A Robust Protocol for Germ Layer Differentiation of Human Pluripotent Stem Cells and Evaluation of Their Chromosome...

Human pluripotent stem cells (hPSCs) can differentiate into any cell type of the human body and represent a powerful in vitro model to recapitulate early human development. However, their use in regenerative medicine and developmental studies is limited by their genomic and chromosomal instability acquired during long-term culture, often driven by the clonal advantage conferred by spontaneous gain or loss of chromosomes. Here, a detailed culture condition that robustly recapitulates early differentiation and describes methods to assess chromosomal and genomic (in)stability in hPSC cultures. First, the cell culture approaches are described that enable controlled exit from pluripotency and homogeneous differentiation into primitive streak, neuroectoderm, paraxial/lateral mesoderm, and definitive endoderm-like cells. Second, a comprehensive set of protocols is described to assess genomic and chromosomal instability during routine pluripotent stem cell culture, including the detection of single- and double-strand breaks during S-phase, mitotic abnormalities (such as ultrafine bridges, lagging chromosomes or micronuclei) and their direct consequence-the emergence of aneuploidy after cell division. In summary, this work aims to standardize protocols to model early human post-implantation lineage commitment and to provide the tools required to assess routine karyotypic stability of hPSCs during in vitro culture.