Undifferentiated Stage Research Articles

Reproductive cycle of the rock oyster Striostrea prismatica in a natural and estuarine-lagoon environment of the Mexican Central Pacific

ABSTRACT Striostrea prismatica is an oyster of commercial importance present in several locations of the Eastern Tropical Pacific. The objective of this study was to evaluate the sex ratio, gametogenic cycle, maturity stages, size at first maturity (SH50) and monthly variation in the condition index of this species over the course of an annual cycle on the coast of Colima, Mexico. The oysters found in the shallow sublittoral areas near Faro de Campos were larger and heavier than those in Cuyutlan lagoon. The sex ratio was significantly shifted towards males at both sites. The gonad showed a prolonged undifferentiated stage lasting from November to April, a spawning period limited to June through October and an extended post-spawning period from August to December. SH50 was higher in females in the lagoon as well as in the shallow sublittoral. Monthly variation in surface temperature and chlorophyll a in both study areas only correlated with the condition index of populations in the shallow sublittoral and inner lagoon in the months of lowest temperatures and highest chlorophyll a. These findings provide basic information on the reproductive cycle of rock oysters that may prove useful for the development of improved harvesting practices in the study region.

Morphogenesis, megagametogenesis, and microgametogenesis in Actinidia arguta flower buds

In dioecious plants, understanding sexual differentiation is important for improving cultivation techniques. Flower bud morphogenesis and gametophyte formation underlie the mechanism of sexual differentiation. Actinidia arguta (hardy kiwi) is a unisexual plant, and there have been few studies on its flower bud differentiation or gametophyte formation. We studied flower bud morphogenesis and gametophyte formation in A. arguta using paraffin sections and found that the morphogenesis of male and female flower buds was divided into six stages: the undifferentiated stage, inflorescence initials differentiation stage, calyx primordium differentiation stage, petal primordium differentiation stage, stamen primordium differentiation stage, and pistil primordium differentiation stage. Morphogenesis in the male inflorescence can reach quaternary flower primordia, and morphogenesis in the female inflorescence can reach the tertiary flower primordia, however, no Stage 4 lateral flowers in the male inflorescence or Stage 3 lateral flowers in the female inflorescence were found in natural situation. The embryo sac in female plants was a monocellular Polygonum type, and the embryo sac development in male plants was incomplete; abortion occurred before the archesporial cells developed. The male gametophyte differentiation process in the male plant was normal, with pollen grains similar in shape to a rugby ball and arranged neatly in the anther. The pollen grain differentiation in female plants was abnormal; abortion occurred at the uninucleate microspore stage, and mature pollen grains were irregularly spherical.

Annual gametogenic phenology of oyster, Magallana bilineata (Röding, 1798) collected from the west coast of Moheshkhali Island, Cox's Bazar, Bangladesh

The current study aimed to describe the annual gametogenic phenology of the oyster Magallana bilineata (Röding, 1798) (=Crassostrea madrasensis), which is found on the west coast of Moheshkhali Island, Cox's Bazar, Bangladesh. Samples were drawn monthly from the intertidal region during low tide, from which 20 adult individuals were selected at random for biometry and histology. The mean condition index (CI), a ratio of tissue wet weight (g) to shell length (cm), varied from 0.58 ± 0.08 to 1.32 ± 0.36. Histology revealed two spawning cycles in the habitat of M. bilineata over the 12 months of the study. Gametogenesis initiated in December and May, and ripe animals principally occurred from July to September and February to May. In the habitat, M.bilineata exhibited two spawning peak periods: April to June and August to October. The undifferentiated stage as a preparatory step for the next spawning extended from November to February for the first spawning cycle and for a brief period in June for the next spawning cycle. The initiation of spawning in March could be associated with the high-level decline of salinity and increased temperature between February and March, in association with the annual rainfall start, whereas the next spawning cycle could be associated with a gradual increase in salinity. No spawning activity was reported from December to February, when the water temperature remained below 22 °C. Further study could be undertaken on the timing of spatfall of M.bilineata in the habitat to harvest spats for commercial farming of this promising species.

Human RFX6 regulates endoderm patterning at the primitive gut tube stage.

