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Blood, Vol. 109, Issue 4, 1460-1471, February 15, 2007
The hypomorphic Gata1low mutation alters the proliferation/differentiation potential of the common megakaryocytic-erythroid progenitor
Blood Ghinassi et al.
109: 1460
Supplemental materials for: Ghinassi et al, Vol 109, Issue 4, 1460-1471
Files in this Data Supplement:
- Table S1. Primer pairs used in the semiquantitative expression profiling of the SN1 cell line (PDF, 24.4 KB)
- Table S2. Frequency of mast, basophilic, erythroid, and megakaryocytic cells generated at day 7 in cultures of the different classes of progenitor cells prospectively isolated from the marrow of wild-type and Gata1low littermates, as indicated -
Cultures were stimulated either with SCF and IL-3 or with a combination of cytokines reported to enhance erythromegakaryocytic differentiation. Because the frequency of the different types of precursor cells was the same under the 2 conditions, the 2 sets of data have been pooled together. Results are reported as mean (± SD) of at least 3 separate experiments per progenitor type. Values observed in culture of Gata1low progenitors that are statistically different (P < .01) from those observed in the cultures of the corresponding wild-type cells are indicated in bold. The frequency of cells expressing neutrophil (Gr1), monomacrophagic (CD11c or Mac3), T-cell (CD4 or CD8 or both), and B-cell (B220) markers is not reported because it is always below 1%, with the exception of some (5%-6%) neutrophils observed in cultures of Gata1low MEPs, and of MPs, both Gata1low and wild type. The majority of the wild-type MCP progeny did not express any of the differentiation markers used in the study and probably represents c-Kitlow T1ST2low mast-cell precursors.
- Table S3. Functional characterization of c-KithighT1/ST2high cells isolated by sorting by the progeny at day 7 of progenitor cells prospectively isolated from the bone marrow of wild-type or Gata1low littermates, as indica -
c-Kithigh/T1/ST2high cells were isolated by sorting (< 95% pure) from the progeny obtained after 7 days of culture of wild-type MCPs and Gata1low MEPs, as indicated (see Figure 4B). The cells (103/mL) were cultured in the presence of SCF and IL-3, and their progeny analyzed, in turn, 14 days later. **Fold increase statistically higher (P < .01) than that observed in cultures of wild-type cells. Results from a representative experiment performed in triplicate are shown.
- Table S4. Calculation of the frequency of immortalization of single Gata1low MEPs (PDF, 54.5 KB) -
Results obtained with MEPs from 4 independent purifications were pooled together. Each well received 100 µL of a 3-cell/mL solution. Therefore, about 30% of the wells in each culture contained at most 1 cell. Under these conditions, if 30% of the wells contain signs of proliferation, all of the seeded cells had multiplied. The frequency of the original MEPs that gave rise to cells that proliferate with 100% of efficiency at the fourth passage was calculated on the tracking of the proliferation of their progeny in each passage, according to the following formula: 12 clones that proliferated in quaternary cultures/total of 27% of primary MEPs analyzed (384 × 27/100 = 104 MEPs) = 12/104 = 11.5%.
- Figure S1. MEPs from Gata1low mice generate as many mast cells as wild-type MCPs when cultured in BMMC conditions (JPG, 97.5 KB)
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(A) Number of cells observed after 14 and 21 days in BMMCs seeded with MCPs (squares) and MEPs (circles) prospectively purified from wild-type (top panels) and Gata1low (bottom panels), as indicated. Many (15 × 106) cells were observed at day 14 starting from 103 wild-type MCPs, but wild-type MEPs did not grow under these conditions. In contrast, Gata1low MEPs generated high numbers of cells (∼107) by days 14 and 21 (in this last case, significantly more cells were generated by Gata1low MEPs than by wild-type MCPs). Results are presented as mean (± SD) number of cells observed in 3 separate experiments per group of mice. Values observed in Gata1low BMMCs that are statistically different from those obtained in the corresponding wild-type BMMCs are indicated by a single asterisk (P < .01). (B) Flow cytometric analyses for CD34/c-Kit and Fc RI expression and May-Grünwald staining of cells present after 14 and 21 days in BMMCs seeded with wild-type MCPs (top panels) and Gata1low MEPs (bottom panels). The differences observed in progression of differentiation in cultures of Gata1low MEPs and wild-type MCPs are equivalent to those described before for the corresponding bulk cultures (Figure 2). Cells at the early stages of differentiation were generated by wild-type MCPs by day 14 and proceeded to CD117highFcRIpos cells by day 21. In contrast, the mast cells generated by Gata1low MEPs remained immature. The dotted curve in the histogram analysis correspond to cells stained with an irrelevant antibody. Original magnification, ×40.
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