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STEM CELL BIOLOGY
Jan-Kan ChenCollege of MedicineChang Gung University
--2
InvariantInvariant asymmetryasymmetry
Populational Populational asymmetryasymmetry
成體幹細胞之分裂與分化模式成體幹細胞之分裂與分化模式 JKC
?
Growth factor
Microenvironment mediate cell differentiation
paracrine
ES-feeder interactions
ES-matrix
Physiol. Rev., 85, 635, 2005
In vitro differentiation of ES cells
Definition of Adult Stem Cells
Property Assay or experimental identification
•High proliferative potential •In vitro expansion and passage(Barrandon Y & Green H, 1987)
•Relatively undifferentiatedphenotype
•Lacking differentiation related protein or
express undifferentiation marker(Coulombe PA, et al., 1989)
•Slow-Cycling •Label-retaining cells (LRCs) ; almost in G0/G1 phase
(Cotsarelis G, et al., 1989)
(5/50)
History of Adult Stem Cell ResearchSince the 1970’s, bone marrow transplants have been used for treatment of immunodeficiencies and leukemias.
Mammalian epidermal stem cells
Nature, 414, 98, 2001
Published Reports on Identification of Human
Adult Stem Cells
Sources of adult stem cells include bone marrow, blood, the cornea and the retina ofthe eye, brain, skeletal muscle, dental pulp, liver, skin, adipocyte, the lining of the gastrointestinal tract, and pancreas.
unipotent ?
Adult Stem Cells
Plasticity
Plasticity is the ability of an adult stem cell from one tissue to generate the specialized cell type of another tissue.
Example: Adult stem cells from bone marrow generated cells that resemble neurons
Different steps in the transition of adult corneal epithelium into an epidermis
(After 21Days)
Cornea epithelium
Epidermis
Embryo dermis
Development 127, 5487-5495 (2000)
N ENGL J MED., 349, 570, 2008
Possible Roles of Bone Marrow–Derived and Circulating Stem Cells in the Repair of Solid-Organ Tissue
Potential Application of Stem Cell Technology
How Does Cell Therapy Work?
Bone marrow transplants are an example of cell therapy in which the stem cells in a donor's marrow are used to replace the blood cells of the victims of leukemia.
Cell therapy is also being used in experiments to graft new skin cells to treat serious burn victims, and to grow new corneas for the sight-impaired.
In all of these uses, the goal is for the healthy cells to become integrated into the body and begin to function like the patient's own cells.
1. Type 1 diabetes mellitus - beta cells of the pancreas
2. Parkinson's disease - dopamine-secreting cells of the brain
3. Spinal cord injuries leading to paralysis of the skeletal
muscles
4. Ischemic stroke where a blood clot in the brain has caused
neurons to die from oxygen starvation
5. Multiple sclerosis - loss of myelin sheaths around axons
6. Myocardium infraction – death of cardiomyocytes
What Diseases Can be Cured by Stem Cell Therapies?
Any disease in which there is tissue degeneration can be a potential candidate for stem cell therapies
Stem cell research / Cell therapyStem cell research / Cell therapy
Tissue or organTissue or organ
Specific cell Specific cell typestypes
Stem cellsStem cellsDifferentiationDifferentiation
EngraftmentEngraftment
DiabetesDiabetes
Parkinson’s diseaseParkinson’s disease
Spinal cord injurySpinal cord injury
BlindnessBlindness
……
DiseaseDisease
Condition Number of Persons AffectedCardiovascular diseases 58 MillionAutoimmune diseases 30 MillionDiabetes 16 MillionOsteoporosis 10 MillionCancer 8.2 MillionAlzheimer's disease 4 MillionParkinson's disease 1.5 MillionBurns (severe) 0.3 MillionSpinal cord injuries 0.25 MillionBirth defects 150,000 (per year)Total 128.4 Million
Data from the Patients' Coalition for Urgent Research, Washington, DC
Persons in the United States affected by diseases that may be helped by human pluripotent stem cell research
STEM CELL BIOLOGY
PART2: The Embryonic Stem Cell
Jan-Kan ChenCollege of MedicineChang Gung University
Embryonic stem cells
Totipotent stem cell
pluripotent stem cell
multipotent stem cell
Early developement in humans
Day 0: Fertilization of the oozyte in the oviduct.
