the pluripotency : lessons from embryonic stem cell properties
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The Pluripotency : Lessons from Embryonic Stem Cell Properties. M. Saifur Rohman , M.D., Ph.D. Cardiologist. Outline. Regenerative medicine Historical Perspective Stem Cells Embryonic Stem Cells (ESC) Pluripotency induction (iPS): A future challenge - PowerPoint PPT PresentationTRANSCRIPT
The Pluripotency: Lessons from
Embryonic Stem Cell Properties
M. Saifur Rohman, M.D., Ph.D.
Cardiologist
Outline
• Regenerative medicine• Historical Perspective• Stem Cells• Embryonic Stem Cells (ESC)• Pluripotency induction (iPS): A future challenge• Adult Stem Cells (ASC): A comparison• The use stem cell in medicine
• Regenerative medicine aims to repair diseased or damaged tissues by replacing the affected cells with healthy, functional cells of the same type.
• The prospects of this discipline have been boosted by the promise of ES cells, which are pluripotent (they can differentiate into any cell type) & which can be maintained in culture to “self-renewal” indefinitely.
Regenerative Medicine
Blelloch, Nature 2008.-
Historical Perspective• Major Changes in regenerative medicine (replacement
of damaged or diseased cell tissues with new cells and tissues) due to advances in stem cell technologies
• First successful bone marrow transplant done in1956 on leukemic patient. Bone marrow contains adult derived hematopoietic stem cells, able to regenerate tissues similar to the specialized tissues in which they are found.
• Embryonic stem cells believed to have greater potential. This line of stem cell research has been the most controversial.
Historical Perspective
Isolation & Culture of Embryonic Stem Cells(Human-1998; Mouse-1981)
Advantages:1) Proliferate indefinitely2) Form any tissue
Method patentedU.S. patent held by Univ. Wisconsin J. Thomson 1998
1 stem cell
Self renewal - maintains the stem cell pool
4 specialized cells
Differentiation - replaces dead or damagedcells throughout your life
Why self-renew and differentiate?1 stem cell
Stem Cells: Definition
• Stem cells: Primitive cells that have the capacity for extensive self-renewal, clonogenic, and the ability to differentiate into multiple cell types
• Embryonic stem cells: Totipotent (pluripotent) cells derived from the inner cell mass of the blastocyst; they give rise to cells of all three germ layers
• Adult stem cells: present in all renewing tissues; these cells divide for self-renewal and differentiate into multiple progenitor cell types.
• Progenitor cells: multipotential intermediate stem cells that serve as the direct precursors for tissue-specific mature cells.
• Endothelial progenitor cells: cells that are present in blood and bone marrow; they are involved in angiogenesis and postnatal neovasculogenesis.
Stem Cells: Definition
• Mesenchymal stem cells: also referred to as marrow stromal cells; these cells differentiate in vitro along multiple pathways that include cardiac myogenesis.
• Hemangioblasts: primitive embryonic cells that give rise to both hemopoeitic stem cells and endothelial progenitor cells; they may also exist in adult bone marrow.
Stem Cells: Definition
121212
Mammalian development
Emerging Technol Platform for SCs, 2010.-
Developmentalpotential
Totipotent
Pluripotent
Multipotent
Oocyte Zygote
Sperm
Trophoblast(extraembryonic)
Inner cellmass Blastocyst
Epiblast
Primitive
Primitive streak
Endoderm(lung, liver,
pancreas, etc.)
Mesoderm(blood, heart, bone,
skeletal muscle, etc.)
Ectoderm(central and peripheral
nervous system,epidermis, etc.)
EmbryonicStem cells
(in vitro)
Stem Cells
• Embryonic Stem Cell
• Adult Stem Cells
Loose definitionStrict definition pluripotent
totipotent
Human development : Stem cell perspective
Generates every cell in the body including the placenta and extra-embryonic tissues
Can form the entire human being
Cannot form the entire human being
Can generate every cell in the body except placenta and extra-embryonic tissues
Become specific cell types; may or may not have plasticity
Characteristic of Stem Cells
• Undifferentiated cells with the capacity for unlimited or prolonged self renewal that can give rise to differentiated cells
• Metaplasia- the formation of one differentiated cell type from another
• Slow cycling in cell division• Contact-insensitive; deficient in gap junction
intercell. communication• Specific gene expression
SCAN – Stem Cell Action Network
Embryonic Stem Cells (ESC)Researchers extract stem cells from a 5-7 days old blastocyst.Researchers extract stem cells from a 5-7 days old blastocyst.
