Talk:Lecture - Stem Cells

From Embryology
Revision as of 13:08, 1 October 2015 by Z8600021 (talk | contribs) (→‎Embryonic Stem Cells)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

2012 - This lecture will be presented by an expert guest lecturer Professor Edna Hardeman Here is a PDF for her lecture. File:Stem Cells E Hardeman orig.pdf


Anthony Lee Lecture 2010: Stem Cells Lecture 2010 Notes: high-resolution (PDF) Part 1 of 2 | Stem Cells Lecture 2010 Notes: high-resolution (PDF) Part 2 of 2


Lecture 22 - Stem Cells Lecture Date: 2012-10-09 Lecture Time: 15:00 Venue: CLB 3 Speaker: Kuldip Sidhu


http://stemcellres.com/content/1/4/28

http://www.biomedcentral.com/1471-213X/11/56/abstract

Introduction

File:Week1 cartoon600.jpg
Week 1 Human Development - Embryonic Stem Cells
Inner cell mass

The term "stem cell" is used so freely these days in many different forums that it is difficult sometimes understand without context what scientists, politicians, ethicists and commentators are discussing. This lecture will focus on the cell biology of stem cells and the current research on growing and differentiating theses cells.

Background information

Why are they in the News?

  • Scientific and Ethical
  • Therapeutic uses
  • Issues relating to human cloning
  • Use of excess human eggs/sperm for research purposes
  • Availability of human stem cell lines

What can they be used for?

  • Generation of “knock out” mice
  • Studying regulation of cell differentiation in development
  • Therapeutic uses?
  • Genetic disease
  • Neurodegenerative
  • Injury

PubMed

  • Medline Search “stem cell” 2002 - 110,920 | 2004 - 128,485 | 2005 - 140,966 | 2006 - 154,176 | 2011 - 182,559

Tissue Stem Cells

  • differentiated cells have short life spans continually replaced
  • blood cells, epithelial cells of skin and digestive tract
  • fully differentiated cells do not proliferate
  • proliferation of less differentiated- stem cells
  • produce daughter cells that either differentiate or remain as stem cells

Blood Cells

  • All different types of blood cells develop from a pluripotent stem cell in bone marrow
  • Precursors of differentiated cells undergo several rounds of cell division as they mature
    • proliferation ceases at terminal stages of differentiation

Embryonic Stem Cells

NIH - What are embryonic stem cells?

Pluripotent Stem Cells

  • What is a stem cell- Pluripotent
  • Pluripotent - to describe stem cells that can give rise to cells derived from all 3 embryonic germ layers
    • Mesoderm
    • Endoderm
    • Ectoderm
  • layers are embryonic source of all cells of the body

Blastocyst

  • hollow structure composed of about 100 cells surrounding an inner cavity
  • Only ES cells, which form inner cell mass, actually form the embryo.
  • ES cells can be removed from the blastocyst and grown on lethally irradiated “feeder cells.” (See E. Robertson et al., 1986, Nature 323:445)

Stem Cell Definition

  • cell that has ability to divide for indefinite periods
  • self replicate
  • throughout life of organism
  • stem cells can differentiate
    • conditions, signals
  • to the many different cell types

Chimeric Mouse

  • ES or teratocarcinoma
  • shows that stem cells can combine with cells of a normal blastocyst to form a healthy chimeric mouse

Embryoid Bodies

  • spheroid cellular tissue culture structure
  • mouse and human ES cells have the capacity to undergo controlled differentiation
  • recapitulate some aspects of early development
    • regional-specific differentiation program
    • derivatives of all three embryonic germ layers

Historic References

Mouse

  • Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Martin GR. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7634-8.
  • Characterization of a pluripotent stem cell line derived from a mouse embryo. Wobus AM, Holzhausen H, Jakel P, Schoneich J. Exp Cell Res. 1984 May;152(1):212-9.
  • Transgenesis by means of blastocyst-derived embryonic stem cell lines Proc Natl Acad Sci U S A. 1986 Dec;83(23):9065-9. Gossler A, Doetschman T, Korn R, Serfling E, Kemler R.

Pig and Sheep

Derivation of pluripotent, embryonic cell lines from the pig and sheep. Notarianni E, Galli C, Laurie S, Moor RM, Evans MJ. J Reprod Fertil Suppl. 1991;43:255-60.

Primate

Isolation of a primate embryonic stem cell line. Thomson JA, Kalishman J, Golos TG, Durning M, Harris CP, Becker RA, Hearn JP. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7844-8.

