Preconditioning CPCs could provide drastic improvements in cardiac structure and function; however, a systematic approach to determining a mechanistic basis for these modifications founded on the physiology of CPCs is lacking. We have identified a novel property of CPCs to respond to electrical stimulation by initiating intracellular Ca2+ oscillations. We used confocal microscopy and intracellular calcium imaging to determine the spatiotemporal properties of the Ca2+ signal and the key proteins involved in this process using pharmacological inhibition and confocal Ca2+ imaging. Our results provide valuable insights into mechanisms to enhance the therapeutic potential in stem cells and further our understanding of human CPC physiology.For nearly a century, the heart was viewed as a terminally differentiated organ until the discovery of a resident population of cardiac stem cells known as cardiac progenitor cells (CPCs). It has been shown that the regenerative capacity of CPCs can be enhanced by Several laboratories have demonstrated the feasibility and utility of ex vivo manipulation of adult stem cells modified by genetic engineering [The heart was considered a postmitotic organ incapable of regeneration until the discovery of a resident population of cardiac stems cells brought about the potential for cardiac tissue regeneration . Known amyocytes , 3–7. Demyocytes , 8, rece in vivo , 5, 7. Hineering –13 or exineering , 14–17. 2+) signaling in adult cells [2+ is an integral second messenger in the heart, regulating the processes of excitation-contraction coupling  and excitation-transcription coupling . Previous reports showed that electrical stimulation of adult cardiac adipose tissue-derived progenitor cells caused changes in cell phenotype and genetic machinery, making them more suitable for cardiac regenerative approaches [ de novo expression of the late cardiac sarcomeric proteins, troponin T and cardiac alpha actinin, and increase the expression of connexin 43 and its relocation to the cell membrane [2+ is involved in assembly of contractile apparatus and localization of key cardiogenic transcription factors, such as myocyte enhancer factor 2c, in CPCs [2+ handling in CPCs would substantially advance our knowledge of CPCs physiology and their potential in cardiac repair, providing fundamental information that could be used for developing new therapeutic approaches to improve myocardial repair and regeneration.Electrical stimulation is one treatment known to enhance cardiogenic potential of various stem cells via activation of calcium  used in this study were obtained under IRB approval which allows for isolation of CPCs from deidentified atrial waste tissue removed during surgical repair of congenital heart defects. Patients were not subject to any additional risks by involvement in this study. Confidentiality of patient information was assured as we did not record the patients' names or demographic information and did not contact the patients. A clinical research nurse obtained needed information such as age, gender, ethnicity, diagnosis, and drug therapy, but tissue samples were deidentified and data obtained from the biopsy were identified by an internal lab number only. No preference as to gender, race, or ethnicity was used for patient enrollment.2+ channel and the inositol 1,4,5-trisphosphate receptor were obtained from Santa Cruz Biotechnology , and primary antibody for the ryanodine receptor and secondary antibodies Alexa Fluor 488 and Alexa Fluor 647 were purchased from Thermo Fisher Scientific .All chemicals and reagents were purchased from Sigma-Aldrich , unless noted otherwise. Stock solutions of tetracaine and 2-aminoethoxydiphenyl borate (2-APB) were made in methanol and ethanol, respectively, and diluted to work concentrations in Tyrode's solution. The vehicle was added to Tyrode's solution for control experiments. Primary antibodies for the L-type Caμm filter. Cells were incubated with Dynabeads  conjugated to a c-kit antibody  prior to magnetic sorting. Sorted cells were plated in a T-75 tissue culture flask and expanded to confluence. Cells were pooled from 4–6 donors to reduce variability. CPC culture media consisted of Ham's F-12 basal media  along with 10% fetal bovine serum , 1% penicillin, 1% streptavidin, 1% L-glutamine, and 0.1 μg/mL basic fibroblast growth factor. Prior to experiments, cells were plated on glass coverslips and allowed to attach overnight at 37°C.Human child (aged 1–5 years) cardiac progenitor cells were isolated from biopsied tissue using c-kit antibody-conjugated magnetic beads and propagated in culture as previously described . BrieflyμM fluo-4/AM for 20 min followed by a 20 min wash in Tyrode's solution for deesterification of the dye. Electrical field stimulation was applied using an IonOptix MyoPacer Cell Stimulator  and fluo-4 was excited at 488 nm and emission collected at 515 ± 15 nm using laser scanning confocal microscopy . Calcium oscillation measurements were acquired from CPCs during 0.5 or 1 Hz stimulation bathed in Tyrode's solution, which contained (in mM) 130 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 D-glucose, and 10 Hepes, pH 7.4 with NaOH. All experiments were performed at room temperature (20–24°C). Data were analyzed using Olympus FV1000 FluoView software and Fiji [Cells were loaded with 10 and Fiji .μM) in Tyrode's solution and the indicator was excited at 488 nm, and emission was measured at >600 nm.Cell membranes and transverse tubular structures were visualized with the membrane-bound fluorescent probe Di-8-ANEPPS  by 2-dimensional confocal microscopy. Cells were loaded for 15 min with Di-8-ANEPPS  and secondary antibodies for 2 hrs at dilution of 1 : 500. After washing with PBS, VECTASHIELD HardSet Antifade Mounting Medium containing DAPI  was used to mount the coverslips onto glass slides for visualization.CPCs were plated on glass coverslips, fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton, and blocked with 3% BSA/PBS for 2 hrs. Cells were treated with the appropriate primary  and incubated in stimulation media. CPC stimulation media consisted of Ham's F-12 basal media along with 10% fetal bovine serum, 1% penicillin, 1% streptavidin, 1% L-glutamine, and 2 mM CaCl2. The C-Pace EP Culture Pacer  was used for culture stimulation at 0.5 Hz, 10 ms duration, 32 V for 72 hrs. Media were changed every 24 hrs.CPCs were seeded at a density of 4 × 102+-free media for 72 hrs were also processed and stained as described below for basal and Ca2+-dependent alignment quantification. Following three washes in 1x PBS, the cells were permeabilized with 0.1% Triton in 1x PBS. Cells were again washed and then blocked in 3% bovine serum albumin for 1 hour at room temperature. Cells were then stained with 10 μg/mL fluorescein-5-maleimide for 1 hour at room temperature in the absence of light, followed by washing. Cells were washed prior to imaging on an Olympus IX70 inverted fluorescent microscope . Two-dimensional fluorescent images were acquired using the 20x objective and analyzed with Fiji software using the directionality function to obtain the primary angle of the cells and the percent of cells within one standard deviation of the direction of the applied electrical field [After 72 hours of electrical stimulation, cells were immediately fixed in 4% paraformaldehyde for 20 minutes at room temperature. Unstimulated cells cultured in stimulation media for 72 hrs or cells stimulated in Caal field .F/F0, where F0 is the basal fluorescence in resting cells prior to electrical stimulation  or the diastolic fluorescence just before an oscillation (for subsequent oscillations). Fluorescent cellular images are presented as two-dimensional images representative of multiple trials. Summary data are presented as the mean ± SD of n measurements, where n is the total number of cells from 4 different pools of cells. Statistical comparisons between groups were performed with Student's t-test. Differences were considered statistically significant at p < 0.05.Fluorescence traces and confocal line scan data are presented as individual observations representative of multiple recordings or as the average of multiple recordings. Fluorescence traces were background subtracted and plotted as + CPCs by a method previously described by our laboratory [2+ dye fluo-4/AM and stimulated at 0.5 or 1 Hz. 2+ oscillations in CPCs elicited by electrical stimulation. A rapid rise in cytosolic Ca2+ was observed followed by frequency-independent cytosolic Ca2+ oscillations . Quantification of the amplitude of the Ca2+ oscillations showed no significant difference between the initial oscillation amplitudes at 0.5 or 1 Hz or the subsequent oscillation amplitudes  in unstimulated cells under our experimental conditions nor did the imaging laser activate Ca2+ release events. Together, these data show that electrical stimulation of CPCs elicits a robust cytosolic and nuclear Ca2+ response from these cells.The amplitude of nuclear Caor cells –22. We d2+ oscillations were further analyzed by two-dimensional and high-speed line scan confocal imaging to determine the spatiotemporal properties of Ca2+ oscillations in CPCs. 2+ oscillation in a CPC. Interestingly, Ca2+ release begins in a focal region in the periphery of the cell and then propagates in a wave-like fashion through the cytosol and the nucleus. Fluorescent traces from a cytosolic region (blue) and the nucleus (red) are shown in 2+ in the region near the site of Ca2+ release activation increases first (blue trace), and the Ca2+ signal propagates outward through the cytosol and eventually reaches the nucleus and an increase in the nuclear Ca2+ is seen (red trace). Again, a larger nuclear Ca2+ oscillation compared to the cytosolic Ca2+ oscillation can be seen in these traces, with the red nuclear trace reaching a higher amplitude than that of the blue cytosolic trace. These traces show the spatiotemporal heterogeneity of Ca2+ release activated by electrical stimulation in CPCs and show that the Ca2+ oscillations in these cells propagate in a wave-like fashion, initiating at the periphery of the cell and propagating through the cytosol and nucleus.The Ca2+ release activated by electrical stimulation starts at the periphery of the cell and propagates across the line in a wave-like fashion over time. We also looked for cell membrane invaginations that may identify preferential sites in the CPC similar to transverse tubules (T-tubules) in adult ventricular myocytes. T-tubules are invaginations of the sarcolemmal membrane in cardiac myocytes that bring the surface membrane in close proximity to the sarcoplasmic reticulum (SR) membrane allowing for efficient coupling of Ca2+ entry through sarcolemmal L-type Ca2+ channels and activation of intracellular Ca2+ release by ryanodine receptors on the SR [2+ release activation in CPCs  was our first candidate for the protein mediating the response of CPCs to electrical stimulation. Pretreatment of cells for 5 min with the LTCC inhibitor nifedipine nearly abolished electrically induced increases in cytosolic Ca2+ in all cells recorded, suggesting that electrically induced Ca2+ oscillations in CPCs are dependent on the activity of the voltage-dependent LTCC . To determine whether the same process was present in CPCs, we first tested the necessity of extracellular Ca2+ for electrically induced Ca2+ oscillations in CPCs. Bathing the cells for 5 min prior to recording in an external solution without Ca2+ (0 Ca2+) prevented electrically induced Ca2+ release in CPCs (2+ release in our CPCs (data not shown); however, pretreatment