Stem cells can divide in culture to form more of their own kind, Stem cells can divide in culture to form more of their own kind, thereby creating a stem cell line.thereby creating a stem cell line.
The research aims to induce these cells to generate healthy tissue needed by patients.
Properties of Human ESC in Culture
• Pluripotent- able-to form any of 200 different types of cells of the body
• Self renewing in vitro- can propagate or proliferate indefinitely in the undifferentiated state
• Express the enzyme telomerase (required to maintain the end of chromosomes) and Oct4 ( a master regulator of ESC pluripotency)
• Maintain normal chromosome structure and complement even after long periods in culture ( unlike many other tissue cell lines)
Characteristics of Human ESC
• Normal Karyotypes• Express high telomerase activity• Express cell surface markers of primate ESC• Maintained undifferentiated proliferation for 4-5
months and development potential to form thropoblast and, endoderm, mesoderm, ectoderm
• Developed teratomas in immune deficient mice
ESC: What they can do
embryonic stem cells
PLURIPOTENT
all possible types of specialized cells
differentiation
neuronsgrow under conditions B
ESC: Challenges
embryonic stem cells
skin
grow under conditions A
blood
grow under conditions C
liver
grow under conditions D
?
SCAN – Stem Cell Action Network
Two Sources of Embryonic Stem Cells
1. Excess fertilized eggs from IVF (in-vitro fertilization) clinics
2. Therapeutic cloning (somatic cell nuclear transfer)
SCAN – Stem Cell Action Network
Tens of thousands of frozen embryos are routinely destroyed when couples finish their treatment.
These surplus embryos can be used to produce stem cells.
Regenerative medical research aims to develop these cells into new, healthy tissue to heal severe illnesses.
SCAN – Stem Cell Action Network
Somatic Cell Nuclear Transfer
The nucleus of a donated egg is removed and replaced with the nucleus of a mature, "somatic cell" (a skin cell, for example).
No sperm is involved in this process, and no embryo is created to be implanted in a woman’s womb.
The resulting stem cells can potentially develop into specialized cells that are useful for treating severe illnesses.
Pluripotency signature
• Pluripotent – distinct cellular marker and
functions• Factors that are expressed in somatic cells
or tissue specific genes must be shut down• Expression of genes for pluripotency
associated factors : octamer binding transcription (Oct4) and Nanog must be initiated
Review : Epigenetic ?
Human ESC remain embryo because of epigenetic factors
• Molecule central that balancing act – H3K4me3 & H3K27me3
• Genes that modified only by H3K4me3 contain DNA recipe to proliferate
• Genes that do not carry both completely silenced in ES
Science daily, Oct,8,2007
Embryonic stem cells regulations
• Maintained by 3 TF genes :– Oct4– Sox2– Nanog
• Feed-forward & feedback maintain pluripotent gene expression
• Oct4 – important TF - regulate a pluripotent gene expression in early embryonic development
• During differentiation – the expression is down regulated
Specific mechanism required to disrupt differentitation ( GCNF – Germ Cell Nuclear factor)
Yamanaka et al, Jaenish et al; Thomson et al, 1998
Oct-4 (octamer-binding transcription factor 4)
• Oct-4 (octamer-binding transcription factor 4) also known as POU5F1 (POU domain, class 5, transcription factor, is a protein that in humans is encoded by the POU5F1 gene
• This protein is critically involved in the self-renewal of undifferentiated embryonic stem cells. As such, it is frequently used as a marker for undifferentiated cells.
• Oct-4 expression must be closely regulated; too much or too little will actually cause differentiation of the cells.
