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Using a recently described serum-free culture system of purified human CD34+ progenitor cells, we show here a critical cooperation of flt3 ligand (FL) with transforming growth factor-beta1 (TGF-beta1) in the induction of in vitro dendritic cell/Langerhans cell (DC/LC) development. The addition of FL to serum-free cultures of CD34+ cells supplemented with TGF-beta1, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha, and stem cell factor strongly increases both percentages (mean, 36% +/- 5% v 64% +/- 4%; P = .001) and total numbers (4.4- +/- 0.8-fold) of CD1a+ dendritic cells. These in vitro-generated CD1a+ cells molecularly closely resemble a particular type of DC known as an epidermal Langerhans cell. Generation of DC under serum-free conditions was found to strictly require supplementation of culture medium with TGF-beta1. Upon omission of TGF-beta1, percentages of CD1a+ DC decreased (to mean, 10% +/- 8%; P = .001) and, in turn, percentages of granulomonocytic cells (CD1a- cells that are lysozyme [LZ+]; myeloperoxidase [MPO+]; CD14+) increased approximately threefold (P.05). Furthermore, in the absence of TGF-beta1, FL consistently promotes generation of LZ+, MPO+, and CD14+ cells, but not of CD1a+ cells. Serum-free single-cell cultures set up under identical TGF-beta1- and FL-supplemented culture conditions showed that high percentages of CD34+ cells (mean, 18% +/- 2%; n = 4) give rise to day-10 DC colony formation. The majority of cells in these DC-containing colonies expressed the Langerhans cell/Birbeck granule specific marker molecule Lag. Without TGF-beta1 supplementation, Lag+ colony formation is minimal and formation of monocyte/macrophage-containing colonies predominates. Total cloning efficiency in the absence and presence of TGF-beta1 is virtually identical (mean, 41% +/- 6% v 41% +/- 4%). Thus, FL has the potential to strongly stimulate DC/LC generation, but has a strict requirement for TGF-beta1 to show this costimulatory effect.

Several studies have demonstrated that dendritic cells (DC) can be generated in vitro from CD34+ hemopoietic progenitor cells. The growth requirements for these cells are poorly characterized, however. In particular, undefined serum/plasma components seem to significantly contribute to in vitro DC development. We report here that the cytokine combination granulocyte-macrophage CSF (GM-CSF) plus TNF-alpha and stem cell factor (SCF) commonly used for the in vitro generation of DC in serum/plasma-supplemented medium is, in the absence of serum supplementation, very inefficient in inducing DC development. We further demonstrate that supplementation with TGF-beta 1 is required for substantial DC development to occur in the absence of serum. Culture of CD34+ cells under serum-free conditions with TGF-beta 1 plus GM-CSF, TNF-alpha, and SCF strongly induces DC differentiation. This culture condition is even more efficient than culturing CD34+ cells with GM-CSF plus TNF-alpha and SCF in the presence of cord blood plasma. The proportions and total yields of cells with typical DC morphology and CD1a molecule expression are higher. The allostimulatory capacity of DC from TGF-beta 1-supplemented, cultures exceeds allostimulation by cells grown in plasma-containing medium. Substantial numbers (21 +/- 7%) of cells grown in TGF-beta 1-supplemented, but not plasma-supplemented, cultures express the Birbeck granule marker molecule Lag and display numerous Birbeck granules. Cells with distinct monocytic features are less frequently observed in TGF-beta 1-supplemented serum-free cultures. The addition of neutralizing anti-TGF-beta 1 Ab abrogates the observed TGF-beta 1 effects.