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1.
J Biochem ; 125(3): 602-12, 1999 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-10050050

RESUMEN

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.


Asunto(s)
Linfocitos B/citología , Linfocitos B/inmunología , Línea Celular , Reordenamiento Génico de Linfocito B , Células del Estroma/citología , Animales , Técnicas de Cocultivo , Genes de Inmunoglobulinas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Ratones , Ratones Endogámicos C57BL
2.
Leuk Lymphoma ; 29(1-2): 61-9, 1998 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-9638976

RESUMEN

Development of hematopoietic stem cells is regulated by stromal cells of the bone marrow. Many stromal cell lines have been established from temperature-sensitive SV40 large T-antigen gene transgenic mice and used to examine regulation of the purified stem cells. When the sorted stem cells were cocultured on the stromal cell layers, cobblestone formation was induced by the stromal cells. The cobblestones were formed by finite cell division (8 divisions on average) of sorted Lin- c-Kit+ Sca1+ stem cells committed to myeloid or lymphoid lineages. These stromal cell lines showed variable activities supporting the stem cell development. In one stromal cell line, TBR59, two waves of cobblestone formation committed to either myeloid lineage or lymphoid lineage were induced. TBR31-1, another bone marrow stromal cell line, induced only the cobblestone formation committed to lymphoid lineage. These results indicate that the bone marrow stromal cells selectively induce lineage-specific commitment of the stem cells. Both cobblestone formations require c-Kit function as well as adhesive interaction through VLA4 and VCAM1.


Asunto(s)
Células de la Médula Ósea/fisiología , Células Madre Hematopoyéticas/fisiología , Sistema Linfático/citología , Animales , Recuento de Células , Línea Celular , Separación Celular , Ratones , Células del Estroma/fisiología
3.
Exp Hematol ; 26(4): 280-7, 1998 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-9546310

RESUMEN

To understand the regulatory mechanisms involved in the development of hematopoietic stem cells, we cultured lineage-negative, c-Kit+ Sca-1+ stem cells sorted from bone marrow cells by a fluorescence-activated cell sorter (FACS) on layers of bone marrow stromal cell lines established from SV40 T-antigen gene transgenic mice. We previously reported that the TBR59 stromal cell line induced two sequential cobblestone formations: the first formation committed to the myeloid and the second to the lymphoid lineage. After examination of many other bone marrow stromal cell lines, we found that TBR31-1 stromal cells supported only lymphoid development of the sorted stem cells. The sorted stem cells proliferated by forming cobblestones and the cells were released from the cobblestones. Most released cell populations were B220-positive lymphoid cells; cell production continued for 2 months. Addition of G-CSF or M-CSF produced only a slight effect on myeloid development. FACS analysis of the released cells showed that the B-lymphoid-committed progenitors developed into mature B-cells by expressing surface immunoglobulin M. These results indicate that TBR31-1 bone marrow stromal cells selectively support B-lymphoid development, whereas TBR59 cells support both myeloid and lymphoid development of stem cells.


Asunto(s)
Linfocitos B/citología , Células de la Médula Ósea/citología , División Celular , Células Madre Hematopoyéticas/citología , Células del Estroma/citología , Animales , Antígenos Ly/análisis , Antígenos Transformadores de Poliomavirus/genética , Línea Celular , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos/farmacología , Granulocitos/citología , Antígenos Comunes de Leucocito/análisis , Factor Estimulante de Colonias de Macrófagos/farmacología , Proteínas de la Membrana/análisis , Ratones , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-kit/análisis
4.
Cell Struct Funct ; 22(3): 357-64, 1997 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-9248999

RESUMEN

Bone marrow is a major granulopoietic organ whose hematopoietic microenvironment is comprised of stromal cells. In the present work, we examined the regulation of in vitro granulopoiesis with an established line of bone marrow stromal cells. In coculture of the progenitor cells on the particular stromal cell lines from bone marrow, large granulocyte (G) colonies consisting of over 200 cells were formed without G-CSF for 5 days. Stromal cells supported development of Gr-1 (granulocyte specific surface marker)-negative progenitors into Gr-1 and myeloperoxidase positive granulocytes. Seventy percent of the large G-colonies were formed on the stromal layers even in the presence of anti-G-CSF antibody, which indicates the G-CSF independent pathway of granulopoiesis. Inhibition of the large G-colony formation by the addition of anti-adhesion molecules, such as very late activation antigen-4 (VLA-4) and CD31 (PECAM-1), suggested the role of cell-to-cell adhesion in stroma-supported granulopoiesis.


