Accessing the Human Pluripotent Stem Cell Translatome by Polysome Profiling.

Polysome profiling is a technique that uses sucrose density gradient ultracentrifugation to separate complexes of mRNAs associated with one or more ribosomes. Here we describe polysome profiling analysis in human pluripotent stem cells (hPSCs) using a continuous ultraviolet spectrophotometer and a gradient fractionator. We provide protocols for processing sucrose gradient fractions for isolation of RNA for RT-qPCR or large-scale sequencing analysis, used to establish the translational status of specific mRNAs and identify the role of noncoding RNA in translation.

Reorganization of Metabolism during Cardiomyogenesis Implies Time-Specific Signaling Pathway Regulation.

Understanding the cell differentiation process involves the characterization of signaling and regulatory pathways. The coordinated action involved in multilevel regulation determines the commitment of stem cells and their differentiation into a specific cell lineage. Cellular metabolism plays a relevant role in modulating the expression of genes, which act as sensors of the extra-and intracellular environment. In this work, we analyzed mRNAs associated with polysomes by focusing on the expression profile of metabolism-related genes during the cardiac differentiation of human embryonic stem cells (hESCs). We compared different time points during cardiac differentiation (pluripotency, embryoid body aggregation, cardiac mesoderm, cardiac progenitor and cardiomyocyte) and showed the immature cell profile of energy metabolism. Highly regulated canonical pathways are thoroughly discussed, such as those involved in metabolic signaling and lipid homeostasis. We reveal the critical relevance of retinoic X receptor (RXR) heterodimers in upstream retinoic acid metabolism and their relationship with thyroid hormone signaling. Additionally, we highlight the importance of lipid homeostasis and extracellular matrix component biosynthesis during cardiomyogenesis, providing new insights into how hESCs reorganize their metabolism during in vitro cardiac differentiation.

Polysome-associated lncRNAs during cardiomyogenesis of hESCs.

Long non-coding RNAs (lncRNAs) have been found to be involved in many biological processes, including the regulation of cell differentiation, but a complete characterization of lncRNA is still lacking. Additionally, there is evidence that lncRNAs interact with ribosomes, raising questions about their functions in cells. Here, we used a developmentally staged protocol to induce cardiogenic commitment of hESCs and then investigated the differential association of lncRNAs with polysomes. Our results identified lncRNAs in both the ribosome-free and polysome-bound fractions during cardiogenesis and showed a very well-defined temporal lncRNA association with polysomes. Clustering of lncRNAs was performed according to the gene expression patterns during the five timepoints analyzed. In addition, differential lncRNA recruitment to polysomes was observed when comparing the differentially expressed lncRNAs in the ribosome-free and polysome-bound fractions or when calculating the polysome-bound vs ribosome-free ratio. The association of lncRNAs with polysomes could represent an additional cytoplasmic role of lncRNAs, e.g., in translational regulation of mRNA expression.

Secretome Analysis Performed During in vitro Cardiac Differentiation: Discovering the Cardiac Microenvironment.

Human pluripotent stem cells are an important tool for the study of developmental processes, such as cardiomyogenic differentiation. Despite the advances made in this field, the molecular and cellular signals involved in the commitment of embryonic stem cells to the cardiac phenotype are still under investigation. Therefore, this study focuses on identifying the extracellular signals involved in in vitro cardiac differentiation of human embryonic stem cells. Using a three-dimensional cardiomyogenic differentiation protocol, the conditioned medium and the extracellular matrix (ECM) of embryoid body cultures were collected and characterized at four specific time points. Mass spectrometry (MS) and antibody array analysis of the secretome identified a number of secreted proteins related to signaling pathways, such as Wnt and TGFß, as well as many ECM proteins. When comparing the proteins identified at selected time points, our data pointed out protein interactions and biological process related to cardiac differentiation. Interestingly, the great changes in secretome profile occurred during the cardiac progenitor specification. The secretome results were also compared with our previous RNAseq data, indicating that the secreted proteins undergo some level of gene regulation. During cardiac commitment it was observed an increase in complexity of the ECM, and some proteins as IGFBP7, FN1, HSPG2, as well as other members of the basal lamina could be highlighted. Thus, these findings contribute valuable information about essential microenvironmental signals working on cardiomyogenic differentiation that may be used in future strategies for cardiac differentiation, cardiomyocyte maturation, and in advances for future acellular therapies.

