Difference between revisions of "2009 Lecture 21"

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* human recombinant leukemia inhibitory factor (LIF) 2,000 units/ml
 
* human recombinant leukemia inhibitory factor (LIF) 2,000 units/ml
  
 +
Establishment of order from 1 cell
 +
clonal
 +
stem cells
 +
Developmental Models
 +
Signals, position, time
 +
differentiation
 +
Patterns, programs
 +
3 Adult Cell Types (Proliferation)
 +
Differentiated cells
 +
no longer capable of cell division
 +
Cardiac muscle cells, neurons
 +
produced during embryonic development, differentiate, then retained throughout life of organism
 +
Cells in G0 stage of cell cycle
 +
resume proliferation when needed to replace cells
 +
skin fibroblasts, smooth muscle cells, endothelial cells, epithelial cells of internal organs, such as the liver, pancreas, kidney, lung, prostate and breast
 +
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
 +
Movie: Neural Stem Cell
 +
PubMed- Stem Cells
 +
Medline Search “stem cell”
 +
2002 - 110,920
 +
2004 - 128,485
 +
2005 - 140,966
 +
2006 - 154,176
 +
 +
NIH - Stem Cells
 +
National Institute of Health (USA)
 +
http://stemcells.nih.gov/index.asp
 +
Stem Cells Report (2001)
 +
http://stemcells.nih.gov/info/scireport/
 +
 +
 +
Stem cells in Development
 +
Blastocyst
 +
 +
 +
 +
 +
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
 +
 +
 +
FourHumanESCell Lines
 +
Long Term ES Cell Culture
 +
Human ES Cell Differentiation
 +
 +
 +
Neural Stem Cells: Adult
 +
Purification of a pluripotent neural stem cell from the adult mouse brain.
 +
Rietze et al. The Walter and Eliza Hall Institute of Medical Research
 +
purified NSCs from the adult mouse brain by flow cytometry, and directly examined the cells' properties
 +
Nature. 2001 Aug 16;412(6848):736-9.
 +
 +
 +
Stem Cell Markers
 +
Every cell surface has specialized proteins (receptors) that can selectively bind or adhere to other “signalling” molecules (ligands)
 +
Different types of receptors differ in structure and affinity for signalling molecules
 +
Cells use these receptors and molecules that bind to them as a way of communicating with other cells and to carry out their proper functions in the body
 +
Covered in Lecture 11, 12
 +
Same cell surface receptors are stem cell markers
 +
Stage-Specific Embryonic Antigen-1,4
 +
Tumor Rejection Antigen (TRA-1-60)
 +
Stem Cell Antigen 1 (Sca-1)
 +
Marker Examples
 +
Stage-Specific Embryonic Antigen-1 (SSEA-1)
 +
role in cell adhesion, migration and differentiation
 +
often differentially expressed during development
 +
Stage-Specific Embryonic Antigen-4 (SSEA-4)
 +
surface embryonic antigen of human teratocarcinoma stem cells (EC), human embryonic germ cells (EG) and human embryonic stem cells (ES)
 +
down-regulated following differentiation of human EC cells
 +
Antigen not expressed on undifferentiated murine EC, ES and EG cells but up-regulated on differentiation of murine EC and ES cells
 +
ES Therapeutics
 +
Possible Therapeutic Uses
 +
Neural
 +
Parkinson’s, ALS, spinal cord injury……..
 +
Cell Replacement
 +
cell death, loss of function
 +
Grafting
 +
where host-graft rejection normally requires substantial ongoing immunosuppression
 +
Repair
 +
Spinal cord and brain injury
 +
Other Diseases
 +
Diabetes, muscular dystrophies, cardiac, vital organs……
 +
 +
 +
Stem Cells and Grafting
 +
Nature paper showed possible therapeutic use of ES cells in grafting
 +
host-graft rejection normally requires substantial ongoing immunosuppression
 +
Pre-implantation stage stem cells induce long-term allogeneic graft acceptance without supplementary host conditioning
 +
Aug02 FANDRICH etal.
 +
Stem Cell Experiments
 +
Human
 +
2 labs in 1998 human embryonic stem cells Gearhart 1998, Thomson et al. 1998
 +
inner cell masses of embryos not implanted into infertility patients
 +
germ cells derived from spontaneously aborted fetuses
 +
Mouse
 +
ES cells cultured in conditions to form glial stem cells
 +
glial stem cells were transplanted into mice that had a genetic deficiency of glial function, and cured defect (Brüstle et al. 1999)
 +
Neural stem cells derived from mouse ES cells
 +
divide and differentiate into functional neurons when injected into a damaged rodent nervous system (McDonald et al. 1999)
 +
Human Stem Cells
 +
Human neural stem cells improve sensorimotor deficits in adult rat brain with experimental focal ischemia
 +
Ischemic stroke caused by interruption of cerebral blood flow leads to brain damage with long-term sensorimotor deficits
 +
intravenous injection
 +
determine migration, differentiation and long-term viabilities of human NSCs in rat brain
 +
Human NSCs were detected in lesion side and labelled with marker for neurons or astrocytes
 +
Post-ischemic hemispheric atrophy noted but reduced in NSCs-ischemia group
 +
 +
== Neural Therapeutic Uses?==
 +
 +
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
 +
* Björklund et al Proc. Natl. Acad. Sci. USA, Vol. 99, Issue 4, 2344-2349, February 19, 2002
 +
 +
===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
  
