ABSTRACT
Human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) are a promising source for cell transplantation into the damaged heart, which has limited regenerative ability. Many methods have been developed to obtain large amounts of functional CMs from hPSCs for therapeutic applications. However, during the differentiation process, a mixed population of various cardiac cells, including ventricular, atrial, and pacemaker cells, is generated, which hampers the proper functional analysis and evaluation of cell properties. Here, we established NKX2-5eGFP/w and MLC2vmCherry/w hPSC double knock-ins that allow for labeling, tracing, purification, and analysis of the development of ventricular cells from early to late stages. As with the endogenous transcriptional activities of these genes, MLC2v-mCherry expression following NKX2-5-eGFP expression was observed under previously established culture conditions, which mimic the in vivo cardiac developmental process. Patch-clamp and microelectrode array electrophysiological analyses showed that the NKX2-5 and MLC2v double-positive cells possess ventricular-like properties. The results demonstrate that the NKX2-5eGFP/w and MLC2vmCherry/w hPSCs provide a powerful model system to capture region-specific cardiac differentiation from early to late stages. Our study would facilitate subtype-specific cardiac development and functional analysis using the hPSC-derived sources.
Subject(s)
Batch Cell Culture Techniques/methods , Cardiac Myosins/metabolism , Cell Tracking/methods , Heart Ventricles/cytology , Homeobox Protein Nkx-2.5/metabolism , Myocytes, Cardiac/cytology , Myosin Light Chains/metabolism , Pluripotent Stem Cells/cytology , Cardiac Myosins/genetics , Cell Differentiation/physiology , Cell Separation/methods , Cells, Cultured , Gene Knock-In Techniques , Genes, Reporter/genetics , Heart Ventricles/metabolism , Homeobox Protein Nkx-2.5/genetics , Humans , Myocytes, Cardiac/metabolism , Myosin Light Chains/genetics , Pluripotent Stem Cells/metabolism , Tissue Engineering/methodsABSTRACT
Embryonic stem cells (ESCs) are useful resources for drug discovery, developmental biology and disease studies. Cellular microenvironmental cues play critical roles in regulating ESC functions, but it is challenging to control them with synthetic components. Nanofibers hold a potential to create artificial cellular cues for controlling cell adhesion and cell-cell interactions. Mouse ESC (mESC) were cultured on electrospun nanofibers made from polymethylglutarimide (PMGI), which is a synthetic thermoplastic polymer stable under culture conditions. Both topology and the density of PMGI nanofibers were key factors. mESCs on nanofibers had a growth rate comparable to those cultured conventionally and retained their pluripotency. Furthermore, self-renewed ESCs differentiated into all three germ layers thereby providing a reliable way to expand mESCs without feeder cells.
Subject(s)
Cell Culture Techniques/methods , Embryonic Stem Cells/physiology , Nanofibers/chemistry , Tissue Scaffolds/chemistry , Animals , Cell Adhesion/physiology , Cell Differentiation/physiology , Cell Proliferation , Culture Media , Embryonic Stem Cells/cytology , Imides/chemistry , Mice , Polymers/chemistryABSTRACT
Cardiomyocytes arise from cells that migrate to the mid-to-anterior region of the primitive streak (PS) during embryogenesis. We previously showed that canonical Wnt/ß-catenin pathway signaling leads to the development of nascent PS populations from human embryonic stem cells (hESCs) and that synergistic activation of the Wnt/ß-catenin pathway and inhibition of bone morphogenetic protein (BMP) signaling by Noggin induced the formation of anterior PS cells. We herein demonstrate that anterior PS cells induced by the activation of ß-catenin with Noggin differentiate into functional cardiomyocytes when cultured in suspension with BMP4 and fibroblast growth factor 2 (FGF2). All aggregates generated from the anterior PS cells developed into contracting cells demonstrating their cardiac potential. More than 30% of the cells in each aggregate were α-actinin-positive cardiomyocytes. In addition, these cardiomyocytes could be easily purified up to 80% by simple size fractionation. In contrast, the posterior PS cells induced by ß-catenin activation without Noggin showed poor cardiac potential. These results show that the commitment to a cardiac lineage in vitro occurs through similar cellular and molecular signaling pathways involved in cardiac development in vivo, thus providing a valuable culture model for studying early cardiac developmental events in hESCs.