Cell cycle genes are downregulated after adipogenic triggering in human adipose tissue-derived stem cells by regulation of mRNA abundance.

The adipogenic process is characterized by the expression of adipocyte differentiation markers that lead to changes in cell metabolism and to the accumulation of lipid droplets. Moreover, during early adipogenesis, cells undergo a strong downregulation of translational activity with a decrease in cell size, proliferation and migration. In the present study, we identified that after 24 hours of adipogenic induction, human adipose tissue-derived stem cells (hASCs) undergo a G1-cell cycle arrest consistent with reduced proliferation, and this effect was correlated with a shift in polysome profile with an enrichment of the monosomal fraction and a reduction of the polysomal fraction. Polysome profiling analysis also revealed that this change in the monosomal/polysomal ratio was related to a strong downregulation of cell cycle and proliferation genes, such as cyclins and cyclin-dependent kinases (CDKs). Comparing total and polysome-associated mRNA sequencing, we also observed that this downregulation was mostly due to a reduction of cell cycle and proliferation transcripts via control of total mRNA abundance, rather than by translational control.

Cardiomyogenic differentiation is fine-tuned by differential mRNA association with polysomes.

BACKGROUND: Cardiac cell fate specification occurs through progressive steps, and its gene expression regulation features are still being defined. There has been an increasing interest in understanding the coordination between transcription and post-transcriptional regulation during the differentiation processes. Here, we took advantage of the polysome profiling technique to isolate and high-throughput sequence ribosome-free and polysome-bound RNAs during cardiomyogenesis. RESULTS: We showed that polysome-bound RNAs exhibit the cardiomyogenic commitment gene expression and that mesoderm-to-cardiac progenitor stages are strongly regulated. Additionally, we compared ribosome-free and polysome-bound RNAs and found that the post-transcriptional regulation vastly contributes to cardiac phenotype determination, including RNA recruitment to and dissociation from ribosomes. Moreover, we found that protein synthesis is decreased in cardiomyocytes compared to human embryonic stem-cells (hESCs), possibly due to the down-regulation of translation-related genes. CONCLUSIONS: Our data provided a powerful tool to investigate genes potentially controlled by post-transcriptional mechanisms during the cardiac differentiation of hESC. This work could prospect fundamental tools to develop new therapy and research approaches.

Polysome profiling followed by RNA-seq of cardiac differentiation stages in hESCs.

The regulation of gene expression acts at numerous complementary levels to control and refine protein abundance. The analysis of mRNAs associated with polysomes, called polysome profiling, has been used to investigate the post-transcriptional mechanisms that are involved in different biological processes. Pluripotent stem cells are able to differentiate into a variety of cell lineages, and the cell commitment progression is carefully orchestrated. Genome-wide expression profiling has provided the possibility to investigate transcriptional changes during cardiomyogenesis; however, a more accurate study regarding post-transcriptional regulation is required. In the present work, we isolated and high-throughput sequenced ribosome-free and polysome-bound RNAs from NKX2-5eGFP/w HES3 undifferentiated pluripotent stem cells at the subsequent differentiation stages of cardiomyogenesis: embryoid body aggregation, mesoderm, cardiac progenitor and cardiomyocyte. The expression of developmental markers was followed by flow cytometry, and quality analyses were performed as technical controls to ensure high quality data. Our dataset provides valuable information about hESC cardiac differentiation and can be used to investigate genes potentially controlled by post-transcriptional mechanisms.

