Efficient Differentiation of Embryonic Stem Cells into Mesodermal Precursors by BMP, Retinoic Acid and Notch Signalling

The ability to direct differentiation of mouse embryonic stem (ES) cells into specific lineages not only provides new insights into the pathways that regulate lineage selection but also has translational applications, for example in drug discovery. We set out to develop a method of differentiating ES cells into mesodermal cells at high efficiency without first having to induce embryoid body formation. ES cells were plated on a feeder layer of PA6 cells, which have membrane-associated stromal-derived inducing activity (SDIA), the molecular basis of which is currently unknown. Stimulation of ES/PA6 co-cultures with Bone Morphogenetic Protein 4 (BMP4) both favoured self-renewal of ES cells and induced differentiation into a Desmin and Nestin double positive cell population. Combined stimulation with BMP4 and all-trans Retinoic Acid (RA) inhibited self-renewal and resulted in 90% of cells expressing Desmin and Nestin. Quantitative reverse transcription-polymerase chain reaction (qPCR) analysis confirmed that the cells were of mesodermal origin and expressed markers of mesenchymal and smooth muscle cells. BMP4 activation of a MAD-homolog (Smad)-dependent reporter in undifferentiated ES cells was attenuated by co-stimulation with RA and co-culture with PA6 cells. The Notch ligand Jag1 was expressed in PA6 cells and inhibition of Notch signalling blocked the differentiation inducing activity of PA6 cells. Our data suggest that mesodermal differentiation is regulated by the level of Smad activity as a result of inputs from BMP4, RA and the Notch pathway

Generation, expansion and functional analysis of endothelial cells and pericytes derived from human pluripotent stem cells

Human endothelial cells (ECs) and pericytes are of great interest for research on vascular development and disease as well as future therapy. This protocol describes the efficient generation of ECs and pericytes from human pluripotent stem cells (hPSCs) under defined conditions. Essential steps for hPSC culture, differentiation, isolation and functional characterization of ECs and pericytes are described. Substantial numbers of both cell types can be derived in only 2-3 weeks: this involves differentiation (10 days), isolation (1 day) and 4 or 10 days expansion of ECs and pericytes, respectively. We also describe two assays for functional evaluation of hPSC-derived ECs: (i) primary vascular plexus formation upon co-culture with hPSC-derived pericytes and (ii) incorporation in the vasculature of zebrafish xenografts in vivo. These assays can be used to test the quality and drug sensitivity of hPSC-derived ECs and model vascular diseases with patient-derived hPSCs

Generation of Hematopoietic Stem and Progenitor Cells from Human Pluripotent Stem Cells.

Human pluripotent stem cells (PSCs) have the potential to provide a virtually unlimited supply of cells for transplantation therapy. When combined with recent advances in genome editing technologies, human PSCs could offer various approaches that enable gene therapy, drug discovery, disease modeling, and in vitro modeling of human development. De novo generation of hematopoietic stem cells (HSCs) from human PSCs is an important focus in the field, since it enables autologous HSC transplantation to treat many blood disorders and malignancies. Although culture conditions have been established to generate a broad spectrum of hematopoietic progenitors from human PSCs, it remains a significant challenge to generate bona fide HSCs that possess sustained self-renewal and multilineage differentiation capacities upon transplantation. In this review, recent promising advances in the efforts to generate HSCs and hematopoietic progenitors from human PSCs in vitro and in vivo or from somatic cells are discussed