no. defined, xeno-free system by temporal modulation of regulators of canonical Wnt signaling. Appropriate differentiation stage-specific application of Gsk3 inhibitor, Wnt inhibitor, then Gsk3 inhibitor is sufficient to produce cells expressing epicardial markers and exhibiting epicardial phenotypes with a high yield and purity from multiple hPSC lines in 16 days. Characterization of differentiated cells is performed via flow cytometry and immunostaining to assess quantitative expression and localization of epicardial cell-specific proteins. differentiation to fibroblasts and smooth muscle cells is also described. In addition, culture in the presence of TGF inhibitors allows long-term expansion of hPSC-derived epicardial cells for at least 25 population doublings. Functional human epicardial cells differentiated via this protocol may constitute a potential cell source for heart disease modeling, drug screening, and cell-based therapeutic applications. INTRODUCTION Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), possess enormous potential for the study and treatment of cardiovascular diseases due to their capacity for unlimited self-renewal and ability to form any somatic cell type1,2. Functional epicardial cells and their progeny differentiated from hPSCs could be beneficial for many applications, including cardiac disease modeling, drug discovery and cellular therapies3. Realization of this potential will require protocols to differentiate hPSCs to cardiovascular cell lineages with high efficiency and reproducibility in a scalable and cost-effective manner. Moreover, therapeutic applications necessitate defined, xeno-free cell manufacturing processes. Over the past decade, there has been significant progress in the generation of cardiomyocytes4C8, endothelial cells9C13, and smooth muscle cells (SMCs)14C16 from hPSCs. However, there have only been a few reports describing the differentiation of hPSCs to epicardial cells. Epicardial Dienestrol cells have been shown to contribute to fibroblast, smooth muscle, and vascular endothelial cell compartments in the developing heart, and also secrete trophic and regulatory factors involved in heart development and maintenance17,18. Initial efforts to differentiate hPSCs into epicardial cells implemented stage-specific application of BMP and Wnt ligands to embryoid bodies (EBs)19. In this approach19, treatment of EBs with BMP4 for 1 day and then BMP4, Activin A, and bFGF for 3 days induced mesoderm differentiation. The EBs were plated and treated with DKK1, VEGF, and SB431542 for 2 days Dienestrol to stimulate cardiovascular specification. Addition of BMP4 during ZBTB16 this stage resulted in epicardial differentiation. Iyer report, including the starting cardiac progenitor cells and exposure to different developmental pathway modulators, may account for the generation of a more homogenous subpopulation of epicardium in Dienestrol our protocol. These findings improve our understanding of epicardial cell specification and self-renewal, and have implications for generating human epicardial cells for therapeutic applications. In this protocol, we provide a detailed step-by-step process for 2D monolayer-based direct differentiation of hPSCs to epicardial cells. This protocol uses a completely defined, growth factor- and xeno-free system and applies temporal modulation of Wnt/-catenin signaling via small molecules. This protocol is based on our earlier reports of cardiac progenitor and epicardial differentiation5,22 and is composed of four major stages: (steps 1C8) induction of cardiac progenitors from hPSCs by temporal modulation of canonical Wnt signaling under defined, albumin-free conditions, (steps 9C14) directed differentiation of cardiac progenitors to pro-epicardial then epicardial cells by Gsk3 inhibitor treatment, (steps 15 A-C) long-term maintenance of hPSC-derived epicardial cells under chemically defined conditions in the presence of a TGF inhibitor, and (steps 15 D) differentiation of epicardial cells to fibroblasts Dienestrol and SMCs. This protocol will enable efficient production of human epicardial cells for development and disease research, drug screening and testing, and advancing cardiac cellular therapies. Experimental design Induction of cardiac progenitors from hPSCs (Steps 1C8) A summary of cardiac progenitor generation (GiWi2 protocol5) is shown in Fig. 1. The hPSCs are initially cultured on Matrigel-coated plates or Synthemax-coated plates in mTeSR1 or E8 medium until fully confluent. For translational applications where fully-defined differentiation is important, a combination of Synthemax and E8 is recommended. The starting hPSC population should contain at least 95% Oct4+ cells with no detectable karyotypic abnormalities. Differentiation is initiated by removing Dienestrol the maintenance medium and adding RPMI basal medium containing a Gsk3 inhibitor, such as CHIR99021. 24 hr of culture in this medium generates a high percentage of brachyury-expressing cells ( 95% by flow cytometry) (Fig. 1). In order to direct these brachyury-expressing mesendoderm progenitor cells to a cardiac progenitor fate, inhibition of canonical Wnt signaling by Wnt signaling inhibitors, such as Porcupine inhibitors IWP2 or IWP4, is.

Related Post