 
== References ==
 
== References ==

Revision as of 00:03, 26 May 2009

Stem Cells

Introduction

Week 1 Human Development - Embryonic Stem Cells
NIH - stem cell cartoon

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 can also be found at UNSW Embryology Stem Cells and Week 1 Development.

Research that led to Stem Cells

  • Human Diseases
    • Generation of “knock out” mice
  • Human Development
    • Studying regulation of cell differentiation in development
  • Human Reproduction
    • Disorders, sterility

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

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.

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

Stem Cell Markers

In order to carry out research on stem cells, it is important to be able to identify them. A number of different research groups in the late 90's generated several antibodies which specifically identified undifferentiated, differentiating or differentiated stem cells from a number of different sources and species. Note that the nomenclature in some cases is based upon the antibody used to identify the cell surface marker.

  • Every cell surface has specialized proteins (receptors) that can selectively bind or adhere to other “signalling” molecules (ligands)
  • Different types of receptors differ in structure and affinity for signalling molecules
  • Cells use these receptors and molecules that bind to them as a way of communicating with other cells and to carry out their proper functions in the body


  • Stage-Specific Embryonic Antigen-1 (SSEA-1) cell surface embryonic antigen which has a role in cell adhesion, migration and differentiation and is often differentially expressed during development. Can be identified by Davor Solter monoclonal antibody MC-480 (SSEA-1).
  • Stage-Specific Embryonic Antigen-4 (SSEA-4) cell surface embryonic antigen of human teratocarcinoma stem cells (EC), human embryonic germ cells (EG) and human embryonic stem cells (ES) which is down-regulated following differentiation of human EC cells. Antigen not expressed on undifferentiated murine EC, ES and EG cells but upregulated on differentiation of murine EC and ES cells. Can be identified by Davor Solter monoclonal antibody MC-813-70 (SSEA-4)
  • Tumor Rejection Antigen (TRA-1-60) Sialylated Keratan Sulfate Proteoglycan expressed on the surface of human teratocarcinoma stem cells (EC), human embryonic germ cells (EG) and human embryonic stem cells (ES).
  • Tumor Rejection Antigen (TRA-1-81) antigen expressed on the surface of human teratocarcinoma stem cells (EC), human embryonic germ cells (EG) and human embryonic stem cells (ES).
    • Both TRA antibodies identify a major polypeptide (Mr 240 kDa) and a minor polypeptide (Mr 415 kDa).
  • Oct-4 (Pou5f1 – Mouse Genome Informatics) gene has an essential role in control of developmental pluripotency (Oct4 knockout embryo blastocysts die at the time of implantation). Oct4 also has a role in maintaining viability of mammalian germline.
  • Stem Cell Antigen 1 (Sca-1) member of the Ly-6 family of GPI-linked surface proteins (Mr 18 kDa) and a major phenotypic marker for mouse hematopoietic progenitor/stem cell subset.
  • CD133, AC133, prominin 5 transmembrane glycoprotein (865 aa) expressed on stem cells with hematopoietic and nonhematopoietic differentiation potential.