for 5 min with 2-aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor, significantly decreased the amplitude of electrically induced Ca2+ release (3R is another intracellular Ca2+ release channel found in adult cardiac myocytes and has also been shown to mediate spontaneous Ca2+ release in multiple types of stem cells including CPCs [3R function, albeit a nonspecific one.In order to determine the proteins involved in the activation of Caent LTCC . In adul in CPCs . Pretrea release . The IP3ing CPCs , and 2-A3R in CPCs, while no RyR was detected consistent with our functional studies (3R are key proteins in electrically induced Ca2+ oscillations from human CPCs and represent a previously undescribed pathway for the activation of Ca2+ signaling in these cells; however, more experiments are necessary to confirm these results in human CPCs.Immunocytochemistry confirmed the presence of the LTCC and the IP studies . In summ2+-free media to serve as a stimulation control. Cells were fixed, permeabilized, incubated with 10 μg/mL fluorescein-5-maleimide, and imaged. Representative images of unstimulated and stimulated CPCs are shown in 2+-free media. These data illustrate an important phenotypic change induced in CPCs with chronic electrical stimulation and also indicated that this change is dependent on the presence of extracellular Ca2+. Our results showing that long-term culture stimulation induces Ca2+-dependent anisotropy in CPCs are important for the tissue architecture necessary for proper alignment of the cells in a tissue-like structure to prevent electrical conduction abnormalities, impaired pump function, or arrhythmias upon CPC implantation.Anisotropy of cardiac tissue is a key characteristic of the structure and function of the heart that facilitates electrical and mechanical activation of the myocardium. Alignment of cells organizes them into a tissue-like structure and has been shown to improve the differentiation of stem cells . Therefo2+ handling linked, directly or indirectly, to almost all properties of cardiomyocytes including excitation-contraction coupling and excitation-transcription coupling, a solid understanding of this process in CPCs is crucial for fully realizing the cardiac therapeutic potential of these cells given recent studies that have shown that these cells do not significantly contribute to cardiac myocyte replenishment in vivo [+ cardiac progenitor cells to respond to electrical stimulation by initiating intracellular Ca2+ oscillations. CPCs were isolated from human pediatric patients and pooled from multiple donors to diminish the effects of patient variability in our experiments. The use of human CPCs gives relevance to our findings for translation into a novel therapeutic strategy for clinical use, and our ability to perform live cell recordings of cytosolic Ca2+ in electrically stimulated CPCs represents a novel approach to understanding the physiology of these cells. As we have shown in this work, pediatric CPCs possess a broad spectrum of functional molecular elements of Ca2+ signaling. Ca2+-dependent regulatory mechanisms can be supposed to influence their differentiation potential, and we speculate that electrical stimulation activates a Ca2+-dependent signaling pathway in which the increased intracellular Ca2+ activates transcription of cardiospecific genes. In summary, we have characterized the electrically induced Ca2+ signal in CPCs by measuring the amplitude of cytosolic and nuclear Ca2+ oscillations elicited by electrical stimulation, identified the mechanism by which Ca2+ oscillations initiate and propagate throughout the cells, and also determined the key proteins involved in this process. Since our experiments were done in human pediatric CPCs, our results may be specific to that population of cells, and further experiments will be necessary to determine whether our results are species- or age-dependent [ ex vivo electrical stimulation on human CPCs. This study furthers our understanding of CPC physiology and may be used to direct future therapeutic strategies with these cells.With Ca in vivo –6. In thependent . To our


STC2 has also been described in mammals, and as platelet function highly depends on both extracellular and intracellular Ca2+, we have explored its expression and function in these cells. STC2−/− mice exhibit shorter tail bleeding time than WT mice. Platelets from STC2-deficient mice showed enhanced aggregation, as well as enhanced Ca2+ mobilization in response to the physiological agonist thrombin (Thr) and the diacylglycerol analog, OAG, a selective activator of the non-capacitative Ca2+ entry channels. Interestingly, platelets from STC2−/− mice exhibit attenuated interaction between STIM1 and Orai1 in response to Thr, thus suggesting that STC2 is required for Thr-evoked STIM1-Orai1 interaction and the subsequent store-operated Ca2+ entry (SOCE). We have further assessed possible changes in the expression of the most relevant channels involved in non-capacitative Ca2+ entry in platelets. Then, protein expression of Orai3, TRPC3 and TRPC6 were evaluated by Western blotting, and the results revealed that while the expression of Orai3 was enhanced in the STC2-deficient mice, others like TRPC3 and TRPC6 remains almost unaltered. Summarizing, our results provide for the first time evidence for a role of STC2 in platelet physiology through the regulation of agonist-induced Ca2+ entry, which might be mediated by the regulation of Orai3 channel expression.Stanniocalcin 2 (STC2) is a fish protein that controls body Ca Agonist might increase [Ca2+]c either by releasing Ca2+ from the intracellular stores or inducing Ca2+ entry from the extracellular medium. Regarding Ca2+ entry, store-operated calcium entry (SOCE) is a major mechanism activated by receptor occupation  junctions in order to communicate with and activates Orai1  channels  and microthrombosis (as detected in certain forms of retinopathy and nephropathy), as well as other cardiovascular disorders  and Ca2+ ionophores, have been found to activate UPR in culture cells  was first identified as a fish protein involved in calcium and phosphate homeostasis . Mouse anti-STIM1 [Clone 44 Clone 44–GOK (RUO)] antibody was from BD Biosciences® . Rabbit polyclonal anti-TRPC1 (against peptide QLYDKGYTSKEQKDC) antibody was supplied by OriGene® (Rockville (MD) USA). Anti-TRPC6 extracellular antibody was from Alomone . Anti-TRPC3 was from Abcam . Mouse and rabbit HRP-conjugated secondary antibodies were from Jackson Immunoresearch® . Rabbit polyclonal anti-Orai1 (against sequence amino acids 288–301) antibody, anti-Orai3 antibody, anti-actin antibody, thrombin (Thr), ADP, thapsigargin (TG), 1-oleoyl-2-acetyl-sn-glycerol (OAG), apyrase, aspirin, as well as other reagents of analytic grade were purchased from Sigma-Aldrich® .−/− mice were kindly provided by Dr. Roger Reddel from the Children's Medical Research Institute (CMRI)   and have been approved by the Local Ethical Committee.Anesthetized animals were subjected to surgical sectioning of the tip of the tail , and a whatman filter paper was imbibed with the blood at regularly periods of 30 s until the bleeding stopped . PRP was supplemented with aspirin (100 μM) and apyrase (40 U/mL) , and PRP was centrifuged again at 600 × g for 5 min. The pellet was suspended in Ca2+-free Tyrode's buffer containing: NaCl 137 mM, KCl 2.7 mM, NaHCO3 12 mM, NaH2PO4 0.43 mM, Glucose 0.1%, HEPES 5 mM, BSA 0.35%, MgCl2 1 mM, pH 7.13 and supplemented with apyrase (40 μg/mL).Isoflurane anesthetized mice were bled from the retroorbital plexus  and ADP (100 μM). Percentage of aggregation at two different time points  as well as the percentage of the slope (in case of Thr) and percentage of delay time to initiate the irreversible phase of aggregation (in case of ADP) were used to compare platelet aggregability in both mice strains.Platelet aggregation was monitored using a Cronologh® aggregometer according to the protocols described elsewhere   or Laemmli's buffer as required . Blots were incubated overnight with 10% (w/v) BSA in Tris-buffered saline with 0.1% Tween 20 (TBST) to block residual protein-binding sites. Detection of STIM1, Orai1, TRPC1, TRPC3, TRPC6, and Orai3 was achieved by incubation for 1 h with anti-STIM1 antibody diluted 1:1,000 in TBST, for 2 h with anti-Orai1, anti-TRPC1, or anti-TRPC6 antibody diluted 1:500 in TBST, and overnight with anti-TRPC3 or anti-Orai3 antibody diluted 1:1,000 and 1:250 in TBST, respectively. To detect the primary antibody, membranes were incubated with the appropriate secondary antibody  washed six times in TBST, and exposed to enhanced chemiluminescence reagents for 1 min. Optical density of the bands was detected using the C-Digit System , and differences between samples were analyzed using the Image J software (N.I.H. USA). Data are normalized using the amount of actin (for Western blotting) or the result obtained after reprobing the membranes with the immunoprecipitating antibody (for the immunoprecipitations).Murine platelets were either mixed with RIPA  combined with the Dunnet's post-test. The significance level was P < 0.05.Analysis of statistical significance was performed using the Student's unpaired −/− mice presented shorter mean bleeding time than WT mice . These findings suggest that STC2 might play a role in the regulation of haemostasia.Wild type (WT) and STC2-deficient mice (KO) were subjected to tail bleeding as described in the Material and Methods section. As depicted in Figure P < 0,05); while the slope was 75 and 95% at 3 min and 63 and 71% at 8 min in platelets from WT and STC2-deficient mice, respectively. Similarly, platelets from STC2−/− mice exhibited a significant difference in the percentage of aggregation 4 min after the addition of the weak agonist, ADP . Next, we have explored the mechanism underlying the exacerbated response to thrombin. First of all, we assessed Ca2+ release from the TG-sensitive stores using the SERCA inhibitor thapsigargin (TG), which induces passive Ca2+ efflux from the stores. The treatment of mice platelets with 1 μM TG in a Ca2+-free medium resulted in a sustained increase in [Ca2+]c due to Ca2+ release from the internal stores. As shown in Figure 2+ efflux from the ER, an event that is influenced by both the Ca2+ gradient across the ER membrane, which provides the driving force for Ca2+ efflux, and also the ER membrane permeability to Ca2+. As the response to TG, estimated as the integral of the rise in [Ca2+]c over the resting level for 2½ min after the addition of TG, was found to be greater in STC2-deficient mice, thus suggesting that these cells have a greater ability to store Ca2+, we have further investigated the Ca2+ leakage rate in platelets from both strains. Our results indicate that slope of the rise in [Ca2+]c upon stimulation with TG was similar in platelets from WT and STC2-deficient mice , thus suggesting that the leakage rate is similar in both strains.As previously described by others, platelet function is regulated by several mechanisms, including intracellular Ca2+-entry in human platelets mainly occurs via two different pathways, SOCE and non-capacitative Ca2+ influx (Jardin et al., 2+ entry was significantly greater in STC2-deficient cells (Figure 2+ entry evoked by Thr. Therefore, our findings indicate that, in addition to the negative regulatory role of STC2 in SOCE, previously documented by others and confirmed by us in mice platelets, the exacerbated Thr-evoked Ca2+ mobilization might involve the enhancement of non-capacitative Ca2+ entry. To assess this possibility, platelets were stimulated with