3. Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, Korea.
Heart failure is the leading cause of death worldwide . Approximately 6 million people suffer from heart failure in the United States every year . Despite this high incidence, existing surgical and pharmacological interventions for treating heart failure are limited because these approaches only delay the progression of the disease; they cannot directly repair the damaged hearts . In the case of large myocardial infarction (MI), patients progress to heart failure and die within short time from the onset of symptoms .
The adult human heart has minimal regenerative capacity, because during mammalian development, the proliferative capacity of cardiomyocytes (CMs) progressively diminishes and becomes terminally differentiated shortly after birth .Therefore, once CMs are damaged, they are rarely restored . When MI occurs, the infarcted area is easily converted to non-contractile scar tissue due to loss of CMs and replacement by fibrosis . Development of a fibroblastic scar initiates a series of events that lead to adverse remodeling, hypertrophy, and eventual heart failure [2, 3, 7].
While heart transplantation is considered the most viable option for treating advanced heart failure, the number of available donor hearts is always less than needed . Therefore, more realistic therapeutic options have been required . Accordingly, over the past two decades, cell-based cardiac repair has been intensively pursued [2, 7]. Several different cell types have been tested and varied outcomes were obtained. Indeed, the key factor for successful cell-based cardiac repair is to find the optimal cell type that can restore normal heart function. Naturally, CMs have been considered the best cell type to repair a damaged heart . In fact, many scientists hypothesized that implanted CMs would survive in damaged hearts and form junctions with host CMs and synchronously contract with the host myocardium . In fact, animal studies with primary fetal or neonatal CMs demonstrated that transplanted CMs could survive in infarcted hearts [9-11]. These primary CMs reduced scar size, increased wall thickness, and improved cardiac contractile function with signs of electro-mechanical integration [9-11]. These studies strongly suggest that CMs can be a promising source to repair the heart. However, the short supply and ethical concerns disallow using primary human CMs. In a patient with ischemic cardiomyopathy, about 40-50% of the CMs are lost in 40 to 60 grams of heart tissue . Even if we seek to regenerate a fairly small portion of the damaged myocardium, a large number of human primary CMs would be required, which is impossible.
Accordingly, CMs differentiated from human pluripotent stem cells (hPSCs) including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have emerged as a promising option for candidate CMs for cell therapy [12, 13]. hPSCs have many advantages as a source for CMs. First, hPSCs have obvious cardiomyogenic potential. hPSC derived-CMs (hPSC-CMs) possess a clear cardiac phenotype, displaying spontaneous contraction, cardiac excitation-contraction (EC) coupling, and expression of cardiac transcription factors, cardiac ion channels, and cardiac structural proteins [14, 15]. Second, undifferentiated hPSCs and their differentiated cardiac progeny display significant proliferation capacity, allowing generation of a large number of hPSC-CMs. Lastly, many pre-clinical studies demonstrated that implantation of hPSC-CMs can repair injured hearts and improve cardiac function [16-19]. Histologically, implanted hPSC-CMs are engrafted, aligned and coupled with the host CMs in a synchronized manner [16-19].
In the last two decades, various protocols for differentiating hPSCs into CMs have been developed to improve the efficiency, purity and clinical compatibility  . The reported differentiation methods include, but are not limited to: differentiation via embryoid body (EB) formation , co-culture with END-2 cells , and monolayer culture [15, 21, 22]. The EB-mediated CM differentiation protocol is one of the most widely employed methods due to its simple procedure and low cost. However, it often becomes labor-intensive to produce scalable EBs for further differentiation, which makes it difficult for therapeutic applications. EB-mediated differentiation also produces inconsistent results, showing beating CMs from 5% to 70% of EBs. Recently, researchers developed monolayer methods to complement the problems of EB-based methods [15, 21, 22]. In one representative protocol, hPSCs are cultured at a high density (up to 80%) and treated with a high concentration of Activin A (100 ng/ml) for 1 day and BMP4 (10 ng/ml) for 4 days followed by continuous culture on regular RPMI media with B27 . This protocol induces spontaneous beating at approximately 12 days and produces approximately 40% CMs after 3 weeks. These hPSC-CMs can be further cultured in RPMI-B27 medium for another 2-3 weeks without significant cell damage . However, these protocols use media with proprietary formulations, which complicates clinical application. As shown, most monolayer-based methods employ B27, which is a complex mix of 21 components. Some of the components of B27, including bovine serum albumin (BSA), are animal-derived products, and the effects of B27 components on differentiation, maturation or subtype specification processes are poorly defined. In 2014, Burridge and his colleagues developed an advanced protocol that is defined, cost-effective and efficient . By subtracting one component from B27 at a time and proceeding with cardiac differentiation, the researchers reported that BSA and L-ascorbic acid 2-phosphate are essential components in cardiac differentiation. Subsequently, by replacing BSA with rice-derived recombinant human albumin, the chemically defined medium with 3 components (CDM3) was produced. The application of a GSK-inhibitor, CHIR99021, for the first 2 days followed by 2 days of the Wnt-inhibitor Wnt-59 to cells is an optimal culture condition in CDM3 resulting in similar levels of live-cell yields and CM differentiation .
Despite remarkable improvement in the generation of hPSC-CMs, obtaining pure populations of hPSC-CMs still remains challenging. Currently available methods can only generate a mixture of cells which include not only CMs but other cell types. This is one of the most critical barriers for applications of hPSC-CMs in regenerative therapy, drug discovery, and disease investigation. For Instance, cardiac transplantation of non-pure hPSC-CMs mixed with undifferentiated hPSCs or other cell types may produce tumors or unwanted cell types in hearts [23-28]. Accordingly, a pure or enriched population of hPSC-CMs would be required, particularly for cardiac cell therapy. Enriched hPSC-CMs would also be more beneficial for myocardial repair due to improved electric and mechanical properties . A pure, homogeneous population of hPSC-CMs would pose less arrhythmic risk and have enhanced contractile performance, and would be more useful in disease modeling as they better reflect native CM physiology. Finally, purified hPSC-CMs would better serve for testing drug efficacy and toxicity. Therefore, many researchers have tried to develop methods to purify CMs from cardiomyogenically differentiated hPSCs.
There are three important topics that are not addressed in this review. First is the beneficial role of other cell types such as endothelial cells and fibroblasts in the integration, survival, and function of CMs [30-32]. We did not discuss this issue because it would need a separate review due to the volume of material. While the roles of such cells are important, the value of having purified hPSC-CMs is not diminished. Although cell mixtures or tissue engineered products can be used, unless purified CMs are employed, they would form tumors or other cells/tissues when implanted in vivo. Our point here is that even if cardiomyocytes are mixed with non-CMs, all cells should be clearly defined and purified as well. If the mixture is made in a non-purified or non-defined manner (for example, an unsophisticated top-down approach), there would be undefined cells that are neither CMs, ECs, nor fibroblasts and these unidentified cells will make aberrant tissues or tumors. Second, we did not deal with maturation of hPSC-CMs because of its broad scope and depth [33, 34]. Third is direct reprogramming or conversion of somatic cells into CMs. There has been another advancement in the generation of CMs by directly reprogramming or converting somatic cells into CM-like cells by introducing a combination of cardiac transcription factors (TFs) or muscle-specific microRNAs (miRNAs) both in vitro and in vivo [35-41]. These cells are referred to as induced CMs (iCMs) or cardiac-like myocytes (iCLMs). While this is an important advancement, we did not cover this topic either due to its size. Accordingly, this review will focus on the various strategies for purifying or enriching hPSC-CMs reported to date (Figure 1).
