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Induction and enhancement of cardiac cell differentiation from mouse and human induced pluripotent stem cells with cyclosporin-A.

ABSTRACT: Induced pluripotent stem cells (iPSCs) are novel stem cells derived from adult mouse and human tissues by reprogramming. Elucidation of mechanisms and exploration of efficient methods for their differentiation to functional cardiomyocytes are essential for developing cardiac cell models and future regenerative therapies. We previously established a novel mouse embryonic stem cell (ESC) and iPSC differentiation system in which cardiovascular cells can be systematically induced from Flk1(+) common progenitor cells, and identified highly cardiogenic progenitors as Flk1(+)/CXCR4(+)/VE-cadherin(-) (FCV) cells. We have also reported that cyclosporin-A (CSA) drastically increases FCV progenitor and cardiomyocyte induction from mouse ESCs. Here, we combined these technologies and extended them to mouse and human iPSCs. Co-culture of purified mouse iPSC-derived Flk1(+) cells with OP9 stroma cells induced cardiomyocyte differentiation whilst addition of CSA to Flk1(+) cells dramatically increased both cardiomyocyte and FCV progenitor cell differentiation. Spontaneously beating colonies were obtained from human iPSCs by co-culture with END-2 visceral endoderm-like cells. Appearance of beating colonies from human iPSCs was increased approximately 4.3 times by addition of CSA at mesoderm stage. CSA-expanded human iPSC-derived cardiomyocytes showed various cardiac marker expressions, synchronized calcium transients, cardiomyocyte-like action potentials, pharmacological reactions, and ultra-structural features as cardiomyocytes. These results provide a technological basis to obtain functional cardiomyocytes from iPSCs.

SUBMITTER: Fujiwara M

PROVIDER: S-EPMC3043062 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Induction and enhancement of cardiac cell differentiation from mouse and human induced pluripotent stem cells with cyclosporin-A.

Fujiwara Masataka M Yan Peishi P Otsuji Tomomi G TG Narazaki Genta G Uosaki Hideki H Fukushima Hiroyuki H Kuwahara Koichiro K Harada Masaki M Matsuda Hiroyuki H Matsuoka Satoshi S Okita Keisuke K Takahashi Kazutoshi K Nakagawa Masato M Ikeda Tadashi T Sakata Ryuzo R Mummery Christine L CL Nakatsuji Norio N Yamanaka Shinya S Nakao Kazuwa K Yamashita Jun K JK

PloS one 20110222 2

Induced pluripotent stem cells (iPSCs) are novel stem cells derived from adult mouse and human tissues by reprogramming. Elucidation of mechanisms and exploration of efficient methods for their differentiation to functional cardiomyocytes are essential for developing cardiac cell models and future regenerative therapies. We previously established a novel mouse embryonic stem cell (ESC) and iPSC differentiation system in which cardiovascular cells can be systematically induced from Flk1(+) common ...[more]

PMID: 21364991

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Project description:Cardiomyocytes differentiated from human induced pluripotent stem cells (iPSCs) are valuable for the understanding/treatment of the deadly heart diseases and their drug screening. However, the very much needed homogeneous 3D cardiac differentiation of human iPSCs is still challenging. Here, it is discovered surprisingly that Rock inhibitor (RI), used ubiquitously to improve the survival/yield of human iPSCs, induces early gastrulation-like change to human iPSCs in 3D culture and may cause their heterogeneous differentiation into all the three germ layers (i.e., ectoderm, mesoderm, and endoderm) at the commonly used concentration (10 μM). This greatly compromises the capacity of human iPSCs for homogeneous 3D cardiac differentiation. By reducing the RI to 1 μM for 3D culture, the human iPSCs retain high pluripotency/quality in inner cell mass-like solid 3D spheroids. Consequently, the beating efficiency of 3D cardiac differentiation can be improved to more than 95 % in ~7 days (compared to less than ~50 % in 14 days for the 10 μM RI condition). Furthermore, the outset beating time (OBT) of all resultant cardiac spheroids (CSs) is synchronized within only 1 day and they form a synchronously beating 3D construct after 5-day culture in gelatin methacrylol (GelMA) hydrogel, showing high homogeneity (in terms of the OBT) in functional maturity of the CSs. Moreover, the resultant cardiomyocytes are of high quality with key functional ultrastructures and highly responsive to cardiac drugs. These discoveries may greatly facilitate the utilization of human iPSCs for understanding and treating heart diseases. Graphical abstract Rock inhibitor (RI) at 10 μM induces early gastrulation-like heterogeneous differentiation of human iPSCs and compromises their cardiac differentiation in 3D. Importantly, reducing RI to 1 μM retains human iPSCs in solid inner cell mass-like spheroids with high pluripotency for efficient and homogeneous cardiac differentiation in 3D. This discovery may greatly facilitate human iPSCs-based therapy and modeling of heart diseases.Image 1 Highlights • Rock inhibitor (RI) at the widely used 10 μM induces early gastrulation-like differentiation of human iPSCs (hiPSCs) in 3D.• Reducing RI to 1 μM retains the high pluripotency of hiPSCs in inner cell mass-like solid spheroids in 3D.• Reducing RI to 1 μM shortens the time needed for 3D cardiac differentiation of hiPSCs by ~1 week.• Reducing RI to 1 μM doubles the percentage of beating cardiac spheroids after 3D cardiac differentiation of hiPSCs.

