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.

Skeletal dysplasia-causing TRPV4 mutations suppress the hypertrophic differentiation of human iPSC-derived chondrocytes

“…No reuse allowed without permission. However, we may be preventing some hypertrophy since our chondrogenic protocol uses a pan-Wnt inhibitor to prevent off-target differentiation and promote a homogenous chondrocyte population (Wu et al, 2021). Nevertheless, our WT chondrocytes, but not TRPV4 mutants, exhibited hypertrophic differentiation with BMP4 treatment, suggesting that DEGs/pathways detected in our sequencing analysis are still robust.…”

“…To confirm if the hiPSCs with dysplasia-causing mutations would undergo proper chondrogenesis, we differentiated CRISPR-Cas9-edited hiPSCs with mutant TRPV4 alongside an isogenic wildtype (WT) using our previously published protocol (S. S. Adkar et al, 2019;Wu et al, 2021). After 12 days of monolayer mesodermal differentiation, the cells underwent 42 days of chondrogenic differentiation, and pellets were collected at days 7, 14, 28, and 42.…”

“…The hiPSCs were differentiated through the mesodermal pathway as previously described (S. S. Adkar et al, 2019;Dicks et al, 2020;Wu et al, 2021). In brief, cells were fed daily with different cocktails of growth factors and small molecules for twelve days in mesodermal differentiation medium and driven through the anterior primitive streak (1 day; 30 ng/ml Activin Louis, MO).…”

“…Cells were differentiated into chondrocytes using a high-density, suspension pellet culture (S. S. Adkar et al, 2019;Dicks et al, 2020;Wu et al, 2021). In summary, cells were resuspended in chondrogenic medium: Dulbecco's Modified Eagle Medium/F12, glutaMAX (DMEM/F12; cat.…”

Comparative evaluation of isogenic mesodermal and ectomesodermal chondrocytes from human iPSCs for cartilage regeneration

“…These results indicate that it has remained challenging to derive a homogenous population of target cells from PSCs ( 36 , 37 ). The approaches that have been applied to address the challenge include inhibiting the formation of undesired cell populations ( 38 ) and improving the efficiency of chondrogenesis ( 15 , 39 ). In this study, we identify the growth factors down-regulated in hiPSC-derived chondrocytes compared to those in reference NCs and then add the identified molecules in culture during chondrogenic differentiation of MCs and NCCs to increase the generation of the chondrocyte population.…”