image from:

2 Translational Medicine: By drawing upon discoveries made in the basic sciences: biochemistry, immunology, engineering and physics, we are "translating" these advances into changes in the management of diseases. Bench to bedside: This collaboration between basic scientists and clinician investigators forges a bridge between the scientific research bench to the patient's bedside.

3 What s is Research for? 求知, 求真 What s is Medicine for? 療癒病患

4 求知 求真? 療癒病患

5 Clinical Research Teaching/Training Non-clinical research Clinical Practice Research Institutes Universities Patient Clinic

6 Clinical Research Basic Research Lab-oriented Research In vitro studies Animal model pathophysiology Disease mechanism research Patient-oriented research Human pathophysiology Dx/Rx disease Public Health Epidemiology Behavioral studies Health Service Clinical Practice Clinical Trials

7 Where can emerging science make an impact in Biopharm pipeline? Systems Biology Pharmaco phore data Imaging Quality by design Biopharm production Biopharmacueticals Virtual Screening Predictive Toxicology Translational science Innovative trial design Target Id. Hit Id. Lead Id. Lead Opt. Prenom. Preclin. Dev. Principle Testing Concept testing Dev. for Launch Launch and PLC Prod. Maint. Cell-based & novel disease models Stem cell tox models Formulation & Nanotech. Pharmacodynamics Stratified medicines Structural biology Absorption models Biomarkers Combination therapies Healthcare information Disease prevention vaccines Microdosing

8 Preface

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10 Embryonic Development ( 胚胎發育 ) From one cell to a whole organism (Example: Zebrafish development) video clip: replay speed ~3000:1 fast-forward

11 Wolpert fig 2.21

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14 Cancer cell international 2007, 7:9

15 Properties of Stem cell

16 Key Definitions: Totipotent: toti-, from Latin totus, means whole 全能 = whole potent can form every cell type, including germ cells Pluripotent: pluri-, from Latin plur-, means more, 多高能 several, more than one can form many different derivatives, but not germ cells Multipotent: from Latin multus- means much or many. 多能 can form a few different cell types toti > pluri > multi

17 Self-Renewal Stem Cell - Definition It can divide without limit ( 可無限分裂 ); at least for the lifetime of animal It is not itself terminally differentiated ( 未終止分化 ) ; Not at the end of differentiation pathway Potency When it divides each daughter cell has a cell fate choice ( 子代細胞擇定分化途徑 ); it can remain a stem cell, or it can embark on a course leading to terminal differentiation

18 Where can you find stem cells?

19 Sources of Stem Cells Stem cell type Embryonic 胚胎 Fetal stem cells 胎兒 Umbilical cord blood stem cells 臍帶 Placenta derived stem cells 胎盤 Adult stem cells 成人 Description Cells from human blastocysts Cells from gonads of aborted fetuses Cells from the umbilical cord blood of newborns Cells from the placenta of newborns Cells from adult tissues, such as bone marrow

20 ADULT STEM CELLS ARE OBTAINED IN A VARIETY OF WAYS Illustration by Matt Bohan, 2007

21 STEM CELLS HAVE ALSO BEEN FOUND IN MATURE ORGANS Illustration by Matt Bohan, 2007

22 Summary Stem cell type Description Examples Totipotent Pluripotent 多高能 Multipotent Each cell can develop into a new individual Cells can form any (over 200) cell types Cells differentiated, but can form a number of other tissues Cells from early embryos (1-3 days) Some cells of blastocyst (5 to 14 days) Fetal tissue, cord blood, and adult stem cells

23 How do you obtain the embryonic stem cells?

24 Cloning ( 複製 ) Reproduced by permission of the NIH Purpose of therapeutic cloning is to harvest ES cells for treatment blastocyst destroyed Purpose of reproductive cloning is to make new person blastocyst implanted in uterus

25 Cloning Dolly 桃莉羊

26 Complications Observed in Cloned Animals Extremely low reproductive efficiency ( 低繁殖率 ) High late fetal losses ( 高死胎率 ) Placental abnormalities ( 胎盤異常 ) Increased birth size ( 巨嬰症 ) Large offspring syndrome Risk to mother and offspring High early neonatal death rates( 高夭折率 ) Respiratory deaths

