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The Clinical Status of Stem Cell Therapy for Ischemic Cardiomyopathy

Ischemic cardiomyopathy (ICM) is becoming a leading cause of morbidity and mortality in the whole world. Stem cell-based therapy is emerging as a promising option for treatment of ICM. Several stem cell types including cardiac-derived stem cells (CSCs), bone marrow-derived stem cells, mesenchymal stem cells (MSCs), skeletal myoblasts (SMs), and CD34+ and CD 133+ stem cells have been applied in clinical researches. The clinical effect produced by stem cell administration in ICM mainly depends on the transdifferentiation and paracrine effect. One important issue is that low survival and residential rate of transferred stem cells in the infracted myocardium blocks the effective advances in cardiac improvement. Many other factors associated with the efficacy of cell replacement therapy for ICM mainly including the route of delivery, the type and number of stem cell infusion, the timing of injection, patient's physical condition, the particular microenvironment onto which the cells are delivered, and clinical condition remain to be addressed. Here we provide an overview of the pros and cons of these transferred cells and discuss the current state of their therapeutic potential. We believe that stem cell translation will be an ideal option for patients following ischemic heart disease in the future.

Introduction
Reduced blood supply in infracted myocardium is the leading cause of morbidity and mortality in patients with ischemic cardiomyopathy (ICM) [1,2]. While approximately 1% of adult cardiomyocytes possess ability of self-renewal, they cannot afford heart tissue impairment from serious or acute myocardial infarction [3][4][5]. Thus, ischemia-induced cardiomyocyte apoptosis and necrosis damage left ventricle geometry undergoing progressive ventricle remodeling, hypertrophy, and fibroblast proliferation resulting in scar information and poor contractility of left ventricle [6][7][8].
The common treatment strategies such as pharmacotherapy, coronary artery bypass grafting (CABG), and coronary artery stent enable the recovery of blood supply to the ischemic regions and relatively alleviate pain and suffering, but they fail to treat the pathophysiological changes following ischemic injury and regenerate novel muscle tissue. Therefore, the ideal treatment effect is to make myocardial cell regeneration resident cardiac progenitor cells or other exogenous multipotent stem cells [9]. Stem cell implantation treatment for ICM has brought a new dawn for patients while it faces a new challenge. Accumulating evidences have reported that stem cells repaired damaged heart by the means of differentiation to cardiac muscle cell, promoting angiogenesis, forcing proliferation of endogenous cardiac stem cells, and secreting cytokines, chemokines, and growth factors to activate endogenous reparative responses, inhibit cell apoptosis and fibrosis, and improve myocardial contraction [10]. In the last 2 Stem Cells International decade, many clinical trials have been implemented to assess the safety, feasibility, and efficacy of stem cell administration in patients with ischemic cardiomyopathy. Different cell types including bone marrow-derived stem cells, mesenchymal stem cells (MSCs), cardiac-derived stem cells (CSCs), skeletal myoblasts (SMs), and hematopoietic stem cells (HSCs) have been used to evaluate the cell-based therapeutic potential. However, promising results from most clinical studies to improve functional parameters have yielded to the few mixed ineffective treatments. Delivery modalities, cell types and dose, cell isolation procedures, and timing of cell transplantation may determine the curative effect on cardiac functional recovery [11][12][13]. Here, the current status of clinical research and future outlook of stem cell-based therapeutics for ischemic cardiomyopathy are elaborated.

Types of Stem Cell and Their Clinical Studies
In the last two decades, many different stem cell populations have been investigated and suggested to enhance cardiac function recovery in clinical trials. These stem cells can be categorized according to their cellular structure, function, origin, or cell surface marker, transcription factor, and specific protein. The simplest and most common way to group them depends on their site of origin. Stem cells isolated from heart are named cardiac-derived stem/progenitor cells and other types of stem cells are known as extracardiac-derived stem cells. Here, recent clinical trials of stem cell replacement therapy for ICM are described in Table 1.

Cardiac-Derived Progenitor/Stem Cells.
In 2003, cardiac stem cells (CSCs) were first discovered by Nadal-Ginard and colleagues [14] which break the traditional idea that heart was terminal differentiated organ. The multipotent and self-renewing characteristics of these cells have been identified in animal models which showed their ability to give rise to cardiomyocytes, endothelial cells, and smooth muscle cells indicating a potential regenerative capacity of adult heart [15]. The experimental studies have reported that cardiomyocyte lineages can be derived from different types of cardiac stem cells including c-kit + cell, isl-1 + cell and sca-1 + cell (restricted to murine hearts), and cardiosphere-derived cells (CDCs) [16]. CDCs are isolated from cardiac biopsies and grow as self-adherent clusters containing a heterogenous cell population of stem cells positive for c-kit (endogenous CSCs), CD105, and CD90 (cardiac MSCs) but negative for CD 45 (hematopoietic stem cell) indicating their capacities of clonogenic, self-renewal, and multilineage differentiation [17]. Accumulating evidences have described the capabilities of CSCs or CDCs in regenerative ability of cardiac muscles without teratoma formation [16,18]. CSCs residing in the myocardial inches are mainly located in left ventricular apex and atrial tissue. Although limited number of CSCs, 1 CSC per 8,000 to 20,000 cardiomyocytes, can generate enough cardiomyocytes a day to maintain the balance of the renewal of myocardial cells in healthy heart, they cannot bear great or acute damage of cardiac tissue.
The reasons for the limitations of CSCs include the following aspects: the number and viability of CSCs decrease with age and further decrease in coronary heart disease [19][20][21][22] and ckit + CSCs nearly lost their homing ability in postnatal heart [23]. Therefore, exploiting practical and effective methods for the isolation and expansion of CSCs will be great impetus for CSC therapy for ischemic heart disease. It is exciting that Smith and colleagues have presented a feasible and safe method for the isolation and expansion of adult CSCs from endomyocardial biopsy specimens [24] which provided possibility to treat ICM.
To date, several clinical trials have been allowed and completed to display the feasibility, safety, and efficacy of CSCs translation therapy in patients with ICM. The first clinical phase I trial recruiting 16 patients with CABG and postinfarction left ventricular (LV) dysfunction (EF ≤ 40%) suggested that intracoronary infusion of 1 million autologous CSCs (c-kit + ) increased LVEF from 30.3% to 38.5% at 4 months and 42.3% at 1 year and decreased infract size in seven patients from 32.6 g to 24.8 g at 4 months ( = 0.004) and 22.8 g at 1 year ( = 0.04) which revealed a striking treatment potential of CSCs [25]. Thereafter, a randomized phase I trial conducted by Makkar assessed the safety of intracoronary infusion of CDCs in patients with LV dysfunction after myocardial infarction (MI) (LVEF = 25%∼45%) [26]. The corresponding results showed reduced scar mass ( = 0.001), increased viable heart mass ( = 0.01), and regional contractility ( = 0.02) at 6 months in CDC treatment group, but there are no different changes in end-diastolic volume, end-systolic volume, and LVEF comparing with standard care patients. Fortunately, no serious adverse effect occurred by 6 months indicating the safety of CDC implantation treatment of MI. Recently, a randomized and controlled CADUCEUS trial (cardiosphere-derived autologous stem cells to reverse ventricular dysfunction) was carried out in patients with LV dysfunction to examine the efficacy of this therapeutic method [27]. The major results suggested that CDC treatment had a significant relationship with the improvement of LVEF and reduction of scar size at 1 year after treatment comparing with routine-care control patients [27]. By now, CSC therapy mostly focus on the patients with MI and LV dysfunction, mainly due to the cardiac fibrosis with no blood supply in the infraction location and this therapy showed more efficacy in MI than chronic ICM. However, these limited clinical results cannot fully prove the practical competence of CSC therapy for ICM. Although these clinical data may not satisfy every patient and clinical investigator, they provide important basis for exploiting more effective cell processing methods and have initiated larger-scale phase I or II clinical studies (NCT01458405 and NCT01758406) to evaluate the curative effect of CDC/CSC transplantation into patients with ICM.    approximately 0.01% MSCs [55]. Although most of BMMNCs are not stem cells, the cardiac repair effect is considered to mainly depend on the significant source of HSCs which contribute to the development of new cardiac tissue and blood vessels. It is commonly accepted that the cardiac repair by HSCs therapy may be largely dependent on secretion of growth factors and other proteins to promote angiogenesis [56,57] and stimulate cell proliferation and migration of endogenous cardiac stem cells or cardiomyocytes [11,58]. A case study that referred to one patient with MI first demonstrated the therapeutic possibility under clinical conditions using intracoronary transplantation of autologous BMMNCs [59]. Afterwards, several clinical trials proved that intracoronary administration of adult bone marrow stem/progenitor cells (BMCs) in patients with acute myocardial infarction (AMI) improved LV contractile function and depressed LV remodeling which have been reported by REPAIR-AMI trials [41,60,61] and TOPCARE-AMI trial [42]. In addition, treatment with BMCs significantly reduced adverse clinical events [41,62]. Therefore, the clinical experimental responses confirm the feasibility and possibility for the BMMNC infusion therapy for ICM though the detailed and solid action mechanisms of BMMNCs have not been concluded. Subsequently, more convincing evidences about the improvement of LVEF, cardiac contractility, and exercise capacity were supported by numerous larger-scale clinical trials.