Human

Embryonic stem cell lines derived from human blastocysts. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Science. 1998 Nov 6;282(5391):1145-7.


Stem Cell Lines ATCC - Embryonic Stem cell lines

Timeline of Human Embryonic Stem Cell Research

  • 1878 First reported attempts to fertilize mammalian eggs outside the body
  • 1959 First report of animals (rabbits) produced through IVF in the United States
  • 1960's Studies of teratocarcinomas in the testes of several inbred strains of mice indicates they originated from embryonic germ cells. The work establishes embryonal carcinoma (EC) cells as a kind of stem cell
  • 1968 Edwards and Bavister fertilize the first human egg in vitro
  • 1970's EC cells injected into mouse blastocysts produce chimeric mice. Cultured SC cells are explored as models of embryonic development, although their complement of chromosomes is abnormal
  • 1978 Louise Brown, the first IVF baby, is born in England
  • 1980 Australia's first IVF baby, Candace Reed, is born in Melbourne
  • 1981 Evans and Kaufman, and Martin derive mouse embryonic stem (ES) cells from the inner cell mass of blastocysts. They establish culture conditions for growing pluripotent mouse ES cells in vitro. The ES cells yield cell lines with normal, diploid karyotyes and generate derivatives of all three primary germ layers as well as primordial germ cells. Injecting the ES cells into mice induces the formation of teratomas. The first IVF baby, Elizabeth Carr, is born in the United States.
  • 1984–88 Andrews et al., develop pluripotent, genetically identical (clonal) cells called embryonal carcinoma (EC) cells from Tera-2, a cell line of human testicular teratocarcinoma. Cloned human teratoma cells exposed to retinoic acid differentiate into neuron-like cells and other cell types
  • 1989 Pera et al., derive a clonal line of human embryonal carcinoma cells, which yields tissues from all three primary germ layers. The cells are aneuploid (fewer or greater than the normal number of chromosomes in the cell) and their potential to differentiate spontaneously in vitro is typically limited. The behavior of human EC cell clones differs from that of mouse ES or EC cells
  • 1994 Human blastocysts created for reproductive purposes using IVF and donated by patients for research, are generated from the 2-pronuclear stage. The inner cell mass of the blastocyst is maintained in culture and generates aggregates with trophoblast-like cells at the periphery and ES-like cells in the center. The cells retain a complete set of chromosomes (normal karyotype); most cultures retain a stem cell-like morphology, although some inner cell mass clumps differentiate into fibroblasts. The cultures are maintained for two passages
  • 1995–96 Non-human primate ES cells are derived and maintained in vitro, first from the inner cell mass of rhesus monkeys, and then from marmosets. The primate ES cells are diploid and have normal karyotypes. They are pluripotent and differentiate into cells types derived from all three primary germ layers. The primate ES cells resemble human EC cells and indicate that it should be possible to derive and maintain human ES cells in vitro.
  • 1998 Thomson et al., derive human ES cells from the inner cell mass of normal human blastocysts donated by couples undergoing treatment for infertility. The cells are cultured through many passages, retain their normal karyotypes, maintain high levels of telomerase activity, and express a panel of markers typical of human EC cells non-human primate ES cells. Several (non-clonal) cell lines are established that form teratomas when injected into immune-deficient mice. The teratomas include cell types derived from all three primary germ layers, demonstrating the pluripotency of human ES cells. Gearhart and colleagues derive human embryonic germ (EG) cells from the gonadal ridge and mesenchyma of 5- to 9-week fetal tissue that resulted from elective abortions. They grow EG cells in vitro for approximately 20 passages, and the cells maintain normal karyotypes. The cells spontaneously form aggregates that differentiate spontaneously, and ultimately contain derivatives of all three primary germ layers. Other indications of their pluripotency include the expression of a panel of markers typical of mouse ES and EG cells. The EG cells do not form teratomas when injected into immune-deficient mice
  • 2000 Scientists in Singapore and Australia led by Pera, Trounson, and Bongso derive human ES cells from the inner cell mass of blastocysts donated by couples undergoing treatment for infertility. The ES cells proliferate for extended periods in vitro, maintain normal karyotypes, differentiate spontaneously into somatic cell lineages derived from all three primary germ layers, and form teratomas when injected into immune-deficient mice.
  • 2001 As human ES cell lines are shared and new lines are derived, more research groups report methods to direct the differentiation of the cells in vitro. Many of the methods are aimed at generating human tissues for transplantation purposes, including pancreatic islet cells, neurons that release dopamine, and cardiac muscle cells.


