PRODUCTION AND MAINTENANCE OF EMBRYONIC STEM CELLS
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Transcript of PRODUCTION AND MAINTENANCE OF EMBRYONIC STEM CELLS
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PRODUCTION AND MAINTENANCE OF EMBRYONIC STEM CELLS
SEMINAR ON
ANKUR SHARMA
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STEM CELLS Unspecialized cells, reproduce indefinitely and under right
conditions develop into wide variety of mature cells with specialized functions.
TYPES OF STEM CELLS
B ased on sou rce o f o rig in B ased on p o ten cy
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• Totipotent Able to differentiate into all cell types
(only zygote and first cleavage blastomeres)
• Pluripotent Ability to form all lineage of body
(except placanta and extra embryonic membrane)
exp- embryonic stem cells
• Multipotent Adult stem cells having ability to form multiple
cell types of one lineage
exp- Hematopoietic stem cells
• Unipotent Cells able to form one cell type only
exp- Spermatogonial stem cells
(develop in sperm only)
STEM CELLS (Based on differentiation potential)
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• Embryonic
(Embryonic Stem Cells, Embryonic germ Cells)
• Fetal stem cells
(Umbilical cord, placenta, amniotic fluid)
• Adult Stem Cells
- Bone marrow (hematopoietic & mesenchymal)- Tissue: neural, skin, skeletal muscle (satellite cells), endothelial progenitors (EPCs)
STEM CELLS (Based on source of origin)
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PROPERTIES :-
• Self renewal
• Pluripotency
• Differentiation to any cell type
• G1 check point absent
• High telomerase activity
• Absence of X chromosomal inactivation
• Teratoma formation
• Germ line transmission
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EMBRYONIC STEM CELLS
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Characteristics Embryonic stem cells Embryonic germ cells
Origin Embryonic (Epiblast of ICM)
Embryonic(Primordial germ cells)
Potency Pluripotent Pluripotent (in vitro)
Isolated from • Pre implantation embryo
• ICM of blastocyst
• Post implantation embryo• Gonadal ridge of 5-9 week
old fetusProliferation capacity Higher (Upto 300
population doubling)Less (maximum reported 70-
80)Embryoid body
formation+ +
Teratoma formation + -
Stable karyotype + +
Chimera formation + -
Growth characteristics (in vitro)
Form flat, loose aggregates
Form rounded, multi-layer clumps
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SOURCE OF EMBRYONIC STEM CELLS
BLASTOCYCST
FERTILIZATION
NUCLEAR TRANSFER
PARTHENOGENESIS
EMBRYONIC STEM CELLS
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DERIVATION OF EMBRYONIC STEM CELLS FROM FERTILIZED OOCYTE
OOCYTE (ovaries of slaughtered animals, ovum pick-up)
IVM
IVFSPERM
ZYGOTE
MORULA (16-32 cells)
BLASTOCYSTICM
TROPHOECTODERM
Epiblast
Hypoblast
Embryonic stem cells
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In NT, the genetic material of the oocyte is removed and replaced with a diploid nucleus from a somatic (body) cell.
This divides to yield an NT blastocyst whose genes are identical with those of the donor somatic cell.
NT blastocysts, like normal blastocysts, can be used to derive embryonic stem cells from their inner cell masses.