Early on, researchers isolated hPSC-CMs manually under microscopy by mechanically separating out the beating areas from myogenically differentiating hPSC cultures [18, 20, 42]. This method usually generates 5-70% hPSC-CMs. Although generally crude, it can enrich even higher percentages of CMs with further culture. This manual isolation method has the advantage of being easy, but while it can be useful for small-scale research, it is very labor intensive and not scalable, precluding large scale research or clinical application.
Currently available strategies for enriching cardiomyocytes derived from human pluripotent stem cells.
Xu et el. reported that hPSC-CMs, due to their physical and structural properties, can be enriched by Percoll density gradient centrifugation . Percoll was first formulated by Pertoft et al  and it was originally developed for the isolation of cells, organelles, or viruses by density centrifugation. The Percoll-based method has several advantages. The procedure for Percoll-based separation is very simple and easy, it is inexpensive, and its low viscosity allows more rapid sedimentation and lower centrifugal forces compared to a sucrose density gradient. Lastly, it can be prepared and kept for a long time in an isotonic solution to maintain osmolarity. Although Percoll separation has resulted in major improvements in hPSC-CM isolation procedures, it has clear limitations with regard to purity and scalability. Previous studies found that Percoll separation is only able to enrich 40 -70% of hPSC-CMs. It is also not compatible with large-scale enrichment of hPSC-CMs.
Another traditional method for purifying hPSC-CMs is based on the expression of a drug resistant gene or a fluorescent reporter gene such as eGFP or DsRed, which is driven by a cardiac specific promoter in genetically modified hPSC lines [45, 46]. Here, enrichment of hPSC-CMs can be achieved by either drug treatment to eliminate cells that do not express the drug resistant gene or with FACS to isolate fluorescent cells [47, 48].
Briefly, enrichment of PSC-CMs by genetically based selection was first reported by Klug et al . The authors generated murine ES cell lines via permanent gene transfection of the aminoglycoside phosphotransferase gene driven by the βMHC (MYH7) promoter. With this approach, highly purified murine ESC-CMs up to 99% were achieved. Next, several studies reported the use of various CM-specific promoters to enrich ESC-CMs such as αMhc (Myh6), Myh7, Ncx (Sodium Calcium exchanger) and Mlc2v (Myl2) [46, 50, 51]. In the case of hESCs, αMHC/EGFP hESCs were generated by permanent transfection of the EGFP-tagged αMHC promoter . Similarly, an NKX2.5/eGFP hESC line was generated to enrich GFP positive CMs . However, since αMHC and NKX2.5 are expressed in general CMs, the resulting CMs contain a mixture of the three subtypes of CMs, nodal-, atrial-, and ventricular-like CMs. To enrich only ventricular-like CMs, Huber et al. generated MLC2v/GFP ESCs to be able to isolate MLC2v/GFP positive ventricular-like cells by FACS  [54-57]. In addition, the cGATA6 gene was used to purify nodal-like hESC-CMs . Future studies should focus on testing new types of cardiac specific promoters and devising advanced selection procedures to improve this strategy.
While fluorescence-based cell sorting is more widely used, the drug selection method may be a better approach to enrich high purity of hPSC-CMs during differentiation/culture as it does not require FACS. The advantage is its capability for high-purity cell enrichment due to specific gene-based cell sorting. These highly pure cells can allow more precise mechanistic studies and disease modeling. Despite its many advantages, the primary weakness of genetic selection is genetic manipulation, which disallows its use for therapeutic application. Insertion of reporter genes into the host genome requires viral or nonviral transfection/transduction methods, which can induce mutagenesis and tumor formation [50, 59-61].
Practically, antibody-based cell enrichment is the best method for cell purification to date. When cell type-specific surface proteins or "marker" proteins are known, one can tag cells with antibodies against the proteins and sort the target cells by FACS or magnetic-activated cell sorting (MACS). The main advantage is its specificity and sensitivity, and its utility is well demonstrated in research and even in clinical therapy with hematopoietic cells . Another advantage is that multiple surface markers can be used at the same time to isolate target cells when one marker is not sufficient. However, no studies have reported surface markers that are specific for CMs, even after many years. Recently, though, several researchers demonstrated that certain proteins can be useful for isolating hPSC-CMs.
In earlier studies, KDR (FLK1 or VEGFR2) and PDGFR-α were used to isolate cardiac progenitor cells . However, since these markers are also expressed on hematopoietic cells, endothelial cells, and smooth muscle cells, they could not enrich only hPSC-CMs. Next, two independent studies reported two surface proteins, SIRPA  and VCAM-1 , which it was claimed could specifically identify hPSC-CMs. Dubois et al. screened a panel of 370 known antibodies against CMs differentiated from hESCs and identified SIRPA as a specific surface protein expressed on hPSC-CMs . FACS with anti-SIRPA antibody enabled the purification of CMs and cardiac precursors from cardiomyogenically differentiating hPSC cultures, producing cardiac troponin T (TNNT2, also known as cTNT)-positive cells, which are generally considered hPSC-CMs, with up to 98% purity. In addition, a study performed by Elliot and colleagues identified another cell surface marker, VCAM1 . In this study, the authors used NKX2.5/eGFP hESCs to generate hPSC-CMs, allowing the cells to be sorted by their NKX2.5 expression. NKX2.5 is a well-known cardiac transcription factor and a specific marker for cardiac progenitor cells [66, 67]. To identify CM-specific surface proteins, the authors performed expression profiling analyses and found that expression levels of both VCAM1 and SIRPA were significantly upregulated in NKX2.5/eGFP+ cells. Flow cytometry results showed that both proteins were expressed on the cell surface of NKX2.5/eGFP+ cells. Differentiation day 14 NKX2.5/eGFP+ cells expressed VCAM1 (71 %) or SIRPA (85%) or both VCAM1 and SIRPA (37%). When the FACS-sorted SIRPA-VCAM1-, SIRPA+ or SIRPA+VCAM1+ cells were further cultured, only SIRPA+ or SIRPA+VCAM1+ cells showed NKX2.5/eGFP+ contracting portion. Of note, NKX2.5/eGFP and SIRPA positive cells showed higher expression of smooth muscle cell and endothelial cell markers indicating that cells sorted solely based on SIRPA expression may not be of pure cardiac lineage. Hence, the authors concluded that a more purified population of hPSC-CMs could be isolated by sorting with both cell surface markers. Despite significant improvements, it appears that these surface markers are not exclusively specific for CMs as these antibodies also mark other cell types including smooth muscle cells and endothelial cells. Furthermore, they are also known to be expressed in the brain and the lung, which raises concerns whether these surface proteins can be used as sole markers for the purification of hPSC-CMs compatible for clinical applications.
More recently, Protze et al. reported successful differentiation and enrichment of sinoatrial node-like pacemaker cells (SANLPCs) from differentiating hPSCs by using cell surface markers and an NKX2-5-reporter hPSC line . They found that BMP signaling specified cardiac mesoderm toward the SANLPC fate and retinoic acid signaling enhanced the pacemaker phenotype. Furthermore, they showed that later inhibition of the FGF pathway, the TFGβ pathway, and the WNT pathway shifted cell fate into SANLPCs, and final cell sorting for SIRPA-positive and CD90-negative cells resulted in enrichment of SANLPCs up to ~83%. These SIRPA+CD90- cells showed the molecular, cellular and electrophysiological characteristics of SANLPCs . While this study makes important progress in enriching SANLPCs by modulating signaling pathways, no specific surface markers for SANLPCs were identified and the yield was still short of what is usually expected for cells purified via FACS.
Hattori et al. developed a highly efficient non-genetic method for purifying hPSC-derived CMs, in which they employed a red fluorescent dye, tetramethylrhodamine methyl ester perchlorate (TMRM), that can label active mitochondria. Since CMs contain a large number of mitochondria, CMs from mice and marmosets (monkey) could be strongly stained with TMRM . They further found that primary CMs from several different types of animals and CMs derived from both mESCs and hESCs were successfully purified by FACS up to 99% based on the TMRM signals. In addition to its efficiency for CM enrichment, TMRM did not affect cell viability and disappeared completely from the cells within 24 hrs. Importantly, injected hPSC-CMs purified in this way did not form teratoma in the heart tissues. However, since TMRM only functions in CMs with high mitochondrial density, this method cannot purify entire populations of hPSC-CMs . While originally TMRM was claimed to be able to isolate mature hPSC-CMs, mounting evidence indicates that hPSC-CMs are similar to immature human CMs at embryonic or fetal stages. Therefore, both the exact phenotype of the cells isolated by TMRM and its utility are rather questionable [33, 34]. Two subsequent studies demonstrated that TMRM failed to accurately distinguish hPSC-CMs due to the insufficient amounts of mitochondria .
