Varying Levels of CD34 Expression Correlate with Maintenance and Differentiation of HSC/MPP-Like Cell Line EML.

The HSC/MPP-like cell line EML has the capacity for multilineage (erythroid, myeloid, lymphoid) differentiation in vitro, and is functionally heterogeneous. Phenotypically, EML cell line consists of Sca-1⁺ c-kit⁺ Flk⁻ CD34⁺ cells (80%) and Sca-1⁺ c-kit⁺ Flk⁻ CD34⁻ cells (20%), and resembles Lin⁻ c-kit⁺ Sca-1⁺ Flk2⁻ BM cells, which are also heterogeneous for CD34 expression. Functional analysis has revealed that purified CD34⁺ EML cells reproducibly contain the majority of progenitors capable of mutilineage differentiation in response to cytokines, whereas CD34⁻ EML cells differentiated poorly or not at all. Since the CD34⁺ EML cell population can be divided into CD34^low, CD34^medium and CD34^high subpopulations we investigated the correlation between the progenitor content and the level of CD34 expression. The CD34^low, CD34^med and CD34^high EML cells were purified by FACS sorting, and their multilineage differentiation analyzed by CFC assays. Multiple CFC experiments have reproducibly shown that the progenitor frequency declines with a decrease in the level of CD34 expression on EML cells, with CD34^high cells producing the highest and the CD34^low cells producing the lowest number of colonies. These findings indicate that CD34⁺ EML cell population is functionally heterogeneous, and that the overall multilineage differentiation of EML cells strongly correlates with the CD34 expression levels. Since the majority of LTR-HSC reside within LKS Flk2⁻ CD34⁻ BM cells, and LKS Flk2⁻ CD34⁺ BM cells are highly enriched for STR-HSC and MPPs, we tested whether CD34⁻ EML cells are more primitive and give rise to CD34⁺ cells. The CD34⁻ and CD34⁺ EML cells were purified (>99% pure) by sorting, cultured separately for 7 days, and cell aliquots from these cultures analyzed daily for CD34 expression. Four separate experiments have revealed that both CD34⁻ and CD34⁺ EML cells can generate each other, suggesting that although the CD34⁻ EML cells could be more primitive, a subpopulation of CD34⁺ cells retains the capacity to give rise to CD34⁻ cells. Previous studies have shown that mouse LTR-HSC can alternate between CD34⁻ “inactive” and CD34⁺ “activated” state, and that the heterogeneous population of LKS CD34⁺ BM cells contains activated LTR-HSC, STR-HSC and MPPs. Thus, we investigated how the level of CD34 expression correlates with the capacity of CD34⁺ EML cells to give rise to CD34⁻ cells. Purified CD34^low, CD34^med and CD34^high EML cells were cultured for 5 days, and the cells from each culture analyzed for CD34 expression. Remarkably, three separate experiments have revealed that CD34^low EML cells have the highest capacity to give rise to CD34⁻ EML cells, whereas CD34^med and CD34^high EML cells had very little capacity to generate CD34⁻ EML cells. These findings suggest a model in which (a) CD34⁻ EML cells are more primitive cells in an “inactive” (differentiate inhibited) state that generate CD34⁺ EML cells, and (b) the population of CD34⁺ EML cells is heterogeneous and consists of CD34^med/high cells that can readily differentiate into multiple lineages, and CD34^low cells that are in “activated” state and can revert back to the CD34⁻ state. Cumulatively, these results support the notion that the maintenance and differentiation of EML cells is linked with the varying levels of CD34 expression. Therefore, EML cell line could represent a powerful in vitro model to study the linear differentiation of CD34⁻ cells into CD34⁺ cells, and the mechanisms of reversible CD34 expression and activation of primitive hematopoietic cells.