Subject(s)
Bone Morphogenetic Proteins/antagonists & inhibitors , Embryonic Stem Cells/metabolism , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Primitive Streak/cytology , Signal Transduction , beta Catenin/metabolism , Bone Morphogenetic Proteins/metabolism , Embryonic Stem Cells/cytology , HumansABSTRACT
We have developed a combined micro-channel and micro-well system for easy cell loading, culture and post-culture operation on a chip. To demonstrate the reliability of the system, on chip cell culture and differentiation were performed with different types of substrates made of culture dish, glass cover slide and polydimethylsiloaxe (PDMS). As expected, mouse embryo fibroblasts (MEF) showed different adhesion and growth rate on different substrates. When embryonic stem (ES) cells were co-cultured with MEFs, the formation of ES colonies is efficient on both glass and Petri dish, although PDMS could also be used. Finally, ES cell differentiation with neuron growth factors was performed on different substrates, showing clear advantages of using culture Petri dish over both glass and PDMS.
Subject(s)
Bioreactors , Cell Culture Techniques/instrumentation , Cell Separation/instrumentation , Embryonic Stem Cells/physiology , Microfluidic Analytical Techniques/instrumentation , Tissue Engineering/instrumentation , Animals , Cell Differentiation , Cell Proliferation , Cells, Cultured , Embryonic Stem Cells/classification , Equipment Design , Equipment Failure Analysis , Mice , Species SpecificityABSTRACT
Naive and primed human pluripotent stem cells (hPSCs) have provided useful insights into the regulation of pluripotency. However, the molecular mechanisms regulating naive conversion remain elusive. Here, we report intermediate naive conversion induced by overexpressing nuclear receptor 5A1 (NR5A1) in hPSCs. The cells displayed some naive features, such as clonogenicity, glycogen synthase kinase 3ß, and mitogen-activated protein kinase (MAPK) independence, expression of naive-associated genes, and two activated X chromosomes, but lacked others, such as KLF17 expression, transforming growth factor ß independence, and imprinted gene demethylation. Notably, NR5A1 negated MAPK activation by fibroblast growth factor 2, leading to cell-autonomous self-renewal independent of MAPK inhibition. These phenotypes may be associated with naive conversion, and were regulated by a DPPA2/4-dependent pathway that activates the selective expression of naive-associated genes. This study increases our understanding of the mechanisms regulating the conversion from primed to naive pluripotency.
Subject(s)
Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Steroidogenic Factor 1/metabolism , Gene Expression Regulation/drug effects , Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3/metabolism , Histones/metabolism , Humans , Principal Component Analysis , Protein Binding/drug effects , Protein Kinase Inhibitors/pharmacology , Steroidogenic Factor 1/genetics , Transcription, Genetic/drug effectsABSTRACT
Chiral N-heterocyclic carbenes, which are derived from C2-symmetric 1,3-bis(1-arylethyl)imidazolium salts, catalyze enantioselective acylation of racemic secondary alcohols.
ABSTRACT
Human induced pluripotent stem cell (hiPSC) lines have a great potential for therapeutics because customized cells and organs can be induced from such cells. Assessment of the residual reprogramming factors after the generation of hiPSC lines is required, but an ideal system has been lacking. Here, we generated hiPSC lines from normal human dermal fibroblasts with piggyBac transposon bearing reprogramming transgenes followed by removal of the transposon by the transposase. Under this condition, we compared the phenotypes of transgene-residual and -free hiPSCs of the same genetic background. The transgene-residual hiPSCs, in which the transcription levels of the reprogramming transgenes were eventually suppressed, were quite similar to the transgene-free hiPSCs in a pluripotent state. However, after differentiation into keratinocytes, clear differences were observed. Morphological, functional, and molecular analyses including single-cell gene expression profiling revealed that keratinocytes from transgene-free hiPSC lines were more similar to normal human keratinocytes than those from transgene-residual hiPSC lines, which may be partly explained by reactivation of residual transgenes upon induction of keratinocyte differentiation. These results suggest that transgene-free hiPSC lines should be chosen for therapeutic purposes.
Subject(s)
Cellular Reprogramming/genetics , Genetic Engineering/methods , Induced Pluripotent Stem Cells/cytology , Keratinocytes/cytology , Tissue Engineering/methods , Blotting, Southern , Cell Differentiation/genetics , Fibroblasts/cytology , Humans , Immunohistochemistry , Real-Time Polymerase Chain Reaction , TransgenesABSTRACT
A small-molecule fluorescent probe specific for human pluripotent stem cells would serve as a useful tool for basic cell biology research and stem cell therapy. Screening of fluorescent chemical libraries with human induced pluripotent stem cells (iPSCs) and subsequent evaluation of hit molecules identified a fluorescent compound (Kyoto probe 1 [KP-1]) that selectively labels human pluripotent stem cells. Our analyses indicated that the selectivity results primarily from a distinct expression pattern of ABC transporters in human pluripotent stem cells and from the transporter selectivity of KP-1. Expression of ABCB1 (MDR1) and ABCG2 (BCRP), both of which cause the efflux of KP-1, is repressed in human pluripotent stem cells. Although KP-1, like other pluripotent markers, is not absolutely specific for pluripotent stem cells, the identified chemical probe may be used in conjunction with other reagents.