Extracardiac-Derived
To date, the largest meta-analysis reviewed by Jeevanantham compiled and systematically evaluated the clinical data from 50 studies enrolling 2625 patients with acute or chronic ICM [63]. Regardless of the detailed design of this study (e.g., path and timing of delivery, number of administrated cells, follow-up time after cell fusion, various populations of BMMNC, evaluation parameter, and imaging modalities) and the specific type of ICM (AMI or chronic ICM), BMMNC-treated patients showed a modest increase of LVEF (∼3.96%) and smaller reduction of infarct size (∼4.03%), LV end-systolic volume (∼8.91 mL), and LV enddiastolic volume (∼5.23 mL) compared to control subjects. This analysis also exhibited that BMMNC administration significantly reduced occurrence of some events including mortality, and the recurrent rate of MI and stent thrombosis. This modest improvement was consistent with another recent meta-analysis published in 2014 [64]. These pooled results from 32 trials comprising 1300 BMMNC-treated patients and 1006 control-treated patients both with acute coronary syndrome or stable coronary disease demonstrated statistically significant increase in LVEF (∼4.6%) and decrease in perfusion defect size (∼9.5%) without influence of baseline ejection fraction or perfusion defect size. Therefore, these outcomes of statistical analysis support the BMMNC therapy for MI to improve cardiac infarction, cardiac remodeling, and patient-important clinical outcomes. However, another recent meta-analysis drew a discrepancy conclusion from 22 randomized controlled trials [65]. In this study, compared with control arm, BMMNC therapy was safe on the whole in patients with ICM but neither promoted the recovery of heart function on MRI-derived parameters nor improved clinical outcome despite slight increase in LVEF (∼2.1%).
The lack of ventricular improvement by BMMNC therapy was paralleled in a more recent clinical trial recruiting 28 patients with advanced ICM [40]. After 6 months, BMMNCtreated patients showed no significant improvement in LVEF, LV end-systolic volume (LVESV), and LV infarct volume indicating ineffective outcome of this cell therapy project.
Another new placebo-controlled study including 65 patients with ICM and LVEF < 50% displayed no statistically significant increase in LVEF and reduction in infarct size as a percentage of LV mass in BMMNC-treated group [30]. By now, it is apparent that BMMNC therapy provided highly variable and inconsistent results which showed mild to modest or no benefit effects in LV function and geometry [43]. Fortunately, no major adverse events were detected in all of the trial participants. On the whole, BMMNC therapy has been widely used in patients with the acute or chronic ICM and showed its importance in the therapy for ICM. However, the discrepant results have come out which may be due to numerous factors mainly including the method of BMMNC isolation and the type and total number of injected cells. The cellular mechanisms affecting the therapy efficacy mainly include efficient homing, proliferation, engraftment, and differentiation of infused BMMNCs which can be influenced by various culture conditions. A large-scale randomized multicenter European clinical trial is currently recruiting AMI patients to investigate intracoronary infusion of BMMNCs for assessing all-cause mortality (NCT01569178 and NCT01693042). It is expected that these data will be launched for larger randomized controlled clinical studies which involve the improvements in cell preconditioning, cell isolation procedure, and selection plan of cell infusion.

Mesenchymal Stem Cells.
MSCs mainly express cell surface antigens of CD73, CD90, CD105, CD44, CD106, and CD166 and negatively express CD34, CD31, CD14, CD45, CD133, and CD105 surface molecules [66,67]. They have advantages of self-renewal ability, multilineage differentiation potential, low immunogenicity, immunosuppressive properties, and low tumorigenicity [68] and face no risk of immune rejection by the host for lacking expression of major histocompatibility complex class II [69]. Under proper stimulation MSCs can differentiate into cardiomyocytes or secrete some growth factors for cardiac repair. However, the survival rate and migration ability in target tissue are two key factors to determine the therapeutic effect of MSCs. Most clinical studies showed improvement in cardiac function after MSC treatment of ICM despite of few ineffective results of MSC replacement therapy [70].
Earlier clinical studies showed positive effect of MSC treatment in ICM. A placebo-controlled trial recruited 69 patients with AMI after percutaneous coronary intervention (PCI) who were treated by intracoronary delivery of autologous MSCs [71]. Three months after transplantation comparing with control group, significant improvement of LVEF and ratio of end-systolic pressure to end-systolic volume indicated effective abilities of cardiac repair and reverse remodeling after autologous MSC administration [71]. Intravenous injection of allogeneic MSCs which reduced ventricular tachycardia episodes and increased LVEF within six months produced a similar effect to the above conclusion [72]. Later, a clinical trial of intramyocardial injection of autologous MSCs in patients with remote myocardial infarction for 1 year showed a decreased infarct size and improved regional LV function by test of peak Eulerian circumferential strain [43]. These conclusions were further confirmed by the latest clinical trials which suggested that patients following AMI showed improvement in LVEF in bone MSC-treated group versus control group [32,73]. But paper published in JAMA in 2014 revealed that although transendocardial stem cell injection with MSCs improved 6-minute walk distance, regional myocardial function, and peak Eulerian circumferential strain at the site of injection and reduced infarct size as a percentage of LV mass, no changes were observed in left ventricular chamber volume and ejection fraction [30]. So it was suggested that either delivery methods or allogeneic/autologous stem cells are associated with the therapeutic effect. There were no serious adverse events emerging across these studies. Two metaanalyses have evaluated bone MSCs therapy for ischemic heart disease and concluded that bone MSC treatment significantly reduced mortality risk, decreased angina episodes per week, and leaded to an even better life quality [74,75]. A recent power meta-analysis involving 1255 patients found moderate quality evidence that bone MSC treatment improved LVEF [76]. Therefore, most of these studies showed to some extent improvements in cardiac function and attenuation in left ventricular dilation and remodeling either in MI patients or ICM patients. Although the curative effect of MSC grafting did not reach the ideal target, considerable prospect has been demonstrated in the treatment of AMI or ICM. To be sure, all of these studies showed safety and no severe adverse events of MSC grafting in patients with MI or acute/chronic ICM diseases. Numerous ongoing clinical trials have been encouraged for further investigation of the safety and efficacy after MSC treatment in patients with ICM (NCT01556022, NCT01216995, and NCT00644410). What influences the efficacy of MSC therapy also needs to be explored and investigated.

