embryonic stemccells: a basic concept

8/14/2019 Embryonic StemcCells: A basic concept

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Embryonic Stem Cells: AEmbryonic Stem Cells: A

Basic ConceptBasic Concept

Prepared by:

Dr. D. Kumar

Ph.D, Animal Biotechnology

National Dairy Research Institute, Karnal (India)-132001


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A cell from the embryo, fetus, or adult that has,under certain conditions, the ability to reproduceitself for long periods

Stem cells are undifferentiated/uncommitted cells

It can also give rise to specialized cells that make

up the tissues and organ of the body

What are stem cells ?


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Why stem cells ?

A unique capacity to renew itself and give rise to

specialized cell types

Uncommitted cells remain uncommitted until

receive a signal to develop into a specialized cell

type

A single cell must be capable of multilineage

differentiation


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Where is Stem Cells

Adult stem cellsAdult tissues

Placenta derived stem cellsPlacenta of newborns

Umbilical cord

stem cells

Umbilical cord blood of

newborns

Gonadal stem cellsGonads of fetuses

Embryonic stem cellsBlastocysts

Stem cell typeSource


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Types of Stem CellsT

ypes of Stem Cells

B a s e d o n s o u r c e o f o r i g i n

B a s e d o n p o t e n c y

Adult Stem Cells

Embryonic Stem Cells

Embryonic germ Cells

Placental stem cells

Totipotent Stem Cells

Pluripotent Stem CellsMultipotent Stem Cells Unipotent Stem Cells


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Defining properties of Stem Cells

(SCs)

Derived from specific region of organism

Exhibit long term self-renewal capacity

Exhibit and maintain a stable, normal geneticmakeup

Have ability to differentiate in different cell types

Have capacity to integrate into all fetal tissuesduring development


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Contd..

Are clonogenic in nature

Give rise to egg or sperm cells

Express pluripotency based markers

Lack the G1 checkpoint

Do not show X inactivation


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Background of ESCs derivationBackground of ESCs derivation

Establishment of mouse ES cell line

(Evans and Kaufman., 1981)

Establishment of first human embryonic pluripotent stem

cells line (Thomson,J.A. et al., 1998)

Isolation of ESC like cells from bovines

(Saito et al.,1992;Cibelli et al.,1998; Iwasaki et al.,2000)

Isolation of ESC like cells from sheep

(Notarianni et al., 1991; Campbell et al.,1996)

Isolation of ESC like cells from buffalo

(Chauhan et al., 2006;V.Verma et al., 2006; Imtiaz et al.,2002)

Isolation of ESC like cells from goat (Vasanth et al.,2004)


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Methods for isolation of ICMMethods for isolation of ICM

Immunosurgery

Intact blastocyst culture

Mechanical isolation

Enzymatic digestion


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Basic needs for culturing ESCs

Feeder cells

Leukemia inhibitory factor

Serum

Growth factors

Overall culture system


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Feeder lay ereeder lay er

Feeder layer are mitotically inactivated cellsused to provide a homely environment for

culturing ES cells

Inactivated by using Mitomycin-C or gamma

irradiation

Provides nutrients to ICM derived cells

Secretes cytokines such as Leukemia InhibitoryFactor-(LIF), which prevents differentiation of

ES Cells


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LIF/gp130/STAT3 PathwayLIF

LIFRgp130

JAKJAK

STAT

STATP

P

PSTAT

STAT

STAT

P

P

Activation oftranscription of genes

STAT

(Williams et al., 1988)


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Dattena et al., 2006STOSheep

Imtiaz, 2002; Chauhan et al., 2006; Verma et al.,

2006

Buffalo fetal fibroblast,MEFBuffalo

Reubinoffet al.,2000; Hong Hyuket al., 2003;