Alpha 6 integrin

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


Current stem cell research

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

Establishment of order from 1 cell clonal stem cells Developmental Models Signals, position, time differentiation Patterns, programs 3 Adult Cell Types (Proliferation) Differentiated cells no longer capable of cell division Cardiac muscle cells, neurons produced during embryonic development, differentiate, then retained throughout life of organism Cells in G0 stage of cell cycle resume proliferation when needed to replace cells skin fibroblasts, smooth muscle cells, endothelial cells, epithelial cells of internal organs, such as the liver, pancreas, kidney, lung, prostate and breast 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 Movie: Neural Stem Cell PubMed- Stem Cells Medline Search “stem cell” 2002 - 110,920 2004 - 128,485 2005 - 140,966 2006 - 154,176

NIH - Stem Cells National Institute of Health (USA) http://stemcells.nih.gov/index.asp Stem Cells Report (2001) http://stemcells.nih.gov/info/scireport/


Stem cells in Development Blastocyst



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


FourHumanESCell Lines Long Term ES Cell Culture Human ES Cell Differentiation


Neural Stem Cells: Adult Purification of a pluripotent neural stem cell from the adult mouse brain. Rietze et al. The Walter and Eliza Hall Institute of Medical Research purified NSCs from the adult mouse brain by flow cytometry, and directly examined the cells' properties Nature. 2001 Aug 16;412(6848):736-9.


Stem Cell Markers Every cell surface has specialized proteins (receptors) that can selectively bind or adhere to other “signalling” molecules (ligands) Different types of receptors differ in structure and affinity for signalling molecules Cells use these receptors and molecules that bind to them as a way of communicating with other cells and to carry out their proper functions in the body Covered in Lecture 11, 12 Same cell surface receptors are stem cell markers Stage-Specific Embryonic Antigen-1,4 Tumor Rejection Antigen (TRA-1-60) Stem Cell Antigen 1 (Sca-1) Marker Examples Stage-Specific Embryonic Antigen-1 (SSEA-1) role in cell adhesion, migration and differentiation often differentially expressed during development Stage-Specific Embryonic Antigen-4 (SSEA-4) surface embryonic antigen of human teratocarcinoma stem cells (EC), human embryonic germ cells (EG) and human embryonic stem cells (ES) down-regulated following differentiation of human EC cells Antigen not expressed on undifferentiated murine EC, ES and EG cells but up-regulated on differentiation of murine EC and ES cells ES Therapeutics Possible Therapeutic Uses Neural Parkinson’s, ALS, spinal cord injury…….. Cell Replacement cell death, loss of function Grafting where host-graft rejection normally requires substantial ongoing immunosuppression Repair Spinal cord and brain injury Other Diseases Diabetes, muscular dystrophies, cardiac, vital organs……


Stem Cells and Grafting Nature paper showed possible therapeutic use of ES cells in grafting host-graft rejection normally requires substantial ongoing immunosuppression Pre-implantation stage stem cells induce long-term allogeneic graft acceptance without supplementary host conditioning Aug02 FANDRICH etal. Stem Cell Experiments Human 2 labs in 1998 human embryonic stem cells Gearhart 1998, Thomson et al. 1998 inner cell masses of embryos not implanted into infertility patients germ cells derived from spontaneously aborted fetuses Mouse ES cells cultured in conditions to form glial stem cells glial stem cells were transplanted into mice that had a genetic deficiency of glial function, and cured defect (Brüstle et al. 1999) Neural stem cells derived from mouse ES cells divide and differentiate into functional neurons when injected into a damaged rodent nervous system (McDonald et al. 1999) Human Stem Cells Human neural stem cells improve sensorimotor deficits in adult rat brain with experimental focal ischemia Ischemic stroke caused by interruption of cerebral blood flow leads to brain damage with long-term sensorimotor deficits intravenous injection determine migration, differentiation and long-term viabilities of human NSCs in rat brain Human NSCs were detected in lesion side and labelled with marker for neurons or astrocytes Post-ischemic hemispheric atrophy noted but reduced in NSCs-ischemia group

Neural Therapeutic Uses?

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
  • Björklund et al Proc. Natl. Acad. Sci. USA, Vol. 99, Issue 4, 2344-2349, February 19, 2002

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

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

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