Richard P. Davis

NKX2-5eGFP/w hESCs for isolation of human cardiac progenitors and cardiomyocytes

NKX2-5 is expressed in the heart throughout life. We targeted eGFP sequences to the NKX2-5 locus of human embryonic stem cells (hESCs); NKX2-5eGFP/w hESCs facilitate quantification of cardiac differentiation, purification of hESC-derived committed cardiac progenitor cells (hESC-CPCs) and cardiomyocytes (hESC-CMs) and the standardization of differentiation protocols. We used NKX2-5 eGFP+ cells to identify VCAM1 and SIRPA as cell-surface markers expressed in cardiac lineages.

Cardiomyocytes Derived From Pluripotent Stem Cells Recapitulate Electrophysiological Characteristics of an Overlap Syndrome of Cardiac Sodium Channel Disease

Background— Pluripotent stem cells (PSCs) offer a new paradigm for modeling genetic cardiac diseases, but it is unclear whether mouse and human PSCs can truly model both gain- and loss-of-function genetic disorders affecting the Na+ current (INa) because of the immaturity of the PSC-derived cardiomyocytes. To address this issue, we generated multiple PSC lines containing a Na+ channel mutation causing a cardiac Na+ channel overlap syndrome. Method and Results— Induced PSC (iPSC) lines were generated from mice carrying the Scn5a1798insD/+ (Scn5a-het) mutation. These mouse iPSCs, along with wild-type mouse iPSCs, were compared with the targeted mouse embryonic stem cell line used to generate the mutant mice and with the wild-type mouse embryonic stem cell line. Patch-clamp experiments showed that the Scn5a-het cardiomyocytes had a significant decrease in INa density and a larger persistent INa compared with Scn5a-wt cardiomyocytes. Action potential measurements showed a reduced upstroke velocity and longer action potential duration in Scn5a-het myocytes. These characteristics recapitulated findings from primary cardiomyocytes isolated directly from adult Scn5a-het mice. Finally, iPSCs were generated from a patient with the equivalent SCN5A1795insD/+ mutation. Patch-clamp measurements on the derivative cardiomyocytes revealed changes similar to those in the mouse PSC-derived cardiomyocytes. Conclusion— Here, we demonstrate that both embryonic stem cell- and iPSC-derived cardiomyocytes can recapitulate the characteristics of a combined gain- and loss-of-function Na+ channel mutation and that the electrophysiological immaturity of PSC-derived cardiomyocytes does not preclude their use as an accurate model for cardiac Na+ channel disease.

A protocol describing the use of a recombinant protein-based, animal product-free medium (APEL) for human embryonic stem cell differentiation as spin embryoid bodies

In order to promote the uniform and reproducible differentiation of human embryonic stem cells (HESCs) in response to exogenously added growth factors, we have developed a method (spin embryoid bodies (EBs)) that uses a recombinant protein-based, animal product-free medium in which HESCs are aggregated by centrifugation to form EBs. In this protocol we describe the formulation of this medium, denoted APEL (Albumin Polyvinylalcohol Essential Lipids), and its use in spin EB differentiation of HESCs. We also describe a more economical variant, BPEL (Bovine Serum Albumin (BSA) Polyvinylalchohol Essential Lipids), in which BSA replaces the recombinant human albumin. The integration of a medium that includes only defined and recombinant components with a defined number of cells to initiate EB formation results in a generally applicable, robust platform for growth factor-directed HESC differentiation.

A method for genetic modification of human embryonic stem cells using electroporation

The ability to genetically modify human embryonic stem cells (HESCs) will be critical for their widespread use as a tool for understanding fundamental aspects of human biology and pathology and for their development as a platform for pharmaceutical discovery. Here, we describe a method for the genetic modification of HESCs using electroporation, the preferred method for introduction of DNA into cells in which the desired outcome is gene targeting. This report provides methods for cell amplification, electroporation, colony selection and screening. The protocol we describe has been tested on four different HESC lines, and takes approximately 4 weeks from electroporation to PCR screening of G418-resistant clones.

Isogenic human pluripotent stem cell pairs reveal the role of a KCNH2 mutation in long‐QT syndrome

Patient‐specific induced pluripotent stem cells (iPSCs) will assist research on genetic cardiac maladies if the disease phenotype is recapitulated in vitro. However, genetic background variations may confound disease traits, especially for disorders with incomplete penetrance, such as long‐QT syndromes (LQTS). To study the LQT2‐associated c.A2987T (N996I) KCNH2 mutation under genetically defined conditions, we derived iPSCs from a patient carrying this mutation and corrected it. Furthermore, we introduced the same point mutation in human embryonic stem cells (hESCs), generating two genetically distinct isogenic pairs of LQTS and control lines. Correction of the mutation normalized the current (IKr) conducted by the HERG channel and the action potential (AP) duration in iPSC‐derived cardiomyocytes (CMs). Introduction of the same mutation reduced IKr and prolonged the AP duration in hESC‐derived CMs. Further characterization of N996I‐HERG pathogenesis revealed a trafficking defect. Our results demonstrated that the c.A2987T KCNH2 mutation is the primary cause of the LQTS phenotype. Precise genetic modification of pluripotent stem cells provided a physiologically and functionally relevant human cellular context to reveal the pathogenic mechanism underlying this specific disease phenotype.

