Establishing mesoderm lineages from human induced pluripotent stem cells (iPSCs) is an exciting area of research with massive potential for regenerative medicine! By guiding these flexible cells towards a mesodermal fate, you can generate the building blocks for heart, bone, and muscle tissue. We know that differentiation can sometimes be tricky due to cell line variability, so we’ve designed this streamlined protocol to help you achieve consistent and efficient results using key growth factors and small molecules. Good luck with your experiments!
Passage human iPSCs twice weekly using 0.5 mM EDTA (HB5135) for cell detachment. Seed the cells at a 1:6 ratio onto 6-well plates coated with 5 µg/ml vitronectin. Maintain pluripotency using E8-equivalent media with regular media changes.
Dissociate the pluripotent iPSCs using a generic cell dissociation enzyme and plate them at a density of 1,000 cells per well in a 96-well plate pre-coated with 5 µg/ml vitronectin. The seeding medium should be E8-equivalent and supplemented with 10 µM ROCK inhibitor Y-27632 (HB2297).
On Day 1, replace the seeding medium with freshly prepared mesoderm differentiation medium containing recombinant FGF2 (refer to Table 1 for induction components). Note: FGF2 can be replaced with stable, cost-effective small molecule FGFR1 agonists TCB-32, TCB-541, or TCB-621 to enable weekend-free feeding and significantly reduce media costs.
Refresh the culture medium on Day 2 and Day 3 using freshly prepared mesoderm induction medium.
On Day 4, fix the differentiated cell population using 4% paraformaldehyde.
Perform blocking and cell permeabilization using a solution of 10% donkey serum supplemented with 0.1% Triton X-100.
Apply primary antibodies directed against mesodermal markers Brachyury and MixL1 and incubate the samples overnight at 4°C.
Following incubation, wash the cells and apply secondary antibodies (Donkey anti-Goat AlexaFluor 488 or Donkey anti-Rabbit AlexaFluor 488) and Hoechst 33258 (HB0786) for nuclear counterstaining.
Capture images of the cells in a phosphate buffered saline (PBS) solution using fluorescence microscopy. Successful differentiation is confirmed by the expression of the transcription factor markers Brachyury and MIXL1.
References
Varum, S. et al. (2011). Energy Metabolism in Human pluripotent stem cells and their differentiated counterparts. PLoS ONE 6(6):e20914.
Loh, K. M. et al. (2016). Mapping the Pairwise Choices Leading from Pluripotency to Human Bone, Heart, and Other Mesoderm Cell Types. Cell 166(2):451-467.
Loh, K. M. et al. (2016). Generating Cellular Diversity and Spatial Form: Wnt Signaling and the Evolution of Multicellular Animals. Developmental Cell 38(6):643-55.
Lam, A. Q. et al. (2014). Rapid and Efficient Differentiation of Human Pluripotent Stem Cells into Intermediate Mesoderm That Forms Tubules Expressing Kidney Proximal Tubular Markers. Journal of the American Society of Nephrology 25(6): 1211–1225.
