CXCL4/PF4 is a predictive biomarker of cardiac differentiation potential of human induced pluripotent stem cells.
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SUBMITTER: Ohashi F
PROVIDER: S-EPMC6420577 | biostudies-literature | 2019 Mar
REPOSITORIES: biostudies-literature
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CXCL4/PF4 is a predictive biomarker of cardiac differentiation potential of human induced pluripotent stem cells.
Ohashi Fumiya F Miyagawa Shigeru S Yasuda Satoshi S Miura Takumi T Kuroda Takuya T Itoh Masayoshi M Kawaji Hideya H Ito Emiko E Yoshida Shohei S Saito Atsuhiro A Sameshima Tadashi T Kawai Jun J Sawa Yoshiki Y Sato Yoji Y
Scientific reports 20190315 1
Selection of human induced pluripotent stem cell (hiPSC) lines with high cardiac differentiation potential is important for regenerative therapy and drug screening. We aimed to identify biomarkers for predicting cardiac differentiation potential of hiPSC lines by comparing the gene expression profiles of six undifferentiated hiPSC lines with different cardiac differentiation capabilities. We used three platforms of gene expression analysis, namely, cap analysis of gene expression (CAGE), mRNA ar ...[more]
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Project description:Mature mammalian hearts possess very limited regenerative potential. The irreversible cardiomyocyte loss after heart injury can lead to heart failure and death. Pluripotent stem cells (PSCs) can differentiate into cardiomyocytes for cardiac repair, but there are obstacles to their clinical application. Among these obstacles is their potential for post-transplant rejection. Although human amniotic fluid-derived stem cells (hAFSCs) are immune privileged, they cannot induce cardiac differentiation. Thus, we generated hAFSC-derived induced PSCs (hAFSC-iPSCs) and used a Wnt-modulating differentiation protocol for the cardiac differentiation of hAFSC-iPSCs. In vitro studies using flow cytometry, immunofluorescence staining, and patch-clamp electrophysiological study, were performed to identify the characteristics of hAFSC-iPSC-derived cardiomyocytes (hAFSC-iPSC-CMs). We injected hAFSC-iPSC-CMs intramuscularly into rat infarcted hearts to evaluate the therapeutic potential of hAFSC-iPSC-CM transplantation. At day 21 of differentiation, the hAFSC-iPSC-CMs expressed cardiac-specific marker (cardiac troponin T), presented cardiomyocyte-specific electrophysiological properties, and contracted spontaneously. Importantly, these hAFSC-iPSC-CMs demonstrated low major histocompatibility complex (MHC) class I antigen expression and the absence of MHC class II antigens, indicating their low immunogenicity. The intramyocardial transplantation of hAFSC-iPSC-CMs restored cardiac function, partially remuscularized the injured region, and reduced fibrosis in the rat infarcted hearts. Therefore, hAFSC-iPSCs are potential candidates for the repair of infarcted myocardium.
| S-EPMC7177657 | biostudies-literature
Project description:Perhaps one of the most significant achievements in modern science is the discovery of human induced pluripotent stem cells (hiPSCs), which have paved the way for regeneration therapy using patients' own cells. Cardiomyocytes differentiated from hiPSCs (hiPSC-CMs) could be used for modelling patients with heart failure, for testing new drugs, and for cellular therapy in the future. However, the present cardiomyocyte differentiation protocols exhibit variable differentiation efficiency across different hiPSC lines, which inhibit the application of this technology significantly. Here, we demonstrate a novel myocyte differentiation protocol that can yield a significant, high percentage of cardiac myocyte differentiation (>85%) in 2 hiPSC lines, which makes the fabrication of a human cardiac muscle patch possible. The established hiPSCs cell lines being examined include the transgene integrated UCBiPS7 derived from cord blood cells and non-integrated PCBC16iPS from skin fibroblasts. The results indicate that hiPSC-CMs derived from established hiPSC lines respond to adrenergic or acetylcholine stimulation and beat regularly for greater than 60 days. This data also demonstrates that this novel differentiation protocol can efficiently generate hiPSC-CMs from iPSC lines that are derived not only from fibroblasts, but also from blood mononuclear cells.