Gene expression analysis of human adipose tissue-derived stem cells during the initial steps of in vitro osteogenesis.

Mesenchymal stem cells (MSCs) have been widely studied with regard to their potential use in cell therapy protocols and regenerative medicine. However, a better comprehension about the factors and molecular mechanisms driving cell differentiation is now mandatory to improve our chance to manipulate MSC behavior and to benefit future applications. In this work, we aimed to study gene regulatory networks at an early step of osteogenic differentiation. Therefore, we analyzed both the total mRNA and the mRNA fraction associated with polysomes on human adipose tissue-derived stem cells (hASCs) at 24 h of osteogenesis induction. The RNA-seq results evidenced that hASC fate is not compromised with osteogenesis at this time and that 21 days of continuous cell culture stimuli are necessary for full osteogenic differentiation of hASCs. Furthermore, early stages of osteogenesis induction involved gene regulation that was linked to the management of cell behavior in culture, such as the control of cell adhesion and proliferation. In conclusion, although discrete initial gene regulation related to osteogenesis occur, the first 24 h of induction is not sufficient to trigger and drive in vitro osteogenic differentiation of hASCs.

Chemical and biological characterization of polysaccharides isolated from Ilex paraguariensis A. St.-Hil.

The potential gastroprotection of polysaccharides (SP) isolated from maté (Ilex paraguariensis) leaves of different growth stages, under different sunlight conditions and of processing methods were evaluated. The SP consist of type I arabinogalactan (AG1) containing a (1→4)-linked ß-Galp chain, with substituents of arabinosyl units at O-6. This arabinogalactan seems to be attached to rhamnosyl units from a RG1, via 1→4 linkage. Oral administration of SP1, SP9, SP10, SP11 and SP12 inhibited the gastric lesions induced by ethanol in rats. Altogether, the present data indicate the therapeutic role of maté polysaccharides against gastric lesion and propose its use or of its crude plant extract as a phytotherapic medicine.

Antiulcer and gastric antisecretory effects of dichloromethane fraction and piplartine obtained from fruits of Piper tuberculatum Jacq. in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Piper tuberculatum Jacq. (Piperaceae) is medicinally used as an analgesic and as a treatment for gastric complaints. Thus, the current study aimed to investigate the gastroprotective and antisecretory properties of the dichloromethane fraction of the fruit of Piper tuberculatum (DFPT) and piplartine, a compound isolated from the DFPT, in rats. MATERIALS AND METHODS: Gastric ulcers were induced in fasted rats by oral administration of absolute ethanol and then mucus content and glutathione (GSH) levels were measured. Mechanisms underlying the antisecretory action were studied through gastric H(+),K(+)-ATPase activity of highly purified rabbit gastric microsomes and pylorus ligature method in rats. RESULTS: In the acute toxicity test the values of estimated LD50 for oral and intraperitoneal administration of DFPT were 1.6266 and 0.2684g/kg, respectively. The DFPT (ED50=29mg/kg, p.o.) and piplartine (4.5mg/kg, p.o.) promoted gastroprotection against acute lesions induced by ethanol, effect that could be related with the maintenance of GSH levels in the gastric mucosa. However, only DFPT stimulated gastric mucus secretion. In vitro, the DFPT and piplartine inhibited the H(+),K(+)-ATPase activity and, in vivo DFPT and piplartine also reduced basal gastric acid secretion, as well as that stimulated by pentagastrin. CONCLUSIONS: These results demonstrate that DFPT and piplatine cause marked gastroprotective effects accompanied by the increase and maintenance of gastric mucus and GSH levels, as well as a reduction in gastric acid secretion through the gastrinergic pathway.

Antiulcer effect of bark extract of Tabebuia avellanedae: activation of cell proliferation in gastric mucosa during the healing process.

