Dual Reporter MESP1mCherry/w-NKX2-5eGFP/w hESCs Enable Studying Early Human Cardiac Differentiation

Abstract

Understanding early differentiation events leading to cardiogenesis is crucial for controlling fate of human pluripotent stem cells and developing protocols that yield sufficient cell numbers for use in regenerative medicine and drug screening. Here, we develop a new tool to visualize patterning of early cardiac mesoderm and cardiomyocyte development in vitro by generating a dual MESP1 mCherry/w -NKX2-5 eGFP/w reporter line in human embryonic stem cells (hESCs) and using it to examine signals that lead to formation of cardiac progenitors and subsequent differentiation. MESP1 is a pivotal transcription factor for precardiac mesoderm in the embryo, from which the majority of cardiovascular cells arise. Transcription factor NKX2-5 is expressed upon cardiac crescent formation. Induction of cardiac differentiation in this reporter line resulted in transient expression of MESP1-mCherry, followed by continuous expression of NKX2-5-eGFP. MESP1-mCherry cells showed increased expression of mesodermal and epithelial-mesenchymal-transition markers confirming their mesodermal identity. Whole-genome microarray profiling and fluorescence-activated cell sorting analysis of MESP1-mCherry cells showed enrichment for mesodermal progenitor cell surface markers PDGFR-a, CD13, and ROR-2. No enrichment was found for the previously described KDR1PDGFR-a1 progenitors. MESP1-mCherry derivatives contained an enriched percentage of NKX2-5-eGFP and Troponin T expressing cells, indicating preferential cardiac differentiation; this was enhanced by inhibition of the Wnt-pathway. Furthermore, MESP1-mCherry derivatives harbored smooth muscle cells and endothelial cells, demonstrating their cardiac and vascular differentiation potential under appropriate conditions. The MESP1-NKX2-5 hESC reporter line allows us to identify molecular cues crucial for specification and expansion of human cardiac mesoderm and early progenitors and their differentiation to specific cardiovascular derivatives. STEM CELLS 2015;33:56-67

Expansion and patterning of cardiovascular progenitors derived from human pluripotent stem cells

“…5). Downregulation of KDR and CD13 is consistent with their being earlier mesodermal markers [28][29][30] , and the absence of CD117 is consistent with cardiac lineage-tracing data 31 .…”

“…Comparison of these cells with earlier MESP1 + mesodermal cells isolated from a MESP1 reporter derivative of the same hESC line 28 showed much lower expression of MESP1 in all sphere populations (Fig. 3c).…”

“…Gene expression values were normalized to the mean expression of the housekeeping genes encoding a human ribosomal protein (RPLP0), glucuronidase (GUSB), and RNF7. RNA from MESP1-expressing sorted cells was obtained from a derivative knock-in reporter line and used as a reference 28 . The hESC-cardiomyocyte control RNA was obtained from eGFP + sorted cells from a standard monolayer differentiation of the NKX2-5 eGFP/w hESC line with maintenance in the same medium as the CPC-CMs.…”

Cardiac differentiation of human pluripotent stem cells in scalable suspension culture

“…1), and it addresses all crucial elements, including cells, media and hardware handling. To facilitate straightforward establishment of the method in the laboratory, we recommend using available reporter cell lines such as the cardiac-specific expression of GFP under the control of the endogenous NKX2.5 promoter 32 (NKX2-5 w/eGFP ) or a recent MESP1 w/mCherry /NKX2-5 w/eGFP double-transgenic line 33 to monitor differentiation efficiency at early and late stages, respectively. By using such control lines and by modifying the concentration of the chemical effector molecules, we show systematic process optimization and cell line-dependent adaptation to a multiwell plate screening format compatible with drug screening assays.…”

Long noncoding RNA Braveheart promotes cardiogenic differentiation of mesenchymal stem cells in vitro

“…lncRNA-Bvht functions via Mesp1 to modulate the expression of cardiac transcription factors and further promote cardiogenic differentiation of ESCs [14]. Previous data show that Mesp1 is capable of initiating the EMT process by regulating EMT-associated genes [20]. …”