Zygote – totipotent
Day 4-5: (16 cells) – morula, soloid mass of cells
Day 6-7: Blastocyst formation - pluripotent
3rd week: Gastrulation, i.e formation of the three germ layers.
Factors associated with early embryogenesis
Inner cell mass: FGF-4 (embryogenesis and
differentiation of trophectoderm)
Trophectoderm: leptin and STAT3 (implantation)
Trophoblast (mouse): Mash 2 (placenta formation)
Epiblast: goosecoid, T, Evx-1, follistatin (primitive
streak formation)
Regulation of body pattern and differentiation
GATA-4, -6: Early differentiation
Hox: Anterior-posterior polarity
Nodal and Lefty: Left-right symmetry
Hex: Anterior-posterior development
Mrg1: Heart formation
BMP-4: Differentiation of mesenchymal cell, primitive streak
migration, CNS development
Wnt3: Formation of the primitive streak and the node
HNF-4, STAT-3: Visceral endoderm differentiation
Thomson et al., (1998) Science 282 : 1145-1147
Culture of human embryonic stem cells
How Many Human Embryonic Stem Cell Lines are There?The actual number of human embryonic stem cell lines is a matter of some debate. To date, more than 100 human embryonic stem cell lines have been derived worldwide. However, most of those lines have not adequately characterized yet. Only 22 cell lines are eligible for federal funding in the USA.
Mouse ES Cells Human ES Cells
Telomerase activity + +
Regulation of self-renewal
Via gp 130 receptors, MEF feeder layer,
Nanog, BMP-4
Feeder cells (MEF or human cells), serum,
bFGF, MatrigelGrowth characteristics in vitro
Tight, rounded, multilayer clusters
Flat, loose aggregates
EB formation Simple and cystic EBs Cystic EBsTeratoma formation in vivo
+ +
MEF, mouse embryonic fibroblasts; EB, embryoid body.
Comparison of some properties of mouse and human embryonic stem cells
Maintaining mouse embryonic stem cells in their undifferentiated state
LIF, either produced by feeder cells or added exogenously, allows mouse ES cells to proliferate without differentiation in vitroLIFR and gp130 are required for LIF binding, which in turn activates STAT3, which is necessary for continued proliferation of ES cellsSTAT3 and Oct-4 may interact and perhaps affect the function of a common set of target genesActivation of ERK and SHP-2 inhibit self-renewal of ES cellsIn mouse ES cells, Oct-4 expression and Gab-1 activation suppress Ras-ERK signalling pathway, and suppress induction of differentiation
Leukemia inhibitory factor (LIF)
Early blastocyst development and implantation
Survival for primordial germ cell
Maintenance of mouse embryonic stem (ES) cell but not human ES cell
Effect of LIF on self-renewal of mouse embryonic stem cells
Nature 336, 684-7 (1988)
+LIF 24h +LIF 48h
-LIF 24h -LIF 48h
Regulation of self-renewal in mouse ES cells by Oct3/4, Nanog, BMP-dependent SMAD, and LIF-dependentJAK/STAT3 signaling pathways
Physiol. Rev. 85: 635-678, 2005