Project description:Human cardiomyocytes (CMs) have potential for use in therapeutic cell therapy and high-throughput drug screening. Because of the inability to expand adult CMs, their large-scale production from human pluripotent stem cells (hPSC) has been suggested. Significant improvements have been made in understanding directed differentiation processes of CMs from hPSCs and their suspension culture-based production at chemically defined conditions. However, optimization experiments are costly, time-consuming, and highly variable, leading to challenges in developing reliable and consistent protocols for the generation of large CM numbers at high purity. This study examined the ability of data-driven modeling with machine learning for identifying key experimental conditions and predicting final CM content using data collected during hPSC-cardiac differentiation in advanced stirred tank bioreactors (STBRs). Through feature selection, we identified process conditions, features, and patterns that are the most influential on and predictive of the CM content at the process endpoint, on differentiation day 10 (dd10). Process-related features were extracted from experimental data collected from 58 differentiation experiments by feature engineering. These features included data continuously collected online by the bioreactor system, such as dissolved oxygen concentration and pH patterns, as well as offline determined data, including the cell density, cell aggregate size, and nutrient concentrations. The selected features were used as inputs to construct models to classify the resulting CM content as being "sufficient" or "insufficient" regarding pre-defined thresholds. The models built using random forests and Gaussian process modeling predicted insufficient CM content for a differentiation process with 90% accuracy and precision on dd7 of the protocol and with 85% accuracy and 82% precision at a substantially earlier stage: dd5. These models provide insight into potential key factors affecting hPSC cardiac differentiation to aid in selecting future experimental conditions and can predict the final CM content at earlier process timepoints, providing cost and time savings. This study suggests that data-driven models and machine learning techniques can be employed using existing data for understanding and improving production of a specific cell type, which is potentially applicable to other lineages and critical for realization of their therapeutic applications.

Project description:BackgroundHuman induced pluripotent stem cells (hiPSC) harbor the potential to differentiate into diverse cardiac cell types. Previous experimental efforts were primarily directed at the generation of hiPSC-derived cells with ventricular cardiomyocyte characteristics. Aiming at a straightforward approach for pacemaker cell modeling and replacement, we sought to selectively differentiate cells with nodal-type properties.MethodshiPSC were differentiated into spontaneously beating clusters by co-culturing with visceral endoderm-like cells in a serum-free medium. Subsequent culturing in a specified fetal bovine serum (FBS)-enriched cell medium produced a pacemaker-type phenotype that was studied in detail using quantitative real-time polymerase chain reaction (qRT-PCR), immunocytochemistry, and patch-clamp electrophysiology. Further investigations comprised pharmacological stimulations and co-culturing with neonatal cardiomyocytes.ResultshiPSC co-cultured in a serum-free medium with the visceral endoderm-like cell line END-2 produced spontaneously beating clusters after 10-12 days of culture. The pacemaker-specific genes HCN4, TBX3, and TBX18 were abundantly expressed at this early developmental stage, while levels of sarcomeric gene products remained low. We observed that working-type cardiomyogenic differentiation can be suppressed by transfer of early clusters into a FBS-enriched cell medium immediately after beating onset. After 6 weeks under these conditions, sinoatrial node (SAN) hallmark genes remained at high levels, while working-type myocardial transcripts (NKX2.5, TBX5) were low. Clusters were characterized by regular activity and robust beating rates (70-90 beats/min) and were triggered by spontaneous Ca2+ transients recapitulating calcium clock properties of genuine pacemaker cells. They were responsive to adrenergic/cholinergic stimulation and able to pace neonatal rat ventricular myocytes in co-culture experiments. Action potential (AP) measurements of cells individualized from clusters exhibited nodal-type (63.4%) and atrial-type (36.6%) AP morphologies, while ventricular AP configurations were not observed.ConclusionWe provide a novel culture media-based, transgene-free approach for targeted generation of hiPSC-derived pacemaker-type cells that grow in clusters and offer the potential for disease modeling, drug testing, and individualized cell-based replacement therapy of the SAN.

Dataset Information

Induction and enhancement of cardiac cell differentiation from mouse and human induced pluripotent stem cells with cyclosporin-A.

Publications

Induction and enhancement of cardiac cell differentiation from mouse and human induced pluripotent stem cells with cyclosporin-A.

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