In vitro differentiated CD56-positive ILC transcriptional profiling via scRNA-seq

The innate cytotoxic Natural Killer (NK) cells emerged during hematopoiesis through a linear model of human NK development, yet how in vitro model of NK differentiation recapitulates in vivo process is largely under-explored. Here, we established that NK cell trajectory in vitro can be divided into 4 stages by sequential acquisition of CD161, CD56 and CD94 in which CD56 bifurcation can separate Stage 3a (CD56-) as ILC-precursor that can further give rise to stage 3b (CD56+) and stage 4 (CD94+). Re-plating results together with clonal tracing between S3b, S4 and ILC3 subsets supported a diverging developmental point between NK and ILC3 lineages occurs at the S3a stage and accompanied by the loss of the ILC3 potential as NK cell maturation progress from S3b toward S4. Single-cell transcriptomic and RNA-velocity connected the NK cytotoxic trajectory with a coordinated network of transcription factors (TFs) that are highly compatible with primary NK cell gene program. CD56-positive ILC generated from in vitro differentiation culture after 4 weeks, further undegone column-purification for Lineage-postive cells removal. Purifed CD56-positive ILC cells are subjected to transcriptomic profiling via single-cell RNA sequencing with 10X Genomics platform. ----------------------------- Authors state: raw FASTQ files cannot be uploaded due to compliance with GDPR guildlines for human samples.