Fluckiger A C, Sanz E, Garcia-Lloret M, Su T, Hao Q L, Kato R, Quan S, de la Hera A, Crooks G M, Witte O N, Rawlings D J
Department of Pediatrics, the Jonsson Comprehensive Cancer Center, Molecular Biology Institute, University of California, Los Angeles, CA, USA.
Blood. 1998 Dec 15;92(12):4509-20.
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 mu 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.
我们描述了一种长期体外培养系统，该系统由CD34(+)或CD34(+)CD38(-)脐带血造血祖细胞起始，可支持正常人类B细胞系发育，包括产生分泌成熟Ig的B细胞。在第一阶段（人类B祖细胞长期培养[HB-LTC]），将CD34(+)造血祖细胞培养在小鼠基质细胞系S17上，导致大量CD10(+)、CD19(+)早期B祖细胞持续产生。采用逆转录聚合酶链反应（RT-PCR）以及针对VpreB（替代轻链）、细胞质μ链和表面IgM表达的三参数流式细胞术来表征这些培养物中存在的CD19(+)B祖细胞。该分析显示了不同的B细胞系亚群，包括前B细胞、循环前B细胞以及IgM+、IgD-/+未成熟B细胞。IgM+ B细胞的有限扩增以及该群体的未成熟表面表型（IgM+、IgD+、CD10(+)、CD38(+)）表明HB-LTC条件无法为进一步分化提供适当信号。使用第二个培养阶段来确定这些未成熟B细胞是否具有功能活性。在存在IL-10和IL-4的情况下，将纯化的CD19(+)细胞转移到表达人CD40配体的成纤维细胞上。这导致细胞增殖，IgM+细胞表面表型转变为与活化成熟B细胞一致的表型，Ig分泌以及同种型转换。值得注意的是，使用由高度纯化的多能CD34(+)CD38(-)造血干细胞祖细胞建立的两阶段培养也产生了产生IgM和IgG的B细胞。这种培养模型应允许对人类B细胞发生过程中的主要转变点进行详细的体外分析和基因操作，从B细胞系的定向分化开始，直至成熟B细胞的发育和活化。
