A Novel Stromal Cell-Dependent B Lymphoid Stem-Like Cell Line That Induces Immunoglobulin Gene Rearrangement

Abstract

A stroma-dependent B lymphoid cell line (B31-1) has been established by coculturing sorted stem cells on a novel bone marrow stromal cell line (TBR31-1). B31-1 cells express B220, but do not express other B lymphoid differentiation markers including CD43, heat stable antigen (HSA), or surface immunoglobulin (Ig) M (sIgM), and their Ig heavy chain (IgH) gene loci are germ-line in configuration. The addition of interleukin (IL)-7 or coculture with another stromal cell line, ST2, induces D-J rearrangement of the IgH gene and B lymphocyte differentiation markers. B31-1 cells restore an in vivo repopulation activity to lethally irradiated mice, and the repopulated cells differentiate to HSA+ pre-B cells.Continuous coculture results in two distinct populations, B220(-) c-Kit+ cells and B220(+) c-Kit+ cells; B220(-) c-Kit+ cells are self-renewed and differentiate to B220(+) c-Kit+ cells, while B220(+) c-Kit+ cells produce only B220(+) c-Kit+ cells. Both B220(-) and B220(+) cells similarly express the IgH germ-line transcript (Imu), mRNAs for recombinase (TdT, Rag-1, and Rag-2), and lymphoid-specific transcription factors (Pax-5, EBF, E12/E47, Oct-2, and Ikaros), but the DNA binding activity of Pax-5, EBF, Oct-2, and E2A are low in B220(-) cells and while high in B220(+) cells. These results suggest the existence of at least two active states in the IgH locus before the induction of IgH gene rearrangement during B lymphopoietic development.

Sox4 Is Required for the Survival of Pro-B Cells

“…4E). Since Src kinase is downstream of c-Kit and plays a critical role in pro-B cell development (28,54), we determined the levels of total Src and phosphorylated Src (p-Y416) in pro-B cells from Sox4 fl/fl Vav-Cre and Sox4 fl/ + Vav-Cre mice by flow cytometric analysis. We found that there was no overt difference in total Src, but phosphorylated Src (p-Y416) was significantly reduced upon Sox4 deletion (median fluorescence intensity [MFI] 29.4 versus 13.2; Fig.…”

The immortalized cell lines with differentiation potentials: Their establishment and possible application

“…We established several stroma-dependent hematopoietic cell lines to examine the regulatory mechanisms of hematopoiesis by stromal cells. (20)(21)(22)(23)(24) Among them, a primitive hematopoietic cell line, THS119, was established from BM Lin -/Sca-1 + cells of the transgenic mice after lengthy passaging by coculture with TBR59 (21) THS119 cells exhibited immature primitive hematopoietic cells such as forming cobblestones and surface phenotypes (Lin -/c-Kit + /Sca1 + ), and multiple expressions of both earlier developmental markers of myeloid, lymphoid and the hematopoietic cell-specific transcription factors. Growth of THS119 cells is ts T-antigen-dependent and stromal celldependent.…”

Possible Applications of Conditionally Immortalized Tissue Cell Lines with Differentiation Functions

“…Thus, it is possible to establish cell lines from tiny tissues containing a small quantity of cells. [4][5][6][7] Unique morphology specific to proximal tubule Vasopressininduced intracellular calcium increase Kidney tubule cell lines from microdissected nephron segments [4][5][6][7] Distal tubule Intrarenal site-specific actions Cortical and outer medullary collecting duct (OMCD) Terminal proximal tubules (S3 segment) Hepatocyte cell lines (TLR) [2] Liver-specific morphological and biochemical properties inducibility of P450IA1 by 3-methylcholanthrene Gastric surface mucous cells GSM06 and GSM10 [11] PAS-and Con A-positive glycoproteins Tracheal epithelial cell lines, TM01 and TM02-3 [12] High-molecular-weight glycoconjugates, intercellular adhesion molecular-1 (ICAM-1) Colonic epithelial cell line MCE301 [13] Microvilli-like structures, tight junctions and desmosomes, cytoskeletal (acidic cytokeratins and actin), basement membrane (laminin and collagen type IV) junctional complex proteins (ZO-1 and desmoplakin I ϩ II), alkaline phosphatase (a brush border-associated enzyme) Retinal pigment epithelium-derived cell line (RPET) [14] T1-tyrosinase production, cytokeratin, microvilli Gingival epithelial cell lines (GE1 and GE6) [15] Keratins 4 and 13, keratohyalin granules and involucrin Vascular smooth muscle cell lines (SVS30 and SVS24 cells) [8][9][10] ␣-Actin, smooth muscle myosin heavy chain-1, and calponin proliferation-dependent expression Bone marrow stromal cell lines Mesenchymal stem cell and progenitor cell lines Bone marrow stromal cell line (TBR31-2) [16,17] Multipotency of mesenchymal cell differentiation Adipocyte (lipoprotein lipase) Osteoblast (type I collagen and osteocalcin) Chondrocyte (type II and X collagen) Bone marrow stromal cell line (TBR-B) [8][9][10] Muscle cell (desmin) Differentiation potential to smooth muscle h1-calponin, h-caldesmon, SM22␣ and ␣-actin Bone marrow stromal cell line (TBR-series) [16,17] Differentiation potentials to mesenchymal lineages of cells Hematopoietic microenvironment [20][21][22][23]…”