The Pluripotency : Lessons from Embryonic Stem Cell Properties

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.


• 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.


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

?


Two Sources of Embryonic Stem Cells

1. Excess fertilized eggs from IVF (in-vitro fertilization) clinics

2. Therapeutic cloning (somatic cell nuclear transfer)


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.


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


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

Thank You

Documents

The Pluripotency : Lessons from Embryonic Stem Cell Properties