the diacylglycerol analog, OAG, a selective activator of non-capacitative Ca2+ entry channels (Hassock et al., 2 resulted in a sustained increase in [Ca2+]c. As expected, platelets from the STC2-deficient mice exhibited a greater increase in [Ca2+]c in response to OAG; while we did not detect changes in the resting [Ca2+]c.Thr-evoked Cas Figure ; however2+ channel Orai1. Previous studies have revealed that STC2 regulates STIM1 expression and function, and subsequently, negatively modulates SOCE. Therefore, we have explored whether STC2 might be involved in the interaction between STIM1 and Orai1. As depicted in Figure 2+]c (Shi et al., 2+ entry might involve STIM1-TRPC1 interaction in platelets from STC2-deficient mice.As mentioned above, SOCE requires physical interaction between the ER sensor STIM1 and the Ca2+ entry in platelets from STC2-deficient mice, we have analyzed the expression of the non-capacitative channels at the protein level in the platelets of both strains. Previous studies have reported that TRPC3, TRPC6 and Orai3 are involved in non-capacitative entry (Berna-Erro et al., −/− mice as detected by Western blotting with the anti-Orai3 antibody. This finding strongly suggests that STC2 modulates Orai3 expression in mice platelets. Furthermore, this observation might provide a mechanism for the enhanced non-capacitative Ca2+ influx in platelets from STC2-deficient mice.In an attempt to assess the mechanism underlying the increase in non-capacitative Ca2+ homeostasis in these cells, as it was previously reported in other mammal cells (Zeiger et al., 2+ in the ER, although the leakage rate across the ER membrane is not significantly enhanced. Furthermore, our results indicate that STC2 modulate agonist-induced Ca2+ entry, so that the deficiency of STC2 results in exacerbated Ca2+ signals and, most likely, platelet hyperaggregability.Here we provide for the first time evidence supporting a role for STC2 in platelet aggregation and haemostasis. Using the murine model lacking STC2, we have found that these mice exhibited enhanced platelet response to strong and weak agonists, such as thrombin and ADP, respectively, and subsequently reduced bleeding time. Our results indicate that STC2 might modulate platelet aggregation through the regulation of Ca2+ influx in platelets via SOCE and non-capacitative Ca2+ entry (Jardin et al., 2+ influx induced by Thr in platelets from STC2-deficient mice is more likely mediated by enhanced non-capacitative Ca2+ entry. Our findings indicate that STC2 deficiency results in an impaired STIM1/Orai1 interaction, but also we have found that TG-induced Ca2+ entry in platelets from STC2 KO mice is significantly enhanced. This apparent discrepancy might be explained by the fact that TG has been found to activate non-capacitative Ca2+ entry in platelets through the release of autocrine factors (Harper et al., 2+ entry induced by TG in platelets from STC2-deficient mice might involve reduced SOCE and enhanced non-capacitative Ca2+ entry. In support of this hypothesis, we have found that non-capacitative Ca2+ entry evoked by OAG, a diacylglycerol analog, is enhanced in STC2-deficient mice, which provides evidence for a role of STC2 in the regulation of non-capacitative Ca2+ influx in these cells. In murine platelets, two main TRPC channels have been found to be activated in response to OAG, TRPC3 and TRPC6 (Harper et al., 2+ entry (Berna-Erro et al., 2+ entry, these findings suggest a possible mechanism for the enhanced activation of non-capacitative Ca2+ entry in these cells. The enhanced Orai3 expression in platelets from STC2-deficient mice is unlikely to be responsible for the enhanced Ca2+ release in response to TG, as a previous study has revealed that Orai3 is not involved in the TG-revealed ER Ca2+ leak (Leon-Aparicio et al., Thrombin evokes Ca2+ entry is enhanced, while SOCE might be attenuated (Jardin et al., 2+ entry in these cells. Summarizing, we provide for the first time evidence for a role of STC2 in platelet aggregation through the modulation of Ca2+ homeostasis.We have recently suggested that STC2 might be involved in the pathophysiology of type 2 diabetes mellitus, where STC2 is downregulated (Lopez et al., EL and LG-G performed most of the experiments with the same grade of responsibility. JP-G, CC, NB, and MG provided us with the mice blood samples, as well as they were in charge of the statistical analysis of data. GS performed a critical review of the manuscript, while JR and PR were in charge of conceptual and protocol design of the manuscript and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Equine infectious anemia is an equine disease caused by equine infectious anemia virus, which was first reported in 1840. Equine infectious anemia virus research in China started in the 1960s, focusing on etiology, pathology, diagnosis, and immunology. Notably, in 1978 an attenuated vaccine was successfully developed for equine infectious anemia virus, effectively preventing equine infectious anemia virus in China. This article will review equine infectious anemia virus in China, including past and recent research, and commemorate scientists who have made great contributions to equine infectious anemia virus prevention. Lentivirus genus of the Retroviridae family. The Lentivirus genus also includes human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and Maedi-visnavirus (MVV) EIAV almost infected equids worldwide. It causes a persistent infection characterized by recurring febrile episodes associated with viremia, fever, thrombocytopenia, and wasting symptoms [Equine infectious anemia virus (EIAV) is a member of the symptoms . EIAV raReports and records of EIAV did not appear in China until the early 20th century. Japan spread EIAV to China during World War II. Due to an incomplete quarantine, horses with a latent infection were traded from the former Soviet Union and Mongolia, which also introduced EIAV to China. From 1954 to 1959, EIAV mainly occurred in Heilongjiang, Jilin, Liaoning, Inner Mongolia, Yunnan, Hebei, and the Shanxi province. EIAV prevalence in Shandong, Gansu, Jiangsu, and the Anhui Province was due to infected horses purchased from Xinjiang. EIAV prevalence in Hebei, Beijing, Tianjin, Shaanxi, and Henan provinces was caused by EIAV-latent horses purchased from Liaoning, Jilin, and Heilongjiang. The Chinese EIAV epidemic was started by horses traded from foreign countries, and gradually spread from north to south, eventually inducing a nationwide outbreak.From 1954 to 1989, EIAV broke out in 23 provinces to varying degrees, contaminating 77% of the country’s total provinces  was widely applied . EIAV coFrom 1974 to 1978, two serological methods dependent on antibody detection were developed to battle EIAV. The first was the complement-fixation (CF) test which detected specific antibodies that fixed complement when interacting with the corresponding antigen. Hemolysis was used as an indicator system to detect residual complement . AnotherIt is impossible to eradicate EIAV merely with quarantine measures. Antibody levels are not constant in the infected animal, so it is not possible to detect all the subclinical cases. An integrated control program including vaccination and quarantine was adopted. This not only eliminated most of the infected animals and reduced EIAV spread, but also enabled most of the healthy horses to obtain immunity. These measures accelerated the control and eradication of EIAV in China Table .For more than a century, the most successful vaccines were live attenuated vaccines which have been widely used to control the epidemics of many viruses, such as smallpox, polio, and measles , 5. SincLN, and attempted to adapt this virus to several other animals, tissues, or cell lines. All these attempts failed, except in donkey. The virus experienced 117 passages (EIAVDV117) in donkey, with enhanced pathogenicity instead of attenuation. After 121 serial passages of EIAVDV117 strain on donkey leukocyte (EIAVDLV121), there was a progressive loss of virulence  antibody and AGID antibody can be detected Figure . Three wThe EIAV-specific cellular immune response was also evaluated. CD8 positive T cells increased after immunization and induced an EIAV-specific CTL response , 23, 24.Furthermore, vaccine-induced TLR3 activation and INF beta production were higher than in the molecular clone strains . Infectiin vitro and in vivo, the molecular strain induced less protection and lower specific neutralizing production [In vivo evolution of the EIAV vaccine was investigated, and the envelope gp90 gene variation was associated with declining diversity after vaccination. This trend coincided with the maturation of immunity to EIAV [The diverse immunogen composition of the EIAV vaccine may induce protective immunity –31. Althoduction . These f to EIAV . DiverseInnate restriction factors are host factors which could intervene against every step of the viral life cycle and inhibit viral replication , 34. ManTetherin  is a type II single-pass transmembrane protein with an unusual topology, including an N-terminal cytoplasmic tail (CT), a single transmembrane domain, an extracellular domain, and a C-terminal glycosylphosphatidylinositol (GPI) membrane anchor . The hosType I interferon (IFN) and the subsequent induction of interferon-stimulated genes (ISGs) promotes resisting viral infection. Viperin, also known as RSAD2, is an endoplasmicreticulum (ER) associated multifunctional protein with broad antiviral activity. Tang et al. demonstrated that equine viperin could distort the ER, inhibit viral Gag production and/or release, Env and Receptor, leading to reduced EIAV replication .Schlafen (SLFN) family proteins are divided into three groups  based on size and structure. All these proteins contain a specific SLFN box upstream of the AAA domain, which has ATP-binding activity. Because of the RNA/DNA helicase function of the AAA domain, SLFNs have RNA structure-modelling activity and participate in RNA metabolism , 42. CodAdenosine deaminases that act on RNA (ADARs) are RNA-editing enzymes that catalyze the hydrolytic C6 deamination of adenosine (A) to produce inosine (I) in double-stranded RNA substrates –47. ADAREIAV often results in a rapid, dynamic, three-stage disease process: acute, chronic, and long-term asymptomatic . The iniBecause viral envelope variants were produced and selected sequentially, the virus could escape the host immune responses, leading to the cyclic nature of chronic EIA , 54. Surin vivo [The long terminal repeat (LTR) enhancer region of EIAV is one of the most variable regions in the EIAV genome , 57. Up in vivo .FDDV13, China has successfully controlled and most areas have eradicated EIAV. However, EIA is still prevalent worldwide, especially in France, Slovenia, Ireland, Belgium, Italy, Japan, and Brazil [FDDV13 is a very effective vaccine to control EIA.After using the attenuated vaccine EIAVd Brazil –65. SomeDeveloping an effective vaccine against lentivirus infections is still needed in both human and veterinarian medicine. EIA is still a severe lentivirus disease in horses of many other countries. In China, because many effective strategies were applied including the EIAV live attenuated vaccine, EIAV was successfully controlled and most areas have eradicated EIAV. While this serves as a model for controlling EIAV in other countries and battling other viral diseases, the attenuation mechanisms in these passages were not well understood. Understanding the attenuation mechanisms in the EIAV vaccine will provide further insight to controlling EIAV and other viruses.