Cell Types Developed •Ectoderm, endoderm, mesoderm, and neural precursors•Cardiomyocytes•Cardiomyocytes, endodermal, hematopoietic, and neuronal cells•Neuronal, epithelial, pancreatic, urogenital, hematopoietic, muscle, bone, kidney, and heart cells•Neural epithelium, embryonic ganglia, stratified squamous epithelium, gut epithelium, cartilage, bone, smooth and striated muscle cells•Cells with properties of pancreatic -like cells•Cardiomyocytes, pigmented and nonpigmented epithelial cells, neural cells, mesenchymal cells, erythroid, macrophage, granulocyte, and megakaryocyte cells•Myeloid, erythroid, megakaryocyte colony-forming cells•Neural precursors, glial and neuronal cells: incorporation into the brain (H1, H9, H9.2 lines)•Neural precursors, glial and neuronal cells: incorporation into the brain (HES-1 line)•Neural progenitor, dopaminergic, GABAergic, glutamatergic, glycinergic neurons, astrocytes•Neural progenitor, neuronal cells•Trophoblast•Hepatocytes
Examples demonstrating the developmental potential of human ES cells in vitro
Directed differentiation of human ES cells in vitro
Human ES cells differentiate spontaneously if removed from feeder cells and grown in suspension culture
bFGF: Epidermal epithelial cells (keratin)
Activin A:Muscle cell-like syncytium (enolase)
Retinoid acid: Neuron (neurofilament H)
Mouse BM stromal cell: Hematopoietic precursor cell (CD34)
Effects of eight growth factors on the differentiation of cells derived from human
embryonic stem cellsNone of the growth factors directs differentiation exclusively to one cell typeActivin A and TGF-1 mainly induce mesodermal cellsRA, EGF, BMP-4 and bFGF activate ectodermal and mesodermal markersNGF and HGF allow differentiation into the three germ layersMost of the factors inhibit differentiation of specific cell types, and this inhibitory effect is more pronounced than an induction effect
(PNAS 97:11307-12, 2000)
Schuldiner et al PNAS 97:11307-12, 2000
+ HGF + activin A
+ RA + bFGF + BMP-4
An ES colony
Schuldiner et al PNAS 97:11307-12, 2000
Schuldiner et al PNAS 97:11307-12, 2000
Nature Biotechnology 25, 1468 - 1475 (2007)
Nature Biotechnology 25, 1468 - 1475 (2007)
Schematic illustration for the isolation and differentiation of hES cell–derived NCS cell
Nature Reviews Molecular Cell Biology 7, 885–896 (2006)
Adipocyte differentiation
Thank YouThank You
Cdx2
Oct4 Gata6
NanogPrecusor(totopotent)Inner cell mass
(pluripotent)
Trophectoderm(multipotent)Primitive endoderm
(multipotent)Epiblast(pluripotent)
Cdx2
Oct4Gata6
Nanog
Precusor(totopotent)
Inner cell mass(pluripotent)
Trophectoderm(multipotent)
Primitive endoderm(multipotent)
Epiblast(pluripotent)
Cdx2
Oct4Gata6
Nanog
Precusor(totopotent)
Inner cell mass(pluripotent)
Trophectoderm(multipotent)
Primitive endoderm(multipotent)
Epiblast(pluripotent)
Self-organizing transcription faactors network for ES cells self-renew
Oct4: Loss of Oct4 causes differentiation of ES cells into trophectoderm.Overexpression of Oct4 results in differentiation into primitive endoderm
and mesoderm.
Sox2: One of the target genes of Oct4 and is required in ES cells with pluripotent sustenance.
Nanog: Nanog can activate Oct4 promoter and also as transcription repressor for cell differentiation genes.
Autoinductive FGF4/Erk signaling poises ESCs for lineage entry and must be resisted to allow self-renewal. A.Oct4 and Sox2 direct expression of fgf4 and poise ES cell from lineage commitment, Elevated Erk activity provides a signal rendering pluripotent cells susceptible to lineage inductive cues.B. Self-renewal of the pluripotent ES cell state requires overcoming the fgf4/Erk signal. The actions of FGF can be 1) blocked by inhibitors; 2)reversed by constitutive Nanog expression; 3) counteracted by LIF and BMP4.