Transcriptional factor RFX6 is known to be a causal gene of Mitchell-Riley syndrome (MRS), an autosomal recessive neonatal diabetes associated with pancreatic hypoplasia and intestinal atresia/malformation. The morphological defects are limited to posterior foregut and mid-hindgut endodermal lineages and do not occur in the anterior foregut lineage; the mechanism remains to be fully elucidated. In this study, we generated RFX6+/eGFP heterozygous knockin and RFX6eGFP/eGFP homozygous knockin/knockout human-induced pluripotent stem cell (hiPSC) lines and performed in vitro endoderm differentiation to clarify the role of RFX6 in early endoderm development. RFX6 expression was found to surge at the primitive gut tube (PGT) stage in comparison with that in the undifferentiated or definitive endoderm stage. At the PGT stage, the expression of PDX1 and CDX2, posterior foregut and mid-hindgut master regulators, respectively, was decreased by the RFX6 deficit. PDX1+ and CDX2+ cells were mostly green fluorescent protein (GFP)+ in RFX6+/eGFP hiPSCs, but their cell number was markedly decreased in RFX6eGFP/eGFP hiPSCs. The expression of SOX2, an anterior foregut marker, was not affected by the RFX6 deficit. In addition, we found a putative RFX6-binding X-box motif using cap analysis of gene expression-seq and the motif-containing sequences in the enhancer regions of PDX1 and CDX2 bound to RFX6 in vitro. Thus, RFX6 regulates the ParaHox genes PDX1 and CDX2 but does not affect SOX2 in early endodermal differentiation, suggesting that defects in early stage endoderm patterning account for the morphological pathology of MRS.

Low-dose Bisphenol A exposure alters the functionality and cellular environment in a human cardiomyocyte model

Early embryonic development represents a sensitive time-window during which the foetus might be vulnerable to the exposure of environmental contaminants, potentially leading to heart diseases also later in life. Bisphenol A (BPA), a synthetic chemical widely used in plastics manufacturing, has been associated with heart developmental defects, even in low concentrations. This study aims to investigate the effects of environmentally relevant doses of BPA on developing cardiomyocytes using a human induced pluripotent stem cell (hiPSC)-derived model. Firstly, a 2D in vitro differentiation system to obtain cardiomyocytes from hiPSCs (hiPSC-CMs) have been established and characterised to provide a suitable model for the early stages of cardiac development. Then, the effects of a repeated BPA exposure, starting from the undifferentiated stage throughout the differentiation process, were evaluated. The chemical significantly decreased the beat rate of hiPSC-CMs, extending the contraction and relaxation time in a dose-dependent manner. Quantitative proteomics analysis revealed a high abundance of basement membrane (BM) components (e.g., COL4A1, COL4A2, LAMC1, NID2) and a significant increase in TNNC1 and SERBP1 proteins in hiPSC-CMs treated with BPA. Network analysis of proteomics data supported altered extracellular matrix remodelling and provided a disease-gene association with well-known pathological conditions of the heart. Furthermore, upon hypoxia-reoxygenation challenge, hiPSC-CMs treated with BPA showed higher rate of apoptotic events. Taken together, our results revealed that a long-term treatment, even with low doses of BPA, interferes with hiPSC-CMs functionality and alters the surrounding cellular environment, providing new insights about diseases that might arise upon the toxin exposure. Our study contributes to the current understanding of BPA effects on developing human foetal cardiomyocytes, in correlation with human clinical observations and animal studies, and it provides a suitable model for New Approach Methodologies (NAMs) for environmental chemical hazard and risk assessment.

Clinical features and outcomes of pediatric nasopharyngeal carcinoma: A SEER database study.