Although data are limited, sperm population studies have indicated an association of poor [Ca2+]i response to progesterone with reduced fertilization ability.Stimulation with progesterone is a widely used method for assessing [CaThis was a cohort study using semen samples from 21 donors and 101 patients attending the assisted conception unit at Ninewells Hospital Dundee who were undergoing ART treatment. Patients were recruited from January 2016 to June 2017.2+]i responses were examined by single cell imaging and motility parameters assessed by computer-assisted sperm analysis (CASA).Semen donors and patients were recruited in accordance with local ethics approval (13/ES/0091) from the East of Scotland Research Ethics Service (EoSRES) REC1. [Ca2+]i oscillations and responses to progesterone could not be analysed. All patient samples loaded with the [Ca2+]i-indicator fluo4 responded to progesterone stimulation with a biphasic increase in fluorescence (transient followed by plateau) which resembled that seen in progesterone-stimulated donor samples. The mean normalized response  was significantly smaller in IVF-FF and ICSI patient groups than in donors. All samples were further analysed by plotting, for each cell, the relationship between resting fluorescence intensity and the progesterone-induced fluorescence increment. In donor samples these plots overlaid closely and had a gradient of ≈ 2 and plots for most IVF(+ve) samples closely resembled the donor distribution. However, in a subset (≈ 10%) of IVF(+ve) samples, 3/8 IVF-FF samples and one-third of ICSI samples the gradient of the plot was significantly lower, indicating that the response to progesterone of the cells in these samples was abnormally small. Examination of the relationship between gradient (regression coefficient of the plot) in IVF samples and fertilization rate showed a positive correlation. In IVF-FF and ICSI groups, the proportion of cells in which a response to progesterone could be detected was significantly lower than in donors and IVF (+ve) patients. Approximately 20% of cells in donor, IVF(+ve) and ICSI samples generated [Ca2+]i oscillations when challenged with progesterone but in IVF-FF samples only ≈ 10% of cells generated oscillations and there was a significantly greater proportion of samples where no oscillations were observed. Levels of hyperactivated motility were lower in IVF(+ve) and IVF-FF groups compared to controls, IVF-FF also having lower levels than IVF(+ve).For analysis, patient samples were divided into three groups IVF(+ve) , IVF-FF  and ICSI (21 samples). A further 10 IVF samples showed large, spontaneous [Cain vitro study and caution must be taken when extrapolating these results in vivo.This is an 2+]i signalling in sperm from sub-fertile men that cannot be detected in population studies.This study reveals important details of impaired [CaThis study was funded by a MRC project grant . Additional funding was provided by Chief Scientist Office/NHS research Scotland. Interestingly, though only one patient had no detectable CatSper function, several patients had more subtle abnormalities of the [Ca2+]i response when challenged with P4  cells.P4 [Ca2+]i imaging provides information on activity of individual sperm that cannot be obtained by studying populations, including the proportion of responsive cells, the presence of sub-populations that respond differently and the nature and complexity of the single cell [Ca2+]i signal. We have used single cell imaging to investigate responses to P4 in sperm samples from sub-fertile men attending an ART clinic, specifically (i) the nature and heterogeneity of single cell [Ca2+]i responses and (ii) the relationship between P4-induced [Ca2+]i responses and fertilization success.Although time consuming and technically more complex, single cell [Ca2+]i imaging of spermatozoa from patients was carried out using an aliquot of the sperm preparation used for ART. Measurements were made on the day of treatment, allowing direct correlation with ART. Computer-assisted sperm analysis (CASA) was done on each aliquot. For analysis, patient samples were divided into three groups IVF(+ve) (successful fertilization), IVF-FF (failed fertilization) and ICSI.Single cell i levels for 3–5 min, cells were stimulated with P4 (3.6 μM). Images were acquired at 0.33 Hz using a ×40 oil objective with a CoolSNAP MYO CCD camera controlled by Metsoftware . Fluorescence was excited at 488 nm and recorded at 520 nm. Illumination and camera gain settings were maintained constant and fluorescence intensity values are therefore directly comparable between all recordings. A region of interest was drawn round the head and neck region of each cell and several areas were also chosen to assess background fluorescence. Those cells where fluorescence levels fell noticeably during the pre-stimulation period (loss of dye indicating that the cell was dead or dying) were excluded from the analysis. After background correction, resting fluorescence intensity (mean of 25–30 consecutive images collected prior to P4 stimulation) and peak fluorescence intensity (mean of 4–5 consecutive images spanning the peak of the P4-induced [Ca2+]i transient) were determined for each cell. P4-induced fluorescence increment for each cell was then calculated by subtracting resting fluorescence from peak fluorescence (F = ((F − Frest)/Frest) × 100%, where ΔF is percentage change in intensity, F is fluorescence intensity at time t, and Frest is the mean of 25–30 determinations of F prior to P4-stimulation. A mean normalized trace was calculated for each experiment by taking the mean ΔF of all cells in the experiment (ΔFmean) at each time point. To assess responsiveness to P4 in each cell, the mean and 95% confidence interval of fluorescence intensity were calculated for the period prior to P4 stimulation (C ± c) and the 4–5 images spanning the peak of the transient response (T ± t). The response of that cell was considered significant and classified as a responder if: T – t > C + c  which persisted in the presence of P4. These oscillations often ‘masked’ the [Ca2+]i response to P4 which could not be assessed. These data are presented and discussed separately and are not included in the 3 patient groups.To assess the occurrence of i levels (fluo4 fluorescence after background correction) were similar in donors, IVF+ (successful fertilization) and ICSI patients, but in the eight IVF-FF (failed fertilization) patients mean resting fluorescence was more than double that in donor cells  and ICSI samples, respectively; Chi-square; Fig. Mean resting [Calls Fig. a. Examin2+]i signal composed of an initial transient followed by a sustained [Ca2+]i plateau  against mean resting [Ca2+]i (resting fluorescence) for each of the 21 donor recordings gave an approximately linear relationship  generated a significant increase in fluorescence upon stimulation with P4  for each of the 749 donor cells (21 samples) gave a straight line relationship with a gradient of ~2 , similarly to that obtained when plotting of mean data for each experiment . In contrast, several of the other IVF-FF samples had distributions much closer to that for donor cells .To assess the variation of single cell responses to P4, Ca+i transicells 98. ± 0.5% gcells 98. ± 0.5% gcells 98. ± 0.5% gor cells  and the cells 98. ± 0.5% gor cells , but thecells 98. ± 0.5% gor cells . OverallP = 0.0004; R2 = 0.14; Fig. n = 55) and 61.8 ± 3.8% (n = 15), respectively (P = 0.0015).Since IVF patient samples varied considerably in their sensitivity to P4, we investigated the relationship between the regression coefficient (P4-induced fluorescence increment:resting fluorescence) for each sample and fertilization rate of that sample at IVF. There was a significant positive relationship between these variables  and ICSI samples was 20–25%, similar to donor controls . Variation between the eight IVF-FF patients was considerable (proportion of oscillating cells ranged from 0 to 54%), but the proportion of samples in which no cells generated [Ca2+]i oscillations (3/8) significantly exceeded that in donors  or IVF(+ve) samples . Plotting of the relationship between generation of [Ca2+]i oscillations (% cells oscillating) and fertilization for all IVF samples (IVF(+ve) and IVF-FF) revealed a weak but significant correlation  compared to 54.8 ± 1.3 s (n = 183 cells) in donors .Single cell imaging allows the detection of complex [Callations /8 signif2+]i oscillations, as described previously (P < 0.001). However, in 10 patient samples, all of which fertilized at IVF (fertilization rate = 60.7 ± 7.4%), we observed large spontaneous [Ca2+]i oscillations similar in amplitude to those induced by P4 . Examination of the relationship between the proportion of spontaneously oscillating cells in each of these 10 patients and fertilization rate at IVF showed a weak, non-significant relationship  we observed spontaneous i responses to P4 in donor and patient samples and to assess how this relates to fertilizing ability (by IVF) of the sperm population. Our data show not only that P4-evoked and spontaneous [Ca2+]i signals vary between cells in a single ejaculate , but that there is clear variation between and within patient types (as assessed by an ART clinic) in regard to the proportion of cells that respond to the CatSper agonist P4 and the nature of the responses elicited.CatSper channels are the main source of Ca2+]i may be characteristic of some sub-fertile men. Increased resting [Ca2+]i could be due to enhanced tonic Ca2+-influx through CatSper, for instance due to unusually high pHi or depolarized Vm -null mice have increased [Ca2+]i, though the loss of motility in such cells is far more severe than the effects observed in this study  fluorescence showed wide variation between samples both within and between patient and donor groups. In particular, in the IVF-FF patient group, half of the samples showed an unusually high resting fluorescence. Though we cannot discount the possibility that this reflects abnormalities of dye loading/behaviour in these samples, it suggests that high resting sperm [Ca2+]i then (i) a ‘normal’ P4-induced CatSper activation/Ca2+ influx will give a smaller proportional increase in fluorescence and (ii) the dye may approach saturation, underestimating the [Ca2+]i signal. Therefore, to analyse the amplitude of [Ca2+]i responses to progesterone we investigated the relationship between resting fluorescence and the P4-induced fluorescence increment. Plotting the data from donor samples either using sample means or individual cells gave a clear, linear relationship that showed no evidence of dye saturation over the range of resting fluorescence observed. For most patient samples a similar relationship between resting fluorescence and the P4-induced fluorescence increment was seen but in ~10% of IVF(+ve) patients and one-third of IVF-FF and ICSI patients the response to P4 fell clearly below the ‘normal’ range.Since resting fluorescence varied between donor/patient groups, simple normalization of fluorescence to pre-stimulus levels is potentially misleading. If the high levels of resting fluorescence in these samples genuinely reflect high [Ca2+]i and/or expression of functional CatSper. Data from patient 2236 produced an intriguing ‘hybrid’ plot including cells that responded ‘normally’ to P4 and cells that gave a negligible/zero response, suggesting that only a sub-population of these sperm express functional CatSper. Significantly, though the response to P4 was impaired in a significant proportion of the 91 patients where analysis was possible, we did not detect any men who were null or ‘functionally null’  and fertilization rate of the sample in IVF. Consistent with previous studies on P4-induced population [Ca2+]i transient is followed, in a subset of cells, by [Ca2+]i oscillations which are dependent on influx of extracellular Ca2+ but appear also to involve repetitive mobilization of Ca2+ stores  group the mean percentage of cells that generated oscillations upon P4 treatment was only half that in donors and in the IVF(+ve) group and the proportion of samples that failed totally to generate oscillations was significantly higher in the IVF-FF group. [Ca2+]i responses to P4 were small in these samples, consistent with the dependence of oscillations on background Ca2+ influx through CatSper. However, in the large IVF(+ve) group (n = 62) generation of oscillations showed no significant relationship to P4-induced fluorescence increment  or to the regression coefficient of the single cell (fluorescence increment:resting fluorescence) scatter plot , suggesting that other aspects of Ca2+-handling, presumably including activity of the Ca2+-store, are also important and may lead to failure of oscillations and reduced fertility.Upon stimulation of human sperm with P4, the initial [Ca2+]i oscillations that persisted in the presence of P4 with no significant change in amplitude or frequency and largely masked the P4-induced [Ca2+]i transient. The occurrence of spontaneous oscillations might indicate attainment of an advanced level of capacitation  CaSupplementary Figure 1Click here for additional data file.Supplementary Figure 2Click here for additional data file.Supplementary Figure 3Click here for additional data file.Supplementary Figure 4Click here for additional data file.Supplementary Figure 5Click here for additional data file.Supplementary Figure 6Click here for additional data file.Supplementary Figure 7Click here for additional data file.


