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Dissociation of GFP and CD45 expression in the hematopoietic system Implications for tracking stem cell fate

, : Dissociation of GFP and CD45 expression in the hematopoietic system Implications for tracking stem cell fate. Blood 102(11): 340a-341a, November 16

Genetically marked donor cells are widely used to evaluate the mutilineage differentiation potential of somatic stem cells. Although the C57BL/6-TgN(ACTBEGFP)1Osb transgenic mouse is frequently described as "ubiquitously" expressing GFP, little is known about tissue specific expression of the GFP transgene. Here we show that GFP expression in the hematopoietic compartment is highly variable. In the bone marrow, only 46+-3% of cells expressed GFP. However, 18+-2% of myeloid cells (Mac-1+) and 67+-3% of erythroid progenitors (Ter-119+) demonstrated no GFP expression. By contrast, 93+-1% of the leukocytes in the peripheral blood expressed GFP. In addition to the anticipated GFP+ CD45+ phenotype (69+-6%), significant numbers of CD45+ cells did not express GFP (6.5+-1%) while a large percentage of GFP+ cells were CD45 negative (25+-7%). Both the GFP+ CD45- phenotype and the GFP- CD45+ phenotype were found in the myeloid (Mac-1+/Gr-1+), B-cell (B220+) and T-cell (CD3+) lineages. Virtually all hematopoietic stem cells in the bone marrow, as defined by the c-Kit+Sca-1+Lin-(KSL) phenotype, expressed GFP (>99%); however, two distinct populations of these cells were identified: GFPmedium KSL cells (one to two logs above the control) and GFPhi cells (three to four logs above the control). The functional activity of these two subsets of KSL cells was evaluated by competitive repopulation using recipients congenic at the CD45 locus. Although the levels of total multilineage donor cell engraftment were similar between the two groups (58+-15% vs. 65+-9%), transplanted GFPmedium KSL cells infrequently gave rise to GFPhigh cells (<10%) and nearly all expressed CD45 (>98%). Conversely, the progeny of GFPhigh KSL cells in the blood were predominately GFPhigh (81+-2%), and remarkably 33+-7% of these donor-derived hematopoietic cells exhibited reduced or absent CD45 expression. Taken together these results demonstrate significant heterogeneity of GFP expression in the hematopoietic compartment and underscore the importance of using two independent markers to reliably assess the developmental potential of stem cells.


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