Employing the unique metabolic properties of CMs, Tohyama et al. developed an elegant purification method to enrich PSC-CMs . This approach is based on the remarkable biochemical differences in lactate and glucose metabolism between CMs and non-CMs, including undifferentiated cells. Mammalian cells use glucose as their main energy source . However, CMs are capable of energy production from different sources such as lactate or fatty acids . A comparative transcriptome analysis was performed to detect metabolism-related genes which have different expression patterns between newborn mouse CMs and undifferentiated mouse ESCs. These results showed that CMs expressed genes encoding tricarboxylic acid (TCA) cycle enzymes more than genes related to lipid and amino acid synthesis and the pentose phosphate cycle compared to undifferentiated ESCs. To further prove this observation, they compared the metabolites of these pathways using fluxome analysis between CMs and other cell types such as ESCs, hepatocytes and skeletal muscle cells, and found that CMs have lower levels of metabolites related to lipid and amino acid synthesis and pentose phosphate. Subsequently, authors cultured newborn rat CMs and mouse ESCs in media with lactate, forcing the cells to use the TCA cycle instead of glucose, and they observed that CMs were the only cells to survive this condition for even 96 hrs. They further found that when PSC derivatives were cultured in lactate-supplemented and glucose-depleted culture medium, only CMs survived. Their yield of CM population was up to 99% and no tumors were formed when these CMs were transplanted into hearts. This lactate-based method has many advantages: its simple procedures, ease of application, no use of FACS for cell sorting, and relatively low cost. More recently, this method was applied to large-scale CM aggregates to ensure scalability. As a follow-up study, the same group recently reported a more refined lactate-based enrichment method which further depletes glutamine in addition to glucose . The authors found that glutamine is essential for the survival of hPSCs since hPSCs are highly dependent on glycolysis for energy production rather than oxidative phosphorylation. The use of glutamine- and glucose-depleted lactate-containing media resulted in more highly purified hPSC-CMs with less than 0.001% of residual PSCs . One concern of this lactate-based enrichment method is the health of the purified hPSC-CMs, because physiological and functional characteristics of hPSC-CMs cultured in glucose- and glutamine-depleted media for a long time may have functional impairment since CMs with mature mitochondria were not able to survive without glucose and glutamine, although they were able to use lactate to synthesize pyruvate and glutamate . In addition, this lactate-based strategy can only be applied to hPSC- CMs, but not other hPSC derived cells such as neuron or β-cells.
Our group also recently reported a new method to isolate hPSC-CMs by directly labelling cardiac specific mRNAs using nano-sized probes called molecular beacons (MBs) [29, 73, 74]. Designed to detect intracellular mRNA targets, MBs are dual-labeled antisense oligonucleotide (ODN) nano-scale probes with a DNA or RNA backbone, a Cy3 fluorophore at the 5' end, and a Black Hole quencher 2 (BHQ2) at the 3' end [75, 76]. They form a stem-loop (hairpin) structure in the absence of a complementary target, quenching the fluorescence of the reporter. Hybridization with the target mRNA opens the hairpin and physically separates the reporter from the quencher, allowing a fluorescence signal to be emitted upon excitation. The MB-based method can be applied to the purification of any cell type that has known specific gene(s) .
In one study , we designed five MBs targeting unique sites in TNNT2 or MYH6/7 mRNA in both mouse and human. To determine the most efficient transfection method to deliver MBs into living cells, various methods were tested and nucleofection was found to have the highest efficiency. Next, we tested the sensitivity and specificity of MBs using an immortalized mouse CM cell line, HL-1, and other cell types. Finally, we narrowed it down to one MB, MHC-MB, which showed >98% sensitivity and > 95% specificity. This MHC-MB was applied to cardiomyogenically differentiated mouse and human PSCs and FACS sorting was performed. The resultant MHC-MB-positive cells expressed cardiac proteins at ~97% when measured by flow cytometry. These sorted cells also demonstrated spontaneous contraction and all the molecular and electrophysiological signatures of human CMs. Importantly, when these purified CMs were injected into the mouse infarcted myocardium, they were well integrated into the myocardium without forming any tumors, and they improved cardiac function.
In a subsequent study , we refined a method to enrich ventricular CMs from differentiating PSCs (vCMs) by targeting a transcription factor which is not robustly expressed in cells. Since vCMs are the main source for generating cardiac contractile forces and the most frequently damaged in the heart, there has been great demand to develop a method that can obtain a pure population of vCMs for cardiac repair. Despite this critical unmet need, no studies have demonstrated the feasibility of isolating ventricular CMs without permanently altering their genome. Accordingly, we first designed MBs targeting the Iroquois homeobox protein 4 (Irx4) mRNA, a vCM specific transcription factor [78, 79]. After testing sensitivity and specificity, one IRX4-MB was selected and applied to myogenically differentiated mPSCs. The FACS-sorted IRX4-MB-positive cells exhibited vCM-like action potentials in more than 98% of cells when measured by several electrophysiological analyses including patch clamp and Ca2+ transient analyses. Furthermore, these cells maintained spontaneous contraction and expression of vCM-specific proteins.
The MB-based cell purification method is theoretically the most broadly applicable technology among the purification methods because it can isolate any target cells expressing any specific gene. Thus, the MB-based sorting technique can be applied to the isolation of other cell types such as neural-lineage cells or islet cells, which are critical elements in regenerative medicine but do not have specific surface proteins identified to date. In addition, theoretically, this technology may have the highest efficiency when MBs are designed to have the maximum sensitivity and specificity for the cells of interest, but not others. These characteristics are particularly important for cell therapy. Despite these advantages, the delivery method of MB into the cells needs to be improved. So far, nucleofection is the best delivery method, but caused some cell damage with < 70% cell viability. Thus, development of a safer delivery method will enable wider application of MB-based cell enrichment.
Recently, Miki and colleagues reported a novel method for purifying cells of interest based on endogenous miRNA activity . Miki et al. employed several synthetic mRNA switches (= miRNA switch), which consist of synthetic mRNA sequences that include a recognition sequence for miRNA and an open reading frame that codes a desired gene, such as a regulatory protein that emits fluorescence or promotes cell death. If the miRNA recognition sequence binds to miRNA expressed in the desired cells, the expression of the regulatory protein is suppressed, thus distinguishing the cell type from others that do not contain the miRNA and express the protein.
Briefly, the authors first identified 109 miRNA candidates differentially expressed in distinct stages of hPSC-CMs (differentiation day 8 and 20). Next, they found that 14 miRNAs were co-expressed in hPSC-CMs at day 8 and day 20 and generated synthetic mRNAs that recognize these 14 miRNA, called miRNA switches. Among those miRNA switches, miR-1-, miR-208a-, and miR-499a-5p-switches successfully enriched hPSC-CMs with purity of sorted cells up to 96% determined by TNNT2 intracellular flow cytometry. Particularly, hPSC-CMs enriched by the miR-1-switch showed substantially higher expression of several cardiac specific genes/proteins and lower expression of non-CM genes/proteins compared with control cells. Patch clamp confirmed that these purified hPSC-CMs possessed both ventricular-like and atrial-like action potentials.
