Lymphohematopoietic progenitors immortalized by a retroviral vector harboring a dominant-negative retinoic acid receptor can recapitulate lymphoid, myeloid, and erythroid development.

Abstract

The lymphohematopoietic progenitors represent <0.01% of nucleated marrow cells. Here, we describe the immortalization of the murine lymphohematopoietic progenitors by a retroviral vector harboring a dominant-negative retinoic acid receptor. The immortalized progenitors proliferate as a stem-cell-factor-dependent clonal line EML that spontaneously generates pre-pro-B lymphocytes and erythroid and myeloid progenitors. Upon stimulation with intedeukin-7 and stromal cells, the pre-pro-B lymphocytes express RAG-1 and undergo D-[ rearrangements of the immunoglobulin heavy-chain genes. With erythropoietin the erythroid progenitors proliferate and differentiate into red cells. Generation of the common progenitors for neutrophils and macrophages is suppressed in EML but is inducible by high concentrations of retinoic acid. An additional block in neutrophil differentiation occurs at the promyelocyte stage but can also be overcome by high concentrations of retinoic acid. These studies demonstrate a reproducible way to immortalize lymphohematopoietic progenitors and implicate specific roles for retinoic acid receptors at two distinct stages of hematopoiesis.

Mouse neutrophils lacking lamin B-receptor expression exhibit aberrant development and lack critical functional responses

“…EML-like cells were generated using whole bone marrow harvested from a 40 day old female C57BL/6J-Lbr ic-J /Lbr ic-J (ic/ic) mouse and a normal littermate essentially as described [23]. To express a dominant negative form of retinoic acid receptor α (RARα), cells were spinfected for 2 hours with supernatants derived from a stable GP+E86 producer line infected with the LRARα403SN vector (a kind gift of Archibald Perkins, Yale University, New Haven) plus polybrene (4 µg/ml; Sigma, St. Louis , MO) [24].…”

“…The original wild-type EML cells (EML-+/+) described by Tsai et al were generated from the bone marrow of BDF1 male mice by transducing the harvested cells with a dominant negative RARα [23]. Cells cultured with SCF, IL-3 and 10µM ATRA for three days were then shown to form myeloid progenitors (CFU-GMs), many of which were developmentally arrested promyelocytes that proliferated continuously in GM-CSF.…”

A cascade of Ca2+/calmodulin-dependent protein kinases regulates the differentiation and functional activation of murine neutrophils

“…In the present study, we investigated the possible roles for CaMKKα and its putative target CKLiK in murine neutrophil differentiation using the well-characterized myeloid EML/EPRO cell line [18,19]. We first evaluated the expression of mouse CaMKKα and CKLiK in EML cells as they differentiate towards promyelocytic EPRO cells and then into mature neutrophils.…”

A function of p21 during promyelocytic leukemia cell differentiation independent of CDK inhibition and cell cycle arrest

“…RARs are RAdependent transcription factors which, in adult hematopoietic cells, are involved in the control of terminal di erentiation (Tsai et al, 1994;Chambon, 1996). Their target genes are, however, mostly unknown.…”