27 Stem Cell Experiments

28 Tissue Culture Hood for Stem Cell Research You need a controlled, clean and sterile working are

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30 Unfertilized egg Glass needle Suction

31 Egg s genetic material

32 Egg nucleus Nucleated egg

33 Nucleus

34 Embyro

35 Hatched Blastocyst Trophoblast Inner cell mass Cavity Blastocyst

36 Membrane-like Placental ESC

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38 Divergent Religious View on Research and Clinical Use of ESC Religions Catholic 天主教徒 Muslim 伊斯蘭教信徒 Jewish 猶太人 Buddhist 佛教徒 Embryonic Stem Cell (ESC) Research Prohibited (life begins at conception) Acceptable (fetus has moral existence only at the end of the 4 th month) Acceptable (embryo has no moral status until 40 days) Prohibited (life begins at conception)

39 Pros and Cons of Using Embryonic Stem Cell Pros Stem cells can potentially develop into all cell types. Cons Require the killing of an embryo. Difficult to develop and maintain. Unstable and mutate in culture. Pure embryonic stem cells cultures are difficult to obtain.

40 Pros and Cons of Using Adult Stem Cell Pros Do not require the killing of an embryo. Adult stem cell research is more advanced because of its four decade head start over embryo stem cell studies. Cons Difficult to obtain, grow slower, and are less robust when compared to cells extracted from embryos. Stem cells for all cell and tissue types have not yet been found in the adult human. Stem cells in adults are often present in only minute quantities, are difficult to isolate and purify, and their numbers may decrease with age.

41 Stem Cells in Bone Marrow ( 骨髓 ) & Umbilical Cord Blood ( 臍帶血 )

42 Hematopoietic Stem Cells ( 造血幹細胞 )

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44 Hematopoietic Stem Cells ( 造血幹細胞 ) Blood Lines by Donald Metcalf

45 Mesenchymal Stem Cells ( 間葉系幹細胞 )

46 Multi-potent Tri-potent Bi-potent SACTO-CFU Mono-potent Stroma Adipocyte Mesenchymal Stem Cells ( 間葉系幹細胞 ) Chondrocyte Tenocyte MSC ACTO-CFU Osteocyte Neuron CFU: colony forming unit S: marrow stroma A: adipocyte C: chondrocyte T: tenocyte O: osteocyte??? Astrocyte Oligodendrocyte Smooth Muscle Cardiac muscle From Bone marrow-derived mesenchymal stem cells Leukemia & Lymphoma, November 2005; 46(11): Skeletal muscle

47 How to collect bone marrow and cord blood

48 Adult Stem Cell: Bone Marrow Extraction

49 Copyright 2002 American Society of Hematology. Copyright restrictions may apply. Figure 1. Bone marrow aspiration needles are smaller than the Jamshidi needles used to obtain bone marrow biopsies 約鉛筆心粗細 Maslak, P. ASH Image Bank 2002;2002:100529

50 Bone marrow extraction

51 Bone Marrow Figure 1. Scanning under low power reveals a heterogenous population of cells Maslak, P. ASH Image Bank 2005;2005: Copyright 2005 American Society of Hematology. Copyright restrictions may apply.

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53 Human Placenta and Umbilical Cord

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55 How to Process and Save Cord Blood Stem Cells

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59 Intermission

60 The Latest Development

61 Is this a reversible process?

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63 Time Magazine : Stem Cells: Japan Gets Ahead of the Curve Thursday, Jun. 14, 2007 By BRYAN WALSH LEADING LIGHT: Yamanaka, a former orthopedic surgeon, knows how to knit research together with practical applications

64 A breakthrough research!

65 How to turn adult skin fibroblasts into embryonic stem cells From the following article: Stem cells: The magic brew Janet Rossant Nature 448, (19 July 2007)

66 The Latest News James Thomson at U. of WI. Different set of Transcription Factors Oct4, Sox2, Nanog, Lin28 More than on GRN is involved Yu, J. et al., Induced pluripotent stem cell lines derived from human somatic cells. Science 318 (5858), 1917 (2007). c-myc minus Can now avoid the oncogene Nakagawa, M. et al., Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol (2007).