Herein, we review the discovery of extracellular Ca2+ in regulation of mGluRs, summarize the recent developments in probing Ca2+ binding and its co-regulation of the receptor based on structural and biochemical analysis, and discuss the molecular basis for Ca2+ to regulate various classes of drug action as well as its importance as an allosteric modulator in mGluRs.Metabotropic glutamate receptors (mGluRs) associated with the slow phase of the glutamatergic signaling pathway in neurons of the central nervous system have gained importance as drug targets for chronic neurodegenerative diseases. While extracellular Ca Ca2+-binding proteins have Ca2+ affinities that vary by 106-fold or more depending on their cellular locations and functions chromen-1a-carboxamide (PHCCC) of group I mGluRs was reported to enhance the potency of an agonist of mGluR4 l-(+)-2-Amino-4-phosphonobutyric acid (l-AP4). PHCCC is also able to reduce the movement activity in a Parkinsonian rat model o) is about 20,000-fold higher than the intracellular Ca2+ concentration ([Ca2+]i). This great difference across the plasma membrane creates the Ca2+ gradient from mM (extracellular) to nM (intracellular). Accumulating evidence suggests the crucial importance of extracellular Ca2+ and its dynamics in the central nervous system. The dynamic change of Ca2+ is localized due to the fact that the synaptic crevices are insulated to serum due to blood–brain barrier. Triggered by presynaptic Ca2+ channel activation, the neurotransmitter released from the presynaptic vesicles o in the nervous system is about 0.8 to 1.7 mM chromen-1a-carboxylate ethyl ester (CPCCOEt) , or 2-Amino-3-phosphonopropionic acid (AP3) (group I mGluR specific antagonist) or decreasing Ca2+ from 2.5 mM to 1 mM. In contrast, Dihydroxyphenylglycine (DHPG) (the group I mGluR agonist) and Ca2+ increase enhanced quantal size i rise in Purkinje cells was detected in responses to extracellular Ca2+ exposure, but not in cells from mGluR1 knockout mice i, while blocking the receptor-operated and P-type Ca2+ channels by antagonist SKF-96365 and ω-agatoxin IVA did not significantly affect the [Ca2+]i responses to extracellular Ca2+ [2+]i is not likely to be a result of Ca2+ influx through Ca2+ channels. In addition to this direct activation, extracellular Ca2+ also augmented the cellular responses evoked by l-Glu or its analog. Back in 1998, the [3H]-InsP1 accumulation evoked by mGluR1 agonist was significantly facilitated by increasing [Ca2+]o in baby hamster kidney cells (BHK) [2+]i increase attributed by mGluR1 activation and found that the initial [Ca2+]i increase resulted from Ca2+ mobilization from the intracellular Ca2+ stores induced by receptor activation, while the sustained phase of [Ca2+]i was related to the extracellular Ca2+ influx through store-operated Ca2+ channels [2+]i increase and IP3 accumulation i due to Ca2+ release from cytosolic Ca2+ store o of 2–3 fold. Gd3+ is also revealed at the hinge region in the Fourier map, where it shares residues D322 and D324 from the loop that contributes to Ca2+ binding o enhances [3H]-l-Quis binding to wild type mGluR1 and consistently induces [Ca2+]i change . Furthermore, we found that (s)-MCPG efficiently antagonizes both l-Glu- and extracellular Ca2+-induced receptor activation at low concentrations, but the increasing concentration of l-Glu or [Ca2+]o can overcome this inhibition. Consistent with our studies, the receptor-bound structure with s-MCPG or LY341495 (PDB ID: 3KS9) is shown as a relaxed state [l-Glu and Ca2+), while the antagonists increased the space between the loops [2+, respectively, and extracellular Ca2+ increases the potency of Ro-674853 but reduces the inhibition of mGluR1 by CPCCOEt [2+ to the predicted Ca2+-binding site in the ECD of mGluR1 modulates not only glutamate-evoked signaling but also the actions of both orthosteric ligands and allosteric modulators on mGluR1. These studies also open up new avenues for developing allosteric modulators of mGluR function that target specific human diseases.The synergism of Caf cGPCR. . Our labCPCCOEt) . We firsed state . The acted state . Two inthe loops . In addi CPCCOEt . Therefo2+ modulating group I mGluRs via dual activation. Extracellular Ca2+ exhibits strong efforts in modulating several classes of agonists or antagonists to group I mGluR. Since the identified Ca2+ binding site residing at hinge region is only conserved in group I mGluRs, this will re-ignite the interest in generating subtype selective orthosteric modulators. Recent structure determinations of CaSR crystal structures also revealed similarities and distinct structural features between mGluRs and CaSR [2+ will enable us to understand the ligand specificity and molecular mechanism of Ca2+ activation of family C of GPCR. Further development of the Ca2+ sensor to probe Ca2+ concentration changes at synapse clefts and ER Ca2+ release will also enable us to capture the dynamic role of Ca2+ in GPCR-mediated signaling. Recent progress using novel approaches has provided insights for understanding molecular basis of Caand CaSR ,139. Fur


MCL-1, MYC, and cyclin D1. As CDK9 inhibitors are nonselective, predictive biomarkers that may help identify patients most likely to respond to CDK9 inhibitors are now being utilized, with the goal of improving efficacy and safety.Current treatment for acute myeloid leukemia (AML) is less than optimal, but increased understanding of disease pathobiology and genomics has led to clinical investigation of novel targeted therapies and rational combinations. Targeting the cyclin-dependent kinase 9 (CDK9) pathway, which is dysregulated in AML, is an attractive approach. Inhibition of CDK9 leads to downregulation of cell survival genes regulated by super enhancers such as The online version of this article (10.1186/s13046-018-0704-8) contains supplementary material, which is available to authorized users. Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by a clonal proliferation of immature myeloid precursor cells in the peripheral blood, bone marrow, and/or other tissues. It is the most common acute adult leukemia, with approximately 21,380 individuals in the United States diagnosed in 2017 [Treatment for AML has been less than optimal. The standard induction regimen, a continuous infusion of cytarabine for 7 days plus 3 days of an anthracycline, usually daunorubicin or idarubicin (7 + 3 therapy), has changed little over the past 40 years. Complete remission (CR) rates rarely top 70% in younger patients and 50% in older patients . Over thFLT3-mutated AML [IDH-2 mutation as detected by an FDA-approved test [An increasing understanding of the pathobiology and genomics of AML has led to clinical investigation of a variety of novel therapeutic approaches, particularly agents targeted against dysregulated enzymes and mutant driver proteins. In addition, investigations into mechanisms of drug resistance in AML have shed light on means of overcoming chemoresistance, such as targeting leukemic stem cells and the bone marrow microenvironment –10. Two ated AML . Enasideved test . In addived test . In addived test .A therapeutic target that has been investigated in AML is cyclin-dependent kinase (CDK)9, one of a large number of CDKs that control cell-cycle progression and gene transcription. Although originally thought to act via cell-cycle regulation, CDK9 is involved in regulating gene transcription elongation and messenger RNA (mRNA) maturation, as well as other physiologic processes , 16. DysMYC, a downstream proto-oncogene involved in cell growth and cell-cycle progression, and MCL-1, an apoptosis regulator. CDK9 also appears to be involved in several physiologic processes in the cell outside of transcription, including differentiation, apoptosis, and signal transduction [Together with regulatory subunits (cyclins), CDKs form functional complexes responsible for the control of cell proliferation, differentiation, apoptosis, and DNA repair . Whereassduction .42) and a minor 55 kDa (CDK955) protein that is translated from an in-frame mRNA that arises from an upstream transcriptional start site [CDK9 was first designated PITALRE based on a characteristic amino acid motif (Pro-Ile-Thr-Ala-Leu-Arg-Glu), and its function was first elucidated in studies of human immunodeficiency virus , 21. CDKart site , 23.Ser5PS) and CDK9 at Ser2 (YSer2PTSPS). Ser5 phosphorylation results in activation of RNA Pol II such that transcription is initiated and Ser2 phosphorylation allows productive transcriptional elongation  complex that stimulates transcription elongation by phosphorylating the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNA Pol II); the CTD contains tandem repeats of a 7 amino-acid sequence that is phosphorylated by CDK7 at Ser5  values for CDK9 compared with other CDKs/enzymes. As described in the sections that follow, CDK9 inhibitors in general exhibit a variety of effects in AML cells and in vivo models, including reduced phosphorylation of RNA Pol II; reduced levels of proteins such as MYC, MCL-1, XIAP, and cyclin D1; induction of apoptosis; and inhibition of tumor growth and prolonged survival in animal models. There is increasing interest in identifying predictive biomarkers of response to conventional and investigational-targeted therapies in AML, including CDK9 inhibitors. For example, measuring the function of B-cell lymphoma 2 (BCL-2) family proteins using BCL-2 homology domain 3 (BH3) profiling has been shown to provide useful information in discriminating AML treatment response with traditional cytarabine-based therapy and investigational AML regimens [CDK9 inhibitors have been investigated as therapeutics for a variety of hematologic cancers and solid tumors. Table regimens –68. The regimens . For exaAlvocidib was the first CDK inhibitor to enter clinical trials and has been the most studied to date. Alvocidib displays potent activity against CDK9 (6 nM), in addition to activity against CDK4, CDK5, CDK7, and CDK11 , 48. AltMCL-1 and a 2-fold reduction in MCL-1 levels with enhanced apoptosis [BCL-2, which contrasts with earlier studies demonstrating downregulation of BCL-2 expression and may represent a protective antiapoptotic response during cell-cycle arrest [In vitro studies in diverse hematologic malignancies and studies on human on AML marrow blasts have shown that alvocidib reduces levels of MCL-1, BCL-2, and cyclin D1 and inhibits phosphorylation of RNA Pol II  . Based opoptosis . Gene exe arrest . Alvocid≥60 years. Tumor lysis syndrome (TLS) has been seen following initial dosing of alvocidib in AML studies , necessitating appropriate prophylaxis and monitoring [Alvocidib was evaluated in combination with cytarabine and mitoxantrone (FLAM) in a TST manner in multiple clinical studies in R/R AML , 76, 77 nitoring .p = 0.0002). An ongoing international biomarker-driven phase 2 study (NCT02520011) is incorporating this predictive information in identifying a subgroup of patients most likely to respond to alvocidib. The study is comparing FLAM vs. cytarabine and mitoxantrone (AM) in patients with MCL-1-dependent R/R AML as demonstrated by NOXA-BH3 priming of ≥40% by mitochondrial profiling of the bone marrow. It includes an exploratory arm evaluating patients with newly diagnosed MCL-1-dependent high-risk AML.There are ongoing efforts to