Skeletal Myoblasts.
SMs are a small population of undifferentiated and inactive cells residing within mature skeletal muscle fiber. Once injury occurs, SMs are activated from static state and rapidly proliferate and differentiate into muscle fibers to replace injured or dying muscle cells. So a question is derived; whether SMs can fuse with cardiac muscle cells to promote regeneration of cardiac muscle needs to be further confirmed. Besides, SMs feature several unique advantages such as autologous origin, being readily available, easy harvest, a high scalability potential, a high resistance to ischemia, and lack of tumorigenicity [77]. Experiment data has proved their ability to migrate into infarct region, differentiate into myotubes [78], and promote angiogenesis [79], thereby improving cardiac function and attenuating cardiac remodeling. These results pave the way for clinical research and application of SM replacement therapy for ICM. SMs were the first cell type to be employed in the clinical trials. Most of the studies mainly pointed out the safety and feasibility of SM infusion therapy [80,81], but the efficacy is quite controversial. Several pilot studies showed to some degree improvement in LVEF and ventricular remodeling by SM fusion treatment. Patients with severe ICM injected with autologous SMs showed improvement of LVEF and New York Heart Association functional class after follow-up of 6 months or 11 months [82,83]. Although these studies showed the feasibility, safety, and modest effect of SMs grafting in ICM, a high incidence of ventricular arrhythmias event after myoblast transplantation requires immediate attention. Besides, a recent study with about 6-year long-term followup after autologous SM transplantation in seven patients with ischaemic heart disease implied that SM engraftment did not improve LV function [54]. Moreover, SM-treated group had higher incidence of interventions in patients fitted with an internal cardioverter defibrillator compared with control group (87% and 13%, resp.) [84]. Thus, SM therapy has shown a more certain therapeutic effect in the patients with chronic ICM than that of acute ICM which will appeal for many scientists to explore the therapeutic effect of SMs in AMI patients. Yet, these conflicting and vague results warrant initiation of larger randomized double-blind trials (NCT00526253) and engineered SMs should be exploited for further efficacy assessment in acute/chronic ICM. However, it is noteworthy that ventricular arrhythmias may be a potential risk during SM grafting treatment. At this time, SMs may not be a primary option for ICM/MI treatment.

Other Types of Stem
Cell. CD34 + cells and CD133 + cells are two distinct cell populations isolated from bone marrow or peripheral blood. Several clinical researches have been completed focusing on the cardiac repair by promoting angiogenesis and neovascularization in ischemic tissues [85]. Transendocardial injection of peripheral blood CD34 + cells into patients with ICM (LVEF < 40%) increased LVEF and 6-minute walk distance which demonstrated improved LV function and better exercise capacity [46]. Besides, CD34 + cell administration in patients with varying degrees of angina and myocardial ischemia showed significant improvements in angina frequency and exercise tolerance [44,45,86,87]. Major adverse effects were not observed in these CD34 + cell therapy researches. These results emphasize the feasibility and safety of CD34 + cell therapy by delivery of either intramyocardial injection or intracoronary infusion and the efficacy has close contact with the number of transplanted cells. CD133 + cell engraftment has also been approved to have positive effect for modest improvement in cardiac function without severe adverse effect in patients with chronic ICM through intramyocardial injections [50,51,88,89]. But, a clinical trial named Cardio133 proved no effect on LV function and clinical symptoms in patients with chronic ischemic heart disease and impaired LV function treated by intramyocardial injection of CD133 + BMCs [52]. Therefore, both of these two types of stem cells have shown more promising potential in chronic ICM treatment than AMI which deserve further investigations in the treatment of acute ICM. Overall, these results preliminarily show the feasibility and effectiveness of CD34 + or/and CD133 + cells engrafting therapy for ICM and present a promising alternative for cell grafting management in ICM patients.
Stem Cells International 7 Several ongoing researches are expected to get more effective results (NCT00950274 and NCT01337011). Further studies are required to develop new technologies to treat ICM. Thus more preclinical and clinical trials are needed to elucidate the effects of stem cell infusion on the progression of heart repair. Numerous other types of stem cells will be exploited to evaluate their practical abilities to treat ICM in the future.

Influence Factors of Stem Cell Therapy for ICM
By now, it is apparent that the current state of stem cell therapy is quite promising but faces severe situations. Firstly, most clinical results show us the benefit effect of stem cell grafting for cardiac repair regardless of the cell types and delivery ways. Secondly, the current results of stem cell therapy for ICM with high variability and diversity display mild to modest or even conflicting results in LV function and geometry and short-lived duration of cardiac improvement. Lastly, some stem cell types and delivery pathways displayed severe adverse effects such as cardiac arrhythmia and vascular restenosis which draw immediate attention of scientist and clinician. Many important factors affecting the efficacy of stem cell infusion in patients with ICM and MI mainly involve the following aspects: the delivery path of injection, the type and number of donor cells, the viability of fusion cells, and also the medical condition of patient. The delivery of stem cells is one of the most important factors to influence the curative effect which has been reviewed by some academics in detail [90][91][92]. Therefore, our discussion focuses on the other influence factors except delivery.

The Number and Phenotype of Stem Cells.
The phenotype of injected cells has much correlation with the safety and efficacy. At present, MSC transplantation in ICM patients is yielding more promising results and fewer adverse effects versus SM and is easier to be acquired versus CSC. Randomeffects meta-analysis performed on 888 animals through 52 studies suggested that BMMNC therapy showed less effect than MSC treatment. Sensitivity analysis described that efficacy was more relevant to higher cell number (≥10 7 ) and late injections (>1 week) [93]. A recent comparative clinical trial supported more efficacy in MSC-treated group rather than BMMNC-treated group [30]. Besides, the number of cells injected in patients with varying degrees of ICM varies from study to study. One clinical trial has shown that lowdose concentration MSCs (20 millions) resulted in greater reduction in LV volumes and increase in LVEF compared to high-dose groups (100 million and 200 million) [29], which was consistent with another comparison results that low-dose intramyocardial injection of CD34 + cells (1 * 10 5 cells/kg) had greater improvement in exercise tolerance and lower weekly angina frequency at both 6 months and 12 months than that of high-dose group (5 * 10 5 cells/kg) [45]. These instances show the importance of the nature and dosage of injected cells which need further grope and exploration in more detailed clinical studies in the future.

