Transcriptome profiling of hiPSC-derived LSECs with nanoCAGE

Liver sinusoidal endothelial cells (LSECs) are vital for liver function due to their role in microvascular transport. Maintaining LSECs in culture is difficult because they lose key traits like fenestration. Immortalized LSEC lines often fail to reflect native cell behavior. This gap motivated researchers to seek alternative models for LSEC study. Human induced pluripotent stem cells (hiPSCs) offer a promising source for generating LSEC-like cells. Prior research has shown that hiPSCs can differentiate into various vascular cell types. However, no prior work had resolved how well hiPSC-derived LSECs mimic native LSECs at the transcriptomic level. This study aimed to address this uncertainty by profiling gene expression in hiPSC-derived LSECs.

The goal was to assess the transcriptomic profile of LSECs derived from hiPSCs. This would help determine whether these cells can serve as a reliable model for LSECs. The study focused on identifying key vascular markers and regulatory networks. Researchers sought to compare the gene expression of hiPSC-derived LSECs to primary LSECs. A specific problem addressed was the lack of stable LSEC models for in vitro liver tissue engineering. The motivation was to develop a more accurate and sustainable LSEC model for research. This model could support the creation of complex liver-like tissues in the lab. The study also aimed to identify transcription factors involved in LSEC differentiation.

Human induced pluripotent stem cells were differentiated into a LSEC-like phenotype. RNA quantification was used to assess the expression of vascular markers. Immunostaining confirmed the presence of Stabilin-1 and Stabilin-2 proteins. NanoCAGE sequencing was employed to perform whole transcriptome analysis. This method allowed the detection of promoter motifs and gene expression levels. The study compared gene expression profiles of hiPSC-derived LSECs to primary LSEC datasets. Transcription factor activity was analyzed using promoter motif data. A regulatory network was compiled based on the interactions between TFs and target genes.

The hiPSC-derived LSECs showed high expression of CD31, CD144, and STAB2. Immunostaining confirmed expression of Stabilin-1 and Stabilin-2. NanoCAGE analysis revealed upregulation of APLN, LYVE1, VWF, ESAM, and ANGPT2 genes. VEGFA gene expression was found to be downregulated in the derived LSECs. Promoter motif analysis identified several transcription factors, including IRF2, ERG, and MEIS2. These TFs were linked to vascular and endothelial functions. The regulatory network suggested interactions between TFs and their target genes. Comparison with primary LSEC datasets confirmed the similarity of the derived model.

The study demonstrated that hiPSC-derived LSECs express key vascular markers. Transcriptomic profiling confirmed the cells' vascular identity and LSEC-like characteristics. The regulatory network involving transcription factors was validated through comparison with primary data. The proposed model aligns with known LSEC features, suggesting its reliability. This model could serve as a tool for liver tissue engineering and disease modeling. The findings suggest that the derived LSECs maintain essential traits of native cells. The regulatory network provides insight into the transcriptional control of LSEC identity. These conclusions are based on the experimental data and comparisons with reference datasets.