determine predictive biomarkers to allow identification of specific subsets of patients who are likely to respond to alvocidib, such as use of BH3 profiling . As NOXAA phase 1, open-label, dose-escalation, safety and biomarker prediction study was recently registered. This study will explore alvocidib and standard 7 + 3 chemotherapy in patients with newly diagnosed AML (NCT03298984). Correlation between the benefit from alvocidib in combination with 7 + 3 therapy and BH3 profiling for MCL-1 dependency will be assessed as a secondary outcome.MLL-rearrangement positive and negative) with a median IC50 of 385 nM and induced apoptosis [MLL-rearranged patient AML samples, including those with mutant NPM1 or internal tandem duplication of the juxtamembrane domain-coding sequence of the FLT3 gene (FLT3-ITD).BAY 1143572 displays potent CDK9/P-TEFb inhibitory activity in the nanomolar range, with inhibitory activity against other CDKs that is at least 50-fold lower . In in vpoptosis . In addiA phase 1 dose escalation study of BAY 1143572 in combination with granulocyte colony-stimulating factor in patients with advanced malignancies  has been completed, but results are yet to be reported. A phase I dose-escalation study designed to determine the safety, pharmacokinetics, and recommended phase 2 dosing of BAY 1143572 in advanced acute leukemia has completed enrollment (NCT02345382).50 values in the low nanomolar range [50 = 4 nM) and completely suppressed retinoblastoma (Rb) phosphorylation, which correlated with induction of apoptosis. Dinaciclib exposure resulted in cell-cycle arrest in more than 100 tumor cell lines of diverse origin and across a broad range of transformed cellular backgrounds as evidenced by based on total inhibition of bromodeoxyuridine incorporation. Broad antiproliferative activity was seen across this panel of tumor cell lines, with median IC50 values of 11 nM. Dinaciclib has also been shown to downregulate expression of MCL-1 and induce apoptosis in primary patient chronic lymphocytic leukemia (CLL) cells, with activity that was independent of high-risk genomic features [Dinaciclib is a novel and potent inhibitor of CDK1, CDK2, CDK5, and CDK9 with ICar range . In in vfeatures .MLL-rearranged AML mouse models [Mcl-1 was seen and overexpression of Mcl-1 protected AML cells from dinaciclib-induced apoptosis. In mice bearing MLL-AF9-driven murine and human leukemias, dinaciclib exhibited potent antitumor activity and significantly prolonged survival.Apoptotic and antitumor effects of dinaciclib were demonstrated in e models . DecreasDinaciclib has been evaluated in clinical trials in various hematologic indications, with varied effectiveness. In a phase 2 study of dinaciclib monotherapy in patients with relapsed multiple myeloma (MM), 11% of patients achieved a partial response or better . The mos≥ 3 treatment-related adverse events, most commonly hematologic toxicities and fatigue. Interestingly, an additional clinical toxicity reported was TLS, where metabolic changes indicative of large-scale tumor cell lysis occur [A phase 2 randomized study comparing dinaciclib and gemtuzumab ozogamicin in R/R AML and evaluating dinaciclib in ALL (NCT00798213) demonstrated short-lived cytoreductive activity with dinaciclib but a lack of objective clinical response in the 20 patients treated . In addiis occur . While tis occur . ClearlyA randomized phase 3 study in which 42 patients with R/R CLL received treatment suggested promising antileukemic activity with dinaciclib relative to ofatumumab, an anti-CD20 monoclonal antibody  . The mosMCL-1 and XIAP expression, and apoptosis) was demonstrated, but limited clinical activity was seen and three-quarters of the patients experienced grade 3 or 4 toxicities, mainly myelosuppression [XIAP, BCL-2, and MCL-1.SNS-032, a potent CDK9 inhibitor (4 nM) with activity against CDK2 and CDK7, was evaluated in a phase 1 and pharmacologic study in patients with advanced CLL or MM , 58. Mecpression . CombiniFLT3 leukemia cells, and induced tumor regression and prolonged survival in murine AML models. In primary AML patient samples, TG02 inhibited transcription by inducing RNA Pol II Ser2 dephosphorylation and downregulated MCL-1 and XIAP, leading to subsequent BAX activation and apoptosis [TG02 is an oral CDK9 inhibitor with activity against several CDKs in the nanomolar range . TG02 expoptosis . Dynamicpoptosis . In addiPhase 1 studies evaluating TG02 in advanced hematologic malignancies  and in R/R CLL or small lymphocytic lymphoma (NCT01699152), have been completed.50 = 6 nM) that also displays strong activity against CDK1, CDK2, and CDK4 [MCL-1 and XIAP, and induced apoptosis, in primary CLL cells and in AML and ALL cell lines [CDKI-73 is a potent CDK9 inhibitor  that also displays activity against CDK8, as well as CDK7 to a lesser degree [LY2857785 is a potent CDK9 inhibitor


IV) produced panoptic retinal rescue. By combining these treatments, we now show that MSCIV supplementation potentiates the MSCSRI‐mediated rescue of photoreceptors and visual function. Electrophysiological recording from superior colliculi revealed 3.9‐fold lower luminance threshold responses (LTRs) and 22% larger functional rescue area from combined treatment compared with MSCSRI alone. MSCIV supplementation of sham  injection also improved LTRs 3.4‐fold and enlarged rescue areas by 27% compared with saline alone. We confirmed the involvement of MSC chemotaxis for vision rescue by modulating C‐X‐C chemokine receptor 4 activity before MSCIV but without increased retinal homing. Rather, circulating platelets and lymphocytes were reduced 3 and 7 days after MSCIV, respectively. We demonstrated MSCSRI‐mediated paracrine support of vision rescue by SRI of concentrated MSC‐conditioned medium and assessed function by electroretinography and optokinetic response. MSC‐secreted peptides increased retinal pigment epithelium (RPE) metabolic activity and clearance of photoreceptor outer segments ex vivo, which was partially abrogated by antibody blockade of trophic factors in concentrated MSC‐conditioned medium, or their cognate receptors on RPE. These data support multimodal mechanisms for MSC‐mediated retinal protection that differ by administration route and synergize when combined. Thus, using MSCIV as adjuvant therapy might improve cell therapies for retinal dystrophy and warrants further translational evaluation. Stem Cells Translational Medicine2017;6:444–457We previously demonstrated that subretinal injection (SRI) of isogenic mesenchymal stem cells (MSCs) reduced the severity of retinal degeneration in Royal College of Surgeons rats in a focal manner. In contrast, intravenous MSC infusion (MSC Despite hundreds of clinical trials, just one stem cell treatment has been approved for the U.S. market. Additional treatments nearing clinical acceptance use bone marrow mesenchymal stem cells for inflammatory and immune‐related conditions. This is because safety has been established over decades of testing, and cell transplants prolong life‐saving organ and tissue grafts. In the present study, the intravenous delivery of mesenchymal stem cells enhanced the vision rescue from primary cell grafts into diseased retinae. This combined transplant strategy could improve functional outcomes for cell‐based therapies, expand their utility, and expedite their clinical acceptance.IV) promotes the tolerance of transplanted tissues, thereby prolonging primary graft survival and the duration of functional benefit IV supplementation can likewise enhance the therapeutic benefit of primary stem cell grafts to treat retinal dystrophies.Retinal degenerative diseases, such as age‐related macular degeneration (AMD) and retinitis pigmentosa (RP) affect more than 10 million people in the U.S.; however, current treatment options provide only modest or transient benefit MERTK) gene MerTK mutation SRI) derived from Wharton's jelly IV in immunocompetent RCS rats For RP patients with recessive mutations in the MER proto‐oncogene tyrosine kinase  to induce the sham effect and create a chemotactic source in which competitive migration and function could be compared within each animal. We found that MSCIV potentiated the retinal protection conferred by MSCSRI or BSSSRI through nonredundant mechanisms. Translational implications from combined MSC therapy suggest that MSCIV might serve as a universal adjuvant to improve clinical outcomes for cell‐based therapies.In the present study, we addressed whether isogenic MSCIV or MSCSRI, or both, at degeneration onset . Unilateral MSCSRI (5 × 104 cells) in 2 μl of BSS  was performed through a scleral incision using a fire‐polished glass pipette , attached by rubber tubing to a 25‐μl syringe . Sham injection consisted of 2 μl of BSSSRI. At 24 hours after SRI, the rats received MSCIV via the tail vein with 1.25–1.8 × 106 RCS rat MSCs in 500 µl of BSS.The institutional animal care and use committee of Cedars‐Sinai Medical Center's comparative medicine department approved animal procedures, which were performed in compliance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Dystrophic, pigmented RCS rats received isogenic MSChttp://diagnosysllc.com). To measure LTR, we recorded the responses of multiunit neuronal activity in superior colliculi (SC) to light flashes of a 3° area placed in the receptive field while varying the spot brightness. The LTR was defined as the minimal spot luminance capable of eliciting reliable responses with twofold amplitude above background activity. Measurements were performed at 14–18 locations (0.3–0.5‐mm apart) along each side of SC for maximal coverage of the visual field represented on the SC. Data were graphically expressed as the percentage of SC sites at which the LTR was lower or equal to the luminance values indicated by the x‐axis and as raw values recorded from individual animals with colored heat map overlay.Optokinetic response (OKR), electroretinography (ERG), and luminance threshold recording (LTR) were performed according to our published reports http://www.sakura‐finetek.com), sectioned to 10 µm via a cryostat and stained with 0.4% cresyl violet acetate to examine retinal lamination. CXCR4 immunolabeling was performed with antibody clone 12G5 at 1:100 dilution .Photoreceptor rescue was determined at P60 and P90. The eyes were removed, fixed for 1 hour in 4% paraformaldehyde, subjected to sucrose‐water exchange, embedded in Tissue‐Tek O.C.T. compound  in multiwell plates . MSC‐conditioned medium (CdM) was produced, as previously described http://www.emdmillipore.com), and frozen at −80°C until use. Multilineage MSC differentiation after CXCR4 modulation was performed, as previously described MSCs were derived from 6–8‐week‐old RCS rats, as previously described http://www.peprotech.com) or inhibited with 10 μM 1,1′‐bis‐1,4,8,11‐tetraazacyclotetradecane  or exposed to BSS (control) for 2 hours. MSCs were washed , cultured for an additional 4 hours, and then enzymatically detached from cultureware , manually quantified, resuspended in BSS, and used for MSCIV or plated (5 × 104) onto Millicell inserts of 12‐µm pore size (EMD Millipore) in triplicate for migration assays. Replicate aliquots from each treatment were fixed or permeabilized , stained for immunofluorescence and flow cytometry intracellular staining . MSCs migrated overnight toward 