Methods in molecular biology (Clifton, N.J.), 2015
Chemical genomics has the unique potential to expose novel mechanisms of complex cellular biology... more Chemical genomics has the unique potential to expose novel mechanisms of complex cellular biology through screening of small molecules in in vitro assays of a biological phenotype of interest, followed by target identification. In the case of disease-specific assays, the cellular proteins identified might constitute novel drug targets, and the small molecules themselves might be developed as drug leads. In cardiovascular biology, a chemical genomics approach to study the formation of cardiomyocyte, vascular endothelial, and smooth muscle lineages might contribute to therapeutic regeneration. Here, we describe methods used to develop high content screening assays implementing multipotent cardiovascular progenitors derived from human pluripotent stem cells and have identified novel compounds that direct cardiac differentiation.
A critical but molecularly uncharacterized step in heart formation and regeneration is the proces... more A critical but molecularly uncharacterized step in heart formation and regeneration is the process that commits progenitor cells to differentiate into cardiomyocytes. Here, we show that the endoderm-derived dual Nodal/bone morphogenetic protein (BMP) antagonist Cerberus-1 (Cer1) in embryonic stem cell cultures orchestrates two signaling pathways that direct the SWI/SNF chromatin remodeling complex to cardiomyogenic loci in multipotent (KDR/Flk1+) progenitors, activating lineage-specific transcription. Transient inhibition of Nodal by Cer1 induces Brahma-associated factor 60c (Baf60c), one of three Baf60 variants (a, b, and c) that are mutually exclusively assembled into SWI/SNF. Blocking Nodal and BMP also induces lineage-specific transcription factors Gata4 and Tbx5, which interact with Baf60c. siRNA to Cer1, Baf60c, or the catalytic SWI/SNF subunit Brg1 prevented the developmental opening of chromatin surrounding the Nkx2.5 early cardiac enhancer and cardiomyocyte differentiation....
The International journal of developmental biology, 2006
Embryonic Stem (ES) cells have the potential to form every cell of the body and thus are of great... more Embryonic Stem (ES) cells have the potential to form every cell of the body and thus are of great promise for tissue transplantation. One of the rising techniques that allows studying the differentiation state of ES cells is quantitative RT-PCR (qRT-PCR). When relative quantification by qRT-PCR is applied, accurate normalization is necessary, since differentiated embryonic stem cells and developing embryos contain heterogeneous cell populations. Corrections for variations in the qRT-PCR reaction are needed to allow comparisons between different samples. We applied the normalization tools geNorm and Normfinder to ten reference genes identifying the most stable ones for relative quantification of gene expression during differentiation of human ES cells, as well as in differentiated mouse ES cells and in the developing mouse embryo. For relative quantification by qRT-PCR in these systems, we advise to use normalization factors based on multiple stable reference genes. However, when the...
The Wnt family of secreted signaling molecules is conserved throughout the animal kingdom. Wnt si... more The Wnt family of secreted signaling molecules is conserved throughout the animal kingdom. Wnt signaling plays critical roles during embryonic development and mutations leading to the overactivation of the Wnt pathway have been linked to cancer. Wnt signals are transduced intracellularly by the Frizzled family of receptors. Moreover, proteoglycans and the co-receptors LRP5 and -6 participate in the transmission of
ABSTRACT In the United States, the need for cardiac cells for transplantation or research applica... more ABSTRACT In the United States, the need for cardiac cells for transplantation or research applications is immense. While over 300,000 individuals per year seek heart transplants, less than 2% will receive hearts. Human cardiomyocytes are also needed to investigate the safety of new chemical entities being developed as drug candidates because cardiac toxicity is a major cause of drug development failure. Despite the need for human cardiac cells, progress to provide human cardiac cells has been challenging. One approach is to utilize human pluripotent stem cells to generate large numbers of cardiomyocytes for transplantation purposes or for use in drug discovery and development work. Currently, however, there are few methods to produce large numbers of human cardiomyocytes on a biotechnology scale to address the need. The hypothesis of our work is that small molecules could be identified that direct human pluripotent stem cell differentiation to cardiomyocytes. The long term goal of this work is to create a toolbox of small molecule reagents useful to direct human stem cell differentiation into cardiomyocytes and thus address the need for human cardiac cells. Therefore, we screened a collection of about 550 pathway modulators in a human embryonic stem cell-based assay that probes cardiomyocyte differentiation. One compound from the screen, (i.e., IWR-1), a very potent “hit” was selected for further medicinal chemical refinement as a Wnt/β-catenin antagonist and as an agent that directs human stem cell differentiation into cardiomyocytes. In an iterative process of chemical synthesis and parallel evaluation for Wnt/β-catenin inhibition and cardiomyogenesis, several potent agents with improved pharmacodynamic and kinetic properties were elaborated. Cardiomyogenesis was verified by quantifying the effects of transcription factors expressed in human stem cells upon differentiation (i.e., Nkx2.5, αMHC, alpha activin) by RT-qPCR, image analysis and for selected compounds, flow cytometry. The results showed that a distinct Wnt/β-catenin inhibition structure-activity relationship emerged from the "Dynamic Medicinal Chemistry" approach. Potent Wnt/β-catenin antagonists that emerged from this optimization campaign were useful toolbox reagents to produce large numbers of human cardiomyocytes from human stem cells. The conclusion is that small molecules can be used to afford human cardiomyocyte differentiation from pluripotent stem cells. Application of this approach to human induced pluripotent stem (iPS) cells could afford cardiomyocytes that could be used in drug discovery and development applications (i.e., drug toxicity testing) as well as provide a new way to address “Disease in a Dish” issues and create cells for therapeutic utility.