One of the major advantages of this technology is its wider applicability to other cell types. miRNA switches have the flexibility to design the open reading frame in the mRNA sequence such that any desired transgene can be incorporated into the miRNA switches to regulate the cell phenotype based on miRNA activity. The authors tested this possibility by incorporating BIM sequence, an apoptosis inducer, into the cardiac specific miR-1- and miR-208a switches and tested whether they could selectively induce apoptosis in non-CMs. They found that miR-1- and miR-208a-Bim-switches successfully enriched cTNT-positive hPSC-CMs without cell sorting. Enriched hPSC-CMs by 208a-Bim-switch were injected into the hearts of mice with acute MI and they engrafted, survived, expressed both cTNT and CX43, and formed gap junctions with the host myocardium. No teratoma was detected. In addition, other miRNA switches such as miR-126-, miR-122-5p-, and miR-375-switches targeting endothelial cells, hepatocytes, and β-cells, respectively, successfully enriched these cell types differentiated from hPSCs. However, identification of specific miRNAs expressed only in the specific cell type of interest and verification of their specificity in target cells will be key issues for continuing to use this miRNA-based cell enrichment method.
Recent advances in biomedical engineering have contributed to developing systems that can isolate target cells using physicochemical properties of the cells. Microfluidic systems have been intensively applied for cell separation due to recent improvements in miniaturizing a cell culture system [81-83]. These advances made possible the design of automated microfluidic devices with cellular microenvironments and controlled fluid flows that save time and cost in experiments. Thus, there have been an increasing number of studies seeking to apply the microfluidic system for cell separation. Among the first, Singh et al. tested the possibility of using a microfluidic system for the separation of hPSC  by preparative detachment of hPSCs from differentiating cultures based on differences in the adhesion properties of different cell types. Distinct streams of buffer that generated varying levels of shear stress further allowed selective enrichment of hPSC colonies from mixed populations of adherent non-hPSCs, achieving up to 95% purity. Of note, this strategy produced hPSC survival rates almost two times higher than FACS, reaching 80%.
Subsequently, for hPSC-CMs purification, Xin et al. developed a microfluidic system with integrated ridge-like flow derivations and fishnet-like microcolumns for the enrichment of hiPSC-CMs . This device is composed of a 250 mm-long microfluidic channel, which has two integrated parallel microcolumns with surfaces functionalized with anti-human TRA-1 antibody for undifferentiated hiPSC trapping. Aided by the ridge-like surface patterns on the upper wall of the channel, micro-streams are generated so that the cell suspension of mixed undifferentiated hiPSCs and hiPSC-CMs are forced to cross the functionalized fishnet-like microcolumns, resulting in trapping of undifferentiated hiPSCs due to the interaction between the hiPSCs and the columns, and the untrapped hiPSC-CMs are eventually separated. By modulating flow and coating with anti-human TRA-1 antibody, they were able to enrich CMs to more than 80% purity with 70% viability. While this study demonstrated that a microfluidic device could be used for purifying hPSC-CMs, it was not realistic because the authors used a mixture of only undifferentiated hiPSCs and hiPSC-CMs. In real cardiomyogenically differentiated hiPSCs, undifferentiated hiPSCs are rare and many intermediate stage cells or other cell types are present, so the idea that this simple device can select only hiPSC-CMs from a complex mixture is uncertain.
Overall, the advantages of microfluidic system based cell isolation include fast speed, improved cell viability and low cost owing to the automated microfluidic devices that can control cellular microenvironments and fluid flows [86-88]. However, microfluidic-based cell purification methods have limitations in terms of low purity and scalability [89-92]. In fact, there have been only a few studies demonstrating the feasibility that microfluidic device-based cell separation could achieve higher than 80% purity of target cells. Furthermore, currently available microfluidic devices allow only separation of a small number of cells (< 1011). To employ microfluidic devices for large-scale cell production, we need to develop a next generation of microfluidic devices that can achieve a throughput greater than 1011 sorted cells per hour with > 95% purity.
Having available a large quantity of a homogeneous population of cells of interest is an important factor in advancing biomedical research and clinical medicine, and is especially true for hPSC-CMs. While remarkable progress has been made in the methods for differentiating hPSCs into CMs, technologies to enrich hPSC-CMs, particularly those which are clinically applicable, have been emerging only over the last few years. Contamination with other cell types and even the heterogeneous nature of hPSC-CMs significantly hinder their use for several future applications such as cardiac drug toxicology screening, human cardiac disease modeling, and cell-based cardiac repair. For instance, cardiac drug-screening assays require pure populations of hPSC-CMs, so that the observed signals can be attributed to effects on human CMs. Studies of human cardiac diseases can also be more adequately interpreted with purified populations of patient derived hiPSC-CMs. Clinical applications with hPSC-CMs will need to be free of other PSC derivatives to minimize the risk of teratoma formation and other adverse outcomes.
Therefore, development of reproducible, effective, non-mutagenic, scalable, and economical technologies for purifying hPSC-CMs, independent of hPSC lines or differentiation protocols, is a fundamental requirement for the success of hPSC-CM applications. Fortunately, new technologies based on the biological specificity of CMs such as MITO-tracker, molecular beacons, lactate-enriched-glucose depleted-media, and microRNA switches have been developed. In addition, technologies based on engineering principles have recently yielded a promising platform using microfluidic technology. While due to the short history of this field, more studies are needed to verify the utility of these technologies, the growing attention toward this research is a welcome move.
Another important question raised recently is how to non-genetically purify chamber-specific subtypes of CMs such as ventricular-like, atrial-like and nodal-like hPSC-CMs. So far, only a few studies have addressed this potential with human PSCs. We also showed that a molecular beacon-based strategy could enrich ventricular CMs differentiated from PSCs . Another study demonstrated generation of SA-node like pacemaker cells by using a stepwise treatment of various morphogens and small molecules followed by cell sorting with several sub-specific surface markers. However, the yield of both studies was relatively low (<85%). Given the growing clinical importance of chamber-specific CMs, the strategies for purifying specific subtypes of CM that are independent of hPSC lines or differentiation protocols should be continuously developed. A recently reported cell surface capture-technology [93, 94] may facilitate identification of chamber specific CM proteins that will be useful for target CM isolation.
In summary, technological advances in the purification of hPSC-CMs have opened an avenue for realistic application of hPSC-CMs. Although initial success was achieved for purification of CMs from differentiating hPSC cultures, questions such as scalability, clinical compatibility, and cellular damage remain to be answered and isolation of human subtype CMs has yet to be demonstrated. While there are other challenges such as maturity, in vivo integration, and arrhythmogenecity, this development of purification technology represents major progress in the field and will provide unprecedented opportunities for cell-based therapy, disease modeling, drug discovery, and precision medicine. Furthermore, the availability of chamber-specific CMs with single cell analyses will facilitate more sophisticated investigation of human cardiac development and cardiac pathophysiology.
This work was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIP) (No 2015M3A9C6031514), the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI15C2782, HI16C2211) and grants from NHLBI (R01HL127759, R01HL129511), NIDDK (DP3-DK108245). This work was also supported by a CityU Start-up Grant (No 7200492), a CityU Research Project (No 9610355), and a Georgia Immuno Engineering Consortium through funding from Georgia Institute of Technology, Emory University, and the Georgia Research Alliance.
Corresponding author: Young-sup Yoon, M.D., Ph.D., Department of Medicine, Division of Cardiology, Emory University, 101 Woodruff Dr. WMB 3009, Atlanta, GA, 30322, USA; Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, Korea, E-mail: yyoon5edu.


