Single cell transcriptomic analysis of human pluripotent stem cell chondrogenesis

Abstract

The therapeutic application of human induced pluripotent stem cells (hiPSCs) for cartilage regeneration is largely hindered by the low yield of chondrocytes accompanied by unpredictable and heterogeneous off-target differentiation of cells during chondrogenesis. Here, we combine bulk RNA sequencing, single cell RNA sequencing, and bioinformatic analyses, including weighted gene co-expression analysis (WGCNA), to investigate the gene regulatory networks regulating hiPSC differentiation under chondrogenic conditions. We identify specific WNTs and MITF as hub genes governing the generation of off-target differentiation into neural cells and melanocytes during hiPSC chondrogenesis. With heterocellular signaling models, we further show that WNT signaling produced by off-target cells is responsible for inducing chondrocyte hypertrophy. By targeting WNTs and MITF, we eliminate these cell lineages, significantly enhancing the yield and homogeneity of hiPSC-derived chondrocytes. Collectively, our findings identify the trajectories and molecular mechanisms governing cell fate decision in hiPSC chondrogenesis, as well as dynamic transcriptome profiles orchestrating chondrocyte proliferation and differentiation.

Transcriptional dynamics of colorectal cancer risk associated variation at 11q23.1 are correlated with tuft cell abundance and marker expression in silico

“…Indeed, our study is also not the first use of WGCNA to detect gene-gene relatedness in scRNAseq data. WGCNA has been utilised to identify gene modules associated with the activation of neuronal stem cells (Luo et al, 2015) and human induced pluripotent stem cells (Wu et al, 2021), however, like our own study, these studies did not utilise expression from individual cells as the input for WGCNA.…”

The temporal transcriptomic signature of cartilage formation

“…The authors identified a chondrogenic gene subset containing 1172 entities, whose functional characterisation promises to harness the potential of MSCs for cartilage tissue engineering. The same laboratory has recently published a paper on chondrogenic differentiation of hiPSCs using bulk and single-cell RNA sequencing and identified gene regulatory networks regulating chondrogenesis (36). Temporal changes in the transcriptome of human embryonic stem cells (ESCs) during key stages of chondrogenic differentiation have also been recently published (37).…”

“…Looking at the collagen expression profile of iPSCs during chondrogenesis, COL1A1 had, by far, the highest levels by day 60, followed by COL1A2 and COL3A1 , with relatively low COL2A1 levels (36). Interestingly, iPSCs also did not express the genes encoding collagen types XVII and XX.…”

“…The adolescent cartilage samples showed very high COL6A1 expression, while COL2A1 was only moderately expressed. A high number of COL2A1 transcripts was mainly present for embryonic and week-17 cartilage samples (36).…”

Differentiation of Induced Pluripotent Stem Cells Into Chondrocytes: Methods and Applications for Disease Modeling and Drug Discovery

“…In case of omission of the cell sorting step, an equally homogeneous iPSC‐chondrocyte population can be obtained by inhibiting WNT and melanocyte inducing transcription factor (MITC) signaling during pellet cultivation, as this prevents the generation of off‐target cells such as neural cells and melanocytes. ( 59 ) Importantly, inhibition of WNT signaling prevents chondrocyte hypertrophy, ( 59 ) which is beneficial for regenerative purposes but unfavorable when creating iPSC‐chondrocyte disease models. Based on the results of a study investigating the transcriptomic landscape of human somitogenesis, a more simplified protocol was proposed by Xi and colleagues ( 60 ) First, WNT/β‐catenin signaling was activated by addition of CHIR99021 to the differentiation medium to differentiate human pluripotent stem cells through primitive streak to posterior presomitic mesoderm.…”