Modified from NIH - Stem Cells: Scientific Progress and Future Research Directions 2001

Cord Blood Stem Cells

  • Blood collected from the placental umbilical cord of a newborn baby shortly after birth
    • total amount of blood about 90 ml
  • blood stem cells that can be used to generate red blood cells and cells of the immune system
  • collected, typed, stored in Cord Blood Bank
    • Both public and private Banks have arisen
    • available for use by the donor and compatible siblings
  • suggested use to treat a range of blood disorders and immune system conditions such as leukaemia, anaemia and autoimmune diseases
  • cells provide a resource for bone marrow replacement therapy in many diseases

Cord Blood - Disease Treatments

  • Acute Lymphoblastic Leukaemia
  • Acute Myeloblastic Leukaemia
  • Adrenoleukodystrophy
  • Blackfan-Diamond
  • Chronic Myeloid Leukaemia
  • Chronic Lymphocytic leukaemia
  • Fanconi's Anaemia
  • Hurler's Syndrome
  • Krabbe's disease
  • Lymphomas
  • Myelodysplastic Syndrome
  • Mucolipopolysaccharide deficiency
  • Osteopetrosis
  • Syndrome Severe Aplastic Anaemia
  • Severe Combined Immunodeficiency Disease
  • Thalassaemia
  • Wiskott-Aldrich Syndrome
  • Miscellaneous
  • Cancer
  • Genetic disorders
  • Immune deficiency
  • Storage disorders

Adult Stem Cells

NIH - What are adult stem cells?

Stem Cells in the Adult

  • Connective Tissue
  • Bone marrow
    • Blood Cells, Osteoclasts, blasts
  • Epithelia
    • Gut
    • Skin
  • Neural?

Epidermis: Immortal Stem Cell

Induced Pluripotent Cells

  • non-pluripotent cells engineered to become pluripotent
    • a cell with a specialized function ‘reprogrammed’ to an unspecialized state


Embryonic vs Adult Stem Cells

Embryonic Stem Cell Advantages

  • Pluripotency - ability to differentiateinto any cell type.
  • Immortal - one cell can supply endless amounts of cells.
  • Easily available - human embryos from fertility clinics.

Embryonic Stem Cell Disadvantages

  • Unstable - difficult to control differentiation into specific cell type.
  • Immunogenic - potential immune rejection when transplanted into patients.
  • Teratomas - tumor composed of tissues from 3 embryonic germ layers.
  • Ethical Controversy - unethical for those who believes that life begins at conception.


Adult Stem Cell Advantages

  • Already ‘specialised’ - induction of differentiation into specific cell types will be easier.
  • Plasticity - Recent evidences suggest wider than previously thought ranges of tissue types can be derived.
  • No Immune-rejection - if used in autologous transplantations.
  • No Teratomas - unlike ES cells.
  • No Ethical Controversy - sourced from adult tissues.

Adult Stem Cell Disadvantages

  • Minimal quantity - number of isolatable cells may be small.
  • Finite life-span - may have limited lifespan in culture.
  • Ageing - stem cells from aged individuals may have higher chance of genetic damage due to ageing.
  • Immunogenic - potential immune rejection if donor cells are derived from another individual.

References

Textbooks

Essential Cell Biology

  • Chapter 19 Tissues p622-627

Molecular Biology of the Cell

Alberts, Bruce; Johnson, Alexander; Lewis, Julian; Raff, Martin; Roberts, Keith; Walter, Peter New York and London: Garland Science; c2002

  • Molecular Biology of the Cell 4th ed. - Chapter 19 Cellular Mechanisms of Development p1037-1039

Molecular Cell Biology

Lodish, Harvey; Berk, Arnold; Zipursky, S. Lawrence; Matsudaira, Paul; Baltimore, David; Darnell, James E. New York: W. H. Freeman & Co.; c1999

The Cell- A Molecular Approach

Cooper, Geoffrey M. Sunderland (MA): Sinauer Associates, Inc.; c2000

  • The Cell - A Molecular Approach - IV. Cell Regulation Chapter 14. Cell Proliferation in Development and Differentiation
  • Stem Cells

Search Online Textbooks

Books

PubMed

Reviews

  • Jensen J, Hyllner J, Björquist P. Human embryonic stem cell technologies and drug discovery. J Cell Physiol. 2009 Jun;219(3):513-9. Review. PMID: 19277978