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West Wales Review of the Year: July to December
We take a look at what made the headlines across the region during the last six months of 2020
By Ian Lewis
There is no doubt 2020 has been a very difficult year in the shadow of the coronavirus pandemic.
But while the crisis has dominated the news, here we take a look back at it and other headlines that have shaped the last 12 months.
In the second part of our review (part one can be read here), we turn the attention to July to December.
In July it was announced that the ferry service between Llansteffan and Ferryside would not run again this year due to the ongoing coronavirus restrictions - but bosses promised it will return in spring 2021.
Since lockdown came into force in March, directors of the Carmarthen Bay Ferries halted all services of the Glanferry across the River Towy at the mouth of its estuary, meaning its income dried up.
A new fundraising group, Friends of Glansteffan Ferry, was established to help the service survive.
However, directors of Carmarthen Bay Ferries decided not to restart services this season.
The Glansteffan ferry which links Llansteffan and Ferryside
In a statement, directors Les Jones, Martin Smith, Celia Strange and Kenton Morgan said: "The decision was difficult and we know this will disappoint many of our supporters.
"After an austere Covid-19 lockdown, there is a need for relaxing and interesting open-air activities in the UK.
"Ferry crossings and boat trips on the River Towy have the potential to provide this for our communities and the increasing numbers of people who are visiting the Welsh coast."
"Uncertainty over the virus ultimately led to plans of restarting the ferry service this summer being scrapped."
The directors added: "Our unique amphibious boat was due to operate from April until the end of September."
The ferry remains at its original home – Robust Boatbuilders in Solva, Pembrokeshire - until next season.
Also by this time, during the summer Carmarthenshire Council's registration services team were working around the clock to catch up with the registration of more than 800 babies born in the county during lockdown.
The team was also assisting couples who intend to marry or form a civil partnership or who have had their big day postponed because of Covid-19 restrictions.
By July the team was the process of contacting new parents inviting them to make an appointment to register their child's birth.
The first steps to creating a new community park in Tyisha, Llanelli were taken as part of a multi-million-pound regeneration plans of the area. Pictured from left are: Llanelli MP Nia Griffith and Tyisha county councillors Andre McPherson and Suzy Curry
In Llanelli the first steps to creating a new community park in Tyisha were taken as part of multi-million-pound regeneration plans for the area.
The 'Tyisha – Making it happen' project which aims to transform the area to create a vibrant, new community continues to be a priority for the council, although the coronavirus pandemic has had an impact on some planned works.
Lockdown restrictions meant that some physical works were delayed, including the construction of the new play park at Ann Street - but that was then started over the summer.
There are also plans to relocate young people's projects from the Station Road area along with temporary accommodation.
The month started with tributes pouring in from Welsh rugby communities and beyond following the sudden death of popular Scarlets backroom figure and broadcaster Andrew 'Tommo' Thomas.
A statement issued by Scarlets Rugby read: "We are devastated to hear that the voice of Parc y Scarlets, Andrew 'Tommo' Thomas, passed away suddenly.
"Tommo was a hugely popular figure as the Parc y Scarlets match-day announcer, a passionate Scarlets fan who brought his huge character and energy to every match."
The statement continued: "Tommo loved the Scarlets and put his heart and soul into his role.
"He was much loved by supporters, players and staff."
Broadcaster Andrew 'Tommo' Thomas who sadly passed away over the summer
Tommo' had worked at the BBC and Nation Broadcasting stations, with spells at BBC Radio Cymru, Radio Ceredigion, Radio Carmarthenshire, Radio Pembrokeshire, Bridge FM and Nation Radio.
Tributes flooded in from rugby and broadcasting circles.
Former Wales and Scarlets scrum-half Rupert Moon was among the first to post his condolences on social media, writing: "Lost a great friend ... my brother by another mother.
"Thoughts with his family and all his many friends. Sleep well buddy. Big boys do cry."
On August 26 a freight train carrying diesel fuel and gas oil in tank wagons derailed at, Llangennech, Llanelli.
The resulting blaze could be seen from miles around and hundreds of people in the area were evacuated from their homes.
The train fire in Llangennech, Llanelli, was captured by residents
Two workers were on the train but were not injured and raised the alarm.
An evacuation zone was put in place and police contacted residents and asked them to go to Bryn School and Llangennech Community Centre.
They were allowed back into their homes at around 5.30am the following day.
The train consisted of 25 wagons each holding up to 76 tonnes of diesel or gas oil.
The Rail Accident Investigation Branch (RAIB) said the derailment and subsequent damage to the wagons "resulted in a significant spillage of fuel and a major fire."
The aftermath of the derailment
An aerial view of the derailed carriages
Local people reported "seeing a plume of flames and smoke, and the strong smell of fuel", the RAIB added.
Ten of the wagons derailed and around 330,000 litres of fuel were spilled.
A huge clean up of the fuel spill was launched.
The derailment and subsequent fire of a freight train in Llangennech is not thought to have been caused by criminal activity, British Transport Police (BTP) have said.
Investigators later said the derailment happened after some of its wheels were damaged by a fault with the brakes.
On December 11, work started on removing and replacing 12,000 cubic metres of diesel-contaminated soil around the site.
Specialist environmental contractors are carrying out the complex operation, and working around the clock for several weeks to complete the work as safely and as swiftly as possible.
August saw some streets pedestrianised in Carmarthenshire as part of a package of measures to encourage shoppers and visitors back into town centres after lockdown was lifted.
Parking spaces were moved, speed limits lowered and one-way pedestrian systems put in place to allow safe queuing and social distancing.
The move was part of the still ongoing Carmarthenshire Council recovery plan to help businesses get back on their feet safely while the pandemic still continues.
The temporary measures took effect in Carmarthen, Llanelli and Ammanford on August 3 and will be kept under review.
Council leader Emlyn Dole said in August: "These changes have been implemented after much consideration and within the boundaries of what the council can do safely and responsibly to protect businesses and visitors whilst also keeping our town centres flowing and operating well.
"We really hope that people will come back to our town centres and help them thrive again by shopping and spending their leisure time locally."
Carmarthen county councillors Alun Lenny and Gareth John, who is also mayor at the top of King Street, Carmarthen
December saw a public consultation take place on the measures to assess how effective they have been and what steps can be taken moving into the new year.
Traders in King Street called for the closure to be lifted claiming it had driven all footfall away and businesses were struggling.