Tabebuia avellanedae (syn. Handroanthus impetiginosus) is popularly known as 'ipê-roxo' and has been used in folk medicine as anti-inflammatory and in the treatment of ulcers, bacterial and fungal infections. This study evaluated the gastric ulcer healing property of the ethanolic extract (EET) of barks from Tabebuia avellanedae and investigated the mechanisms that may underlie this effect. Rats were treated with EET (twice a day for 7 days) after induction of chronic gastric ulcers by 80% acetic acid. Following treatment, histological and immunohistochemical analysis were performed in gastric ulcer tissues. Oral administration of EET (100 and 300 mg/kg) significantly reduced the gastric lesion induced by acetic acid in 44 and 36%, respectively. Histopathological evaluation demonstrated a contraction of gastric ulcer size, increase of mucus layer (periodic acid-Schiff stained mucin-like glycoproteins) and cell proliferation (proliferating cell nuclear antigen immunohistochemistry) in animals treated with EET (100 and 300 mg/kg). The results demonstrate that EET significantly accelerates healing of acetic acid induced gastric ulcer in rats through increase of mucus content and cell proliferation, indicating a potential usefulness for treatment of peptic ulcer diseases.

Evaluation of the antinociceptive, anti-inflammatory and gastric antiulcer activities of the essential oil from Piper aleyreanum C.DC in rodents.

ETHNOPHARMACOLOGICAL RELEVANCE: Piper aleyreanum is a small tree that is widely distributed in tropical and subtropical regions, mostly in North and South America, and is used as an immunomodulator, analgesic and antidepressant in folk medicine. AIM OF THE STUDY: This study was designed to investigate the antinociceptive, anti-inflammatory and gastric antiulcer activities of the essential oils from the aerial parts of Piper aleyreanum (EOPa) in rodents. MATERIALS AND METHODS: The antinociceptive and anti-inflammatory effects of orally administered EOPa were evaluated in mice subjected to the formalin and pleurisy models, respectively. We also pretreated the rats with EOPa before acute ethanol-induced gastric lesions and measured gastric lesion extension and mucus and glutathione (GSH) levels in the gastric mucosa. Finally, we performed a phytochemical analysis of EOPa. RESULTS: The chemical composition of EOPa was analyzed by gas chromatography and mass spectrometry (GC/MS), which identified 35 compounds, representing 81.7% of total oil compounds. Caryophyllene oxide (11.5%), ß-pinene (9%), spathulenol (6.7%), camphene (5.2%), ß-elemene (4.7%), myrtenal (4.2%), verbenone (3.3%) and pinocarvone (3.1%) were the major oil constituents. The oral administration of EOPa (10-1000 mg/kg) significantly inhibited the neurogenic and inflammatory phases of formalin-induced licking, with ID50 values of 281.2 and 70.5 mg/kg, respectively. The antinociception caused by EOPa (100 mg/kg, p.o.) was not reversed by naloxone (1 or 5 mg/kg, i.p.) in the formalin test. EOPa (100-300 mg/kg, p.o.) did not affect animal motor coordination in an open-field model. In carrageenan-induced pleurisy, EOPa (1-100 mg/kg, p.o.) significantly decreased the total cell count, neutrophils and mononuclear cells with mean ID50 values of 53.6, 21.7 and 43.5 mg/kg, respectively. In addition, EOPa (1-30 mg/kg, p.o.) protected the rats against ethanol-induced gastric lesions with an ID50 value of 1.7 mg/kg and increased the mucus and GSH levels of the gastric mucosa to levels similar to those of the non-lesioned group. CONCLUSIONS: These data show for the first time that EOPa has significant antinociceptive and anti-inflammatory actions, which do not appear to be related to the opioid system. EOPa also has interesting gastroprotective effects related to the maintenance of protective factors, such as mucus production and GSH. These results support the widespread use of Piper aleyreanum in popular medicine and demonstrate that this plant has therapeutic potential for the development of phytomedicines with antinociceptive, anti-inflammatory and gastroprotective properties.