50 ng/ml CXCL12 in basal medium (BM) containing Dulbecco's modified Eagle's medium (DMEM) with 100 U/ml penicillin/streptomycin (Thermo Fisher). in the bottom chamber. Transwell membranes were washed with PBS, nonmigrated cells were removed via a cotton‐tipped applicator, fixed/permeabilized , and the membranes were removed and coverslipped on glass slides  using mounting media containing nucleic acid fluorescent dye 4′,6‐diamidino‐2‐phenylindole (DAPI) to identify migrated cells . Migration was quantified by averaging the cell number from 10 random fields of view at ×40 magnification using Java‐based imaging software . BM served as the negative control, and BM with 30% fetal bovine serum  served as the positive control.CXCR4 was stimulated in MSCs at passages 4 to 5 with 50 ng/ml recombinant rat CXCL12 . Fluorescent cells were detected via LSR Fortessa Analyzer (BD Biosciences). To determine whether MSC‐CdM rescued retinal function, the rats received unilateral SRI of serum‐free 35× CdM (CdMSRI) collected from passage 3 to 5 RCS‐MSCs. A complete blood count (CBC) was performed on days 1, 3, 7, and 14 on peripheral blood extracted via the tail vein using the Hemavet 950FS . Tissues were evaluated for migration of PKH+ MSCs on day 1, 3, or 7 in retinal cryosections.Fluorescent cell membrane dyes PKH26 and PKH67 (Sigma‐Aldrich) were used to label MSCs immediately before injection. Retinal imaging was performed on flat‐mount dissections or 10‐µm cryosections. For flow cytometry, the eyes were removed and stored at 4°C in PBS (pH 7.4) and dissociated into single‐cell suspension by 20 minutes of incubation at 37°C with enzymatic solution consisting of Cahttp://www.promega.com).RPE cultures were established from P10 RCS rat retinae, seeded at 1,000 per well in 96‐well clear‐bottom tissue culture fluorescence plates (Corning Inc.), and expanded in growth medium (GM) containing Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 100 U/ml penicillin/streptomycin for 2 weeks before bioassays. RPE metabolic activity was measured by cellular dehydrogenase enzyme conversion of 3‐‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium substrate to formazan by colorimetric assay  were labeled with fluorescein‐5‐isothiocyanate isomer‐I  and fed to RPE. RPE were washed (2× PBS), cultured overnight in BM, exposed to 5× MSC‐CdM for 24 hours, and incubated for 18 hours following FITC‐POS addition at 1.44 μg per well. Total fluorescence was measured after three PBS washes to remove unbound POSs . Internalized POSs were quantified by ImageJ colocalization function after fixation/permeabilization  via lysosomal maker immunolabeling for cathepsin‐D 1:250 , and normalized by cell number (DAPI). RPE unexposed to FITC‐POS served as baseline fluorescence control. Samples were measured in triplicate or quadruplicate.To determine RPE phagocytosis after CdM exposure, bovine POSs  neutralizing (Thermo Fisher), fibroblast growth factor (FGF)‐2, vascular endothelial growth factor (VEGF)‐A, CXCL12, glial‐derived neurotrophic factor (GDNF), BDNF, ciliary neurotrophic factor (CNTF) p75 nerve growth factor receptor , connective tissue growth factor , nerve growth factor (NGF)/proNGF, tropomyosin receptor kinase (Trk)A, TrkB, TrkC, glial cell‐derived neurotrophic factor receptor α1, rat p75NTR . Recombinant peptides CXCL12, basic FGF , and BDNF (Sigma‐Aldrich) were used at 100 ng/ml in BM containing 0.1% NaN3 for ex vivo assays.For trophic factor and receptor blocking/neutralization studies, the following antibodies were used at 5 µg/ml: mouse immunoglobulin (Ig) Gt tests were performed using two‐tailed distribution and two‐sample unequal variance (heteroscedastic) to compare LTR values averaged for each eye and raw OKR values from individual eyes. To determine the relative treatment efficacy, values from treated and contralateral (untreated) eyes were compared in individual animals and are expressed as the percentage of improvement, with the animals grouped by treatment. Error bars in animal experiments represent SEM and in ex vivo assays, SD. Statistical significance has been indicated according to convention: ∗, p ≤ .05; ∗∗, p ≤ .01; ∗∗∗, p ≤ .001; †, p ≤ .05; ††, p ≤ .01; †††, p ≤ .001.Student's IV administration increases the vision rescue conferred by MSCSRI transplantation. We first compared subretinal treatments alone, in which rats received MSCSRI (n = 5), BSSSRI , or no treatment (n = 6). As singular treatments, visual acuity was higher from MSCSRI than either BSSSRI (p < .001) or untreated controls .We determined whether MSCIV with MSCSRI (n = 6) produced the highest visual acuity . This represented the greatest functional improvement from MSCIV supplementation.Using the combined treatment strategy, MSCIV supplementation . Bilateral electrophysiological recordings from SC represented approximately 80% of the animal's visual field. The cumulative LTR distribution curve is logarithmic sigmoidal in shape and becomes horizontally exaggerated toward the y‐axis as light sensitivity increases. Untreated RCS rats at P90 showed LTR values of 2.7 log units or higher above background (0.02 log unit). In contrast, congenic wild‐type rats typically show ≤0.5 log unit SRI with MSCIV treatment significantly increased retinal sensitivity to light  compared with MSCSRI or MSCIV singular treatments at the threshold values indicated. LTR did not differ between MSCSRI alone versus BSSSRI combined with MSCIV.Detailed analysis of visual function rescue by LTR indicated synergistic improvement from MSCentation . BilateSRI‐treated eyes increased from 17 of 83 positions (20%) to 36 of 84 positions (43%) by MSCIV supplementation (p < .05). The larger rescue area corresponded to a 3.9‐fold increase in total retinal sensitivity . MSCIV supplementation of BSSSRI increased the hot spot area from 8 of 50 (16%) to 21 of 47 positions  and increased sensitivity 3.4‐fold . LTR consistently showed an increased size of rescue areas from MSCIV supplementation . Photoreceptor rescue areas from MSCSRI alone  to high (red), with hot spots of focal rescue defined as values less than 33% of the mean from both colliculi of each animal . The hotRI alone  were larlone  or antagonist (AMD3100) and confirmed altered migration toward CXCL12 ex vivo and controlled for additional changes to MSC function that might affect graft potency. We first confirmed that MSCs can respond to CXCL12 by their expression of the CXCR4 via immunocytochemistry (p < .05) and decrease with AMD3100 treatment , AMD3100 (n = 6), or BSS  eyes in each animal. Visual acuity increased 18% from CXCL12‐conditioned MSCIV .To determine whether MSC chemotaxis contributed to vision rescue, we stimulated or inhibited CXCR4 activity before MSCIV  and testp < .05; . AMD3100p < .01) or AMD3100 conditioning  on day 3 used for illustration  or BSSSRI (control) and assessed vision at P60. ERG b‐wave amplitudes were 50% higher in CdMSRI‐treated eyes  compared with a 16% decrease with BSSSRI  compared with the contralateral eyes (blue) and compared with BSSSRI  RPE increased during 24 hours of culture in GM compared with BM and was highest in MSC‐CdM (Bound (green), colocalized in silico with phagolysosome marker cathepsin‐D (red), demonstrated greater POS internalization in MSC‐CdM versus BM when normalized to cell number . Spleen weights normalized to body weight did not differ among the rats from the two treatment groups (data not shown). Two of the three rats that received MSCIV had thrombocytopenia on day 3, with platelet counts of 306 × 109/l and 254 × 109/l, well below the normal range . Monocyte counts were also reduced on days 3 and 7 after MSCIV  also produced minor focal rescue  was greater than that from BSSSRI, the repertoire of MSC‐derived trophic factors likely differed from those induced by the sham effect or differed in quantity (SRI or BSSSRI was similarly increased by MSCIV supplementation (3.9‐fold with MSCSRI and 3.4‐fold with BSSSRI). Rescue was greater from MSCSRI by all functional tests, indicating that trophic support from BSSSRI with MSCIV did not compensate for the absence of MSCSRI in retinae .Because the MSC secretome is environment‐dependent IV . We did not examine this possibility; however, in such a scenario, intra‐arterial infusion might increase the number of aggregated MSCs and improve rescue. The intent of enhancing MSCIV migration, targeting, adherence, or integration to specific tissues is to improve therapeutic efficacy IV in the presence of vision rescue suggests that the site of injury is not the only site of MSC action. Accordingly, enhanced MSCIV migration to the retina might reduce vision rescue if it is at the expense of recruitment to the appropriate destination from which MSCs confer therapeutic efficacy. Additionally, the duration of vision rescue greatly exceeded that of MSCSRI graft survival. It is therefore of interest to characterize the temporal window during which synergistic rescue can be achieved and whether this timeframe can be extended by altering the transplant strategy .It is possible that MSCIV supplementation did not facilitate MSCSRI persistence in the subretinal space. This was unlikely to be due to rejection, because MSCs are hypoimmunogenic, mitigate inflammatory responses MerTK in macrophages, because engulfment can occur through alternate pathways IV has been shown to reduce microglia/macrophage infiltration to ischemic sites SRI disappearance from dystrophic retinae is specific to MSCs. Yet unclear is whether this can be advantageous for cotransplant paradigms that necessitate transient support.MSCIV as adjuvant therapy is particularly attractive because MSCs are a renewable resource that can be readministered noninvasively to minimize retinal damage in patients. MSCIV could also reduce immunosuppression toxicity by permitting lower dosing regimens and help avoid complications from patient noncompliance. The multiple and complex mechanisms described for MSC‐mediated retinal protection require further clarification. It is not yet clear how MSC coadministration synergizes to protect dystrophic retinae. However, this observation underscores the possibility that yet unidentified transplant strategies might improve MSC therapy. Of clinical relevance is whether MSCIV supplementation can improve vision rescue from different disease etiologies. More generally, MSCIV adjuvant therapy may enhance the therapeutic outcomes of cell‐based therapies for other medical conditions.Using MSCIV has gained momentum for clinical adoption with their immunosuppressive properties serving as the preeminent rationale. The vision rescue from MSCSRI in RCS rats was dramatically increased by MSCIV supplementation. Thus, cell therapies for progressive degenerative diseases, such as AMD and RP, might be more effective when combined with MSCIV. The translational implications of potentiating treatments currently deemed insufficient warrants further investigation of MSCIV adjuvant therapy.MSCB.B. and S.W.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript, financial support; S.G.: collection and/or assembly of data, data analysis and interpretation; B.L.: conception and design, collection and/or assembly of data, data analysis and interpretation.The authors indicated no potential conflicts of interest.Supporting InformationClick here for additional data file.