Exam results rolled around once again and it was a very different year for many pupils picking up their GCSE and A Level results after months of studying at home in lockdown.
Pupils across Carmarthenshire, Pembrokeshire and Ceredigion receiving their results were congratulated for their hard work and determination in what education leaders have described as an been an exceptionally challenging year.
Due to the coronavirus pandemic, students were unable to sit final exams and instead their grades reflect predicted results based on coursework and mock examination results.
Councillor Glynog Davies, Carmarthenshire Council's executive board member for education and children's services, said everyone who has received their exam results should be proud of what they've achieved.
Mr Davies said: "All of our students should be proud of what they have achieved, and on behalf of the council I would like to congratulate them and wish them well for their onward journey.
"This year has been unlike any other and has presented exceptional challenges for our students and our teachers."
Pupils celebrating A Level exam success at Carmarthen's Ysgol Bro Myrddin School
Bank holiday traffic was brought to a halt on Carmarthen's busiest roundabout when climate change protesters stood in the carriageway of the busy A40.
Drivers were forced to step on the brakes as a group of Extinction Rebellion protesters brandishing coloured flags emblazoned with their logo stepped into two lanes of traffic at the eastbound entrance of the A40.
The disruption – at what is known locally as the 'B&Q roundabout' – stopped the flow of traffic for several minutes before letting drivers continue on their journeys.
Despite beeping horns and some disgruntled motorists, protesters approached vehicles to speak to drivers and hand out leaflets about why they were taking action.
Extinction Rebellion campaigners blocking the A40 in Carmarthen over August bank holiday
Campaigners were claiming that part of Carmarthen near the River Towy and its floodplain would be under water in three decades due to climate change.
Extinction Rebellion called on Carmarthenshire Council and the Welsh Government to take steps now.
Protesters even had a large octopus made of material blocking the road and one campaigner sat in a kayak at the roundabout entrance.
The start of the new school term saw a Carmarthen bus firm suspend four school routes because of Covid-19 social distancing restrictions.
The decision by Johnstown-based Morris Travel left children having to find alternative transport as the new term began.
However, the company's suspension of its school routes meant children who usually pay £1 each way a day to travel to QE High School and Ysgol Bro Myrddin have been left without transport.
All those pupils live within the three mile radius of the town's two secondary schools and are not eligible for school transport from Carmarthenshire Council.
In contrast, pupils catching school buses provided by Carmarthenshire Council were not required to social distance but must wear masks.
Morris Travel's depot in Johnstown, Carmarthen
In Pembrey Country Park more than 200 youths were moved on by police after a mass gathering in the forest with reports of underage drinking and antisocial behaviour.
Officers from Dyfed- Powys Police were called to break up the huge group of youngsters which had descended on the area after being advertised on social media.
Locals said the group was made up of people from as far as Whitland and Swansea and had swelled to more than 200 by around 8.30pm.
As autumn approached, a Chinese takeaway owner from Carmarthen won Richard Madeley and Judy Finnigan's Search For A Bestseller book competition.
Julie Ma won the prize for her book Happy Families and will receive a publishing deal worth at least £10,000, with her work published by Welbeck in February 2021.
Born and raised in Carmarthen, Ma's grandfather arrived in Wales from China in the 1920s. Her parents set up a Chinese takeaway in the 1970s and she took over the business with her brother in 2008.
Julie Ma won the prize for her book Happy Families
The book is the story is of three generations of a Chinese immigrant family in rural Wales
Her love of reading and writing emerged from school and local bookshop, Derrick Williams.
Julie said: "I feel I should be pinched to wake me up.
"As most writers starting out know, it's worth building up a thick skin to deal with all of the rejections, so when I entered I just expected to add another layer to my hide.
"But secretly I was hoping Richard and Judy would enjoy hearing about the folk in the Chinese takeaway and the quirky town they live in. And they did."
Richard Madeley said: "We thought Happy Families was great, absolutely bloody great.
"It's a wonderful story and she's such a talented, funny writer.
"We think this has real appeal to a wide audience and are thrilled to choose it as the winner, Julie Ma is a rare find."
October also saw the announcement that a new development blueprint for Carmarthenshire was likely to be delayed by several months.
The local development plan (LDP) sets out the requirement for 8,835 new homes in the county over a 15-year period, and must be approved by the Welsh Government before it is implemented.
Carmarthenshire's LDP was expected to be adopted at the end of next year, when the current development plan expires.
A report to the council's executive board said a delay of at least six months was required due to the impacts of the coronavirus pandemic.
It said: "Taking into account the local elections in 2022 it is likely that any extension would be seven months, consequently allowing the plan to be adopted ahead of the summer recess in 2022."
Policing was also under the spotlight this month.
Dyfed-Powys Police officer numbers are set to rise to around 1,200 by the end of 2021, but that only takes the force back to 2010 levels of staff.
At a meeting of the Powys Public Service Board (PSB), Dyfed-Powys Police and crime commissioner Dafydd Llywelyn was asked how the recruitment drive was going.
A year ago, the UK Government revealed a commitment to pump £750 million into police forces around the UK.
This would be to recruit 20,000 police officers over three years.
Dyfed Powys Police Crime Commissioner Dafydd Llywelyn announced in October that officers numbers will increase by the end of next year
At the PSB meeting, community councillor Derrick Pugh asked: "Are you happy with their response from the UK Government as far as recruitment is concerned?"
Mr Llywelyn said: "The Prime Minister has been keen to recruit 20,000 additional officers, but we must remember in comparison to 2010, this just brings us back to that level.
"The increase for us is around 70 over the next three years.
"We're on track with that and the money is available in a grant, which we receive every six months from the Home Office."
He added that by the end of the three-year cycle Dyfed-Powys would have around 1,200 officers.
Mr Llywelyn said: "That is the level we had in 2010. There is an element of giving with one hand and taking away with the other.
"We are crying out for more core funding and over the last decade we have relied on the council tax precept to plug any gaps."
The grade two-listed bandstand at Llanelli's Parc Howard in the early 1920s
In Llanelli, it was announced that a bandstand in the town, built before the First World War is to be restored.
Some people in the town will remember the Parc Howard structure as an aviary containing exotic birds.
Carmarthenshire Council is behind the plans for the grade two-listed bandstand, which will require a new floor, the repair and redecoration of existing columns and balustrades, some new replica balustrades and a handrail.