Isolation
Procedure. Cell preparation technologies are basically mature but to some extent variable because of the different incubation periods for screening and expansion. BMMNCs are readily available and injected into patients on the same day of harvest but other types of stem cells experience a process of selection, culture, and expansion which would take several days or weeks. Common methods of MSCs and CSCs in flasks involve a large number of open procedures and require prolonged culture times. Different centrifugation speed and washing buffer composition during the cell processing correlate much with therapeutic effect of ischemic heart disease [94]. Different protocols and regent components for cell isolation and storage held great distinctions in response to the functional capacity of bloodflow-recovery after BMMNC transplantation [95]. Red blood cell contamination during BMMNC isolation process also reduced cell viability, migratory function, colony-forming unit capacity, and neovascularization capacity [96]. A novel method, named Quantum Cell Expansion System, has been developed to generate higher numbers of MSCs in less time and at lower passages leading to a substantial reduction in pollution risk comparing with expansion in the flasks [97]. Thus, studies on establishing new technologies are underway to improve cell quality and quantity. Combination of preclinical and clinical studies will produce great amazing results in developing the best and simplest way to generate ideal stem cells.

Other Influence Factors.
In addition to the aspects above, the cell activity, the injection time, the period of followup, and patient's physical differences are also relevant to the therapeutic effectiveness of cell-based treatment. Infusion time varies from several days to several weeks after AMI. A study of infusion timing showed no differences in LV function improvement at 4 months after intracoronary infusion of BMMNCs at either 5 to 7 days or 3 to 4 weeks among 200 patients with ST-segment elevation myocardial infarction comparing with control group [39]. The TIME randomized trial showed no significant improvement in global or regional EF in patients with left ventricular dysfunction after intracoronary infusion of 150 × 10 6 BMMNCs at either 3 days or 7 days [98]. However, a pooled analysis of 7 randomized controlled trials enrolling 660 patients with AMI stated that bone MSC infusion at 4 to 7 days after AMI held greater improvement in LVEF and reduction of LV end-systolic dimensions compared to cell infusion within 24 hours which might link to the occurrence of acute inflammatory reactions immediately after AMI [99]. Besides, five-year follow-up study of intracoronary BMMNC infusion in 8 patients with stable severe ICM indicated no significant change in systolic and diastolic function [100], but 7-year follow-up of the Dan-Cell study proved a beneficial effect of bone marrow-derived stem cells engrafting in patients suffering from chronic ischemic heart failure [31]. The cardiac improvement was also associated with the seriousness of patient's condition. A better improvement occurred in the patients with greater degree of segmental left ventricular dysfunction after transendocardial injection of MSCs [33]. Besides, it has also been proved 8 Stem Cells International that CDCs derived from heart failure patients displayed a greater therapeutic benefit than those from MI hearts and healthy hearts [101]. Therefore, cell replacement therapeutics is a complex process with many comprehensive elements. The way to achieve the best outcome requires optimum balance conditions of these influence factors.

Low Survival and Residential Rate of Transferred Stem Cells in the Infarcted Myocardium and Strategies for Increasing Efficacy
Although stem cell therapy is emerging as a promising approach to benefit cardiac healing and prevent cardiac heart failure after MI, the low efficacy issue still deserves further concern. That is because of not only significant loss of transplanted cells due to blood circulation, myocardial contraction, and leakage from the injection site but also low survival rate of resident cells due to harsh environment resulting from ischemic, hypoxia, oxidative stress, or inflammatory response [90]. A variety of methods have been emerging to improve cell survival rate and enhance functional capacity of these stem cells for promising application. The improvement projects include cell pretreatment by environment conditions and pharmacological, genetic manipulation prior to stem cell delivery, cotransplantation of stem cells with extracellular matrix molecules, nanofibers, hydrogels, or fibrin glues, and combination therapy using two types of stem cells. Firstly, preconditioned cells can maintain a standby state by activating the cell survival signaling pathways to make them resistant to hostile environment. Hypoxic or shock wave preconditioning have been demonstrated effectively to prevent extensive apoptosis of transplanted cells through several processes including modification of cell phenotype, secretion of various cytokines, and increasing the transcription and translation of antiapoptotic genes [102][103][104][105]. Preclinical studies have shown that pretreating MSCs with transforming growth factor-alpha, basic fibroblast growth factor, interleukin-1 , or transforming growth factorimproved MSC-mediated myocardial protection and stimulated angiogenesis [106][107][108]. It has also been proved that lysophosphatidic acid and atorvastatin can rescue rat bone MSCs from hypoxic or hydrogen peroxide induced apoptosis [109][110][111][112]. Secondly, genetic engineering of MSCs has become a promising approach to improve the MSC performance to protect them from apoptosis, increase their retention rate, and enhance their ability of migration and differentiation and paracrine effect. Overexpression of prosurvival, antiapoptotic, proangiogenic genes or homing receptors has generated significant increase in the survival rate of MSCs and improved their migratory behavior which has been reviewed in detail by Park et al. [113] and Yin et al. [114]. Thirdly, codelivery of stem cells with extracellular matrix molecules, nanofibers, hydrogels, or fibrin glues could directly increase the retention rate of grafted cells and also promote cell survival and differentiation. Encapsulation of CSCs within matrixenriched hydrogel capsules prevented cell death and boosted the retention of CSCs [115]. Two latest studies have reported that the transplantation of MSCs cultured on nanomatrix or cardiac fibroblast-derived 3D extracellular matrix can successfully increase the retention rate of MSCs and promote cell proliferation, adhesion, and migration which demonstrated the potential application in regenerative therapy for ICM [116,117]. Another experimental study demonstrated the formation of thick stratum and angiogenesis of engrafted monolayered MSCs through paracrine pathways [118]. However, grafting of engineered sheet into patients has not been implemented in clinical study due to the unpredictable consequences of transplant rejection and arrhythmias, propelling further investigation for the future. Lastly, combined cell transplantation of autologous SMs and BMMNCs has been proved feasible and effective in patients with severe ICM without generating fatal arrhythmias and complications [53]. After combined transplantation of SMs and bone marrow cells, the patients showed significant improvement in cardiac function and angiogenesis and decreased fibrosis size [119]. Moreover, codelivery of human CSCs and human MSCs through intramyocardial infusion created a greater reduction of infarcted size, a stronger improvement in left ventricular contractility, and 7-fold enhanced engraftment of stem cells compared to each cell therapy group and placebo group which illustrated an important biological interaction between c-kit + CSCs and MSCs [120].
Thus, these preclinical and clinical data provide an important basis for the deeper research direction in the improvement of life quality and cardiac function. Therefore, so many fuzzy and unknown fields deserve further exploration for developing promising treatment methods of ICM.

