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Título: | In Vitro Reconstitution of Human B-Cell Ontogeny: From CD34+ Multipotent Progenitors to Ig-Secreting Cells |
Autor: | Fluckiger, Anne-Catherine; Sanz, Eva; Garcia-Lloret, Maria; Su, Thomas; Hao, Qian-Lin; Kato, Roberta; Quan, Shirley; Hera, Antonio de la CSIC ; Crooks, Gay M; Witte, Owen N; Rawlings, David J | Palabras clave: | Human B-progenitor long-term culture [HB-LTC] Ig-secreting B cells Reverse transcriptase-polymerase chain reaction (RT-PCR) Genetic manipulation Human B ontogeny Lymphopoiesis |
Fecha de publicación: | 15-dic-1998 | Editor: | American Society of Hematology | Citación: | Blood, 92:4509-4520 (1998) | Resumen: | We describe a long-term, in vitro culture system initiated with CD34+ or CD34+CD38−umbilical cord blood hematopoietic progenitors that supports normal human B-lineage development, including the production of mature Ig-secreting B cells. In the first stage (human B-progenitor long-term culture [HB-LTC]), CD34+ hematopoietic progenitors are cultured on the murine stromal cell line, S17, leading to the sustained production of large numbers of CD10+, CD19+early B progenitors. Reverse transcriptase-polymerase chain reaction (RT-PCR) and three-parameter flow cytometry for VpreB (surrogate light chain), cytoplasmic μ chain, and surface IgM expression were used to characterize the CD19+ B progenitors present within these cultures. This analysis showed distinct B-lineage subpopulations, including pro-B cells, cycling pre-B cells, and IgM+, IgD−/+ immature B cells. The limited expansion of IgM+ B cells and the immature surface phenotype of this population (IgM+, IgD+, CD10+, CD38+) suggested that HB-LTC conditions were unable to provide appropriate signals for further differentiation. A second culture stage was used to determine if these immature B cells were functionally competent. Purified CD19+ cells were transferred onto fibroblasts expressing human CD40-ligand in the presence of IL-10 and IL-4. This lead to cell proliferation, modulation of the IgM+ cell surface phenotype to one consistent with an activated mature B cell, secretion of Ig, and isotype switching. Notably, IgM and IgG producing B cells were also generated using two-stage cultures established with highly purified multipotent CD34+CD38−hematopoietic stem cell progenitors. This culture model should permit detailed in vitro analysis and genetic manipulation of the major transition points in human B ontogeny, beginning with commitment to the B lineage and leading to development and activation of mature B cells. | Descripción: | Rapid Communication; 6 Figs. 2 Tables | Versión del editor: | http://bloodjournal.hematologylibrary.org/content/92/12/4509.full.pdf+html | URI: | http://hdl.handle.net/10261/39943 | DOI: | 0006-4971/98/9212-0048$3.00/0 | ISSN: | 0006-4971 | E-ISSN: | 1528-0020 |
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