Cell, 132:532,2008
Maintainingpluripotency
Signaling Transduction Pathways Signaling Transduction Pathways Involved in Maintaining Mouse ESCInvolved in Maintaining Mouse ESC
LIF-STAT3 pathway:
LIF (leukemia inhibitory factor) stimulates mESC through the gp130, which works as a heterodimer together with LIFR.
Activation of gp130 leads to the activation of the JAK and STAT.
Wnt pathway:
Wnt/b-catenin signaling involved in the maintenance of pluripotency of ESC. Wnt signaling activation can upregulate c-Myc and STAT3 expression.
BMP4 pathway:
BMP4 phosphorylates Smad1/5 in mouse ES cells.
Smad1/5 activation results in the expression of inhibitor of differentiation (ld) protein, which blocks the neural differentiation.
Induced pluripotent stem cells, iPS cellsInduced pluripotent stem cells, iPS cells
Man-made pluripotency can be achieved through induced reprogramming of somatic cells
Cell stem cell 2,2,151-9,2888
Startrgies for the generation of pluripotent Startrgies for the generation of pluripotent stem cells from somatic cellsstem cells from somatic cells
Mouse gene combinations for iPS inductionMouse gene combinations for iPS induction
Nature review molecular biology 9,725,2008
The relation of ES cell And iPS cell is unclear, they may beSimilar but not identical.
Klf4: Serves as upstream regulator of Oct4, Sox2, Nanog, and c-Myc.
C-Myc: A major downstream target for the LIF/STAT3 and the Wnt signalling pathways that support maintenance of pluripotency.
Lin28: RNA binding protein. Play a central role in blocking miRNA mediated differentiation in stem cells.
Putative Role of the Four Factors in the Putative Role of the Four Factors in the Induction of iPS CellsInduction of iPS Cells
Pluripotent stem cells are immortal and have open and active chromatin structure. Myc induces these two properties.Myc also induces apoptosis and senescence , which are suppressed by KLF4.Oct3/4 change the cell fate from tumor cells to ES cells.Forced expression of c-Myc and KLF4 alone would result in the generation of tumor cells, but not pluripotent stem cells.
Putative Role of the Four Factors in the Putative Role of the Four Factors in the Induction of iPS CellsInduction of iPS Cells
.
Oct-3/4 and Sox2 activate multiple target genes synergistically.
KLF4 may also function as cofactor of Oct-3/4 and Sox2.
KLF4 : Kruppel-like factors, are zinc-finger proteins.
Following injection into blastocysts, iPS cells Following injection into blastocysts, iPS cells contributed to mouse embryonic developmentcontributed to mouse embryonic development
Mouse E 7.5
Mouse E 13.5
Induction of Pluripotent Stem Cells from Induction of Pluripotent Stem Cells from Fibroblast CulturesFibroblast Cultures
Kazutoshi Takahashi , Keisuke Okita , Masato Nakagawa & Shinya Yamanaka
Nature Protocols 2:3081-9, 2007
Cell 131: 861-72, 2007
Generation of iPS cells from adult human dermal fibroblasts with the same four factors: Oct3/4,Sox2,Klf4 and c-Myc.
