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STEM CELL BIOLOGY Jan-Kan Chen College of Medicine Chang Gung University --2.

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1 STEM CELL BIOLOGY Jan-Kan Chen College of Medicine Chang Gung University --2

2 Invariant asymmetry asymmetry Populational 成體幹細胞之分裂與分化模式 JKC ?

3 Growth factor Microenvironment mediate cell differentiation paracrine ES-feeder interactions ES-matrix

4 Physiol. Rev., 85, 635, 2005 In vitro differentiation of ES cells

5 Definition of Adult Stem Cells PropertyAssay or experimental identification High proliferative potentialIn vitro expansion and passage (Barrandon Y & Green H, 1987) Relatively undifferentiated phenotype Lacking differentiation related protein or express undifferentiation marker (Coulombe PA, et al., 1989) Slow-CyclingLabel-retaining cells (LRCs) ; almost in G 0 /G 1 phase (Cotsarelis G, et al., 1989) (5/50)

6 History of Adult Stem Cell Research Since the 1970’s, bone marrow transplants have been used for treatment of immunodeficiencies and leukemias.

7 Mammalian epidermal stem cells Nature, 414, 98, 2001

8 Published Reports on Identification of Human Adult Stem Cells Sources of adult stem cells include bone marrow, blood, the cornea and the retina of the eye, brain, skeletal muscle, dental pulp, liver, skin, adipocyte, the lining of the gastrointestinal tract, and pancreas. unipotent ?

9 Adult Stem Cells

10 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

11 Different steps in the transition of adult corneal epithelium into an epidermis (After 21Days) Cornea epithelium Epidermis Embryo dermis Development 127, (2000)

12 N ENGL J MED., 349, 570, 2008 Possible Roles of Bone Marrow–Derived and Circulating Stem Cells in the Repair of Solid-Organ Tissue

13 Potential Application of Stem Cell Technology

14 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.

15 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

16 Stem cell research / Cell therapy Tissue or organ Specific cell types Stem cells Differentiation Engraftment Diabetes Parkinson’s disease Spinal cord injury Blindness… Disease

17 ConditionNumber of Persons Affected Cardiovascular diseases58 Million Autoimmune diseases30 Million Diabetes16 Million Osteoporosis10 Million Cancer8.2 Million Alzheimer's disease4 Million Parkinson's disease1.5 Million Burns (severe)0.3 Million Spinal cord injuries0.25 Million Birth defects150,000 (per year) Total128.4 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

18 STEM CELL BIOLOGY PART2: The Embryonic Stem Cell Jan-Kan Chen College of Medicine Chang Gung University

19 Embryonic stem cells Totipotent stem cell pluripotent stem cell multipotent stem cell

20 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.

21 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)

22 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

23 Thomson et al., (1998) Science 282 : Culture of human embryonic stem cells

24 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.

25 Mouse ES CellsHuman 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, Matrigel Growth characteristics in vitro Tight, rounded, multilayer clusters Flat, loose aggregates EB formationSimple and cystic EBsCystic EBs Teratoma formation in vivo ++ MEF, mouse embryonic fibroblasts; EB, embryoid body. Comparison of some properties of mouse and human embryonic stem cells

26 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 vitro LIFR and gp130 are required for LIF binding, which in turn activates STAT3, which is necessary for continued proliferation of ES cells STAT3 and Oct-4 may interact and perhaps affect the function of a common set of target genes Activation of ERK and SHP-2 inhibit self-renewal of ES cells In mouse ES cells, Oct-4 expression and Gab-1 activation suppress Ras-ERK signalling pathway, and suppress induction of differentiation

27 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

28 Effect of LIF on self-renewal of mouse embryonic stem cells Nature 336, (1988) +LIF 24h + LIF 48h - LIF 24h - LIF 48h

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30 Regulation of self-renewal in mouse ES cells by Oct3/4, Nanog, BMP-dependent SMAD, and LIF-dependent JAK/STAT3 signaling pathways Physiol. Rev. 85: , 2005

31 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

32 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)

33 Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells None of the growth factors directs differentiation exclusively to one cell type Activin A and TGF-  1 mainly induce mesodermal cells RA, EGF, BMP-4 and bFGF activate ectodermal and mesodermal markers NGF and HGF allow differentiation into the three germ layers Most of the factors inhibit differentiation of specific cell types, and this inhibitory effect is more pronounced than an induction effect (PNAS 97: , 2000)

34 Schuldiner et al PNAS 97: , HGF+ activin A + RA + bFGF+ BMP-4 An ES colony

35 Schuldiner et al PNAS 97: , 2000

36

37 Nature Biotechnology 25, (2007) Schematic illustration for the isolation and differentiation of hES cell–derived NCS cell

38 Nature Reviews Molecular Cell Biology 7, 885–896 (2006) Adipocyte differentiation

39

40 Thank You

41 Cdx2 Oct4 Gata6 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.

42 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 Maintaining pluripotency

43 Signaling Transduction Pathways Involved 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.

44 Induced 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

45 Startrgies for the generation of pluripotent stem cells from somatic cells

46 Mouse gene combinations for iPS induction Nature review molecular biology 9,725,2008 The relation of ES cell And iPS cell is unclear, they may be Similar but not identical.

47 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.

48 Putative Role of the Four Factors in the Induction 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.

49 Putative Role of the Four Factors in the Induction 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.

50 Following injection into blastocysts, iPS cells contributed to mouse embryonic development Mouse E 7.5 Mouse E 13.5

51 Induction of Pluripotent Stem Cells from Fibroblast Cultures Kazutoshi Takahashi, Keisuke Okita, Masato Nakagawa & Shinya Yamanaka Nature Protocols 2:3081-9, 2007 Cell 131: , 2007 Generation of iPS cells from adult human dermal fibroblasts with the same four factors: Oct3/4,Sox2,Klf4 and c-Myc.

52 Science 318, 1917, 2007 Generation of iPS cells from adult somatic cells with four factors: Oct4, Sox2, nanog and Lin28

53 Safety and ethic issues Use of ES cells

54 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

55 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

56 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。

57 Summary of Policies Defined Around the World Countries Human Embryo Cloning (=creating embryo) Use of Stem Cell Lines Use of Superfluous Embryos France, SpainProhibitedAuthorized Italy, Austria, IrelandProhibited U.K. DenmarkAuthorized Israel, Sweden, Belgium, India ProhibitedAuthorized GermanyProhibitedAuthorized (imported) Authorized U.S.A.Prohibited (public) Free (private) Authorized under restricted condition (public) Free (private) Authorized (in most states) CanadaProhibitedUnder consideration Japan, Netherlands, Korea Authorized TaiwanAuthorized

58 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)

59 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。

60 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 ” 。

61 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

62 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。

63 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」。

64 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。


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