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: www.msnbc.msn.com/id/14535678/site/newsweek/

http://www.msnbc.msn.com/id/14535678/site/newsweek/

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.

http://en.wikipedia.org/wiki/Translational_research

What’s is Research for?求知，求真

What’s is Medicine for?

療癒病患

?求知

求真療癒病患

Clinical ResearchTeaching/Training

Non-clinical research

Research InstitutesUniversities

Clinical Practice

Patient Clinic

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

Where can emerging science make an impact in Biopharm pipeline?

Prod.Maint.

Launch and PLC

Dev. for Launch

Concepttesting

PrincipleTesting

Preclin.Dev.

Pre-nom.

LeadOpt.

LeadId.

HitId.

TargetId.

VirtualScreening

Predictive Toxicology

Stem cell tox models

Cell-based & novel disease

models

Translational science

Biopharm-acueticals

Innovative trial design

Pharmaco-dynamics

Formulation & Nanotech.

Stratified medicines

Combination therapies

Quality by design

Healthcare information

Imaging

Biomarkers

SystemsBiology

Microdosing

Structural biology

Absorptionmodels

Pharmacophore data

Biopharmproduction

Disease prevention vaccines

Preface

Embryonic Development (胚胎發育)

From one cell to a whole organism

(Example: Zebrafish development)video clip: replay speed ~3000:1 fast-forward

Wolpert fig 2.21

Cancer cell international 2007, 7:9

Properties of Stem cell

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

Stem Cell - Definition• Self-Renewal

– 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

Where can you find stem cells?

Sources of Stem CellsStem cell type Description

Embryonic 胚胎 Cells from human blastocysts

Fetal stem cells胎兒

Cells from gonads of aborted fetuses

Umbilical cord blood stem cells 臍帶

Cells from the umbilical cord blood of newborns

Placenta derived stem cells 胎盤 Cells from the placenta of newborns

Adult stem cells成人

Cells from adult tissues, such as bone marrow

“ADULT” STEM CELLS ARE OBTAINED IN A VARIETY OF WAYS

Illustration by Matt Bohan, 2007

STEM CELLS HAVE ALSO BEEN FOUND IN “MATURE” ORGANS

Illustration by Matt Bohan, 2007

SummaryStem cell

type Description Examples

Totipotent Each cell can develop into a new individual

Cells from early embryos(1-3 days)

Pluripotent多高能

Cells can form any (over 200) cell types

Some cells of blastocyst(5 to 14 days)

Multipotent

Cells differentiated, but can form a number of other tissues

Fetal tissue, cord blood, and adult stem cells

How do you obtain the embryonic stem cells?

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

Cloning Dolly桃莉羊

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

Stem Cell Experiments

Tissue Culture Hood for Stem Cell Research

You need a controlled, clean and sterile working are

Unfertilized egg

Suction

Glass needle

Egg’s genetic material

Egg nucleus

Nucleated egg

Nucleus

Embyro

Inner cell mass

Trophoblast

Cavity

Hatched Blastocyst

Blastocyst

ESC

Placental

Membrane-like

Divergent Religious View on Research and Clinical Use of ESC

Religions Embryonic Stem Cell (ESC) Research

Catholic天主教徒

Prohibited (life begins at conception)

Muslim伊斯蘭教信徒

Acceptable (fetus has moral existence only at the end of the 4th month)

Jewish猶太人

Acceptable (embryo has no moral status until 40 days)

Buddhist佛教徒

Prohibited (life begins at conception)

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.

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.

Stem Cellsin

Bone Marrow (骨髓) &Umbilical Cord Blood (臍帶血)

Hematopoietic Stem Cells（造血幹細胞）

Blood Lines by Donald Metcalf

Hematopoietic Stem Cells（造血幹細胞）

Mesenchymal Stem Cells(間葉系幹細胞)

Mono-potentMulti-potent Tri-potent Bi-potent

StromaAdipocyte

MSC

SACTO-CFU

??

?

ACTO-CFU

CFU: colony forming unitS: marrow stromaA: adipocyteC: chondrocyteT: tenocyteO: osteocyte

Mesenchymal Stem Cells(間葉系幹細胞)

Chondrocyte

Tenocyte

Osteocyte

Neuron

Astrocyte

Oligodendrocyte

Smooth Muscle

Cardiac muscle

Skeletal muscleFrom Bone marrow-derived mesenchymal stem cellsLeukemia & Lymphoma, November 2005; 46(11): 1531 – 1544

How to collectbone marrow and cord blood

Adult Stem Cell: Bone Marrow Extraction

Maslak, P. ASH Image Bank 2002;2002:100529

Figure 1. Bone marrow aspiration needles are smaller than the Jamshidi needles used to obtain bone marrow biopsies

約鉛筆心粗細

Copyright ©2002 American Society of Hematology. Copyright restrictions may apply.

