Engineered niches support the development of human dendritic cells in humanized mice.

Classical dendritic cells (cDCs) are rare sentinel cells specialized in the regulation of adaptive immunity. Modeling cDC development is crucial to study cDCs and harness their therapeutic potential. Here we address whether cDCs could differentiate in response to trophic cues delivered by mesenchymal components of the hematopoietic niche. We find that mesenchymal stromal cells engineered to express membrane-bound FLT3L and stem cell factor (SCF) together with CXCL12 induce the specification of human cDCs from CD34+ hematopoietic stem and progenitor cells (HSPCs). Engraftment of engineered mesenchymal stromal cells (eMSCs) together with CD34+ HSPCs creates an in vivo synthetic niche in the dermis of immunodeficient mice driving the differentiation of cDCs and CD123+AXL+CD327+ pre/AS-DCs. cDC2s generated in vivo display higher levels of resemblance with human blood cDCs unattained by in vitro-generated subsets. Altogether, eMSCs provide a unique platform recapitulating the full spectrum of cDC subsets enabling their functional characterization in vivo.

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Life sciences study design
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Sample size
For both in vitro and in vivo experiments, at least 3 independent donors were used to account for biological variation of cord blood samples Data exclusions No data were excluded from analysis.

Replication
All the experiments supporting major conclusions were preformed at least 2 times to account for technical variations. All attempts of replication were successful.
Randomization In each in vivo experiment, animals of the same age and background (NSG) were randomly assigned to each group. In experiments performed in vitro, each cord blood donor was split and allocated to both control and tested conditions (eMSCs).

Blinding
Investigators were not blinded to group allocation. Blinding was not relevant for this study since differences between groups were determined by the physical measurement of cell frequency within each sample by flow cytometry Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.

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The cell lines used were not authenticated.

Mycoplasma contamination
The cell lines used in this study (MS5, OP9 and engineered stromal cells) were regularly tested and resulted negative for mycoplasma contamination.

nature research | reporting summary
October 2018 Flow Cytometry Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
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Methodology
Sample preparation Extracellular staining of cells was preformed by incubating the samples for 30 minutes at 4C in antibody mixes prepared using FACS buffer (PBS, 1% BSA, 2 mM EDTA). For intracellular staining, samples were fixed and permeabilized using the Cytofix/ CytopermTM kit (BD Biosciences) according to manufacturers' instructions. Cell population abundance Purity of FACS-sorted samples was assessed at the sorter and frequency of sorted cells was above 95% of live cells.

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The gating strategy used to analyze and FACS-sort human DC subsets are exemplified in Supplementary Fig.2 and Supplementary  Fig.8.
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