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Project description:Stem cells are promising cell source for treatment of multiple diseases as well as myocardial infarction. Rabbit model has essentially used for cardiovascular diseases and regeneration but information on establishment of induced pluripotent stem cells (iPSCs) and differentiation potential is fairly limited. In addition, there is no report of cardiac differentiation from iPSCs in the rabbit model. In this study, we generated rabbit iPSCs by reprogramming rabbit fibroblasts using the 4 transcription factors (OCT3/4, SOX2, KLF4, and c-Myc). Three iPSC lines were established. The iPSCs from all cell lines expressed genes (OCT3/4, SOX2, KLF4 and NANOG) and proteins (alkaline phosphatase, OCT-3/4 and SSEA-4) essentially described for pluripotency (in vivo and in vitro differentiation). Furthermore, they also had ability to form embryoid body (EB) resulting in three-germ layer differentiation. However, ability of particular cell lines and cell numbers at seeding markedly influenced on EB formation and also their diameters. The cell density at 20,000 cells per EB was selected for cardiac differentiation. After plating, the EBs attached and cardiac-like beating areas were seen as soon as 11 days of culture. The differentiated cells expressed cardiac progenitor marker FLK1 (51 ± 1.48%) on day 5 and cardiac troponin-T protein (10.29 ± 1.37%) on day 14. Other cardiac marker genes (cardiac ryanodine receptors (RYR2), α-actinin and PECAM1) were also expressed. This study concluded that rabbit iPSCs remained their in vitro pluripotency with capability of differentiation into mature-phenotype cardiomyocytes. However, the efficiency of cardiac differentiation is still restricted.
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Project description:IntroductionHuman induced pluripotent stem cells (hiPSCs) have been derived from various somatic cell types. Granulosa cells, a group of cells which surround oocytes and are obtained from the (normally discarded) retrieved egg follicles of women undergoing infertility treatment, are a possible cell source for induced pluripotent stem cell (iPSC) generation. Here, we explored the possibility of using human granulosa cells as a donor cell type for iPSC reprogramming, and compared granulosa cell-derived iPSCs (iGRAs) with those derived from other cell sources, to determine the potential ability of iGRA differentiation.MethodsGranulosa cells were collected from egg follicles retrieved from women undergoing infertility treatment. After short-term culture, the granulosa cells derived from different patients were mixed in culture, and infected with retroviruses encoding reprogramming factors. The resulting iPSC clones were selected and subjected to microsatellite DNA analysis to determine their parental origin. IGRAs were subjected to RT-PCR, immunofluorescence staining, and in vitro and in vivo differentiation assays to further establish their pluripotent characteristics.ResultsMicrosatellite DNA analysis was used to demonstrate that hiPSCs with different parental origins can be simultaneously reprogrammed by retroviral transfection of a mixed human granulosa cell population obtained from multiple individuals. The iGRAs resemble human embryonic stem cells (hESCs) in many respects, including morphological traits, growth requirements, gene and marker expression profiles, and in vitro and in vivo developmental propensities. We also demonstrate that the iGRAs express low levels of NLRP2, and differentiating iGRAs possess a biased differentiation potential toward the trophoblastic lineage. Although NLRP2 knockdown in hESCs promotes trophoblastic differentiation of differentiating hESCs, it does not result in exit from pluripotency. These results imply that NLRP2 may play a role in regulating the trophoblastic differentiation of human pluripotent stem cells.ConclusionsThese findings provide a means of generating iPSCs from multiple granulosa cell populations with different parental origins. The ability to generate iPSCs from granulosa cells not only enables modeling of infertility-associated disease, but also provides a means of identifying potential clinical interventions through iPSC-based drug screening.
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Project description:Stem cell therapy has emerged as a promising strategy for cardiac and vascular repair. The ultimate goal is to rebuild functional myocardium by transplanting exogenous stem cells or by activating native stem cells to induce endogenous repair. CS/PCs (cardiac stem/progenitor cells) are one type of adult stem cell with the potential to differentiate into cardiac lineages (cardiomyocytes, smooth muscle cells and endothelial cells). iPSCs (induced pluripotent stem cells) also have the capacity to differentiate into necessary cells to rebuild injured cardiac tissue. Both types of stem cells have brought promise for cardiac repair. The present review summarizes recent advances in cardiac cell therapy based on these two cell sources and discusses the advantages and limitations of each candidate. We conclude that, although both types of stem cells can be considered for autologous transplantation with promising outcomes in animal models, CS/PCs have advanced more in their clinical application because iPSCs and their derivatives possess inherent obstacles for clinical use. Further studies are needed to move cell therapy forward for the treatment of heart disease.
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| S-EPMC8581226 | biostudies-literature
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.
| S-EPMC7390976 | biostudies-literature
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.
| S-EPMC5644063 | biostudies-literature