STEM CELLS

STEM CELLS
a project presentation for immunology
sinnisky tan
Fyrås, Jämtland
Scope of presentation
Introduction
Techniques in the laboratory
Influence of cytokines and transcription factors
Summary
Introduction
What are stem cells?
Unspecialised
Long-term self-renewal
Ability to differentiate into specialised cells
e.g. heart muscle cells, neurons, liver cells, etc.
Why study stem cells?
To understand SC properties and mechanisms
Manipulate
Quantify
Many applications:
Basic research, e.g. developmental biology, birth defects, cancer
Transplantation research
Therapeutic delivery system
Drug screenings
Types of stem cells
ES cells
Blastocyst (inner cell mass)
Pluripotent
Easily grown in cultures
Adult stem cell
bone marrow, liver, pancreas, gastro. tract
Homeostasis
limited differentiation capacities; plasticity?
Rare; hard to isolate and purify
Potential transplant source; low risk of rejection
Hematopoietic stem cells (HSC)
A stem cell from which all red and white blood cells evolve
Bone marrow, peripheral blood and umbilical cord blood
HSC markers
HSCs (cont’d)
development, maintenance and regeneration of blood tissues for life
Bone marrow transplant: clinical use
Leukemia
cancer chemotherapy (autologous)
http://stemcells.nih.gov/info/scireport/
Laboratory methods for SC isolation and analysis
FACS
Cell culture
Colony forming assays
Cell staining
Immunofluorescence
Immunocytochemistry
PCR
FACS
http://stemcells.nih.gov/info/scireport/
http://stemcells.nih.gov/info/scireport/
Growing ES cells
Colony forming assays
Quantitate and characterise hematopoietic progenitors
HSC immobilised in semi-solid matrix
Supplement with nutrients and cytokines
Proliferation and differentiation
Allows clonal progeny of a single progenitor cells to stay together
Blood (1999) 94:2333
Mast cell colony
Mixed mast cell/ monocyte
Monocyte colony
Cell staining - morphology
E.g. mast cell
Wright-Giemsa
Blood (1999) 94:2333
PNAS (2000) 97:10509
Immunofluorescence
Mol. Biol. Cell. (2005) 16: 1777
http://stemcells.nih.gov/info/scireport/
http://occawlonline.pearsoned.com/
Immunocytochemistry
CD34+ differentiation to DC
Stem cells (2004) 22: 725
SCF/FL/TPO
+ IL-6, 7 days
+ GM-CSF/ IL-4, 5 days
+ TNF-α, 2 days
Model systems & Timing
Mouse IL-3 → mast cell
Human IL-3 → basophils & eosinophils

Addition of IL-4, in presence of SF / IL-6:
Beginning of culture
Inhibit mast cell development via downregulation of c-kit
After 10 weeks of culture
Induces differentiation of chymase-positive mast cells
Monocytes and hMast cells arising from
CD34+/c-Kit+/CD13+ precursors
Blood (1999) 94:2333
Monocytes
hMC
Monocytes
hMC
IL-7 activation in lymphoid precursors
Curr Opin Imm (2004) 16: 180
Staying Undifferentiated: That simple?
Cell fate dependent on contacts betw. HSCs and stromal cells
Notch activation keeps HSC or progenitor cells from embarking on differentiation
Abs used to disrupt contact via VLA-4 / VCAM-1
Cells undergo apoptosis
Wang MW et al. (1998) Cell Growth Differ. 9:105
Alberts Bruce et al.: Molecular biology of the cell.4 th edition.
Notch signaling
Maintenance or enhance HSC self-renewal by activating Hes1 (Notch target)
Oncogene (2002) 21:3295
Notch signaling during T cell development
Imm. Rev. (2002) 187: 65
Integration of Notch and Wnt
Parallel pathways in HSC
Wnt enhance proliferation and survival
Notch preventing differentiation
Wnt represses GSK3β, which can promote Notch (NIC) degradation
Nat. imm. (2005) 6: 314
Lineage commitment by transcriptional factors
determination of HSC fate:
Renewal or
Commitment to CLP / CMP
Transcriptional regulation of
CMP commitment
Oncogene (2002) 21:3295
Transcriptional regulation of
CLP commitment
Oncogene (2002) 21:3295
Oncogene (2002) 21:3295
Cytokine production in basal and induced hematopoiesis
Summary
Stem cell decision is dependent on many factors
HSC differentiation is regulated by a network of cytokines
Transcription factors
govern self-renewal or lineage commitment
also initiated by regulatory cytokines
acknowledgements
elsa björn
jyothi ravi
Gäddede, Jämtland