Conclusion and Perspective
To summarize, the safety and feasibility of stem cell replacement therapy in ICM patients have been widely investigated in numerous clinical studies. MSC or BMMNC replacement therapy has been widely studied in the patients with acute or chronic ICM and shown a certain therapeutic effect. At present, MSC therapy shows more promising results in MI patients and makes a good foundation for the clinical application in the near future. CSCs is an emerging stem type which have been mainly investigated in AMI patients, so it owns a more developing space in ICM patients. Although SMs, CD34 + , and CD133 + have been grafted in patients with ICM, they show less application potential than MSC or CSC treatment. Similar results to the transplantation of these types of stem cells are modest or discrepant improvement. However, the inconsistent results of efficacy evaluation are not a surprise to us due to different techniques and doses of cells delivered via multiple routes and patients with different conditions. On the whole, MSCs and CSCs appear to have greater application potential from the results of comprehensive analysis. As to cell delivery routes, intramyocardial and intracoronary show more promising future than intravenous injection. The action mechanisms of administrated cells are mostly inclined to paracrine effect by releasing of cytokines, chemokines, and growth factors to inhibit cell apoptosis and fibrosis and activating endogenous regenerative system. Other elements need to be further investigated to achieve Stem Cells International 9 the maximum effect regarding the optimal separation process, ideal cell type, cell dose, timing and route of delivery, and clinical indications. While a large number of comparative analysis studies provide very important outcomes, they are still not enough to offer the best choice of therapeutic program. Larger-scale randomized clinical studies are underway to optimize the therapeutic effect. Moreover, other cell types like human umbilical blood MSCs, umbilical cord MSCs, and adipose tissue-derived MSCs show a good prospect in the application of ICM treatment according to large amounts of preclinical data. This detailed summary will stimulate profound interest in pursuing additional and larger-scale clinical trials. We believe that through continuing and collaborative efforts multipotent stem cells transplantation will produce a satisfactory reply to ICM patients.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Engineering

text:
Synergistic function of four novel thermostable glycoside hydrolases from a long-term enriched thermophilic methanogenic digester

In biofuel production from lignocellulose, low thermostability and product inhibition strongly restrict the enzyme activities and production process. Application of multiple thermostable glycoside hydrolases, forming an enzyme “cocktail”, can result in a synergistic action and therefore improve production efficiency and reduce operational costs. Therefore, increasing enzyme thermostabilities and compatibility are important for the biofuel industry. In this study, we reported the screening, cloning and biochemical characterization of four novel thermostable lignocellulose hydrolases from a metagenomic library of a long-term dry thermophilic methanogenic digester community, which were highly compatible with optimal conditions and specific activities. The optimal temperatures of the four enzymes, β-xylosidase, xylanase, β-glucosidase, and cellulase ranged from 60 to 75°C, and over 80% residual activities were observed after 2 h incubation at 50°C. Mixtures of these hydrolases retained high residual synergistic activities after incubation with cellulose, xylan, and steam-exploded corncob at 50°C for 72 h. In addition, about 55% dry weight of steam-exploded corncob was hydrolyzed to glucose and xylose by the synergistic action of the four enzymes at 50°C for 48 h. This work suggested that since different enzymes from a same ecosystem could be more compatible, screening enzymes from a long-term enriching community could be a favorable strategy.

Introduction
Enzymes catalyzing the hydrolysis of cellulose and hemicellulose are a source of worldwide interest for alternative bio-energy development and other environmental applications, such as treating wastewater from the dying, textile and pulping industries. Many enzymes are used in these Abbreviations: SHF, separate hydrolysis and fermentation; SSF, simultaneous saccharification and fermentation; GH, glycoside hydrolase; CMC, carboxymethylcellulose; pNPX, p-nitrophenyl-β-D-xyloside; pNPG, p-nitrophenyl-β-D-glucoside; MIC, microcrystalline cellulose; pNPA, pNP-α-L-arabinofuranoside; pNPC, pNP-β-D-cellobioside; ORF, open reading frame; FPA, filter paper activity; TLC, thin-layer chromatography. processes, including exoglucanase (EC 3.2.1.91), endoglucanase (EC 3.2.1.4), and β-glucosidase (EC 3.2.1.21) for cellulose hydrolysis; xylanase (EC 3.2.1.8) and β-xylosidase (EC 3.2.1.37) to hydrolyze xylan; and α-L-arabinofuranosidase (EC 3.2.1.55) and α-glucuronidase (EC 3.2.1.39) to hydrolyze side chains in hemicellulose (Beg et al., 2001). To utilize these enzymes in biofuel production, two processes are generally used: separate hydrolysis and fermentation (SHF), and simultaneous saccharification and fermentation (SSF). Temperatures of hydrolysis in SHF and SSF are around 50 and 32 • C, respectively (Tomas-Pejo et al., 2008). SHF has been reported to be more efficient and cost-effective than SSF (Magnus et al., 2012) because it uses thermostable enzymes, which improves the solubility of substrates and enhances the mass transfer rate, while reducing cooling requirements and the probability of bacterial contamination (Krahe et al., 1996). However, product inhibition can be a major source of inefficiency in SHF, especially the inhibition on cellulase and xylanase (Zhang and Lynd, 2004). Therefore, combinations of relevant enzymes, forming a so-called enzyme cocktail, have been intensively investigated in recent decades (Zhou et al., 2009;Banerjee et al., 2010). For example, the combination of exoglucanase, endoglucanase, and β-glucosidase is used to increase cellulose degradation efficiency (Gusakov et al., 2007), and the combination of xylanase and β-xylosidase has been shown to enhance hemicellulose hydrolysis (Fan et al., 2009). In general, these cocktails are formed from widely used commercial enzymes, such as the cellobiohydrolases I and II and endoglucanases I and II from the fungus Trichoderma reesei (Henrissat et al., 1985). However, since the host microorganisms from which these enzymes were originally isolated are from different habitats and have different optimal growth conditions, the isolated enzymes generally also have different optimal conditions, and may not be compatible in the same cocktail.
Metagenomic approaches for screening novel enzymes have the potential to increase the compatibility of the candidate enzymes in a cocktail, because these metagenome-oriented enzymes can be from the same microbial community, living in the same habitat, and even in the same niche. For example, hundreds of lignocellulose hydrolases were screened from metagenomic libraries of the rumen (Hess et al., 2011), the gut system of termites (Warnecke et al., 2007), soil (Mcandrew et al., 2013), and biogas digesters (Yan et al., 2013). Nevertheless, more hydrolases are believed to be awaiting discovery in the "microbial dark matter" (Rinke et al., 2013). So far, most of this research has focused on the desirable properties of the discovered enzymes, such as thermostability, ionic strength, alkaline tolerance, activity at cold temperatures, and wide substrate specificity. Comparatively few efforts have been made to investigate their compatibility and synergistic actions (Del Pozo et al., 2012;Geng et al., 2012;Gruninger et al., 2014).
For obtaining thermostable enzymes, we constructed a metagenomic library from a dry thermophilic methanogenic digester community enriched mainly with paper for over 10 years (Tang et al., 2011). We isolated and identified four novel thermostable hydrolases and found them to have high compatibility as a synergistic cocktail for hydrolyzing cellulose, xylan, and steam-exploded corncob. This work further proved that metagenomic screening is an effective way for finding compatible enzymes.

