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

Abstract

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.

Lef1 Contributes to the Differentiation of Bulge Stem Cells by Nuclear Translocation and Cross-Talk with the Notch Signaling Pathway

“…Estrach et al found that the deletion of jagged1 inhibited hair growth and prevented new hair follicle formation, revealing the interaction of the Wnt and Notch signaling pathways in regulating hair follicle cycle maintenance 31. In general, Notch signaling plays a key role in regulating the self-renewal, proliferation, and differentiation of stem cells 32-34. Previous studies have documented that Notch was widely expressed in developing or differentiating hairs, led to the aberrant differentiation of the hair cortex 35-36, and inhibited the terminal differentiation of the epidermis 37.…”

Identification of embryonic stem cell activities in an embryonic cell line derived from marine medaka (Oryzias dancena)

“…As an agent used to induce in vitro lineage-specific differentiation of ESCs, RA induces in vitro differentiation of ESCs into a number of cell types, including neural (Kim et al 2009;Wichterle et al 2002), mesodermal (Kennedy et al 2009;Torres et al 2012), epithelial (Metallo et al 2008), pancreatic (Shim et al 2007), and germ cell lineages (Chen et al 2012). Our results showed that the differentiation patterns of O. dancena ESC-like cells were distinct at the morphological level depending on the presence or absence of RA.…”

Bones Morphogenic Protein‐4 and retinoic acid combined treatment comparative analysis for in vitro differentiation potential of murine mesenchymal stem cells derived from bone marrow and adipose tissue into germ cells

“…Kashani, et al () suggested that RA can induce differentiation of mouse BM‐derived CD15+, Oct4+, and CXCR4+ cells into male germ cells in vitro. Moreover, BMPs play an important role in regulation of stem cell fate in mammals (Torres, Prieto, Durupt, Broad, & Watt, ). PGC formation in epiblast cultures can be induced by human recombinant BMP4 (Okamura, Hayashi & Matsui, ), and Bmp4 null mutants have the most severe defect in germ cell development, with a near‐complete absence of PGCs (de Sousa et al, 2004).…”