Transcriptional profile of LPCs and HSCs following co-culture or conditioned-medium experiments
The aim of this project was to co-culture HSCs (LX-2 cells) with wildtype or c-MET deleted LPCs (BMOL-TAT cells). Additionally, BMOL-TAT cells were indirectly co-cultured by the addition of LX-2-conditioned medium. Bulk RNA-sequencing was then performed to assess transcriptional changes during cellular crosstalk. The results identified that LX-2 cells were not greatly affected at the transcriptional level following 48-hour co-culture with BMOL-TAT cells. However, wildtype and c-MET deleted BMOL-TAT cells were greatly affected at the transcriptomic level by signalling molecules present in LX-2-conditioned medium. This further reinforces the communicative relationship of LPCs and HSCs in diseased liver states. Upon addition of LX-2 conditioned medium on wildtype BMOL-TAT cells, BMOL-TAT cells upregulated expression of ECM remodelling genes, while downregulating expression of ECM deposition genes and cell adhesion genes. This was contrary to c-MET deleted BMOL-TAT cells in LX-2-conditioned medium experiments, as BMOL-TAT cells without the presence of the c-MET receptor decreased expression of ECM remodelling genes, while increasing expression of ECM deposition genes and cell adhesion genes. The data suggests that upon HSC stimulus, LPCs degrade ECM, to migrate to sites of injury and potentially aid in regeneration of the liver. However, without c-MET regulation, this process is hindered.
Steps to reproduce
LX-2 cells were co-cultured in Boyden chambers for 48 hours with either wildtype or c-MET knockout BMOL-TAT cells in the insert above. LX-2 cell RNA was isolated using the Bioline RNA column extraction kit, following the manufacturer’s instructions. Four biological replicates were collected for each experimental group, and RNA was tested for appropriate quality and quantity using the Nanodrop 1000 spectrophotometer. Wildtype and knockout BMOL-TAT cells were seeded in 6-well plates, incubated until the cultures reached approximately 80% confluency and LX-2 cell-conditioned medium was added for either 30 minutes or 6 hours, to assess various timepoints for transcriptional events. RNA was collected using the Bioline RNA column extraction kit following the manufacturers instructions. Four biological replicates were collected for each sample group, and all RNA samples were tested for appropriate quality and quantity using the Nanodrop 1000 spectrophotometer. Samples were shipped to Azenta Life Sciences, Suzhou, China, for quality control, Poly A selection, non-strand specific library preparation and sequencing using Illumina NovaSeq PE150 at 20 M pair reads and 6 Gb/sample. Raw FASTQ files along with standard bioinformatic analysis (quality control, alignment to reference genome, PCA, differential gene expression and enrichment analysis by gene ontology and KEGG pathway analysis) was delivered through Alicloud and provided here. Abbreviations: SC = single culture CC = co-culture CM = conditioned medium 6 = 6 hours 30 = 30 minutes WT or NT = wildtype BMOL-TAT cells KO = c-MET knock out BMOL-TAT cells