Fencing was put up around the building in 2016 as the floor had rotted.
Many bandstands fell out of use in the 1950s and 1960s, according to a heritage impact assessment submitted as part of the council's change of use application, but the one at Parc Howard continued to play a role in civic events.
In later days the bandstand was converted into an aviary.
Bold plans were announced for Llanelli town centre with a £3 million regeneration scheme.
The scheme, by Cygnus Holdings (Llanelli) Ltd, is named Y Linc as it will connect the town centre to Eastgate with walkways and provide new restaurants, apartments and offices to the people of Llanelli, with brands such as Juniper Place and Old Havana due to take up units at the development.
The scheme, which has been backed by the Welsh Government, has been designed by the town's Darkin Architects and will see the former Tom Peppers nightclub and Captain Nemo pubs demolished and a new development built on the site covering 8-16 Market Street next year, if planning permission is approved.
An artist impression of the £3m regeneration scheme Y Linc for Llanelli town centre
David Darkin, owner of Darkin Architects, personally designed the scheme and said the project has been a labour of love for him for the past year.
Mr Darkin, who is also president of Llanelli Chamber of Trade and Commerce and a former mayor of the town, said: "As someone from Llanelli, this scheme is close to my heart and to have been able to design it and meet the needs of what I think Llanelli needs is great.
"It shows that people are looking to the future and investment in Llanelli town centre. If all goes well with the planning, the aim is to get this built next year."
Early November is usually a crucial fundraising periods for the Royal British Legion's annual Poppy Appeal in the run up to Remembrance Sunday and Armistice Day.
However, with Wales in the grip of a two week firebreak lockdown the coffers were difficult to fill for the charity this year.
With non essential retail stores shut and people urged to stay home as much as possible to curb the rising cases of coronavirus, fundraising needed a fresh approach.
The Royal British Legion was asking everyone to back the 2020 Poppy Appeal in a new way.
Like so many things this year, the appeal had to adapt to the threat of coronavirus and the charity is asking the public to support it like never before, as every poppy counts.
Remembrance Sunday saw no parades through the streets of Carmarthen, Llanelli, Ammanford and other towns.
In the Carmarthen area, fundraiser Roy Watkins said this year is like no other.
He said: "The pandemic has hit us hard and of course this second lockdown really couldn't have come at a worse time for us.
"It is normally our busy period for selling poppies out on the streets, door to door and fundraising in the last push towards Remembrance Sunday and Armistice Day."
Across Carmarthenshire people raised money with virtual events and bought poppies online to help the appeal as much as possible.
While the laying of wreaths involved very few people.
In the Gwendraeth Valley a house was severely damaged after it was hit by lightning.
Fire crews, ambulance service and officials from Carmarthenshire Council were at the scene of the incident in Rhosnewydd in Tumble, after residents said they heard something that sounded like an explosion at around 6am.
Two people were taken to Glangwili Hospital while one property sustained severe damage to its roof and another property has suffered minor damage.
Residents said they were woken by something that sounded "like a bomb" which could also be heard in neighbouring villages.
One resident, who did not want to be named, said at the time: "I've never heard anything like it.
"It sounded like a bomb had gone off. There was this huge bang and everyone's car alarms went off. We didn't know what it was and then the fire trucks pulled up."
The year ended as it had began in Carmarthen - with the loss of a major retailer.
While January saw announcements by the Arcadia Group that the town's Topshop/ Topman and Miss Selfridge would close in three months' time, so it was that the Arcadia Group went into liquidation in December.
Arcadia now has no stores in Carmarthen but does have a branch of Outfit in at Parc Trostre Retail Park in Llanelli.
The future of Outfit there is unclear at this stage.
Debenhams has been the anchor store at St Catherine's Walk, Carmarthen for a decade (photo: Alun Lenny)
In addition to Arcadia's demise, the Debenhams chain collapsed meaning its store which anchors St Catherine's Walk in Carmarthen will close unless a rescue package can be found.
Also at risk is Llanelli's Debenhams store at Parc Trostre Retail Park and further afield Swansea's branch in the Quadrant shopping centre.
Parts of west Wales saw flooding as heavy rain swept in over the weekend of December 19.
Areas of Carmarthen town were severely flooded after the River Towy burst its banks and the main A484 road was impassable at Llechryd, in Ceredigion, after the River Teifi flowed across the valley floor.
The River Towy burst in banks in Carmarthen (photo: Dafydd Williams)
The bridge in Llechryd, Ceredigion was completed submerged by flood water (photo: Claire Lewis)
Dafydd Williams, from Llangunnor, said: "Businesses have already been badly hit by Covid. Now they're clearing stock from where they've been flooded."
The coronavirus is still very much at the forefront of the headlines as the year comes to a close with cases skyrocketing and Wales placed in Tier 4 lockdown days before Christmas to try and curb the sharp increase in cases and ease pressure on an NHS at breaking point.
However, the light at the end of the tunnel everyone hopes for during this pandemic started to break through the gloom in December, as the first Covid-19 vaccines were rolled out to frontline workers and the most vulnerable.
Among the first was 108-year-old Mary Keir who lives at the Awel Tywi care home in Llandeilo.
She was given a first dose of the Pfizer/BionTech vaccine shortly after 2.30pm on Thursday, December 17.
As Wales' second oldest woman, Mrs Keir became the first care home resident in the Hywel Dda University Health Board region and indeed across south Wales to be vaccinated.
The vaccine was administered as part of a pilot being carried out by the health board - who manage health care in Carmarthenshire, Ceredigion and Pembrokeshire.
Mary Keir, a resident at Awel Tywi care home in Llandeilo, was the first care home resident to receive the Covid-19 vaccine in south Wales
Mrs Keir, who is a retired nurse, was more than willing to be immunised, and she was equally keen to discuss the process once she had received the vaccine.
She said: "I was ever so pleased because we all feel safer and better for having the opportunity."
Adding: "It didn't hurt a bit and I'm used to injections anyway, both getting them and giving them!"
"It's for our own good so I think we should be thrilled because people have worked so hard to get the vaccine ready and we are all very thankful that it's here and ready to be used."
There we have it, that was the year in headlines across west Wales.
As we stand at the end of a extremely difficult year, that none of us could have predicted, we will all look towards the clock striking midnight on New Year's Eve with the hope that life will slowly return to normal in 2021.
We have been overwhelmed this year, reading about how our communities are helping each other during these difficult times.
We want YOU to continue sharing and shouting about what you are doing for your neighbourhoods as we enter 2021.
Send us your stories via this link.