Lee et al., 2004

MEF, STO

hEF, HUEcells

Human

First et al., 1994; Cibelli et al., 1998; Iwasaki et

al., 2000; Yadav et al., 2005

STO,MEF,BFF,bovine uterus

epithelial cells, human lung

fibroblast

Bovine

Piedrahita et al., 1990 ; Li et al., 2003; Li et al.,

2004

STO,MEF,PEFPorcine

Evans and Kaufman 1981STO, MEFMurine

ReferencesFeeder layersSpecies

Available feeder layers for culturing ES

Cells in different species


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Advantages of allogenetic feeder cells

Allow prolonged growth of undifferentiated ESCs

Eliminate transfer of pathogens

Allow screening for viral contamination

Provide better support for growth

Cell-to-cell contact and identification of isolated

soluble factor significantly improve cell culture


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Characterization of ES cells

Morphology of ES cells/ colony

Expression of surface and transcription markers

Pluripotency nature

Karyotyping

Epigenetic status


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Transcription based markers

OCT4

NANOG

SOX2

GBX2

TERF1

HOXA11

PITH2

UNG

PEA3

KLF2

KLF3/5/9TTF1

JMJ

TOP2A

AND1SALL1

ZIC3

FOXD3

ZNF43PSIP1

RUVBL1

MSH2

ESGREX1


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Alkaline Phosphatase

SSEA-1

SSEA-3

SSEA-4

TRA-1-60

TRA-1-81

TRA-2-49

TRA-2-54

Thy-1

CD9

Podocalyxin

HLA-Class 1TG30

TG343

GCTM-2

E-cadherin

Surface markersSurface markers


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What transcription factors do?

Play an essential role in early development

Help in propagation of undifferntiated embryonic stemcells

Regulate other transcription factors through feedback

loops

Regulate downstream target genes involved in the

maintenance and differentiation of ESCs


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OCT4OCT4

A homoeodomain transcription factor of the POU class

Binds to consensus octameric region ATGCAAAT and

activates several genes involved in pluripotency

Expressed in undifferentiated ESCs, ECCs and EGCs

Expressed in ICM and is downregulated upon differentiationinto trophoblast


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Nanog

The gene named after Tir nan Og or Tir Na Nog means

the land of the ever young

Codes for a homoeobox protein-305 aa long

Expressed in ESCs, EGCs and ECCs but not in

hematopoietic cells, fibroblasts, adult tissues anddifferentiated cells

Expressed in ICM cells but is downregulated atimplantation stage


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SOX2

Member of the SRY sub-family of HMG box

Bind the sequence CT/ATTGT/AT/A

Expressed in ICM and germ cells

Its down regulation leads to differentiation of cells

It forms a ternary complex with OCT4 and activatesgenes


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Important Transcription factors involved in self-

renewal of ES Cells

OCT4

NANOG

SOX2

OCT4-SOX2

Stem cell Genes Differentiation Genes


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Al kal ine Phosphatase An enzyme in blood, intestine, liver and bone cells

Exists as membrane-bound isoforms of

glycoproteins sharing a common protein structure

but differing in carbohydrate content


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Differentiation of ES cells

A process by which an unspecialized cell becomes

specialized into one of the many cells that make up the

body

Certain genes become activated and others get

deactivated

Cells develop a specific structure and perform certain

function


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Method of differentiation

In vitro

SpontaneousFormation of embryoid bodies which contain ectoderm,

mesodrm and endoderm cells

DirectedFormation of a specific cell type in response to a

particular stimulus

In vivoformation of teratoma which contain ectoderm,

mesoderm and endoderm cells


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Protocols for differentiation of ES Cells

Initiate differentiation in the absence of LIFand feeder layer

Cells differentiated asEmbryoid bodies

(Suspension culture)

Directed DifferentiationIn the presence of certain

Growth factors

Analyze Differentiatedcells


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Applications o f ES Csin f arm animals

Transgenesis

Cloning

Generation of chimerasObtaining germ cells to treat infertility

Studies on early embryonic development

Rapid screening of chemicals and drug discovery

Examining complex processes such as genomicimprinting

Gene targeting

embryonic stemccells: a basic concept

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