1. --2 Stem cell biology Jan-Kan Chen College of Medicine
Chang Gung University

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

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

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

5. Definition of Adult Stem Cells
Property
Assay or experimental identification
High proliferative potential
In 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-Cycling
Label-retaining cells (LRCs) ;
almost in G0/G1 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.
Adult stem cell research on humans began in the 1960's, first achieving success in the treatment of a patient with severe combined immunodeficiency disorder in Since the early 1970's, adult stem cells have been successfully used for treatment of immunodeficiencies and leukemias.

7. Mammalian epidermal stem cells
A hair follicle and a segment of adjacent skin is illustrated. The dermal papilla (DP) signals to matrix stem cells (red) located across a basement membrane (green). Matrix cell daughters (yellow) differentiate into a variety of cell types, including the medulla, cortex and cuticle of the hair shaft (brown), the inner root sheath (IRS) and the outer root sheath (ORS). About two-thirds of the way up an anagen follicle lies the bulge — an expanded region that contains long-term stem cells (red). These cells periodically replenish (arrows) the matrix cells, and also help maintain the sebaceous gland (SG) and the epidermal stem cells (red, top layer) that lie against the basement membrane (not shown) overlying the basal layer in interfollicular regions. b, A mammalian gut crypt is a tube of cells arrayed on a basement membrane (green). Stem cells (red) are located in the basal region along with Paneth cells, but their exact location is variable and both types account for only a fraction of the cells present in the regions shown. A portion of the basement membrane in the stem cell region may be specialized (dark green). Stem cell progeny (yellow) known as transit amplifying cells (TA) move upwards and differentiate. Underlying mesenchymal cells (green) send signals that help regulate stem cell activity.
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
Cornea epithelium
Embryo dermis
Epidermis
Development
127, (2000)
(After 21Days)

12. Possible Roles of Bone Marrow–Derived and Circulating Stem Cells in the Repair of Solid-Organ Tissue
After tissue injury, stem cells that are intrinsic to the tissue replace necrotic cells as a first line of defense. If the pool of endogenous stem cells
is exhausted, exogenous circulating stem cells are signaled to replenish the pool and participate in tissue repair. Thus, circulating stem cells
may serve as a backup rescue system.
N ENGL J MED., 349, 570, 2008

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. 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
Type 1 diabetes mellitus - beta cells of the pancreas
Parkinson's disease - dopamine-secreting cells of the brain
Spinal cord injuries leading to paralysis of the skeletal muscles
Ischemic stroke where a blood clot in the brain has caused neurons to die from oxygen starvation
Multiple sclerosis - loss of myelin sheaths around axons
Myocardium infraction – death of cardiomyocytes

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

17. Number of Persons Affected
Persons in the United States affected by diseases that may be helped by human pluripotent
stem cell research
Condition
Number of Persons Affected
Cardiovascular diseases
58 Million
Autoimmune diseases
30 Million
Diabetes
16 Million
Osteoporosis
10 Million
Cancer
8.2 Million
Alzheimer's disease
4 Million
Parkinson's disease
1.5 Million
Burns (severe)
0.3 Million
Spinal cord injuries
0.25 Million
Birth defects
150,000 (per year)
Total
128.4 Million
Data from the Patients' Coalition for Urgent Research, Washington, DC

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

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. Comparison of some properties of mouse and human embryonic stem cells
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, Matrigel
Growth characteristics
in vitro
Tight, rounded, multilayer clusters
Flat, loose aggregates
EB formation
Simple and cystic EBs
Cystic EBs
Teratoma formation
in vivo
MEF, mouse embryonic fibroblasts; EB, embryoid body.

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
+LIF 24h
+LIF 48h
-LIF 24h
-LIF 48h
Nature 336, (1988)

30. JAK/STAT3 signaling pathways
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. Examples demonstrating the developmental potential of human ES cells in vitro
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

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. An ES colony + HGF + activin A + RA + bFGF + BMP-4
Schuldiner et al PNAS 97: , 2000

35. Schuldiner et al PNAS 97:11307-12, 2000
DS: differentiated embryonic cells; ES:embryonic stem cell:
Schuldiner et al PNAS 97: , 2000

36. Schuldiner et al PNAS 97:11307-12, 2000
Thick arrow and large fonts indicate an induction compared with the control(no GF)
Dashed arrow indicates a decrease in expression.
Schuldiner et al PNAS 97: , 2000

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

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

40. Thank You

41. Self-organizing transcription faactors network for ES cells self-renew
Cdx2
Oct4
Gata6
Nanog
Precusor
(totopotent)
Inner cell mass
(pluripotent)
Trophectoderm
(multipotent)
Primitive endoderm
Epiblast
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. Maintaining pluripotency
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

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

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

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

53. Use of ES cells
Safety and ethic issues

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, Spain
Prohibited
Authorized
Italy, Austria, Ireland
U.K. Denmark
Israel, Sweden, Belgium, India
Germany
(imported)
U.S.A.
Prohibited (public)
Free (private)
Authorized under
restricted condition (public)
(in most states)
Canada
Under consideration
Japan, Netherlands, Korea
Taiwan

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： 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。 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。
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。