Science 318, 1917, 2007
Generation of iPS cells from adult somatic cells with four factors:
Oct4, Sox2, nanog and Lin28
Safety and ethic issues
Use of ES cells
研究所需要的胚胎幹細胞其來源:
一、人工流產後的胚胎組織
二、治療不孕症人工受孕過程中所剩餘的胚胎
三、為研究用而由捐贈配子製造出來的胚胎
四、以體細胞細胞核轉植( somatic cell nuclear transfer, SCNT )方式製造的人類胚胎
一、人工流產後的胚胎組織
胚胎是生命的起源,墮胎本身就是一種殺人的行為,所以使用其胚胎遺體來進行 研究在根源上根本就是不道德的行為
二、治療不孕症人工受孕過程中所剩餘的胚胎
人工授精在幹細胞的研究上 在人工授精中有多餘的受精卵,支持胚胎幹細胞研究
的人認為這些多餘的胚胎本來就會被棄置,以其如此,還不如在獲得當事人同意的情形下將其利用來從事幹細胞的研究培養,以應用於臨床醫療。
引發的道德爭議 反對胚胎幹細胞研究的人士認為,儘管幹細胞來源已
獲得當事人同意,但是因為從具有生命的胚胎中取出幹細胞後,整個胚胎也就會跟著死亡,因而幹細胞的研究本質上就是終止生命,就是不道德的。
Summary of Policies Defined Around the WorldCountries Human Embryo Cloning
(=creating embryo)Use of Stem Cell Lines
Use of Superfluous Embryos
France, Spain Prohibited Authorized Authorized
Italy, Austria, Ireland Prohibited Prohibited Prohibited
U.K. Denmark Authorized Authorized Authorized
Israel, Sweden, Belgium, India
Prohibited Authorized Authorized
Germany Prohibited Authorized
(imported)
Authorized
U.S.A. Prohibited (public)
Free (private)
Authorized under
restricted condition (public)
Free (private)
Authorized
(in most states)
Canada Prohibited Under consideration Under consideration
Japan, Netherlands, Korea
Authorized Authorized Authorized
Taiwan Authorized Authorized Authorized
胚胎幹細胞研究規範
第一級 : 絕對禁止 ( 法、瑞士、西、冰島、波蘭 )第二級 : 現有細胞株 , 不得再以受精卵製造 新細胞株 ( 美國 )第三級 : 可使用人工流產與人工生殖之多餘胚胎 ( 日本、加、澳洲、以色列、台灣 )第四級 : 可為研究目的製造新胚胎 ( 大陸、美私人經費贊助者 )
(Science 2001)
在美國幹細胞的研究,聯邦基金可以用來支持胚胎幹細胞的研究,但只限於利用早期建立,現存的六十個幹細胞株。
胚胎幹細胞研究伴隨來的複製人倫理問題
複製技術應用到人類,其問題將要嚴重得多。把複製人的器官當作另一些人的工具是不道德的。各國政府和科學界,國際人類基因組組織( HUGO),歐盟理事會,紛紛表達反對複製人的試驗。聯合國教科文組織 1997年 11月透過了《世界人類基因組與人權宣言》規定 “︰ 基於相互尊重人的尊嚴、平等這一民主原則,不允許進行與人類尊嚴相違背的做法,比如 Reproductive cloning”。2005年 3 月 8 日聯合國大會以 84票贊成, 34票反對, 37票棄權,透過了禁止複製人的決議,決議敦促成員國透過立法“禁止違背人的尊嚴和對人的生命造成傷害的各種形式複製”。
我國幹細胞研究的相關法規
人體器官移植條例 人體器官移植條例施行細則 人體器官組織細胞輸入輸出管理辦法研究用人體檢體採集與使用注意事項胚胎幹細胞研究的倫理規範
胚胎幹細胞研究的倫理規範 -1
研究使用的胚胎幹細胞來源限於:自然流產的胚胎組織、符合優生保健法規定之人工流產的胚胎 組織、
施行人工生殖後,所剩餘得銷毀的胚 胎,但以受精後未逾十四天的胚胎為限。
不得以捐贈之精卵,透過人工受精方式製造胚胎供研究使用。
胚胎幹細胞研究的倫理規範 -2
以「細胞核轉植術」製造胚胎供研究使 用,因牽涉層面較廣,需再作進一步之審慎研議。供研究使用的胚胎幹細胞及其來源,應為無償提供,不得有商業營利行為,且應經當事人同意,並遵守「人體檢體採集與使用注意事項」。
胚胎幹細胞研究的倫理規範 -3
胚胎幹細胞之研究,不得以複製人為研究目的。胚胎幹細胞若使用於人體試驗之研究,應以治療疾病和改善病情為目的,但應遵守醫療法規定,由教學醫院提出人體試驗計畫經核准後方可施行。