Analysis of Fosmid Metagenomic Library
The genomic DNA of the thermophilic methanogenic digester community, as described previously (Tang et al., 2011), was extracted using FastDNA R SPIN Kit for soil (MP, Santa Ana, America). The DNA purification, ligation, and transformation were performed according to the manufacturer's instructions of Copy Control ™ Fosmid Library Production Kit (Epicentre, Madison, America). A fosmid library was constructed, and the analysis of insert DNA fragments was carried out as described previously (Hu et al., 2010). Activities of cellulase and xylanase were screened from the library with the Congo red method (Wood et al., 1988). To screen β-glucosidase activity, the ammonium ferric citrate-esculin hydrate method (Eberhart et al., 1964) was used, while pNPX (p-nitrophenyl-β-D-xyloside) was used to identify β-xylosidase (Poutanen and Puls, 1988). Fosmid clones with positive activities were sequenced using the GS FLX system (Roche, Basel, Switzerland). The sequences were then assembled with the putative open reading frames (ORFs) predicted using SoftBerry fgenesb (Warnecke et al., 2007). The deduced proteins were then annotated against the NCBI nonredundant database. The Pfam (Finn et al., 2014) and CAZY (Lombard et al., 2014) databases were used to identify conserved domains and glycoside hydrolase families, respectively. Signal peptides were identified using SignalP 4.1 (Petersen et al., 2011). Homology models of the proteins were predicted from the SWISS-MODEL server (Arnold et al., 2006) and visualized using PyMOL (Schrodinger, 2010). Active sites of the proteins were identified by multiple sequence alignment (Mcwilliam et al., 2013). The composition of amino acid residues was analyzed online (http://www.bio-soft.net/sms/). In total, four genes from three fosmid clones were selected for further study: Xyl522, coding for β-xylosidase; Xyn526, coding for xylanase; Bgl8520, coding for β-glucosidase; and Cel1753, coding for cellulase. Phylogenetic analysis was performed by MEGA5.1 with the best hits in BLASTP and characterized protein sequences from the same protein family, using the neighbor-joining algorithm.

Cloning, Expression, Purification, and Characterization of Target Enzymes
The four target genes were PCR amplified from the corresponding fosmids with the primers listed in Supplementary  Table 1 using PrimeSTAR HS DNA Polymerase (Takara, Dalian, China) according to the manufacturer's instructions. After purification and double digestion, the PCR amplicons were cloned into pET-28a (+) (Qiagen, Hilden, Germany). Recombinants of Xyl522, Xyn526, Bgl8520, and Cel1753 were further transformed into E. coli BL21 (DE3) (CoWin Biotech, Beijing, China). Recombinant cells were cultured in Lysogeny Broth medium with 50 µg/mL kanamycin and induced by 1 mM IPTG at 37 • C for 4 h. Cells were harvested and subjected to freeze-thaw lysis. Next, the cell lysate was centrifuged at 4 • C, 12,000 × g for 30 min, and the supernatant was collected for protein purification though Ni-NTA resin (GE Healthcare, Uppsala, Sweden) according to the manufacturer's instructions. The protein concentration was determined using the BCA Protein Assay Kit (Tiangen, Beijing, China).
Enzyme activities were detected as follows. pNPX (25 mM final concentration) and pNPG (p-nitrophenyl-β-D-glucoside, 25 mM final concentration) were mixed with 1 µg Xyl522 and 1 µg Bgl8520, respectively, each to a final volume of 40 µL (pH 5.0). After incubation at 50 • C for 5 min, 50 µL 1 M Na 2 CO 3 was added to terminate the reaction. The concentrations of p-nitrophenol product were determined by detecting the absorbance at 410 nm using spectrophotometer UV1700 (Shimadzu, Kyoto, Japan). The specific activities (U) of β-xylosidase and β-glucosidase were shown in micromole pnitrophenol produced from pNPX and pNPG per minute per milligram of protein. Similarly, beech wood xylan (1%, w/v final concentration) and CMC (carboxymethylcellulose, 1%, w/v final concentration) were mixed with 5 µg Xyn526 and 5 µg Cel1753, respectively, to a final volume of 200 µL (pH 5.0). After incubation at 50 • C for 15 min, the reaction mixtures were added to 500 µL dinitrosalicylic acid and boiled for 5 min to terminate the reactions (Miller, 1959). Concentrations of released xylose or glucose were determined by analyzing the absorbance of the reaction mixtures at 540 nm. Activities of cellulase and xylanase were defined as micromole xylose and glucose produced per minute per milligram of protein. The determination of filter paper activity (FPA) was performed as previously described (Zhang et al., 2010); FPA was defined as micromole glucose produced per minute per milligram of protein.
The pH and temperature optima of the four enzymes were determined in the range of pH 3.0-12.0 (Xyl522, pH 3.5-7.0; Xyn526, pH 3.5-11.0; Bgl8520, pH 4.0-9.0; and Cel1753, pH 3.5-9.0) and 20-90 • C (Xyl522 and Bgl8520, 20-85 • C; Xyn526, 20-90 • C; and Cel1753, 20-80 • C). To maintain suitable pH values, citric acid-Na 2 HPO 4 buffer (pH 3.0-8.0), Tris-HCl (pH 8.0-10.0) and Na 2 HPO 4 -NaOH (pH 10.0-12.0) were used. For determination of optimal temperatures, pH was maintained at 5.0, for consistency with conditions in the methanogenic digester community from which the enzymes originate. To test for pH stability, the four enzymes were incubated in the buffer solutions mentioned above with corresponding pH values at 4 • C for 24 h, after which their residual enzyme activities were measured as described above. For example, Xyn526 was incubated at 4 • C in buffer solutions ranging from pH 3.5 to 12.0 for 24 h, after which mixtures containing 5 µg xylanase protein were used for activity determination as described above. To test the thermostability of the four enzymes, Xyl522 and Bgl8520 were incubated at 50, 55, 60, and 75 • C; Xyn526 incubated at 50, 65, 70, and 75 • C; and Cel1753 incubated at 50, 60, 65, and 70 • C for 2 h in buffer solutions of pH 4.5, 6.0, 5.0, and 5.0, respectively. The residual activities of all enzymes were tested at 50 • C and pH 5.0. Briefly, mixtures containing 1 µg Xyl522 and Bgl8520 each, or 5 µg Xyn526 and Cel1753 each were sampled every 20 min until 120 min, and enzyme activity was determined for samples taken at each time point. In addition, CMC, microcrystalline cellulose (MIC), beech wood xylan, pNPG, pNPC (pNP-β-D-cellobioside), pNPX, and pNPA (pNP-α-L-arabinofuranoside) were used to investigate the substrate specificity of the enzymes. K m and V max were calculated by Lineweaver-Burk plot based on enzyme activities at their optimal conditions with different substrate concentrations (1-50 mM for pNPG, pNPX, pNPA, and pNPC; 1-50 g/L for xylan beech wood, xylan oat spelt, CMC, and MIC).
Steam-exploded corncob was prepared according to a previously described protocol (Teng et al., 2010) with a dry weight of 32%, which was composed of cellulose (∼35%), hemicellulose (∼38%) and lignin (∼18%). The corncob biomass (2%, w/v) were incubated with Cel1753 (40 FPA/g, dry weight, as follows), Xyn526 (200 U/g), a cocktail of Cel1753 (40 FPA/g), and Bgl8520 (50 U/g), a cocktail of Xyn526 (200 U/g) and Xyl522 (50 U/g), and a cocktail of all four enzymes at 50 • C, pH 5.0 for 72 h. Enzyme dosages used above were the minimum ones to achieve the saturated hydrolysis of corncobs by itself. The concentrations of glucose, cellobiose, xylose, and xylobiose were measured using a Prominence Liquid Chromatograph-20A (Shimadzu) according to the manufacturer's instructions. Briefly, a fluorometric detector and a shim-pack ISA-07 column were used. The system was operated at 65 • C and a flow rate of 0.6 mL/min using H 3 BO 3 -KOH as the mobile phase with 1% w/v L-arginine for product derivatization. Calculation of saccharification efficiency was performed as described previously (Del Pozo et al., 2012).
The predicted models of enzyme structure (Supplementary Figure 4) suggested that the four enzymes all had a classical (α/β) 8 TIM barrel fold structure. Xyl522, a member of the GH 3 family, also contained an immunoglobulin-like region, which has been proven to contribute to both substrate binding and dimerization in this family (Mcandrew et al., 2013). Additionally, the distance between the two active sites in Xyl522 was calculated as 5.4 Å, In Bgxa1, a multifunctional β-glucosidase/β-xylosidase/αarabinosidase, the same distance is 6.1 Å (Gruninger et al., 2014). Although Bgxa1 and Xyl522 are from the same GH 3 family, this distinction might suggest different substrate specificities.
Cloning, Expression, Purification, and Characterization of Xyl522, Xyn526, Bgl8520, and Cel1753 Xyl522, Xyn526, Bgl8520, and Cel1753, without their signal peptides, were heterologously expressed and purified. Plate-based function screening confirmed each protein was responsible for the assumed activities, further supporting the results of sequence alignment. Xyn526 and Cel1753 showed 79.6 U/mg and 174.0 U/mg specific activities for the hydrolysis of beech wood xylan and CMC, respectively. However, Xyn526 had relatively weak hydrolyzing activity against pNPX, pNPG, and CMC (Table 1). Cel1753 also exhibited hydrolyzing activity for MIC and filter paper, suggesting that it has potential for hydrolyzing cellulose alone. These results revealed that Cel1753 harbored a high proportion of exoglucanase activity relative to its endoglucanase activity (21.8%). This is in contrast to most cellulases, which do not possess both activities (Kim et al., 2008;Ko et al., 2011). Xyl522 could hydrolyze pNPX more effectively than pNPA or pNPG. Bgl8520 could hydrolyze pNPG 50 times more efficiently than pNPC (Table 2), which combined with its relatively low K m for pNPG indicated a higher affinity and efficiency for glycosidic bonds in cellobiose. In addition, Bgl8520 has a higher affinity toward pNPC than other β-glucosidases whose K m are normally between 4.8 and 17.6 mM (Del Pozo et al., 2012), indicating its strong capacity on cellotriose hydrolysis. It is notable that no obvious activities of Xyl522 and Bgl8520 were detected against CMC, MIC, or xylan. Our results therefore suggest that pNPG, xylan from beech wood, pNPX, and CMC were the optimal substrates for Xyl522, Xyn526, Bgl8520, and Cel1753, respectively.
range, with over 80% hydrolysis activity between pH 4.0 and 5.0, and dramatically lower activity at pH values outside this range. In contrast, Xyn526, Cel1753, and Bgl8520 kept over 80% hydrolysis activity within the pH ranges 4.0-11.0, 3.5-8.0, and 4.5-7.5, respectively. Xyn526 and Cel1753 had high pH stabilities, retaining greater than 80% enzyme activity after incubation at 4 • C for 24 h in pH 4.0-12.0 and pH 3.5-8.0, respectively ( Figure 1B). Xyn526 had the highest optimal temperature of 75 • C (Figure 2A); the corresponding values for Xyl522, Bgl8520, and Cel1753 were 60, 65, and 60 • C, respectively. These optimal temperatures were generally higher than lignocellulose hydrolases isolated from other metagenomic libraries so far, the majority of which were below 60 • C (Duan and Feng, 2010;Yan et al., 2013). In addition, Xyl522, Xyn526, Bgl8520, and Cel1753 had notably high temperature stabilities, retaining over 80% residual activity after incubation at 50 • C for 2 h (Figure 2), with thermal half-life being 14, 48, 10, and 32 h, respectively (Supplementary Table 3). Even after 2 h incubation at 70 • C, Xyn526 still had 82.3% residual activity toward xylan, whose thermal half-life was impressively 8 h under the same condition. In comparison, the thermostabilities of many other metagenomic enzymes are relatively weak. For example, cellulase En1 from a 40 • C biogas digester metagenome (Yan et al., 2013) and glucoside hydrolase Bgxa1 from a dairy cow rumen metagenome (Gruninger et al., 2014) could only retain 80 and 10% activities respectively after incubation at 50 • C for 1 h. Xylanase Xyn10N18 from a dairy cow rumen metagenome (Gong et al., 2013) and β-xylosidase RuBG3B from a yak rumen metagenome (Bao et al., 2012) lost all of their activity under the same conditions. Similarly, the alanine, proline, arginine, and glutamic acid content in these proteins were also generally less than those of our four enzymes, while the glycine, serine, lysine, and aspartic acid content was higher. For instance, the percentages of alanine, proline, and arginine residues in cellulase En1 were 4, 2, and 2% less respectively than those in Cel1753; glutamic acid content was the same between the two enzymes. In contrast, the percentages of aspartic acid and lysine residuals in cellulase En1 were 1 and 3% higher respectively than those in Cel1753, although they shared the same percentage of glycine and serine content.