Challenges in Using Stem Cells for Cardiac Repair

Stem cell–derived cardiomyocytes offer promise for treating damage caused by heart attacks, but through different mechanisms than originally believed. Of the many diseases discussed in the context of stem cell therapy, those concerning the heart account for almost one-third of the publications in the field. However, the long-term clinical outcomes have been disappointing, in part because of preclinical studies failing to optimize the timing, number, type, and method of cell delivery and to account for shape changes that the heart undergoes during failure. In situations in which cardiomyocytes have been used in cell therapy, their alignment and integration with host tissue have not been realized. Here we review the present status of direct delivery of stem cells or their derivative cardiomyocytes to the heart and the particular challenges each cell type brings, and consider where we should go from here.

Contractile Defect Caused by Mutation in MYBPC3 Revealed under Conditions Optimized for Human PSC-Cardiomyocyte Function

Summary Maximizing baseline function of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) is essential for their effective application in models of cardiac toxicity and disease. Here, we aimed to identify factors that would promote an adequate level of function to permit robust single-cell contractility measurements in a human induced pluripotent stem cell (hiPSC) model of hypertrophic cardiomyopathy (HCM). A simple screen revealed the collaborative effects of thyroid hormone, IGF-1 and the glucocorticoid analog dexamethasone on the electrophysiology, bioenergetics, and contractile force generation of hPSC-CMs. In this optimized condition, hiPSC-CMs with mutations in MYBPC3, a gene encoding myosin-binding protein C, which, when mutated, causes HCM, showed significantly lower contractile force generation than controls. This was recapitulated by direct knockdown of MYBPC3 in control hPSC-CMs, supporting a mechanism of haploinsufficiency. Modeling this disease in vitro using human cells is an important step toward identifying therapeutic interventions for HCM.

A protocol for removal of antibiotic resistance cassettes from human embryonic stem cells genetically modified by homologous recombination or transgenesis

The first step in the generation of genetically tagged human embryonic stem cell (HESC) reporter lines is the isolation of cells that contain a stably integrated copy of the reporter vector. These cells are identified by their continued growth in the presence of a specific selective agent, usually conferred by a cassette encoding antibiotic resistance. In order to mitigate potential interference between the regulatory elements driving expression of the antibiotic resistance gene and those controlling the reporter gene, it is advisable to remove the positive selection cassette once the desired clones have been identified. This report describes a protocol for the removal of loxP-flanked selection cassettes from genetically modified HESCs by transient transfection with a vector expressing Cre recombinase. An integrated procedure for the clonal isolation of these genetically modified lines using single-cell deposition flow cytometry is also detailed. When performed sequentially, these protocols take ∼1 month.

Transcriptome of human foetal heart compared with cardiomyocytes from pluripotent stem cells

Differentiated derivatives of human pluripotent stem cells (hPSCs) are often considered immature because they resemble foetal cells more than adult, with hPSC-derived cardiomyocytes (hPSC-CMs) being no exception. Many functional features of these cardiomyocytes, such as their cell morphology, electrophysiological characteristics, sarcomere organization and contraction force, are underdeveloped compared with adult cardiomyocytes. However, relatively little is known about how their gene expression profiles compare with the human foetal heart, in part because of the paucity of data on the human foetal heart at different stages of development. Here, we collected samples of matched ventricles and atria from human foetuses during the first and second trimester of development. This presented a rare opportunity to perform gene expression analysis on the individual chambers of the heart at various stages of development, allowing us to identify not only genes involved in the formation of the heart, but also specific genes upregulated in each of the four chambers and at different stages of development. The data showed that hPSC-CMs had a gene expression profile similar to first trimester foetal heart, but after culture in conditions shown previously to induce maturation, they cluster closer to the second trimester foetal heart samples. In summary, we demonstrate how the gene expression profiles of human foetal heart samples can be used for benchmarking hPSC-CMs and also contribute to determining their equivalent stage of development. Summary: The analysis of gene expression in the human foetal heart reveals stage- and chamber-specific genes and provides a dataset that can be used for benchmarking human PSC-derived heart cells.

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

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 MESP1mCherry/w‐NKX2‐5eGFP/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‐α, CD13, and ROR‐2. No enrichment was found for the previously described KDR+PDGFR‐α+ 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