Articles

  • Allen ND, Baird DM. Telomere length maintenance in stem cell populations. Biochim Biophys Acta. 2009 Feb 11. [Epub ahead of print] PMID: 19419691
  • Kenji Matsumoto, Takayuki Isagawa, Toshinobu Nishimura, Takunori Ogaeri, Koji Eto, Satsuki Miyazaki, Jun-ichi Miyazaki, Hiroyuki Aburatani, Hiromitsu Nakauchi, and Hideo Ema Stepwise Development of Hematopoietic Stem Cells from Embryonic Stem Cells PLoS ONE. 2009; 4(3): e4820. Published online 2009 March 16. doi: 10.1371/journal.pone.0004820. PMCID: PMC2653650
  • Tesar PJ. Derivation of germ-line-competent embryonic stem cell lines from preblastocyst mouse embryos. Proc Natl Acad Sci U S A. 2005 Jun 7;102(23):8239-44. Epub 2005 May 25. PMID: 15917331

Search Entrez

Links


References

StemBook Cambridge (MA): Harvard Stem Cell Institute; 2008.

"StemBook is an open access collection of invited, original, peer-reviewed chapters covering a range of topics related to stem cell biology written by top researchers in the field at the Harvard Stem Cell Institute and worldwide. StemBook is aimed at stem cell and non-specialist researchers."

Template:2011ANAT3231



Template:Lecture Header

File:Stem cell therapy cartoon.jpg

Embryonic vs Adult Stem Cells

Embryonic Stem Cell Advantages

  • Pluripotency - ability to differentiateinto any cell type.
  • Immortal - one cell can supply endless amounts of cells.
  • Easily available - human embryos from fertility clinics.

Embryonic Stem Cell Disadvantages

  • Unstable - difficult to control differentiation into specific cell type.
  • Immunogenic - potential immune rejection when transplanted into patients.
  • Teratomas - tumor composed of tissues from 3 embryonic germ layers.
  • Ethical Controversy - unethical for those who believes that life begins at conception.


Adult Stem Cell Advantages

  • Already ‘specialised’ - induction of differentiation into specific cell types will be easier.
  • Plasticity - Recent evidences suggest wider than previously thought ranges of tissue types can be derived.
  • No Immune-rejection - if used in autologous transplantations.
  • No Teratomas - unlike ES cells.
  • No Ethical Controversy - sourced from adult tissues.

Adult Stem Cell Disadvantages

  • Minimal quantity - number of isolatable cells may be small.
  • Finite life-span - may have limited lifespan in culture.
  • Ageing - stem cells from aged individuals may have higher chance of genetic damage due to ageing.
  • Immunogenic - potential immune rejection if donor cells are derived from another individual.

Stem Differentiation

Epithelium

  • each generation at least 1 "immortal" stem cell
    • descendants present in patch in future
  • Other basal cells
    • leave basal layer and differentiate
  • Committed, born different

or may be stem cells equivalent to immortal stem cell in character mortal in sense that their progeny jostled out of basal layer and shed from skin

Amplifying Cells

  • Stem cells in many tissues divide only rarely
  • give rise to transit amplifying cells
  • daughters committed to differentiation that go through a limited series of more rapid divisions before completing the process.
  • each stem cell division gives rise in this way to eight terminally differentiated progeny

Stem Cell Production - Stem Cell Daughter Fates

  • Environmental asymmetry
    • daughters are initially similar
    • different pathways according to environmental influences that act on them after they are born
    • number of stem cells can be increased or reduced to fit niche available
  • Divisional asymmetry
    • stem cell has an internal asymmetry
    • divides in such a way two daughters are already have different determinants at time of their birth

Human embryonic stem cell Differentiation in Culture

File:Human embryonic stem cell defined conditions.jpg

Characterization of teratomas from H9 cells cultured on an hE-cad-Fc-coated surface. Hematoxylin and eosin staining of paraffin sections through teratomas identified the differentiation huES cells into various tissues:

  • a - immature neuroblastic tissue with neuronal rosettes
  • b - neuroepithelium with pigment
  • c - immature sebaceous tissue
  • d - cartilage
  • e - columnar epithelium
  • f - gut-like epithelial structures
  • g - contains neural tissue (1), cartilage (2), bone parenchyma (3), and epithelial tissue (4).

Bar indicates 100 μm.

Figure from: <pubmed>20525219</pubmed>| BMC Dev Biol.