Oct4
• Upon differentiation Oct4 is non expressed
• GCNF is high expressed
• Methylation of Oct4 gene and histone modifications silencing Oct4 gene during differentiation
• Loss of DNA methylation & chromatin remodelling no effect of repression
Oct4
• DNA methylation machinary :
DNA Methyltranferase &
Methyl DNA binding domain protein (MBDs-
MBD1 & MBD2) MBD2 binds to CpG
dinucleotide (CpG" is shorthand for "—C—phosphate—G—", that is, cytosine and guanine separated by a phosphate)
MBD3 bind to unmethylated CpG
dinucleotide
Oct4 and Nanog
Cells
ESC manage their pluripotent status by PcG mediated repressive histone lock
Lysine methylation recrut spec.Binding prot HP1 to H3 lysine 9 Histone code PRC1 (Protein regulator of cytokinesis 1) t o methylated Histon H3 lysine 27Acetylation nucleosome looser, > accesible to transcription factors
Spivakov & Fischer, Nature, April 2007Polycomb-group proteins
Pluripotency properties
• Embryoid body ) ball like embryo)- embryoid bodies
• Chimeric mouse
• Promoter demethylationof oct3/4, Nanog and Rex1,
• Histon demethylation
From pluripotent to differentiated cells
• Neural differentiation : AADC, DAT, ChAT, LMX1b, MAP; b-tubulin, tyrosine hydrolase
• Cardiac differentiation : TnTc, MEF2C, MYL2a, MyhcB , NKX2.5
• Teratoma formation – teratoma is landmark for pluripotent
ESC in the lab, an example : mPer2 conditional knock out generation
loxlox FLPFLP
SacI-blSalI
SalI KpnI-bl
Intron2 Exon2 DT-A
Pgk-neo
Saifur Rohman, et. Upublished data,
Stem cell selection and implantation
Potential embryonic stem cell problems:
• Difficult to establish and maintain
• Difficult in obtaining pure cultures in the dish
• Potential for tumor formation and tissue destruction
• Questions regarding functional differentiation
• *Hansson M et al., Diabetes 53, 2603-2609, 2004• *Sipione S et al., Diabetologia 47, 499-508, 2004 • *Rajagopal J et al.; Science 299, 363; 2003 • *Zhang YM et al.; Circulation 106, 1294-1299; 2002
SCAN – Stem Cell Action Network
The Ethical DebateIn favor of ESCR:
Embryonic stem cell research (ESCR) fulfills the ethical obligation to alleviate human suffering.
• Since excess IVF embryos will be discarded anyway, isn’t it better that they be used in valuable research?
• SCNT (Therapeutic Cloning) produces cells in a petri dish, not a pregnancy.
Against ESCR:
In ESCR, stem cells are taken from a human blastocyst, which is then destroyed. This amounts to “murder.”
• There is a risk of commercial exploitation of the human participants in ESCR.
• Slippery slope argument: ESCR will lead to reproductive cloning.
44
Cellular Plasticity
• The discovery of mammalian cellular plasticity raises the possibility of reprogramming restricted cell fate, & may provide an alternative to many of the obstacles associated with using embryonic & adult stem cells in clinical applications.-
• With a safe & efficient dedifferentiation process, healthy, abundant & easily accessible adult cells from a given individual could be used to generate different functional cell types to repair damaged tissues & organ.-
Lyssiatis et al, Emmerging Techno Platform for SCs, 2009.-
Induced pluripotent stem cells (iPS)
Pluripotent stem cell artificially derived from a non pluripotent cell
by inducing a “forced “ expression of certain genes
Yamanaka et al, 2006, 2007,
Thomson et al, 2007, 2008
• Yamanaka : cell lines with some of the properties of ES cells by introducing just four transcription factors associated with pluripotency – Oct3/4, Sox2, c-Myc & Klf4 – into mouse skin fibroblast then selecting cells that expressed a marker of pluripotency, Fbx15, in response to these factors, these cells were called iPS cells.-
Induced Pluripotent Stem (iPS) Cells
Rossant, Nature 2007.-
Induced pluripotent stem cells (iPS cells)
adult cell
‘genetic reprogramming’= add certain genes to the cell
induced pluripotent stem (iPS) cellbehaves like an embryonic stem cell
Advantage: no need for embryos! all possible types ofspecialized cells
culture iPS cells in the lab
differentiation
Induced pluripotent stem cells (iPS cells)
adult cell (skin)
genetic reprogrammingpluripotent stem cell
(iPS)
differentiation
Properties of iPS identic to natural pluripotent stem cells
• Expression of certain stem cell genes and protein