CELLS DERIVED FROM NUCLEAR TRANSFER
Replace with nucleus of Donor cell
Donor cell
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CELLS DERIVED FROM PARTHENOGENESIS
IVM oocyte
Calcium ionophore (5μM;5min)
6-DMAP (4hr)
IVC (in vitro culture)
Blastocyst
ICM (inner cell mass)
Embryonic stem cells
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METHODS OF ISOLATION
• Immunosurgery
• Enzymatic
• Mechanical
• Intact blastocyst culture
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IMMUNOSURGERY
• Blastocyst is treated with pronase to dissolve the zona pellucida
• Preincubated with antiserum (rabbit anti-mouse serum)
• Exposure to complement (Guinea-pig complement serum)
• Resulting cytotoxicity selectively kills trophoblasts
• Large number of ICMs can be isolated simultaneously
• May cause cytotoxicity to ICM also
• Protocol work best with high quality embryos containing intact trophoectoderm, as only structural integrity of blastocyst prevents ICM from being susceptible to immunological reaction
• u
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Solter and Knowles, 1975
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ENZYMETIC ISOLATION
• Expanded Blastocysts
Pronase : for Zona digestion
Trypsin : for T.E. digestion
Trypsin inhibitors• Hatched Blastocysts
Trypsin : for T.E. digestion
Trypsin inhibitors• Enzymes like Collagenase, Trypsin, Dispase are commonly being used
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MECHENICAL/ MICRODISSECTION
• ICM cells derived mechanical dissection and partial removal of trophoblast layer
• 27G needle is used
• Isolation is performed under Zoom stereomicroscope
• Method suitable for hatched blastocyst (ICM visible)
• Laser assisted dissection to isolate ICM
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Frietas et al., 2011
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INTACT BLASTOCYST CULTURE
• Hatched Blastocyst seeded as such on feeder layer
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Whole embryo with intact zonaPronase digestion
Embryo on mitomycin C-treated feeder layer
(2-3days)
Trophectoderm expansion
ICM dome shaped surrounded by trophectoderm
ICM isolation
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LASER DISSECTION
• Two holding pipettes holds blastocyst with ICM being positioned at 9o’clock.
• 10 infrared laser pulses are fired to split the blastocyst into two unequal portions-the smaller consisting of ICM,the larger consisting exclusively of trophoblast.
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Laser dissection. Blastocyst secured by two holding pipettes with inner cell mass (ICM) being positioned at 9 o'clock before (a) and after (b) being sectioned by laser with (b) and without (c) zona pellucida. Arrows (b, c) indicate the resected area by laser energy. The smaller blastocyst fragment (white arrow) contains the ICM while the larger (yellow arrow) is exclusively trophoblast (d). Scale = 30 μm.
Tanaka N. et al.,2006
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MAINTENANCE OF EMBRYONIC STEM CELLS
ES Cells require meticulous care , need to be maintained carefully and frozen, thawed and trypsinised with
reasonable survival rates.
TWO TYPES OF CULTURE SYSTEMS
COCULTURE WITH FEEDER LAYER (fetal muscle and fetal epithelial, adult epithelial, fallopian tube, marrow, fibroblast,
and placental cells.)
FEEDER FREE CULTURE
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PREPARATION OF FEEDER LAYER
• 60-70% confluent fibroblast culture is either treated with mitomycin C (10ug/ml) or gamma irradiated for mitotic inactivation
• Mitomycin C covalently cross links the complementary strands of DNA both in vitro and in vivo thus preventing DNA replication
• Secretes cytokines such as Leukemia Inhibitory Factor (LIF) preventing differentiation of stem cells
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CULTURE OF BOVINE ICM ON FEEDER LAYER
DMEM supplemented with10-20% FCS.1mM 2-MercaptoethanolhLIF – 10ng/mlStreptomycin- 50ug/mlPenicillin- 100 IU/ml2mM L- Glutamine
Maintained at 370C, 5% CO2 and 95% humidity
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Frietas et al., 2011
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LIF/gp130/STAT3 PATHWAYLIF
LIFRgp130
JAKJAKSTAT
STATP
PP
STAT
STATSTAT
PP
Activation of transcription of genes
STAT
(Williams et al., 1988)
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BASIC PRINCIPLES OF PLURIPOTENCY
LIF/STATBMP
Wnt Signaling PI3KRas/Raf/ERK
PLURIPOTENCY GENES DIFFERENTIATION GENES
Secondary Messengers
Transcription factors
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FEEDER FREE CULTURE SYSTEM (DEFINED MEDIUM)
• Matrigel or Laminin coated plastic plates• MEF conditioned medium • KOSR Fibronectin matrix Supplements- In hESC: KOSR Transforming growth factor-b (TGF-b1), Fibroblast growth factor (bFGF), In mESC: Leukemia inhibitory factor (LIF)