Current stem cell research

File:NIH stem cell cartoon.jpg
NIH - stem cell cartoon

How to:

  • Isolate
  • Grow
  • Maintain, store
  • Differentiate
  • Therapeutic uses

Growth of Embryonic Stem Cells

  • Mouse blastocyst-derived ES cell line D3
    • from American Type Culture Collection (ATCC)
  • Undifferentiated ES cells
    • maintained on gelatin-coated dishes
    • earlier studies, feeder layer

Growth Media

  • DMEM (dulbecco’s modified essential media)
  • 2 mM glutamine (essential amino acid)
  • 0.001% beta-mercaptoethanol (reducing agent)
  • 1x nonessential amino acids (amino acids for growth)
  • 10% donor horse serum (source of growth factors etc)
  • human recombinant leukemia inhibitory factor (LIF) 2,000 units/ml

Inducible Stem Cell

Human induced pluripotent stem cells 01.jpg

(A) Morphology of human iPS cell clones (2a, 3a and 6a) on surfaces coated with Matrigel or hE-cad-Fc. Scale bar indicates 100 μm. (B) Characterization of teratomas from iPS cells (clone 2a) cultured on an hE-cad-Fc-coated surface. Hematoxylin and eosin staining of teratomas showed the differentiation into various tissues, including immature neuroblastic tissue with neuronal rosettes (a), striated muscle (b), and columnar epithelium (c). Bar indicates 100 μm.

Figure from: <pubmed>20525219</pubmed>| BMC Dev Biol.

Mouse- embryonic stem cell signaling regulation.jpg

Yamanaka Factors

A set of 4 transcription factors when introduced into cells induces stem cell formation. These four transcription factors can be expressed from doxycycline (dox)-inducible lentiviral vectors. The only culture difference in iPS cells and human embryonic stem cell culture is that iPS cell culture require 100ng/ml of bFGF in the culture media.

Outline of the MEF reprogramming protocol 1 Outline of the MEF reprogramming protocol 2 | stained with anti-Rex1, Sox2 and SSEA1 antibodies


  • OCT4 Transcription factors containing the POU homeodomain
  • MYC The MYC protooncogene encodes a DNA-binding factor that can activate and repress transcription. Ectopic expression of c-Myc can also cause tumorigenicity in offspring.
  • SOX2 SRY-RELATED HMG-BOX GENE 2
  • KLF4 Kruppel-like factor 4, zinc finger protein, transcription factor which acts as both an activator and repressor.


<pubmed>16904174</pubmed>


Links: Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

More recently shown that Oct4 together with either Klf4 or c-Myc is sufficient to generate iPS cells from neural stem cells.

<pubmed>18594515</pubmed>


Thompson Factor

Neural Therapeutic Uses?

File:Stem cell therapy cartoon.jpg
Stem cell therapy cartoon

NIH - Use of Genetically Modified Stem Cells in Experimental Gene Therapies


Embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model

  • Implantation of fetal dopamine (DA) neurons can reduce parkinsonism in patients
  • current methods are rudimentary
  • lacking a reliable donor cell source

Transplanted ES cells can develop spontaneously into dopamine (DA) neurons

  • Such DA neurons can restore cerebral function and behavior in an animal model of Parkinson's disease

<pubmed>11782534</pubmed>| PNAS

Parkinson Rat Model

Embryonic stem cell Transplant

  • transplanting low doses of undifferentiated mouse embryonic stem (ES) cells into rat striatum
  • results in a proliferation of ES cells into fully differentiated DA neurons
  • ES cell-derived DA neurons caused gradual and sustained behavioral restoration of DA-mediated motor asymmetry


Staining of a Graft

  • 16 weeks after implantation of D3 ES cells into adult 6-OHDA lesioned striatum
    • TH-positive neurons were found within the graft (A and B, green)
    • All TH-positive profiles coexpressed the neuronal marker NeuN (A, red)
    • TH (B) also was coexpressed with DAT (C, red) and AADC (D, blue), shown by white triple labelling (E)

Rotation response to Amphetamine

  • 6-OHDA-lesioned animals were selected for transplantation by quantification of rotational behaviour in response to amphetamine
  • response was examined post-transplantation at 5, 7, and 9 weeks
  • Animals with ES cell-derived DA neurons showed recovery over time from amphetamine-induced turning behavior


