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Blood, Vol. 111, Issue 12, 5562-5570, June 15, 2008
Flk2+ common lymphoid progenitors possess equivalent differentiation potential for the B and T lineages
Blood Karsunky et al.
111: 5562
Supplemental materials for: Karsunky et al
Files in this Data Supplement:
- Figure S1. Effect of incubation period and serum lot on CLP function (JPG, 50.7 KB)
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(A–B) 103 CLPF were either immediately after sorting i.v. injected into sublethally irradiated congenic mice, or after 8 hours of culture in plain media (complete IMDM, 10% FBS) at 37°C without any cytokines. The chimerism and lineage readout was assessed 2 weeks after transplant in thymus and spleen. As maybe expected the (A) total chimerism in mice that had received the incubated CLPF (black bar) was decreased in the spleen compared to mice that had received CLPF without holding (white bar). Interestingly, no thymic contribution or mature T cells in the spleen (not shown) were detectable from incubated CLPF. Bars represent average chimerism in percent of 4–5 transplanted mice and error bars are based on standard deviation. (B) Graphs represent average percentage of B and dendritic cells among CLPF donor derived cells without holding (white bars) or after 8 hours of incubation (black bars). Whereas B cell chimerism in the spleen was comparable the number of dendritic cells from incubated CLPF was more dramatically reduced. These data demonstrates that the lineage readout from CLPF is affected by cell processing steps and that the T cell and DC lineages are more sensitive than the B cell lineage to holding periods. 5 mice were analyzed for the fresh CLPF, and 4 mice for the cultured CLPF. Bars labeled with a double asterisk means p=<0.02. (C) During long sorts cells are often incubated in the sorting tube at room temperature for long periods of time. To compare the viability of MPP and CLPF in room temperature media for long periods of time, we sorted MPP and CLPF into cold PBS with 2% FBS and 10mM EDTA, then incubated these cells for 3 hr on ice or at room temperature. (Left) 3000 MPP or CLPF were then reanalyzed by FACS for viability, and the number of live cells recovered was measured. While we observed a slight decrease in viability between MPP incubated at room temperature versus on ice, the effect in CLPF was much more dramatic, with a nearly 8-fold decrease in viability between the two temperatures. After 3 hours at room temperature, less than 10% of the originally sorted CLPF remained alive, demonstrating a sensitivity of these cells to long exposures at room temperature. (Right) To examine the effects of room temperature incubation on engraftment, we transplanted 5000 MPP or CLP after 3 hours at room temperature or on ice by i.v. injection into sublethally irradiated congenic mice (n = 5). 3 weeks after transplant spleens were analyzed for donor chimerism. Plotted are the total numbers of donor cells observed in 2.5 × 105 events analyzed. We observed a statistically significant decrease in chimerism in both MPP and CLPF following incubation at room temperature compared to on ice, though the dropoff was more severe with CLPF (* = p < 0.05 by t-test). (D) To examine the sensitivity of CLPF to serum lots, we cultured CLPF and a B cell progenitor population (Fraction B, or Fr. B) in liquid culture with cytokines that promote B differentiation (SCF, Flt3L, and IL7), with different brands and lots of serum. 10 cells were sorted per well, and the fraction of wells with colonies after 14 days was measured. CLPF displayed a greater sensitivity to the brand and lot of serum than did Fr. B cells. Thus, multiple sera should be tested before use in CLPF cultures. Serum 1: Omega Scientific lot# 104075. Serum 2: Hyclone SH30070.03, lot# APA20504. Serum 3: Hyclone SH30070.03 lot# ARH27209.
- Figure S2. Phenotypic comparison of CLP populations in C57Bl/6 and Balb/c mouse strains (JPG, 72.8 KB)
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Flow cytometric analysis of bone marrow from C57Bl/6 and Balb/c mice. All events shown are pre-gated on lineage negative cells. A similar population of Flk2+IL7R + cells can be detected in both strains. Histogram plot on the right shows overlay of CD93/AA4.1 expression levels of Flk2+IL7R+ cells from both strains.
- Figure S3. Clonal lymphoid assay (JPG, 142 KB)
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(A) Schematic presentation of the experimental layout. Single CLPF cells were sorted into Terasaki wells using the clone sorter function of a BD FACSAria cytometer. Terasaki plates were previously seeded with OP9 stroma cells and medium contained 100 ng/ml SCF, 20 ng/ml Flt3L, and 10 ng/ml IL-7. Successful deposition of exactly one cell was confirmed by microscopy. After 3 days of culture the number of lymphoid cells was counted again under the microscope. Wells containing 4 or more cells derived from a single CLPF were distributed to equal parts into 96-well plates either containing OP9 stroma cells supplemented with 20 ng/ml SCF, 20 ng/ml Flt3L, and 10 ng/ml IL-7 to test for B cell potential or into wells seeded with OP9-DL1 stroma supplemented with 20 ng/ml SCF, 20 ng/ml Flt3L, and 10 ng/ml IL-7 to test for T cell potential. Aliquots of CLPF clones from Terasaki wells that contained 8 or more cells were in addition seeded into 96 well plates containing OP9 stroma cells supplemented with 10 ng/ml SCF, 10 ng/ml Flt3L, 100 IU/ml IL-2, 10 ng/ml IL-15, and 5 ng/ml IL-7 to test for NK cell potential or into wells seeded with AC6 stroma supplemented with 20 ng/ml SCF, 20 ng/ml Flt3L, 10 ng/ml M-CSF, 20 ng/ml GM-CSF, and 20 ng/ml IL-3 to test for DC and myeloid potential. After 8 days of culture wells were harvested and analyzed by flow cytometry. (B) Examples of contour plots of all 4 lymphoid lineages as detected by flow cytometry. B cells were identified by co-expression of CD19 and B220, T cells by Thy1.1 and CD25, NK cells by NK1.1 alone since co-expression of D×5 was usually restricted to a very small subpopulation of NK1.1+ cells, and DC by CD11c and I-Ab/MHC II. To test for the presence of myeloid cells samples were also stained for CD11b/Mac-1 and Gr-1/Ly6G but no Mac-1+Gr-1+ progeny cells from CLP were detected in any of the wells. The few Mac-1+Gr-1− cells co-stained with CD11c and I-Ab and were thus DC. MPPs were used as a positive control to demonstrate that the DC/myeloid cell culture condition actually supports myeloid development including the generation Mac-1+Gr-1+ granulocytes (lower panel, outer right plot).
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