redpajama_set_name: RedPajamaCommonCrawl


TRAVELING by air has always been an inspiring thing for me. On the ground, one is all too aware of human failings and annoyances. But at 30,000 feet, gazing down on the manicured farms, row after row of lovely houses, complex highways, airports, railroads, city parks, baseball diamonds and towering skyscrapers, one cannot escape a certain awe at what humans have managed to achieve.
For the past week and a half, we have had to endure the crushing realization that while it takes years and years to build something, it takes only seconds to destroy it. A few evil men armed with little more than hate can wreak almost incomprehensible destruction.
The terrorists have broken our hearts -- but they haven't come close to breaking our spirit. In fact, they've done the reverse. As with one will, we are resolved to fight them. It is bracing to discover what resilience lay beneath the placid surface of the average American.
The Bush administration is wise not to characterize this as a war between Christendom and Islam. We have many allies in the Moslem world, and we will need more if we are to find and destroy the terrorist networks. We will also need the territory of friendly Moslem nations as staging areas. Moreover, if nations teetering between Islamism and modernity -- like Egypt, Pakistan and Jordan -- sense that this is a religious war, they will be swept into the camp of our enemies.
Similarly, President Bush was wise to visit the mosque in Washington, D.C., and stress that most Moslems living in the United States are loyal and peaceable.
On the other hand, it would be foolish to permit "sensitivity" or an exaggerated worry about giving offense to inhibit authorities from tracking suspected terrorists. Let's not pretend that "ethnic profiling" is out of the question. It is absolutely necessary. If a young unmarried man from Iraq, Egypt, Syria, Lebanon or half a dozen other nations buys a ticket on a plane, boat or train in the next 10 years and does not receive a thorough background check and pat down at the gate, we are not defending ourselves.
While we cannot and must not fight a religious war against Islam, we do have to take seriously the ideology of Islamism. Only a population thoroughly indoctrinated with hate could yield suicide bombers with the ferocity of those we have recently suffered. We can search out and kill individual terrorists, but if we do not confront the ideological battle, we will achieve only partial success.
William Safire has wisely urged that Radio Free Europe and Radio Liberty be redirected to the nations of the Arab and Moslem world. That would be a start. They are getting a nonstop diet of lies about America. And this raises the delicate matter of Arab "allies." Even those commonly called "moderates" permit the most vicious slanders against the West to circulate in their countries. Much of it is Hitlerian in its anti-Semitism and a great deal is anti-American.
A professor at the University of Lebanon wrote "Most Arabs and perhaps most of the Third World did rejoice, not because of the killing of innocent Americans, but because of the penetration of the bastion of American colonialism and the offensive within its home turf. ... The meaning of terror according to the American is known. It refers to any resistance to the new colonialism..."
Many columnists in the Arab media have dismissed United States claims that Arabs perpetrated the Sept. 11 massacre. They offer other suspects, including the Mossad, President Bush (in order to boost his sagging popularity and unify the country behind him), Colin Powell, the CIA and American terrorists.
There is no acknowledgment in the Arab world -- not among journalists, academics, or even clerics -- that targeting civilians (usually Israelis) is an illegitimate tactic of war (far less of the "peace process"). There were no Arab voices raised in condemnation when suicide bombers blew up Israeli teen-agers at a disco or Israeli families at a pizzeria. To the contrary, the murderers were hailed as martyrs. Those same words are now being attached to the men who killed more than 5,000 Americans.
While we're not at war with Moslems, nor with the Arab world, we must recognize that the climate of opinion in the Arab world is our enemy.


text:
Nuvva Nena () is a 2012 Telugu-language romantic comedy film directed by debutante P. Narayana and produced by Vamsi Krishna under SVK Cinema banner. The film stars Shriya Saran, Allari Naresh, Sharvanand, and Vimala Raman in lead roles with Brahmanandam, Ali, Jeeva, Kovai Sarala, Srinivasa Reddy, and Narsing Yadav appearing in other significant roles. Bheems Ceciroleo composed the film's soundtrack with Mani Sharma providing the background score for the film. It is based on Hindi film Deewana Mastana.

Plot
Avinash is a small-time crook who creates havoc in the town of Amalapuram along with his friend Ali. After a big robbery, they escape to Hyderabad along with 100000 crores in cash. Unexpectedly, Avinash comes across the gorgeous Dr. Nandini, and it is love at first sight for him. He sets about trying to get closer to Nandini and starts hatching plans to that effect. Into this scenario enters Anand who is an extremely timid guy. Anand is extremely frightened about very minor things,

Friction develops between Avinash and Anand as they set about hatching plans to destroy each other's image in front of her. Unexpected competition comes in the form of local Don Aaku Bhai. After much drama, it is revealed to Anand and Avinash that Nandini is already in love with Raja when she invites them to their marriage. Although heartbroken, Avinash and Anand both move on in life after deciding to forget Nandini.

Cast

 Shriya Saran as Dr. Nandhini
 Allari Naresh as Avinash
 Sharvanand as Anand
 Brahmanandam as Aaku Bhai
 Ali as Chanti
 Kovai Sarala as Pushpa
 Jeeva as Inspector
 Srinivasa Reddy as Hotel Waiter
 Narsing Yadav as Narsing Yadav
 Prabhas Sreenu as Thopu Seenu
 Fish Venkat as Aaku Bhai's henchman
 Raghu as Aaku Bhai's henchman
 Raja Abel as Raja (guest appearance)
 Vimala Raman in a cameo appearance

Production
Nuvva Nena was the second film which had Allari Naresh and Sharvanand together after Gamyam (2008). The film is a remake of 1997 Deewana Mastana which had Govinda, Anil Kapoor and Juhi Chawla in the lead.

Release
The film was released on 16 March 2012.

Soundtrack
The audio of the film was released on 19 February 2012 through Aditya Music in the market. The launch of audio was held at Jayabheri Club in Hyderabad on same day. The soundtrack of the film was composed by Bheems Ceciroleo and its consists of six songs. Lyrics for the three songs were written by Krishna Chaitanya and remaining three songs were written by Bheems Ceciroleo, Anantha Sreeram, Srimani.

References

External links

2012 films
Telugu remakes of Hindi films
Films scored by Mani Sharma
2010s Telugu-language films
2012 directorial debut films
Films scored by Bheems Ceciroleo

redpajama_set_name: RedPajamaWikipedia


Enjoy our spacious warm comfortable rooms with panoramic views of the mountains and oak forest with red deer. Our B&B is considered a walkers paradise. It is just a short stroll to Muckross House and the Killarney Lakes.
From Killarney, take the N71 towards Kenmare and continue for approx 4km. On your left you will pass Molly DArcys pub and hotel, take the first immediate left and continue up this steep road approx 1.5km, till you arrive at the sign for Muckross Stables on your left.