Study of Dendritic Cell Development by Short Hairpin RNA-Mediated Gene Knockdown in a Hematopoietic Stem and Progenitor Cell Line In vitro

Dendritic cells, or DCs, are key immune cells linking the innate and adaptive immune system. While DCs are heterogeneous, this method help us understand which genes, including transcription factors, may regulate DC development. This technique provides a simple and quick way to identify a gene that controls DC development from their progenitor in vitro.

To begin, maintain the immortalized hematopoietic stem and progenitor cells or iHSPCs in complete RPMI 1640 medium, supplemented with 100 nanograms per milliliter of the FLT3 ligand and one micromolar beta estradiol. For lentiviral transduction, plate the iHSPCs in 12 well plates at a density of one times 10 to the fifth cells per well and one milliliter of complete medium containing the FLT3 ligand, beta estradiol, and polybrene. Then, add the lentivirus carrying the short hairpin RNA in each well at a multiplicity of infection of 100.

Next, spin the plate at 1100 times g and 37 degrees Celsius for 90 minutes, then incubate the infected cells overnight at 37 degree Celsius. The following day, remove the polybrene by collecting the cells into 15 milliliter tubes in centrifugation, following replacing the media with fresh complete medium containing the FLT3 ligand and beta estradiol. After an additional 24 hours, add six micrograms per milliliter of puromycin to the medium to select the infected cells.

Replace the selection medium every three days and maintain the cells for at least one week to expand the stably transduced iHSPCs. Generate and maintain stable knockdown iHSPCs for LacZ, Tcf4, and Id2 in complete medium supplemented with the FLT3 ligand and beta estradiol. To initiate in vitro differentiation collect the undifferentiated iHSPCs into 15 milliliter tubes and pellet the cells by centrifugation.

After centrifugation, discard the supernatant and wash the cells twice with 10 milliliters of PBS. After the second wash, resuspend the cells in complete medium containing only the FLT3 ligand then seed them at a density of two times 10 to the fifth sells per milliliter into a 12 well plate. After three days, add one milliliter of fresh complete medium containing the FLT3 ligand.

After an additional two days, proceed to analyze the differentiated cells by flow cytometry. For flow cytometric analysis, pipette the cells up and down two to three times in the plate then collect the cells into 1.5 milliliter tubes. Centrifuge the tubes and discard the supernatant before resuspending the cells in 50 microliters of fax buffer.

Next, add 50 microliters of anti-CD16 by 32 hybridoma supernatant and incubate for five to 10 minutes on ice. Then, add fluorescent dye-conjugated antibodies directly to the cells and incubate for 15 minutes on ice in the dark. After incubation, wash the cells with one milliliter of fax buffer and centrifuge the sample.

After centrifugation, resuspend the cells in 100 microliters of fax buffer and analyze the samples by flow cytometry. After short hairpin RNA-mediated knockdown of LacZ, Tcf4, and Id2 in iHSPCS, confirmation of knockdown efficiency by RTQ-PCR showed that the expression of Tcf4 and Tcf4 knockdown iHSPCs was reduced compared to that in LacZ knockdown iHSPCs. Similar results were observed for Id2 expression in the Id2 knockdown iHSPCs.

After culturing LacZ knockdown iHSPCs for five days, the frequency of CD11c positive cells, which represent the dendritic cell population, was around 95%However, knocking down Tcf4 or Id2 slightly decreased the generation of CD11c-positive dendritic cells. Further analysis of CD11c-positive dendritic cells derived from the LacZ knockdown iHSPCs revealed that 70%were conventional dendritic cells while 22%were plasmacytoid dendritic cells. The Tcf4 knockdown iHSPCs generated a significantly lower percentage of plasmacytoid dendritic cells that the LacZ knockdown controls.

In contrast, the Id2 knockdown iHSPCs generated a significantly lower percentage of conventional dendritic cells than the LacZ knockdown controls but a higher percentage of plasmacytoid dendritic cells. Spin infections increase the transduction efficiency of lentivirus-carrying shRNA into the iHSPCs. This technique addresses the role of transcription factors and facilitates the study of other genes in cytokine signaling transduction or metabolism, which are likely to be involved in DC development.