• Chromatin methylation patterns• Doubling time• Embryoid body formation• Teratoma formation• Viable chimera formation• Potency differentiability
Identity of iPS
• Morphology : similar to ESC, sharp edged, flat, tightly pcked
• Doubling time – same• Stem cell markers: SSEA-3, SSEA-4, TRA-1-60,
TRA – 1 -81, TREA -2-49/6E, Nanog
• Telomerase activity:• Stem cell genes: oct3/4, Nanog, GDF4, REXi, FGF4,
ESG1, DPPA2, DPPA4 and hTERT
Methode iPS - transfection
• Retrovirus ( Yamanaka et al, 2006)• Mouse fibroblast into iPS – by retroviral, 2007
– Harvard Team• iPS from adult human (James Thomson,
Junying yu et al, 2008• Adenovirus to transport 4 genes ( Konrad
Hochedlingler – Harvard Universitym 2008)• Yamanaka 2009 – iPS without retrovirus but
plasmid
Genes of induction
1. Oct 3 , 4 - maintain pluripotency2. Sox family – maintain pluripotency3. Klf family – ( Thomson et al, Yamanaka )
controversial4. Myc family – proto-oncogene5. Nanog + Oct 3 , 4 promoting pluripotency6. LIN28 : mRNA binding protein
Yamanaka et al, 2006, 2007, 2009 James Thomson 2006, 2007
Molecular mechanism of iPS generation
Molecular mechanism of iPS generation
Plasmid for cell reprogramming
iPS Cells generation steps
IPS cell generation ways
Application in animal
The use of iPS cells using patient fibroblasts
Parkinson’s disease (Wernig and Jaenisch, 2008, Maehr and Melton PNAS 2009).
Amyopathic Lateral Sclerosis, (Dimos and Eggan Science 2008)
Type I diabetes (Maehr and Melton PNAS 2009)Duchenne and Becker Muscular dystrophin,
Parkinson’s disease, Huntington disease, Down syndrome, Lesch-Nyhan syndrome. (Park and Daley Cell 2008).
• Blood cells (Loh and Daley 2009). B-cells (Hanna and Jaenisch Cell 2008)
• Blood stem cells (Emiinli and Hochedlinger Nat Genet 2009)
• Pancreatic b-cells (Stadtfeld and Hochedlinger Cell Stem Cell2008)
• Hepatic and gastric endoderm (Aoi and Yamanaka Science 2008)
• Neural stem cells (Kim and Scholar, Nature 2008)
61Boenjamin Setiawan, dr.,PhD
The use of iPS cells using patient fibroblasts
Adult stem cells
– Found in adult tissue – Can self-renew many times– Multipotent – they can differentiate to become
only the types of cells in the tissue they come from.• hematopoietic stem cells – give rise to blood
cells• mesenchymal stem cells – give rise to cells
of connective tissues and bones• umbilical cord stem cells – a rich source of
hematopoietic stem cells• stem cells found in amniotic fluid – might be
more flexible than adult stem cells
Differentiation pathway The differentiation pathways of adult stem cells.
– Hematopoietic stem cells give rise to all types of blood cells: red blood cells, B lymphocytes, T lymphocytes, natural killer cells, neutrophils, basophils, eosinophils, monocytes, macrophages, and platelets.
– Bone marrow stromal cells (mesenchymal stem cells) give rise to a variety of cell types: bone cells (osteocytes), cartilage cells (chondrocytes), fat cells (adipocytes), and other kinds of connective tissue cells such as those in tendons. 63
Where are adult stem cells found and what do they normally do?
• An adult stem cell is an undifferentiated cell found among differentiated cells in a tissue or organ, can renew itself, and can differentiate to yield the major specialized cell types of the tissue or organ.
Where adult stem cells are found?
Adult stem cells have been identified in many organs and tissues. However, there are a very small number of stem cells in each tissue.
• Stem cells are thought to reside in a specific area of each tissue where they may remain quiescent (non-dividing, STEM CELL NICHE) for many years until they are activated by disease or tissue injury.
• The adult tissues reported to contain stem cells include Brain, Bone marrow, Peripheral blood, Blood vessels, Skeletal muscle, Skin, hUCB, Umbilical Cord, Amniotic liquid, Adipose Tissue, liver etc.
737373SC Technol, Basic Applic 2010.-
Gene Therapy
Cell Transplantation
TissueEngineering
DNA transfection
e.g., Neuronal bundles orPancreatic Islets, etc.
Neurones
Tissue stem cells
Adult stem cells
Reprogrammed
Ectoderm
Tissuestem cells
Endoderm
Mesoderm
Embryonicstem cells
Muscle
Blood cellsLung/Gut/Liver
Therapeutic Challenge
Germ layers &Tissue
differentiation
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