CCAAT/enhancer binding protein ␣ (C/EBP␣) is an integral factor in the granulocytic developmental pathway, as myeloblasts from C/EBP␣-null mice exhibit an early block in differentiation. Since mice deficient for known C/EBP␣ target genes do not exhibit the same block in granulocyte maturation, we sought to identify additional C/EBP␣ target genes essential for myeloid cell development. To identify such genes, we used both representational difference analysis and oligonucleotide array analysis with RNA derived from a C/EBP␣-inducible myeloid cell line. From each of these independent screens, we identified c-Myc as a C/EBP␣ negatively regulated gene. We mapped an E2F binding site in the c-Myc promoter as the cis-acting element critical for C/EBP␣ negative regulation. The identification of c-Myc as a C/EBP␣ target gene is intriguing, as it has been previously shown that down-regulation of c-Myc can induce myeloid differentiation. Here we show that stable expression of c-Myc from an exogenous promoter not responsive to C/EBP␣-mediated downregulation forces myeloblasts to remain in an undifferentiated state. Therefore, C/EBP␣ negative regulation of c-Myc is critical for allowing early myeloid precursors to enter a differentiation pathway. This is the first report to demonstrate that C/EBP␣ directly affects the level of c-Myc expression and, thus, the decision of myeloid blasts to enter into the granulocytic differentiation pathway.

Adult human bone marrow-derived stem cells, having the ability to differentiate into cells of multiple lineages, have been isolated and propagated by varied protocols, including positive (CD105(+))/negative (CD45(-)GlyA(-)) selection with immunomagnetic beads, or direct plating into selective culture media. Each substratum-adherent cell population was subjected to a systematic analysis of their cell surface markers and differentiation potential. In the initial stages of culture, each cell population proliferated slowly, reaching confluence in 10-14 days. Adherent cells proliferated at similar rates whether cultured in serum-free medium supplemented with basic fibroblast growth factor, medium containing 2% fetal bovine serum (FBS) supplemented with epidermal growth factor and platelet-derived growth factor, or medium containing 10% FBS alone. Cell surface marker analysis revealed that more than 95% of the cells were positive for CD105/endoglin, a putative mesenchymal stem cell marker, and negative for CD34, CD31, and CD133, markers of hematopoietic, endothelial, and neural stem cells, respectively, regardless of cell isolation and propagation method. CD44 expression was variable, apparently dependent on serum concentration. Functional similarity of the stem cell populations was also observed, with each different cell population expressing the cell type-specific markers beta-tubulin, type II collagen, and desmin, and demonstrating endothelial tube formation when cultured under conditions favoring neural, cartilage, muscle, and endothelial cell differentiation, respectively. On the basis of these data, adult human bone marrow-derived stem cells cultured in adherent monolayer are virtually indistinguishable, both physically and functionally, regardless of the method of isolation or proliferative expansion.

CCAAT/enhancer binding proteins (C/ EBPs) are a family of factors that regulate cell growth and differentiation. These factors, particularly C/EBP␣ and C/EBP⑀, have important roles in normal myelopoiesis. In addition, loss of C/EBP activity appears to have a role in the pathogenesis of myeloid disorders including acute myeloid leukemia (AML). Acute promyelocytic leukemia (APL) is a subtype of AML in which a role for C/EBPs has been postulated. In almost all cases of APL, a promyelocytic leukemia-retinoic acid receptor ␣ (PML-RAR␣) fusion protein is expressed as a result of a t(15;17)(q22; q12) chromosomal translocation. PML-RAR␣ inhibits expression of C/EBP⑀, whereas all-trans retinoic acid (tRA), a differentiating agent to which APL is particularly susceptible, induces C/EBP⑀ expression. PML-RAR␣ may also inhibit C/EBP␣ activity. Thus, the effects of PML-RAR␣ on C/EBPs may contribute to both the development of leukemia and the unique sensitivity of APL to tRA. We tested the hypothesis that increasing the activity of C/EBPs would revert the leukemic phenotype. C/EBP␣ and C/EBP⑀ were introduced into the FDC-P1 myeloid cell line and into leukemic cells from PML-RARA transgenic mice. C/EBP factors suppressed growth and induced partial differentiation in vitro. In vivo, enhanced expression of C/EBPs prolonged survival. By using a tamoxifen-responsive version of C/EBP⑀, we observed that C/EBP⑀ could mimic the effect of tRA, driving neutrophilic differentiation in leukemic animals. Our results support the hypothesis that induction of C/EBP activity is a critical effect of tRA in APL. Furthermore, our findings suggest that targeted modulation of C/EBP activities could provide a new approach to therapy of

SummaryMesenchymal stem cell (MSC) therapy has shown promise clinically in graft-versus-host disease and in preclinical animal models of T helper type 1 (Th1)-driven autoimmune diseases, but whether MSCs can be used to treat autoimmune disease in general is unclear. Here, the therapeutic potential of MSCs was tested in the New Zealand black (NZB) ¥ New Zealand white (NZW) F1 (NZB/W) lupus mouse model. The pathogenesis of systemic lupus erythematosus involves abnormal B and T cell activation leading to autoantibody formation. To test whether the immunomodulatory activity of MSCs would inhibit the development of autoimmune responses and provide a therapeutic benefit, NZB/W mice were treated with Balb/c-derived allogeneic MSCs starting before or after disease onset. Systemic MSC administration worsened disease and enhanced anti-double-stranded DNA (dsDNA) autoantibody production. The increase in autoantibody titres was accompanied by an increase in plasma cells in the bone marrow, an increase in glomerular immune complex deposition, more severe kidney pathology, and greater proteinuria. Co-culturing MSCs with plasma cells purified from NZB/W mice led to an increase in immunoglobulin G antibody production, suggesting that MSCs might be augmenting plasma cell survival and function in MSC-treated animals. Our results suggest that MSC therapy may not be beneficial in Th2-type T cell-and B cell-driven diseases such as lupus and highlight the need to understand further the appropriate application of MSC therapy.