Acute myeloid leukemia (AML) primary cells can be isolated from peripheral blood, suspended with media containing bovine serum and cryoprotectant, and stored in liquid nitrogen before being processed for proteomic analysis by mass spectrometry (MS). The presence of bovine serum and human blood proteins in AML samples can hamper the identifications of proteins, and thereby reduce the proteome coverage of the study. Herein, we have established the effect of phosphate buffered saline (PBS) washing on AML patient samples stored in media. Although PBS washes effectively removed serum and blood contaminants, the saline wash resulted in cell burst and remarkable protein material loss. We also compared different methods to preserve the AML proteome from THP-1 and Molm-13 cell lines before MS analysis: (1) stored in media containing bovine serum and dimethyl sulfoxide (DMSO); (2) stored as dried cell pellets; and (3) stored as cell lysates in 4% sodium dodecyl sulfate (SDS). MS analysis of differently preserved AML cell samples shows that preservation with DMSO produce a high number of fragile cells that will burst during freezing and thawing. Our studies encourage the use of alternative preservation methods for future MS analysis of the AML proteome. Acute myeloid leukemia (AML) is an aggressive hematopoietic cancer that is characterized by limited differentiation and uncontrolled proliferation of myeloid progenitor cells . There iWithin the field of MS-based proteomics, large efforts have been put into optimization of sample preparation workflows, including cell lysis and protein digestion ,6, liquiPrimary AML cells may be obtained from the bone marrow aspirate or from the peripheral blood of patients with a high number (>80%) of circulating blasts ,16. AfteIn the current study, we have studied the effect of phosphate buffered saline (PBS) wash on cell pellets from AML patients and evaluate its impact on the AML proteome coverage and quantification. Furthermore, the effect of three storage and preparation protocols on two AML-derived cell lines: (1) liquid nitrogen storage in media containing bovine serum and DMSO; (2) liquid nitrogen storage as dried cell pellets; and (3) −80 °C storage as cell lysates in 4% sodium dodecyl sulfate (SDS) has been investigated for future applicability on the preservation of the AML primary cell material for MS analysis. Cryopreserved AML patient samples in liquid nitrogen contain DMSO to protect the cells from rupture and serum in order to maintain cellular viability and function. Freezing medium containing 20% FBS and 10% DMSO is our standard for primary AML samples destined to cell culture assays, but should be removed prior to proteomic analysis ,17. HereTo examine the changes in the proteome after the PBS washing steps, we performed enrichment analyses with the web-based software a GO tool c. We fouWe have previously demonstrated a significant difference in expression levels of the AML patient proteome between cryopreserved (DMSO samples from now onwards) and freshly processed cells (SDS samples from now onwards), i.e., lysed in 4% SDS directly after blast isolation . Here, wt- and Z-tests ), when comparing DMSO vs. pellet, DMSO vs. SDS, and pellet vs. SDS samples, were analyzed with the WebGestalt enrichment web tool and the GO Slim classification  and Runt-related transcription factor 1 (RUNX1) transcription factors d. While When we compared the FC of proteins significantly regulated in the DMSO vs. SDS samples of the THP-1 cell line to AML patient samples, previously quantified by our group according to the stable isotope labeling with amino acids in cell culture (SILAC) and label-free procedures , we obtaA similar WebGestalt GO enrichment to the one obtained with THP-1 samples was observed when comparing the most abundant proteins in the SDS and DMSO Molm-13 samples .Here, we demonstrate that frozen/thawed primary AML cells should not be washed with PBS after liquid nitrogen storage. An extensive reduction of the protein amount was observed after washing the cell pellet with PBS. Several contaminants , but also fragile cells (both leukemic and red blood cells) were lost during the first wash. Furthermore, the cells surviving the second wash are probably the most robust cells and/or subpopulation of leukemic cells from the first wash. We found a higher loss of proteins in samples with low cell number, which indicates that 1× PBS usage is not suitable for AML samples with low protein amounts. However, samples containing a higher number of cells appear to better resist the PBS-induced bursting. Moreover, the protein loss during the PBS wash might restrict phosphoproteomic sample preparations, which require higher amount of proteins. Enrichment analysis showed overrepresented proteins associated to organelle, nucleus, and mitochondrion after one wash, and chromosome, organelle, peroxisome, and mitochondrion after two washes. This suggested a cell bursting, presumably due to the osmotic shock produced when cells are placed abruptly in buffer and to cell susceptibility after exposure to PBS, altogether affecting subsequent AML proteome analysis.We have previously evaluated the freezing effect on the proteome of AML patients. We observed that mitochondrial proteins involved in the respiratory chain transport and proteins involved in mRNA splicing were more abundant in samples frozen with media containing 10% DMSO than in samples lysed with 4% SDS before freezing . In ordeThe comparison of the different preservation conditions of THP-1 and Molm-13 revealed that the number of quantified proteins in the samples frozen in 4% SDS at −80 °C, frozen as dried pellets, and frozen with media containing 10% DMSO was very similar. A functional enrichment analysis of the significantly expressed proteins found in DMSO vs. SDS or DMSO vs. pellet shows similar enrichment in the categories of biological processes and cellular components. This was expected as both SDS and pellet samples were, respectively, lysed and frozen immediately. The enrichment analysis of proteins more abundant in the DMSO samples showed proteins associated to mitochondrion and nucleus, which probably reflects cell bursting during freezing, thawing, and centrifugation of leukemic cells, as we have previously reported. Moreover, the expression of AML protein biomarkers, such as CEBPA and RUNX1, appeared to be influenced by preservation methodologies . AML cells preserved in DMSO/FBS-containing media are used for cell culturing and flow cytometry assays. The highest leukocyte viability has been observed in patient samples cryopreserved in the presence of 4–5% DMSO . This mi9/L). At least 95% of circulating mononuclear cells being leukemic blasts after a density gradient separation  were stored in liquid nitrogen [AML patient samples were collected, after written informed consent, from subjects with high peripheral blood blast counts  contaminations after the density gradient separation, were selected for the study of PBS wash (1× solution) of AML primary cells . After t2 and 6.5% CO2. Cells were split into three identical aliquots and spun at 140× g at RT for 5 min. One aliquot was lysed with 4% SDS in 0.1 M Tris·HCl pH 7.6 buffer and denatured at 95 °C for 7 min before storage at −80 °C, while another one was resuspended in RPMI media containing 20% FBS and 10% DMSO and frozen at −80 °C for 24–48 h before preservation in liquid nitrogen as previously described [g at 4 °C for 5 min, both sets of samples were lysed with 4% SDS in 0.1 M Tris·HCl pH 7.6 buffer.Two AML cell lines, THP-1 and Molm-13, were selected for the study of different sample preservation methodologies. The cells were cultured with 10% fetal calf serum (FCS) in a medium based on RPMI, with some metabolites adjusted to better reflect the concentrations encountered in circulating human plasma. Thus, alanine was 0.25 mM (0 in RPMI), glutamine was 0.5 mM (2.5 mM in RPMI), and glucose was 5 mM (11 mM in RPMI). The proline concentration was adjusted to 0.12 mM to reflect the level reported in leukemic bone marrow . A low cSDS-lysed patient and cell line samples were processed and digested according to the filter-aided sample preparation (FASP) method ,24. All Dried peptides were dissolved in 20 μL of 2% acetonitrile (ACN) and 0.5% formic acid (FA). Differently preserved THP-1 and Molm-13 samples were analyzed on an Orbitrap Elite mass spectrometer equipped with a nanospray Flex ion source coupled to an Ultimate 3000 Rapid Separation LC system . Approximately 0.5 μg peptides were pre-concentrated and separated, as previously described [Raw data were processed with MaxQuant version 1.5.5.1 ,26. The t-tests and Z-statistics, both run in Microsoft Excel, were applied to compare groups for statistical differences and to obtain fold change significance, respectively [Paired ectively . Functioectively ,32. The Regulated proteins were imported into StringDB software version 10.5 for the analysis of protein interactions . ProteinScatter plots and Spearman correlation were done using with GraphPad Prism v7.03 (GraphPad Software).The raw MS data files of both the PBS wash and preservation experiments are available via ProteomeXchange with identifier PXD008361.The objective of this study was to select optimized preparation conditions of AML samples for MS-based proteomic studies. The use of PBS wash for media and blood contaminant removal showed a highly modified proteome, especially for samples with low cell amounts. Enrichment analysis showed overrepresented proteins that were associated to organelle, nucleus, and mitochondrion, suggesting cell burst after the PBS washing step. The current use of 20% FBS/10% DMSO in the freezing medium of our AML cell line samples affect the quantification of AML proteins when compared to samples lysed and stored in 4% SDS and to samples stored as a dried pellet. We found several proteins with GO terms involved in cell proliferation, regulation of phosphorylation and signal transduction underrepresented in samples cryopreserved in 20% FBS/10% DMSO when compared to dried pellet and 4% SDS storage conditions. A universal preservation method of the AML samples cannot be selected for all subsequent cell-based, genomics and proteomics studies. However, a fraction of the AML blasts after isolation could be immediately frozen as a dried cell pellet or lysed with 4% SDS for future MS-based proteomic characterization.