Methods in molecular biology (Clifton, N.J.), 2015
Chemical genomics has the unique potential to expose novel mechanisms of complex cellular biology... more Chemical genomics has the unique potential to expose novel mechanisms of complex cellular biology through screening of small molecules in in vitro assays of a biological phenotype of interest, followed by target identification. In the case of disease-specific assays, the cellular proteins identified might constitute novel drug targets, and the small molecules themselves might be developed as drug leads. In cardiovascular biology, a chemical genomics approach to study the formation of cardiomyocyte, vascular endothelial, and smooth muscle lineages might contribute to therapeutic regeneration. Here, we describe methods used to develop high content screening assays implementing multipotent cardiovascular progenitors derived from human pluripotent stem cells and have identified novel compounds that direct cardiac differentiation.
A critical but molecularly uncharacterized step in heart formation and regeneration is the proces... more A critical but molecularly uncharacterized step in heart formation and regeneration is the process that commits progenitor cells to differentiate into cardiomyocytes. Here, we show that the endoderm-derived dual Nodal/bone morphogenetic protein (BMP) antagonist Cerberus-1 (Cer1) in embryonic stem cell cultures orchestrates two signaling pathways that direct the SWI/SNF chromatin remodeling complex to cardiomyogenic loci in multipotent (KDR/Flk1+) progenitors, activating lineage-specific transcription. Transient inhibition of Nodal by Cer1 induces Brahma-associated factor 60c (Baf60c), one of three Baf60 variants (a, b, and c) that are mutually exclusively assembled into SWI/SNF. Blocking Nodal and BMP also induces lineage-specific transcription factors Gata4 and Tbx5, which interact with Baf60c. siRNA to Cer1, Baf60c, or the catalytic SWI/SNF subunit Brg1 prevented the developmental opening of chromatin surrounding the Nkx2.5 early cardiac enhancer and cardiomyocyte differentiation....
The International journal of developmental biology, 2006
Embryonic Stem (ES) cells have the potential to form every cell of the body and thus are of great... more Embryonic Stem (ES) cells have the potential to form every cell of the body and thus are of great promise for tissue transplantation. One of the rising techniques that allows studying the differentiation state of ES cells is quantitative RT-PCR (qRT-PCR). When relative quantification by qRT-PCR is applied, accurate normalization is necessary, since differentiated embryonic stem cells and developing embryos contain heterogeneous cell populations. Corrections for variations in the qRT-PCR reaction are needed to allow comparisons between different samples. We applied the normalization tools geNorm and Normfinder to ten reference genes identifying the most stable ones for relative quantification of gene expression during differentiation of human ES cells, as well as in differentiated mouse ES cells and in the developing mouse embryo. For relative quantification by qRT-PCR in these systems, we advise to use normalization factors based on multiple stable reference genes. However, when the...
The Wnt family of secreted signaling molecules is conserved throughout the animal kingdom. Wnt si... more The Wnt family of secreted signaling molecules is conserved throughout the animal kingdom. Wnt signaling plays critical roles during embryonic development and mutations leading to the overactivation of the Wnt pathway have been linked to cancer. Wnt signals are transduced intracellularly by the Frizzled family of receptors. Moreover, proteoglycans and the co-receptors LRP5 and -6 participate in the transmission of
ABSTRACT In the United States, the need for cardiac cells for transplantation or research applica... more ABSTRACT In the United States, the need for cardiac cells for transplantation or research applications is immense. While over 300,000 individuals per year seek heart transplants, less than 2% will receive hearts. Human cardiomyocytes are also needed to investigate the safety of new chemical entities being developed as drug candidates because cardiac toxicity is a major cause of drug development failure. Despite the need for human cardiac cells, progress to provide human cardiac cells has been challenging. One approach is to utilize human pluripotent stem cells to generate large numbers of cardiomyocytes for transplantation purposes or for use in drug discovery and development work. Currently, however, there are few methods to produce large numbers of human cardiomyocytes on a biotechnology scale to address the need. The hypothesis of our work is that small molecules could be identified that direct human pluripotent stem cell differentiation to cardiomyocytes. The long term goal of this work is to create a toolbox of small molecule reagents useful to direct human stem cell differentiation into cardiomyocytes and thus address the need for human cardiac cells. Therefore, we screened a collection of about 550 pathway modulators in a human embryonic stem cell-based assay that probes cardiomyocyte differentiation. One compound from the screen, (i.e., IWR-1), a very potent “hit” was selected for further medicinal chemical refinement as a Wnt/β-catenin antagonist and as an agent that directs human stem cell differentiation into cardiomyocytes. In an iterative process of chemical synthesis and parallel evaluation for Wnt/β-catenin inhibition and cardiomyogenesis, several potent agents with improved pharmacodynamic and kinetic properties were elaborated. Cardiomyogenesis was verified by quantifying the effects of transcription factors expressed in human stem cells upon differentiation (i.e., Nkx2.5, αMHC, alpha activin) by RT-qPCR, image analysis and for selected compounds, flow cytometry. The results showed that a distinct Wnt/β-catenin inhibition structure-activity relationship emerged from the "Dynamic Medicinal Chemistry" approach. Potent Wnt/β-catenin antagonists that emerged from this optimization campaign were useful toolbox reagents to produce large numbers of human cardiomyocytes from human stem cells. The conclusion is that small molecules can be used to afford human cardiomyocyte differentiation from pluripotent stem cells. Application of this approach to human induced pluripotent stem (iPS) cells could afford cardiomyocytes that could be used in drug discovery and development applications (i.e., drug toxicity testing) as well as provide a new way to address “Disease in a Dish” issues and create cells for therapeutic utility.
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Papers by Erik Willems