67 In this example, scientists can now avoid some ethical problems in obtaining the stem cells.

68 Taiwan NSC 97 年度幹細胞優先推動計畫徵求公告 2008/04/28 一 97 年度 幹細胞優先推動計畫 徵求重點說明摘要 幹細胞重點徵求領域範圍, 係由 95 年度科發基金補助之 幹細胞研發重點策略藍圖計畫 規劃而來, 並考量幹細胞技術發展快速, 本年度特別新增 誘導式多能性幹細胞 (ips cell) 相關研究項目 科技顧問組與國科會生物處業於 96 年度進行第ㄧ次幹細胞優先推動計畫徵求作業, 共入選四件計畫, 其內容多與幹細胞的分離 分化技術 表現機制與流程控制 基因外 ( epigenetic ) 調節等研究領域相關 為有效運用研究資源, 本年度徵求主題將以未獲補助之研究領域為優先 計畫之期程約為三年 (97/12/01~100/12/31), 每個團隊每年經費以 2000 萬元為上限

69 幹細胞研發重點領域徵求項目 1. 五項重點研究 : 幹細胞的分離 培養與分化技術 幹細胞的表現機制與流程控制 基因外 ( epigenetic ) 調節與體細胞核核轉移的技術研發 動物移植模型 生醫奈米 與組織工程相關技術的建置 誘導式多能性幹細胞 (ips cells) 相關之分析研究 * 2. 三種應用技術 : 以動物複製與異體移植模型 藥物研發 細胞治療的試驗

70 Research done in my laboratory

71 How to repair a damaged heart?

72

73 Heart Regeneration Lepillina et al., (2006) Cell

74 Zebrafish Core Facilities Collaboration Scheme NTHU Core 750 tanks (Expandable 1500 Tanks) Education & Basic Research Mutual Support Preservation NHRI Core 850 tanks Preclinical Research & Basic Research

75 Nature Reviews Genetics, Vol 8, May 2007 新興的人類疾病模式生物

76 發育快速生長週期短

77 胚胎透明利於觀察

78 與其他模式生物之+ 比較Nature Reviews Genetics, Vol 8, May 2007

79 Transgenic zebrafish allow analysis of endothelial cells in living embryos Vascular EC promoter egfp fli1-egfp transgenic embryo at 2 dpf Dorsal Longitudinal Anastomotic Vessel Intersegmental Vessels (DLAV) (Se) Posterior Cardinal Vein Dorsal Aorta (PCV) (DA) Caudal Vein Capillary Plexus 血管螢光魚

80 Advantages of Studying Circulation in Zebrafish Microangiography: Analysis of blood flow in living embryo by injecting nano-fluorphore into blood circulation 顯微血管造影

81 Our first transgenic line Heart 心臟螢光魚 Nu myl7(cmlc2)-egfp/nuclear mcherry zebrafish

82 神經圖譜 Brainbow zebrafish 5dpf Albert Pan of Harvard University

83 Zebrafish as an effective model system for Human Diseases Research and Pharmaceutical R&D Forward Genetics: ENU mutagenesis Insertional mutagenesis Genomics: Sequenced Genome cdna projects Microarrays Reverse Genetics: Transgenic fish Tilling with ENU Morpholino injection Small Molecule Screens: Predictive of higher vertebrates Delivery by injection or soaking Carcinogenesis: Aqueous delivery Similar to human tumors

84 Three layers no melanocyte no iridophore Transparent Zebrafish Casper Xanthophore only Cell Stem Cell (2008) 2:

85 What type of stem cells is the best?

86 Challenges to stem-cell therapy for cardiac disease

87 Challenges to stem-cell therapy for cardiac disease

88 Stem cell therapy and research requires Cross-disciplinary collaboration and expertise. ( 跨領域專長與合作 )