Asunto(s)
Células de la Médula Ósea , Hematopoyesis , Células del Estroma/citología , Animales , Médula Ósea/fisiología , Células Cultivadas , Epítopos de Linfocito T/genética , Factor Estimulante de Colonias de Granulocitos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células del Estroma/fisiología
6.
Exp Hematol ; 24(8): 883-7, 1996 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-8690046

RESUMEN

A mouse spleen stromal cell line, MSS62, can create an in vitro erythropoietic microenvironment in which development of erythropoietin-responsive progentor cells is stimulated by cell-cell contact via stem cell factor (SCF)/c-Kit and vascular cell adhesion molecule-1 (VCAM-1)/very late activation antigen-4 (VLA-4) interactions between stromal and erythroid cells. To find out the effect of src on the erythropoietic microenvironment, MSS62 cells were transfected with v-src oncogene, and its effect on erythropoietic stimulatory activity was measured. Transfectants with high v-Src activity showed reduction in erythropoietic stimulatory activity. A decrease in cell-surface VCAM-1 and SCF mRNA was accompanied by high v-Src activity. These results suggest that v-Src interferes with the erythropoietic stimulatory activity of the stromal cells through repression of VCAM-1 and SCF.


Asunto(s)
Eritropoyesis , Genes src , Células Madre Hematopoyéticas/citología , Proteína Oncogénica pp60(v-src)/fisiología , Factor de Células Madre/fisiología , Molécula 1 de Adhesión Celular Vascular/fisiología , Animales , Adhesión Celular , División Celular , Línea Celular , Técnicas de Cocultivo , Ensayo de Unidades Formadoras de Colonias , Medios de Cultivo Condicionados , Citometría de Flujo , Leucemia Eritroblástica Aguda/fisiopatología , Ratones , Proteína Oncogénica pp60(v-src)/biosíntesis , Fosforilación , Proteínas Recombinantes/biosíntesis , Bazo/citología , Células del Estroma , Transfección , Células Tumorales Cultivadas
7.
Blood ; 86(7): 2590-7, 1995 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-7545461

RESUMEN

Regulation of development of hematopoietic stem cells was examined by culturing Lin- c-Kit+ Sca1+ stem cells sorted from bone marrow (BM) cells by fluorescence-activated cell sorting on a layer of TBR59, a BM stromal cell line established from simian virus 40 T-antigen gene transgenic mice. The sorted stem cells did not show self-renewal, but two waves (at 7 and 13 days) of a cobblestone formation were induced by the stromal cell layer. The cobblestones were formed by finite cell division (eight divisions on average) of sorted Lin- c-Kit+ Sca1+ stem cells, and divided cells were still immature. The c-Kithigh stem cell population was induced to form the first wave of cobblestone formation committed to myeloid lineage, whereas c-Kitlow population was induced to form the second wave of this formation committed to lymphoid lineage. Both cobblestone formations require c-Kit function, but very late activation antigen-4-vascular cell adhesion molecule-1 interaction plays different parts in the two lineages.


Asunto(s)
Células de la Médula Ósea , Separación Celular , Granulocitos/citología , Células Madre Hematopoyéticas/citología , Linfocitos/citología , Células del Estroma/fisiología , Animales , Anticuerpos Monoclonales/farmacología , Antígenos Ly/análisis , Moléculas de Adhesión Celular/inmunología , Moléculas de Adhesión Celular/fisiología , Células Cultivadas , Citometría de Flujo , Células Madre Hematopoyéticas/química , Proteínas de la Membrana/análisis , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas/análisis , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-kit , Proteínas Tirosina Quinasas Receptoras/análisis , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptores del Factor Estimulante de Colonias/análisis , Receptores del Factor Estimulante de Colonias/antagonistas & inhibidores , Receptores del Factor Estimulante de Colonias/fisiología , Receptores de Antígeno muy Tardío/antagonistas & inhibidores , Receptores de Antígeno muy Tardío/fisiología , Molécula 1 de Adhesión Celular Vascular
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