More Recent papers

Rhee YH, Ko JY, Chang MY, Yi SH, Kim D, Kim CH, Shim JW, Jo AY, Kim BW, Lee H, Lee SH, Suh W, Park CH, Koh HC, Lee YS, Lanza R, Kim KS, Lee SH.Protein-based human iPS cells efficiently generate functional dopamine neurons and can treat a rat model of Parkinson disease. J Clin Invest. 2011 May 16. pii: 45794. doi: 10.1172/JCI45794. PMID: 21576821


Shindo A, Nakamura T, Matsumoto Y, Kawai N, Okano H, Nagao S, Itano T, Tamiya T. Seizure suppression in amygdala-kindled mice by transplantation of neural stem/progenitor cells derived from mouse embryonic stem cells. Neurol Med Chir (Tokyo). 2010;50(2):98-105; discussion 105-6. PMID: 20185872 | Neurol Med Chir (Tokyo)

Cytoskeleton Disease?

Targeted Gene Correction of Laminopathy-Associated LMNA Mutations in Patient-Specific iPSCs

Cell Stem Cell. 2011 May 18.

Liu GH, Suzuki K, Qu J, Sancho-Martinez I, Yi F, Li M, Kumar S, Nivet E, Kim J, Soligalla RD, Dubova I, Goebl A, Plongthongkum N, Fung HL, Zhang K, Loring JF, Laurent LC, Izpisua Belmonte JC. Source Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.

Abstract

Combination of stem cell-based approaches with gene-editing technologies represents an attractive strategy for studying human disease and developing therapies. However, gene-editing methodologies described to date for human cells suffer from technical limitations including limited target gene size, low targeting efficiency at transcriptionally inactive loci, and off-target genetic effects that could hamper broad clinical application. To address these limitations, and as a proof of principle, we focused on homologous recombination-based gene correction of multiple mutations on lamin A (LMNA), which are associated with various degenerative diseases. We show that helper-dependent adenoviral vectors (HDAdVs) provide a highly efficient and safe method for correcting mutations in large genomic regions in human induced pluripotent stem cells and can also be effective in adult human mesenchymal stem cells. This type of approach could be used to generate genotype-matched cell lines for disease modeling and drug discovery and potentially also in therapeutics.

Copyright © 2011 Elsevier Inc. All rights reserved.

PMID: 21596650 | Cell Stem Cell.

Human embryonic stem cells (hESCs) without the use of additional human embryos

  1. Reprogramming of adult cell nucleus - use existing hESCs to fuse with an adult somatic cell, generating a cell line that retains ESC specific properties and yet has the genotype of the somatic cell donor. However, there is no technology available to selectively remove all the ESC chromosomes while retaining the somatic cell chromosomes.
  2. ESCs from embryo like entities - use of somatic cell nuclear transfer (SCNT) to produce developmentally compromised embryo-like structures, with the help of genetically premodified deficient nuclei which cannot support development. The zygote produced by such nuclear transfer undergoes cleavage in-vitro and produce ICM cells, which would be used for deriving ESCs, but would not proceed further in development. A proof of principle to this was accomplished by generating mouse ESCs, using a donor nucleus which was silenced for Cdx2 gene. This is ethically correct for those who believe that fetal life begins only after the embryo implants. However, one need not go for creating a mutation to achieve this target, as a blastocyst cannot develop into a complete human life in vitro, irrespective of the presence or absence of any kind of genetic alterations.
  3. ESC lines from single blastomeres - a single cell can be isolated from the cleavage stage embryo, a technique well established for preimplantation genetic diagnosis (PGDs), and used to create a cell line from it; the rest of the embryo can be transferred back to the uterus to give rise to a fetus. Robert Lanza's group has shown that ESC lines could be established from single cell biopsies of the mouse and human embryos. However, this technique is very difficult to translate to human being. Also, the fate of the residual embryos if they are transferred is largely unknown, as there is a lack of long term studies supporting the health of babies born following PGD.
  4. ESC lines from induced somatic cell dedifferentiation - adult somatic cells are genetically modified and reprogrammed to undergo a process of dedifferentiation, by inducing the expression of pluripotency related genes. Recently, induced pluripotent stem cell lines have been derived by allowing trans-acting factors present in the mammalian oocytes to reprogram somatic cell nuclei to an undifferentiated state. They have demonstrated that four factors OCT-4, SOX-2, Nanog and LIN28 are sufficient to reprogram human somatic stem cells to pluripotent stem cells. Whereas, Takahashi and Yamanaka (2006) induced somatic cells into pluripotent stem cells by introducing four factors OCT-4, SOX-2, c-Myc and KLF-4. These cells designated as ind