Blood -- Human embryonic stem cells: a source of mast cells for the study of allergic and inflammatory diseases

Blood, Vol. 115, Issue 18, 3695-3703, May 6, 2010

Human embryonic stem cells: a source of mast cells for the study of allergic and inflammatory diseases
Blood Kovarova et al. 115: 3695

Supplemental materials for: Kovarova et al

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Document 1. Supplemental materials and methods (PDF, 79.5 KB)
Figure S1. Hematopoietic progenitors derived from hES cell line H9 (JPG, 99.2 KB) -
CD45 and CD43 surface expression. hES cell were differentiated to embryoid bodies and subsequently to hematopoietic progenitors as describe in Methods. Collected non-adherent cells were analysed by FACS. Isotype control (left panel), CD45 and CD43 antibody (right panel). Cells were also stained with anti-CD34 antibody (lower panel), isotype control (transparent), anti-CD34 (blue). Results shown are representative of 3 experiments.
Figure S2. Phenotype of ESMC derived by co-culture with OP-9 cells (JPG, 45.3 KB) -
Human mast cells differentiated by OP-9 co-culture were stained with anti-tryptase (upper-left panel), and anti-chymase antibody (lower-left panel), and Toluidine blue (lower right panel). Nuclei were stained with DAPI (upper right panel). Images were captured using an Olympus BX61 upright fluorescence microscope with objectives 40× (anti-chymase, anti-tryptase staining) and 60× (Toluidine blue staining). Bars in insets equal 10 µm. Results shown are representative of 4 experiments.
Figure S3. Human mast cells differentiated from neo-resistant hES cells (JPG, 171 KB) -
A plasmid carrying the neomycin gene was introduced to the hES cell line H1 by electroporation. G418 resistent clones were identified, individually picked and expanded. DNA was prepared from the clones (#2 ans #3) and integration of neo gene was verify by Southern blot analysis using a ³²P-labled neomycin specific probe. DNA from a mouse ES cell line carrying a single copy of the neomycine gene was used as a positive control.(A). Arrows indicate position of DNA fragments hybridizing to the probe. Neo-hES cells were differentiated to hematopoietic progenitors by EBs formation. Collected progenitors were analyzed by FACS for expression of CD43 and CD45. (B). Isotype control (left panel), anti-CD45 and anti-CD43 antibody (right panel). Progenitors were further differentiated to mast cells expressing c-Kit (C). Isotype control (transparent), anti–c-Kit antibody (blue), n=2. Both clones (#2 and #3) were differentiated, results are from clone #3 differentiation.