Synergistic Hydrolysis of CMC, Xylan, and Steam-exploded Corncob
Cel1753 could hydrolyze CMC to produce mainly cellooligosaccharides along with small amounts of glucose and cellobiose, whereas the Bgl8520 could hardly hydrolyze CMC (Figure 3A). When a cocktail of Bgl8520 and Cel1753 was added to CMC, production of glucose increased markedly compared with Cel1753 working alone, whereas the production of cellobiose and other cello-oligosaccharides decreased, suggesting the efficient degradation of CMC. Similarly, the cocktail of Xyn526 and Xyl522 resulted in higher xylose production from beech wood xylan, with xylobiose completely hydrolyzed ( Figure 3B).
Synergistic action of these enzymes was also detected when steam-exploded corncob was used as substrate. Compared with Cel1753 alone, addition of Bgl8520 significantly increased the yield of glucose from steam-exploded corncob from 11.8 to 52.3%, while the yield of cellobiose in this cocktail decreased dramatically ( Figure 4A). Moreover, glucose production was much quicker in the first 12 h, revealing the synergistic action of the two enzymes and its benefit. Similarly, the supplementation of Xyn526 with Xyl522 resulted in a 56.2% increase in the yield of xylose. This supplementation also simultaneously accelerated the hydrolysis of xylan by consuming xylobiose, which might be attributed to a reduction of Xyn526 inhibition (Figure 4B). When all four enzymes were pooled in a cocktail, xylose and glucose production increased by around 8.3 and 20.5%, respectively, resulting in total hydrolysis efficiency for cellulose and hemicellulose of 71.5 and 87.1% in 48 h at 50 • C, respectively (Figure 4). This incremental effect on catalytic efficiency was higher than seen in previous studies of metagenomic enzymes. For example, supplying a metagenomic β-glucosidase into commercial Trichoderma reesei cellulase resulted in a relative increase of 20% glucose yield from corn stover at 50 • C (Del Pozo et al., 2012). The mixture of two metagenomic enzymes, β-xylosidase and xylanase, resulted in a relative increase of 37% Enzymes activities were determined with pNPX, xylan, pNPG, and CMC as substrates for Xyl522, Xyn526, Bgl8520, and Cel1753, respectively. Standard deviations are shown from triplicate measurements.