e22011 Background: Nasopharyngeal carcinoma (NPC) is a rare malignant tumor. Little is known about the clinical, histological and survival characteristics of this disease in pediatric patients. Methods: We searched 17 registries of the surveillance, epidemiology, and end results (SEER) database for pediatric patients (£20 years old) with NPC who were diagnosed between 2000 to 2019. Data obtained included the demographics, histological subtype, stage, median household income, place of residence, cause specific survival (CSS) and overall survival (OS). We describe the clinical features and survival outcomes by race of pediatric patients. Results: A total of 373 primary pediatric NPC patients were reported between 2000-2019. Median age of diagnosis was 16 years (inter-quartile range 11.5 – 19 years). Of these, 68% were males, 59% were white and 27% were black. Undifferentiated non-keratinizing carcinoma group (WHO type III) was the most frequent (70%) histological sub-type. WHO type I and II disease was found to be present in 12% and 18% cases respectively. Nearly, 4 in 5 patients (83%) had stage III – IV NPC at presentation. Stage I and II disease at presentation was found to be present in 5% and 12% of NPC patients respectively. The median follow-up was 10.1 years. The 5 and 10 year survival estimates were 83% (95% CI 78.8% to 86.9%) and 79% (95% CI 73.9% to 83.1%) respectively. On multivariate Cox regression analysis, gender (Male vs Female: HR: 1.00, 95% CI 0.39-2.57; p = 0.998) and race (White vs Black: HR: 0.81 95% CI 0.311-2.10; p = 0.663) were not found to be significant predictors of survival. Conclusions: Pediatric patients at presentation with NPC were more commonly male, had undifferentiated non-keratinizing carcinoma (WHO type III) and stage III-IV disease. Gender and race were not found to be associated with an increased risk for mortality in pediatric patients presenting with NPC.

PmAGAMOUS recruits polycomb protein PmLHP1 to regulate single-pistil morphogenesis in Japanese apricot.

Japanese apricot (Prunus mume Sieb. et Zucc.) is a traditional fruit tree with a long history. Multiple pistils (MP) lead to the formation of multiple fruits, decreasing fruit quality and yield. In this study, the morphology of flowers was observed at 4 stages of pistil development: undifferentiated stage (S1), predifferentiation stage (S2), differentiation stage (S3), and late differentiation stage (S4). In S2 and S3, the expression of PmWUSCHEL (PmWUS) in the MP cultivar was significantly higher than that in the single-pistil (SP) cultivar, and the gene expression of its inhibitor, PmAGAMOUS (PmAG), also showed the same trend, indicating that other regulators participate in the regulation of PmWUS during this period. Chromatin immunoprecipitation-qPCR (ChIP-qPCR) showed that PmAG could bind to the promoter and the locus of PmWUS, and H3K27me3 repressive marks were also detected at these sites. The SP cultivar exhibited an elevated level of DNA methylation in the promoter region of PmWUS, which partially overlapped with the region of histone methylation. This suggests that the regulation of PmWUS involves both transcription factors and epigenetic modifications. Also, the gene expression of Japanese apricot LIKE HETEROCHROMATIN PROTEIN (PmLHP1), an epigenetic regulator, in MP was significantly lower than that in SP in S2 to 3, contrary to the trend in expression of PmWUS. Our results showed that PmAG recruited sufficient PmLHP1 to maintain the level of H3K27me3 on PmWUS during the S2 of pistil development. This recruitment of PmLHP1 by PmAG inhibits the expression of PmWUS at the precise time, leading to the formation of 1 normal pistil primordium.

Comparative transcriptome analysis of early sexual differentiation in the male and female gonads of common carp (Cyprinus carpio)

The growth rate of allotetraploid common carp (Cyprinus carpio) is sex dimorphic. Thus, intervention for controlling sex is crucial for farming this fish. However, few studies have investigated the spatiotemporal interaction of interaction of various genes related to sex determination/early gonadal differentiation in carp. In this study, comparative gonadal transcriptomic analyses were conducted during 4 key sex differentiaton stages for common carp of both sexes including sex determination stage, pre-gonadal, gonadal differentiation, and maturation stages. Altogether, 305.66 Gb raw reads were obtained form 48 libraries. Differentially expressed genes (DEGs) associated with sex differentiation and gonadal development were identified at each time point. Male-specific DEGs including dmrt1, amh, hmga2, sox3, dmrt2, wnt2b, and wnt9b, and female-biased DEGs including foxl2, cyp19a1a, zp3, zp4, and gper1 were identified. The trend analysis of the key genes of sex determination and gonadal differentiation indicated that 35 dph or earlier is the male sex determination period, 90–100 dph is the key window period of male sex differentiation, and 90–100 dph may be the key window period of female sex differentiation. Functional enrichment analysis showed that DEGs at the testicular undifferentiated stages were mainly enriched in androgen receptor signaling, while DEGs from the onset of testicular differentiation to maturity were enriched in male sex differentiation, spermatogenesis, and male gonadal development. In genetic females, DEGs at the undifferentiated ovarian stage are mainly enriched in insulin-like growth factor (IGF) transport, whereas DEGs from the onset of ovarian differentiation to maturation are mainly enriched in ovarian steroidogenesis and oocyte meiosis. Protein-protein interaction (PPI) analysis revealed that 8 DEGs, including Foxl2, Src, Ncor1, and Smad4, were identified as hub proteins. Additionally, plasma 17β-estradiol and 11-Ketotestosterone levels were sexually dimorphic in C. carpo. The study provide insights into the genetic network of the underlying mechanisms of early sex determination and gonadal differentiation in C. carpo for intervening all-female production in better way.

