[Shiau-ROC TAIWAN-O-4] Inverse genetics to trace the differentiation pathway of human chondrocytes and re-discover a regulator of chondrogenesis by single cell RNA-seq
[Abstract]
[Objective]
Mammalian somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by the forced expression of Yamanaka’s reprogramming factors1. However, only a limited population of the cells that pass through a particular pathway can metamorphose into iPSCs, while the others do not. Objectives of this study is clarifying the specific pathway chondrocytes can successfully transition into iPSCs and the route that iPSCs interfered2 by SOX9.
[Method]
The fate of human articular chondrocytes (LONZA, USA) under reprogramming were chased by a time-coursed single-cell transcriptomic analysis (10X GENOMICS, Pleasanton, CA, USA), which we entitled inverse genetic approach. To verify that chondrocytes go back to pluripotency following the proper differentiation route in an inversed fashion, iPSCs-interference technique was also employed with SOX9.
[Results and Discussion]
We confirmed that human chondrocytes could be converted into cells with an iPSC phenotype. Moreover, it was clarified that a quite limited population of the cells that underwent the silencing of SOX9, a master gene for chondrogenesis, at a particular point during proper transcriptome transition could eventually become iPSCs. Furthermore, by lentiviral transduction to overexpress SOX9, the efficiency of reprogramming chondrocytes into iPS cells was reduced by 68.6% compared to that without SOX9 overexpression (n=3, Student t-test, p<0.001). These findings suggest that those chondrocytes under reprogramming follow the differentiation pathway backwards. Interestingly, the other cells, which failed to be reprogrammed, followed a distinct pathway towards surface zone chondrocytes with stem cell characteristics. We here established the transcriptomic transition route from chondrocytes to iPSCs, and by chasing the reprogramming route of chondrocytes backwards, SOX9 was re-discovered as a regulator of chondrogenesis. This inverse genetics is expected to be utilized for the identification of master genes of the differentiation of chondrocytes and the establishment of more natural and efficient differentiation protocols of iPSCs into chondrocytes.
[References]
1, Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006; 126:663-76
2, Hikichi T et al. Transcription factors interfering with dedifferentiation induce cell type-specific transcriptional profiles. Proc Natl Acad Sci 2013; 110:6412-7
[Objective]
Mammalian somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by the forced expression of Yamanaka’s reprogramming factors1. However, only a limited population of the cells that pass through a particular pathway can metamorphose into iPSCs, while the others do not. Objectives of this study is clarifying the specific pathway chondrocytes can successfully transition into iPSCs and the route that iPSCs interfered2 by SOX9.
[Method]
The fate of human articular chondrocytes (LONZA, USA) under reprogramming were chased by a time-coursed single-cell transcriptomic analysis (10X GENOMICS, Pleasanton, CA, USA), which we entitled inverse genetic approach. To verify that chondrocytes go back to pluripotency following the proper differentiation route in an inversed fashion, iPSCs-interference technique was also employed with SOX9.
[Results and Discussion]
We confirmed that human chondrocytes could be converted into cells with an iPSC phenotype. Moreover, it was clarified that a quite limited population of the cells that underwent the silencing of SOX9, a master gene for chondrogenesis, at a particular point during proper transcriptome transition could eventually become iPSCs. Furthermore, by lentiviral transduction to overexpress SOX9, the efficiency of reprogramming chondrocytes into iPS cells was reduced by 68.6% compared to that without SOX9 overexpression (n=3, Student t-test, p<0.001). These findings suggest that those chondrocytes under reprogramming follow the differentiation pathway backwards. Interestingly, the other cells, which failed to be reprogrammed, followed a distinct pathway towards surface zone chondrocytes with stem cell characteristics. We here established the transcriptomic transition route from chondrocytes to iPSCs, and by chasing the reprogramming route of chondrocytes backwards, SOX9 was re-discovered as a regulator of chondrogenesis. This inverse genetics is expected to be utilized for the identification of master genes of the differentiation of chondrocytes and the establishment of more natural and efficient differentiation protocols of iPSCs into chondrocytes.
[References]
1, Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006; 126:663-76
2, Hikichi T et al. Transcription factors interfering with dedifferentiation induce cell type-specific transcriptional profiles. Proc Natl Acad Sci 2013; 110:6412-7