Discussion
In the biofuel industry, increasing the thermostability of enzymes is important for reducing operational cost. Many attempts have been made to isolate thermostable enzymes, from organisms such as Clostridium absonum (Rani and Nand, 2000) and Thermoascus aurantiacus (Hong et al., 2007). Because of product inhibition, using of a single enzyme usually leads to low efficiency in the lignocellulose hydrolysis process (Gusakov and Sinitsyn, 1992). However, enzyme cocktails composed of different enzymes can form a "working team", which is similar as the different enzymes in a single cell (Nie et al., 2014), to sequentially degrade lignocellulose and its intermediates. These enzyme cocktails have proven to be a suitable solution to product inhibition and have therefore drawn worldwide research interest Gao et al., 2011). To form an efficient cocktail, candidate enzymes need to have compatible pH and temperature optima as well as compatible specific activities. Cocktails have generally been based on commercial enzymes isolated from different microorganisms, which may have different optimal growth conditions. Recently, the isolation of novel enzymes, including thermostable enzymes from metagenomic libraries, has been extensively studied, leading to the isolation of many enzymes with special characteristics such as being cold- (Wierzbicka-Wos et al., 2013) or heat-active (Schroder et al., 2014), and ionic liquid- (Ilmberger et al., 2012) or product-tolerant (Lu et al., 2013). Microorganisms in the same metagenome  generally live together for a relatively long time, and should have similar tolerance for environmental conditions, such as temperature, pH, and oxidation-reduction potentials, which may result in isolation of enzymes with similar optimal conditions. Metagenomic screening may therefore offer a way of increasing the compatibility of different candidate enzymes in a cocktail. However, to date there have been few reports of successful trials using a cocktail of enzymes from the same metagenome. Therefore, we identified four novel thermostable enzymes and reported the first trial of a combination of compatible enzymes from the same metagenome.
The four enzymes we isolated from a long-term enriched thermophilic microbial community had higher thermostability (Figure 2) than those from metagenomes sampled from mesophilic soil, sea sediment, and animal gastrointestinal tracts, whose optimal temperatures were generally under 60 • C (Duan and Feng, 2010), and whose thermostabilities were relatively poor (Feng et al., 2007;Gong et al., 2012). Although a β-glucosidase has been previously found (from screening a hydrothermal spring metagenome) with an optimal temperature of 90 • C (Schroder et al., 2014), its observed relative activity at 50 • C was only 20%. Similarly, although one β-glucosidase (LAB25g2 from cow rumen) retains as much as 82% activity after 5 days of incubation at 50 • C, it required several days to consume cellobiose completely, suggesting its high thermostability with relatively low hydrolysis efficiency (Del Pozo et al., 2012). We attribute the high thermostabilities of our four enzymes to the fact that they were isolated from a thermophilic methanogenic digester community. This community has been enriched with paper at 53 • C for longer than 10 years (Tang et al., 2011), which should have provided a stronger high-temperature selective pressure than that found in the gastrointestinal tracts of ruminants (Yan et al., 2013). Furthermore, the unusual amino acid residue composition of these enzymes may also contribute to their thermostabilities. Lu and colleagues, 1998 reported that there are remarkable differences in amino acids composition between mesophilic and thermophilic proteins. In this research, although the thermostable enzymes we isolated share high amino acid sequence similarities with the mesothermal stable enzymes ( Supplementary Figures 5-8), the thermostable enzymes have higher relative ratios of alanine, proline, arginine, and glutamic acid residues and lower ratios of glycine, serine, lysine, and aspartic acid residues (Supplementary Table 4). Alanine tends to form a stable helix, while the reduced number of glycine residues could lessen flexibility of loops and therefore provide heat resistance (Yip et al., 1995). The structural entropy of proline is less than other amino acids as it requires more energy to unfold; its presence can therefore enhance the thermostability of a protein (Vogt et al., 1997). The presence of arginine, which has a large side chain with a delocalized positive charge, can promote the formation of salt bridges and hydrogen bonds to enhance protein stability (Kumar et al., 2000). Lower levels of hydrophilic amino acids such as serine and lysine have also been linked with thermostability (Argos et al., 1979;Lu et al., 1998). Therefore, we propose that the unusual amino acid composition of the four enzymes we isolated did not change their molecular conformation, but did increase their thermostability (Figure 2).
Using a cocktail composed of the four lignocellulose hydrolases, we observed enhanced hydrolysis of steam-exploded corncob after incubation at 50 • C for 72 h, suggesting the compatible thermostabilities and synergistic cooperation of these enzymes. It is reasonable that Xyl522 and Bgl8520 could prevent product inhibition of Xyn526 and Cel1753, respectively. As cellulose is often embedded in hemicellulose and lignin (Jeoh et al., 2007), the hydrolytic activity of Xyn526 on hemicellulose could expose more cellulose to Cel1753, along with Xyl522 and Bgl8520 in the four-enzyme cocktail, which could further improve the yield of monosaccharide and stimulate lignocellulose hydrolysis in this community (Figure 4). Indeed, Xyl522 and Xyn526 were cloned from the same fosmid clone (F52), suggesting they might be from the same host organism and potentially hydrolyze xylan synergistically in situ. The presence of a signal peptide in Xyn526 indicates that it is secreted out of cells to hydrolyze xylan. The resulting oligosaccharides, such as xylo-oligosaccharides and arabinooligosaccharides, could be then transported into the cytoplasm by carbohydrate ABC transporters, which were found to be enriched in F52. Glucoside hydrolases including Xyl522 could then degrade these oligosaccharides into monosaccharides like xylose and arabinose for further metabolism (Supplementary Figure 1). In addition, the extracellular Cel1753 could hydrolyze cellulose outside cells, which might also be transported into the cells with enriched ABC transporters, such as those found in the F85 fosmid clone (Supplementary Figure 2). These could then be hydrolyzed by non-signal peptide β-glucosidases, such as Bgl8520. It is reasonable to assume that such a complex cooperation can only be achieved in a cocktail of enzymes from the same well-enriched metagenome, because all the derived enzymes had similar optimal temperatures, thermostabilities, and pH values, as well as compatible specific activities, and this favorable compatibility was the basis of their synergism and high efficiency. It is notable that our enzyme cocktail was relatively weak in hydrolysis capacity compared with cocktail Accellerase R Trio ™ , which is composed of cellulase, xylanase, and β-glucosidase, and can hydrolyze 75-90% of glucan and xylan at 50 • C in 72 h. In order to improve the catalytic efficiency of our cocktail to reach industrial standards, we also need to use the strategy of developing industrial enzymes, such as molecular modification (Banerjee et al., 2010). In addition, since the original thermophilic methanogenic digester community could nearly completely convert paper to methane within 72 h (Tang et al., 2011), there should be more powerful enzymes, for more powerful enzyme cocktail, still in the dark matter waiting to be explored.
In summary, four novel thermostable glycoside hydrolases were isolated and identified from a metagenomic library constructed from a long-term dry thermophilic methanogenic digester community. The isolated enzymes and their mixed cocktail were highly effective in the hydrolysis of lignocellulose such as steam-exploded corncob. Since enzymes from the same metagenome may have high compatibilities and act synergistically, these experiments offer a way of screening compatible enzymes to form highly efficient enzyme cocktails.

Author Contributions
MW, YN, ZS, and XW designed and coordinated the study and write the manuscript. MW, GS, GL, and BZ carried out the experiments and analyzed the results. LL and XW contributed preparing the manuscript. All authors read and approved the final manuscript. ZS, YN, and XW contributed equally to this work.