Group_5_Presentation - Mast Cell

Report
GROUP 5
IN VITRO MODELS FOR
HUMAN MAST CELLS
Trainer:
Hammel
Trainees:
Ilan
Patricia Valentin
Mansour Seaf
Adi Efergan
Iva Polakovicova
Rosa Torres
Introduction
• Differences between human and mouse mast cells.
• Limitation of HuMCs
AIM: To make a comparison between different Human mast cell models
MCs Cell lines-
HMC-1
LAD-2
Primary cultured MCs-
HLMCs
sMCs
Peripheral derived CD34+ cells
ESMCs
CBMCs
Introduction
TO BE DISCUSSED:
• Similarities vs differences
• Gene profile and mRNA expression
• Proteins composition and protein levels
• Stage of maturation
• Granule content
• Cell surface receptors expression
• Modes of activation and secretion
Vascular endothelial growth factors
synthesized by human lung mast cells exert
angiogenic effects
Aikaterini Detoraki, MD, PhD,a Rosaria I. Staiano, PhD,a Francescopaolo Granata, MD, PhD,a Giorgio Giannattasio, MD,a
•Nella Prevete, PhD,a Amato de Paulis, MD,a Domenico Ribatti, MD,b Arturo Genovese, MD,a Massimo Triggiani, MD,
•PhD,a and Gianni Marone, MDa
Human mast cells express and synthesize
VEGFs
Expression of VEGFs in human mast cells.
A. VEGF-A, B, C, D and PlGF RT-PCR amplification products from
HLMCs, LAD-2 cells, and HMC-1 cells.
B. Immunoblot with anti–VEGF-B, C, D and anti–GAPDH antibodies of
HLMC, LAD-2, and HMC-1 lysates. MCF-7 and RAW 264.7 cells were
used as positive controls.
PGE2 and NECA affect expression and release of
VEGF in a time-dependent manner
LAD-2
HLMCs
Effects of PGE2 on VEGF production from
human mast cells.
A. Expression of VEGF-A, B, C and D was
determined in LAD-2 cells by qPCR.
B. VEGF-A release was determined in
HLMCs by using ELISA.
HMC-1
HLMCs
Effects of NECA on VEGF production from
human mast cells.
A. Expression of VEGF-A, B, C and D was
determined in HMC-1 cells by qPCR.
B. VEGF-A release was determined in
HLMCs by using ELISA.
Human mast cells express both VEGFR-1 and
VEGFR-2
HLMCs
Expression of VEGFRs in human mast
cells.
A. VEGFR-1, soluble VEGFR-1, VEGFR-2, and
GAPDH RT-PCR amplification products
from HLMCs, LAD-2 cells, and HMC-1
cells.
B. The cell surface expression of VEGFRs in
HLMCs was determined by FACS.
Human mast cells synthesize and release
angiogenin, a member of the ribonuclease A
(RNase A) superfamily
Marianna Kulka,*,1 Nobuyuki Fukuishi,† and Dean D. Metcalfe†
Human mast cells synthesize angiogenin
Analysis of ANG expression.
A. HMC, HMC-1, LAD2, and CD34+derived HuMC expression of
ANG and actin by qRT-PCR.
Human embryonic stem cells: a source of
mast cells for the study of allergic and
inflammatory diseases
Martina Kovarova, Anne M. Latour, Kelly D. Chason, Stephen L. Tilley and Beverly H. Koller
ESMCs produce tryptase and chymase in the
same manner as CBMCs
Phenotype of Embryonic Stem
cell-derived mast cells.
A. Cells were stained with αtryptase, α-chymase , and
Toluidine blue.
B. Quantification of tryptase- and
chymase- positive cells in
culture during mast cell
differentiation.
C. Enzymatic activity of tryptase
measured in mast cell lysates.
D. Inhibition of tryptase activity by
tryptase antagonist.
ESMCs and CBMCs express FcεRI and c-Kit on
the cell surface
Expression of FcRI and c-Kit on ESMCs.
ESMCs
- OP9
ESMCs
- EB
CBMCs
FACS analysis of ESMCs.
A. ESMCs differentiated by co-culture
with OP9.
B. ESMCs differentiated by EB
formation.
C. CBMCs.
D. Expression of c-Kit in ESMCs
differentiated by EB formation.
E. Double staining of ESMCs by α-IgE
and α-c-Kit antibody.
Activation of ESMC by crosslinking of FcεRI
ESMCs
ERK phosphorylationfollowing FcεRI activation
Intracellular Ca 2+ releasefollowing FcεRI activation
Histamine secretion
TNF-α secretion
PGD2 release
CBMCs
ESMCs with the same genetic modification can
be generated
Human mast cells differentiated from genetically modified hES cells.
Mast cells derived from GFP-hES express tryptase.
Top panel: GFP-hES cells.
Bottom panels: Mast cells derived from non-transfected H1 clone.
Supporting Papers
Mast cell lines HMC-1 and LAD2 in comparison
with mature human skin mast cells
Skin MCs
LAD-2
HMC-1
FCεRI
Histamine secretion
Tryptase
Chymase
* Histamine content OK
*
Cultured Mast Cells from Patients with Asthma and
Controls Respond with Similar Sensitivity to IgEMediated Activation
Table 2 Mast cell numbers obtained from asthmatics and control persons (median and range).
The CD63 MFI and the %
CD63+ mast cells cultured
from patients with asthma
and controls were similar.
A. The maximal CD63 MFI
B. The median % CD63+ mast
cells
measured on allergen-specific
activated mast cells from
patients with asthma and
controls.
Gene Expression Profiling of Human Mast Cell
Subtypes: An In Silico Study
Gene Expression Profiles of 16 Samples
2 basophil samples,
2 eosinophil samples,
2 neutrophil samples,
3 lung-derived MC samples,
3 tonsil-derived MC sample,
3 peripheral blood progenitor-derived
MC samples,
1 cord blood-derived MC sample
Summary
• What are the biological questions that are best addressed by the model that you
discussed?
To analyze the function and development of human mast cells.
Mast cells play a pivotal role in bronchial asthma, allergic diseases and are also implicated
in other chronic inflammatory conditions and tumor growth. Therefore, human MCs are
the most appropriate model for the investigation of human MCs related disease.
•
Summary
If more than one model exist- do the distinct models yield similar results?
If not, point out the discrepancies. Criticism of the model discussed.
Human mast cells can be derived from isolated CD34+ or CD133+ hematopoietic precursors
from either cord blood or peripheral blood – need to be cultured several weeks
Alternatively, mature human mast cells can be isolated from a few human tissues, including
lung, skin and gut – can be used immediately
Limitations:
1. Low number of cells
2. Cannot be cultured indefinitely- continuous source is required
3. Genetic differences are present between each population
4. Primary mast cells cannot be easily genetically manipulated
5. Long culturing in the same condition affects the fenotype
Suggestions:
Differentiating cells by using co-culture on supporting cell types such as fibroblast feeder
layer to provide beneficial microenvironment for the differentiation and proliferation
Human Embryonic stem cell derived MCs offer an alternative – e.g. to follow mast cell
differentiation and tracking at in vitro and in vivo experiments
Summary
• If more than one model exist- do you distinct models yield similar results?
If not, point out the discrepancies.
• Criticism of the model discussed.
Also, there are 2 human MC lines: HMC-1 and LAD2
Limitations:
1.
2.
3.
4.
cell-lines
immature MCs
low amount of tryptase and chymase
HMC-1 don’t express FceRI
Summary
• Nothing is perfect in life 
• None of the model is suitable
• The choice of the model depends on the interest of
your study
THANK YOU!!

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