Human CD34⁺CD11b⁻ cord blood stem cells generate in vitro a CD34⁻CD11b⁺ subset that is enriched in langerin⁺ Langerhans dendritic cell precursors

Objective

We investigated whether the expression of CD11b on precursors derived in vitro from CD34⁺ hematopoietic stem cells was related to their ability to generate CD11b⁻ and CD11b⁺ Langerhans dendritic cells (LC).

Methods

Human CD34⁺ cells purified from cord blood were cultured with FLT3 ligand, thrombopoietin, and stem cell factor (FTS) for 2 weeks, analyzed, and sorted by FACS. Sorted fractions were cultured as above, or differentiated into LC with GM-CSF, IL-4, and TGF-β1 (G4-TGF) for 6 days. The capacity of LC to internalize langerin and dextran was assessed.

Results

Ex vivo, human CD34⁺ cells were CD11b⁻ and mostly CLA⁺. After 2 weeks of culture with FTS, CD34⁻CLA⁻CD11b⁻ and CD34⁻CLA⁻CD11b⁺ cells emerged. CD11b⁻ cells were the most ancestral because they were the only ones to proliferate with FTS, and constantly generated CD11b⁺ cells. Both CD11b⁻ and CD11b⁺ sorted cells generated E-cadherin⁺langerin⁺ LC after incubation with G4-TGF. The former fraction contained 46% ± 15% of E-cadherin⁺ and 10% ± 5% of langerin⁺ cells, whereas in the latter fraction these values reached respectively 66% ± 23% and 30% ± 16% (mean ± SD, n = 7, p < 0.056). Looking at functional properties, CD11b⁻ and CD11b⁺ LC were similar in terms of langerin and dextran endocytosis. By contrast, only CD11b⁺ LC internalized fluorescent LPS.

Conclusion

Human CD34⁺CD11b⁻ cells differentiate in FTS culture into a CD34⁻CD11b⁻ precursor that in turn generates CD34⁻CD11b⁺ cells. These cells are enriched in LC precursors compared to CD34⁻CD11b⁻ cells. Both CD11b⁻ and CD11b⁺ LC are generated in vitro, and each fraction may assume different functions in inflammatory situations.