Bone marrow extraction

http://www.emedicine.com/med/topic2971.htm#target1

http://www.emedicine.com/cgi-bin/foxweb.exe/makezoom@/em/makezoom?picture=\websites\emedicine\med\images\Large\1799image01.jpg&template=izoom2http://www.emedicine.com/cgi-bin/foxweb.exe/makezoom@/em/makezoom?picture=\websites\emedicine\med\images\Large\1800image02.jpg&template=izoom2http://www.emedicine.com/cgi-bin/foxweb.exe/makezoom@/em/makezoom?picture=\websites\emedicine\med\images\Large\1802image06.jpg&template=izoom2http://www.emedicine.com/cgi-bin/foxweb.exe/makezoom@/em/makezoom?picture=\websites\emedicine\med\images\Large\1806image09.jpg&template=izoom2http://www.emedicine.com/cgi-bin/foxweb.exe/makezoom@/em/makezoom?picture=\websites\emedicine\med\images\Large\1807image10.jpg&template=izoom2http://www.emedicine.com/cgi-bin/foxweb.exe/makezoom@/em/makezoom?picture=\websites\emedicine\med\images\Large\1808image11.jpg&template=izoom2

Bone Marrow Figure 1. Scanning under low power reveals a heterogenous population of cells

Maslak, P. ASH Image Bank 2005;2005:101401Copyright ©2005 American Society of Hematology. Copyright restrictions may apply.

Human Placenta and Umbilical Cord

How to Process and Save Cord Blood Stem Cells

Intermission

http://science.howstuffworks.com/enlarge-image.htm?terms=Cloning+-desoto+-bower+-ford+-video+-corvette+-retrospective+-oldsmobile+-buick+-yew+-pecan+-bsa+-mercury+-planting+-holly&page=0

The Latest Development

Is this a reversible process?

Time Magazine : http://www.time.com/time/

Stem Cells: Japan Gets Ahead of the CurveThursday, Jun. 14, 2007 By BRYAN WALSH

LEADING LIGHT: Yamanaka, a former orthopedic surgeon, knows how to knit research together with practical applications

http://www.time.com/time/

A breakthrough research!

How to turn adult skin fibroblasts into embryonic stem cells

From the following article:Stem cells: The magic brewJanet RossantNature 448, 260-262(19 July 2007)

http://www.nature.com/nature/journal/v448/n7151/full/448260a.html

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).

In this example, scientists can now avoid some ethical problems

in obtaining the stem cells.

Taiwan NSC97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

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

Research done in my laboratory

How to repair a damaged heart?

Heart Regeneration

Lepillina et al., (2006) Cell

Zebrafish Core FacilitiesCollaboration Scheme

NTHU Core750 tanks

(Expandable 1500 Tanks)

Education&

Basic Research

NHRI Core850 tanks

Preclinical Research&

Basic Research

MutualSupport

Preservation

Nature Reviews Genetics, Vol 8, May 2007

新興的人類疾病模式生物

發育快速生長週期短

胚胎透明 利於觀察

+

與其他模式生物之比較

Nature Reviews Genetics, Vol 8, May 2007

Transgenic zebrafish allow analysis of endothelial cells in living embryos

eGFPVascular EC promoter

fli1-egfp transgenic embryo at 2 dpf

Dorsal AortaDorsal Aorta(DA)(DA)

Posterior Cardinal Vein(PCV)

Intersegmental VesselsIntersegmental Vessels(Se)(Se)

Dorsal Longitudinal Anastomotic VesselDorsal Longitudinal Anastomotic Vessel(DLAV)(DLAV)

Caudal Vein Caudal Vein Capillary PlexusCapillary Plexus

血管螢光魚

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

Our first transgenic line

Heart心臟螢光魚

Nu

myl7(cmlc2)-eGFP/nuclear mCherry zebrafish

神經圖譜

Brainbow zebrafish – 5dpfAlbert Pan of Harvard University

Zebrafish as an effective model system for Human Diseases Research and Pharmaceutical R&D

Genomics:Sequenced GenomecDNA projectsMicroarrays

Reverse Genetics:Transgenic fishTilling with ENUMorpholino injection

Forward Genetics:ENU mutagenesisInsertional mutagenesis

Small Molecule Screens:Predictive of higher vertebratesDelivery by injection or soaking

Carcinogenesis: Aqueous deliverySimilar to human tumors

Three layers

no melanocyte no iridophore

Transparent ZebrafishCasper

Xanthophore onlyCell Stem Cell (2008) 2:183-189

What type of stem cells is the best?

Challenges to stem-cell therapy for cardiac disease

Challenges to stem-cell therapy for cardiac disease

Stem cell therapy and research requires Cross-disciplinary collaboration and expertise.

(跨領域專長與合作)

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Translational Medicine:By drawing upon discoveries made in the basic sciences: biochemistry, immunology, engineering and phyWhat’s is Research for?PrefaceProperties of Stem cellStem Cell - DefinitionWhere can you find stem cells?Sources of Stem Cells“ADULT” STEM CELLS ARE OBTAINED IN A VARIETY OF WAYSSTEM CELLS HAVE ALSO BEEN FOUND IN “MATURE” ORGANSHow do you obtain the embryonic stem cells?Cloning (複製)Cloning Dolly桃莉羊Complications Observed in Cloned AnimalsStem Cell ExperimentsDivergent Religious View on Research and Clinical Use of ESCPros and Cons of Using Embryonic Stem Cell Pros and Cons of Using Adult Stem Cell Stem Cellsin Bone Marrow (骨髓) & Umbilical Cord Blood (臍帶血)Hematopoietic Stem Cells（造血幹細胞）Mesenchymal Stem Cells(間葉系幹細胞)How to collect bone marrow and cord bloodHuman Placenta and Umbilical CordHow to Process and Save Cord Blood Stem CellsIntermissionThe Latest DevelopmentIn this example, scientists can now avoid some ethical problems in obtaining the stem cells.Taiwan NSC97年度幹細胞優先推動計畫徵求公告幹細胞研發重點領域徵求項目Research done in my laboratoryHow to repair a damaged heart?Heart Regeneration