Subject(s)
Fluorescent Dyes/chemistry , Induced Pluripotent Stem Cells/chemistry , Induced Pluripotent Stem Cells/cytology , Molecular Probes/chemistry , Pluripotent Stem Cells/chemistry , Pluripotent Stem Cells/cytology , Animals , HEK293 Cells , Humans , Mice , Microscopy, Fluorescence/methodsABSTRACT
The field of drug testing currently needs a new integrated assay system, as accurate as systems using native tissues, that will allow us to predict arrhythmia risks of candidate drugs and the relationship between genetic mutations and acquired electrophysiological phenotypes. This could be accomplished by combining the microelectrode array (MEA) system with cardiomyocytes (CMs) derived from human embryonic stem cells (hESC) and induced pluripotential stem cells. CMs have been successfully induced from both types, but their maturation process is not systematically controlled; this results in loss of beating potency and insufficient ion channel function. We generated a transgenic hESC line that facilitates maintenance of hESC-CM clusters every 2 weeks by expressing GFP driven by a cardiac-specific alphaMHC promoter, thereby producing a compact pacemaker lineage within a ventricular population over a year. Further analyses, including quantitative RT-PCR, patch-clamp, and MEA-mediated QT tests, demonstrated that replating culturing continuously enhanced gene expression, ionic current amplitudes, and resistance to K(+) channel blockades in hESC-CMs. Moreover, temporal three-dimensional (3D) culturing accelerated maturation by restoring the global gene repressive status established in the adhesive status. Replating/3D culturing thus produces hESC-CMs that act as functional syncytia suitable for use in regenerative medicine and accurate drug tests.
Subject(s)
Embryonic Stem Cells/cytology , Myocytes, Cardiac/physiology , Anti-Arrhythmia Agents/pharmacology , Calcium Channel Blockers/pharmacology , Calcium Channels, L-Type/metabolism , Electrophysiological Phenomena , Gene Expression Regulation , Green Fluorescent Proteins/metabolism , Humans , Induced Pluripotent Stem Cells/cytology , Myocytes, Cardiac/cytology , Myocytes, Cardiac/drug effects , Nifedipine/pharmacology , Patch-Clamp Techniques , Potassium Channels/metabolism , Pyrimidinones/pharmacologyABSTRACT
BACKGROUND: Mouse embryonic stem (ES) cells can differentiate into female and male germ cells in vitro. Primate ES cells can also differentiate into immature germ cells in vitro. However, little is known about the differentiation markers and culture conditions for in vitro germ cell differentiation from ES cells in primates. Monkey ES cells are thus considered to be a useful model to study primate gametogenesis in vitro. Therefore, in order to obtain further information on germ cell differentiation from primate ES cells, this study examined the ability of cynomolgus monkey ES cells to differentiate into germ cells in vitro. METHODS AND FINDINGS: To explore the differentiation markers for detecting germ cells differentiated from ES cells, the expression of various germ cell marker genes was examined in tissues and ES cells of the cynomolgus monkey (Macaca fascicularis). VASA is a valuable gene for the detection of germ cells differentiated from ES cells. An increase of VASA expression was observed when differentiation was induced in ES cells via embryoid body (EB) formation. In addition, the expression of other germ cell markers, such as NANOS and PIWIL1 genes, was also up-regulated as the EB differentiation progressed. Immunocytochemistry identified the cells expressing stage-specific embryonic antigen (SSEA) 1, OCT-4, and VASA proteins in the EBs. These cells were detected in the peripheral region of the EBs as specific cell populations, such as SSEA1-positive, OCT-4-positive cells, OCT-4-positive, VASA-positive cells, and OCT-4-negative, VASA-positive cells. Thereafter, the effect of mouse gonadal cell-conditioned medium and growth factors on germ cell differentiation from monkey ES cells was examined, and this revealed that the addition of BMP4 to differentiating ES cells increased the expression of SCP1, a meiotic marker gene. CONCLUSION: VASA is a valuable gene for the detection of germ cells differentiated from ES cells in monkeys, and the identification and characterization of germ cells derived from ES cells are possible by using reported germ cell markers in vivo, including SSEA1, OCT-4, and VASA, in vitro as well as in vivo. These findings are thus considered to help elucidate the germ cell developmental process in primates.