Abstract
The mesoderm is one of the three germ layers produced during gastrulation from which muscle, bones, kidneys, and the cardiovascular system originate. Understanding the mechanisms that control mesoderm specification could inform many applications, including the development of regenerative medicine therapies to manage diseases affecting these tissues. Here, we used human pluripotent stem cells to investigate the role of cell cycle in mesoderm formation. To this end, using small molecules or conditional gene knockdown, we inhibited proteins controlling G1 and G2/M cell cycle phases during the differentiation of human pluripotent stem cells into lateral plate, cardiac, and presomitic mesoderm. These loss-of-function experiments revealed that regulators of the G1 phase, such as cyclin-dependent kinases and pRb (retinoblastoma protein), are necessary for efficient mesoderm formation in a context-dependent manner. Further investigations disclosed that inhibition of the G2/M regulator cyclin-dependent kinase 1 decreases BMP (bone morphogenetic protein) signaling activity specifically during lateral plate mesoderm formation while reducing fibroblast growth factor/extracellular signaling-regulated kinase 1/2 activity in all mesoderm subtypes. Taken together, our findings reveal that cell cycle regulators direct mesoderm formation by controlling the activity of key developmental pathways.
Highlights
The mesoderm is one of the three germ layers produced during gastrulation from which muscle, bones, kidneys, and the cardiovascular system originate
Characterization of mesoderm subtypes generated from Human pluripotent stem cells (hPSCs)
HPSCs were induced to generate lateral plate mesoderm (LPM), cardiac mesoderm (CM), and presomitic mesoderm (PSM) mesoderm for 36 h followed by the addition of another mixture of growth factors and small molecules to generate functional cell types such as smooth muscle cells, cardiomyocytes, and chondrocytes (Fig. 1A) (20 –22)
Summary
We took advantage of established protocols for differentiating hPSCs into different mesoderm subtypes. The strongest effect was observed in LPM and CM induction, whereas reduction in PSM was less severe, recapitulating the phenotype obtained by inhibiting CDK1 with small molecule Taken together, these results demonstrate that CDK1 is necessary for the activity of BMP and FGF signaling during mesoderm specification. Presence of this small molecule resulted in the complete loss of expression of T during LPM (Fig. 4I) and PSM induction (Fig. 4K) and loss of EOMES during CM induction (Fig. 4J) Taken together, these results demonstrate that mesoderm subtype induction is controlled by the interplays between CDK1 and FGF/ERK1/2, which are necessary for the induction of key mesoderm markers and the down-regulation of pluripotency factors
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