Retinoic acid induced meiosis initiation in female germline stem cells by remodelling three-dimensional chromatin structure.

ObjectivesThis study aimed to clarify the regulation and mechanism of meiotic initiation in FGSC development.Materials and MethodsFGSCs were induced to differentiate into meiosis in differentiation medium. RNA sequencing was performed to analysis the difference of transcription level. High‐through chromosome conformation capture sequencing (Hi‐C) was performed to analysis changes of three‐dimensional chromatin structure. Chromosome conformation capture further confirmed a spatial chromatin loop. ChIP‐qPCR and dual luciferase reporter were used to test the interaction between Stimulated by retinoic acid gene 8 (STRA8) protein and Trip13 promoter.ResultsCompared with FGSCs, the average diameter of STRA8‐positive germ cells increased from 13 μm to 16.8 μm. Furthermore, there were 4788 differentially expressed genes between the two cell stages; Meiosis and chromatin structure‐associated terms were significantly enriched. Additionally, Hi‐C results showed that FGSCs underwent A/B compartment switching (switch rate was 29.81%), the number of topologically associating domains (TADs) increasing, the average size of TADs decreasing, and chromatin loop changes at genome region of Trip13 from undifferentiated stage to meiosis‐initiation stage. Furthermore, we validated that Trip13 promoter contacted distal enhancer to form spatial chromatin loop and STRA8 could bind Trip13 promoter to promote gene expression.ConclusionFGSCs underwent chromatin structure remodelling from undifferentiated stage to meiosis‐initiation stage, which facilitated STRA8 binding to Trip13 promoter and promoting its expression.

Untangling the Web of Protein Kinase C Mediated Regulation of Naïve Vs Primed State of Pluripotency

Introduction‐Pluripotency of stem cells is associated with the dynamicity of the embryo’s developmental stage from which they are derived. Stem cells derived from the pre‐implantation embryo, called embryonic stem cells (ESCs), are naïve in nature, whereas post‐implantation embryo‐derived stem cells, termed epiblast stem cells (EpiSCs), represent the primed state of pluripotency. Maintaining these stem cells in a naïve state is an essential arena of stem cell research since naïve ESCs are capable of clonal propagation and germline transmission, making them a crucial research tool in organogenesis and disease modeling. Inhibition of PKC (PKCi) signaling was found to maintain an undifferentiated stage of pluripotency in both mouse and rat. Interestingly, when used along with a cocktail of inhibitors, the same inhibitor facilitated the derivation of naïve state pluripotency in human ESC. These findings lead us to a hypothesis that PKC signaling inhibition alone has a role in maintaining the naïve state of pluripotency. Hence, it has intrigued us to explore the complex network of mechanisms exerted by PKC signaling in dictating the transition of naïve to primed state of pluripotency.Methodology‐In that pursuit, we first observed the morphology of the mouse ESCs (mESCs) by phase‐contrast microscopy across passages and performed MTT assay to optimize the concentration used for further assay. Quantitative Real‐time PCR and western blotting were performed to determine the expression of transcription factors associated with naïve and primed states of pluripotency. Other techniques, including FACS, TiO2 phospho‐enrichment followed by Mass‐spectrometric (MS) analysis, Immunofluorescence assay, Immunoprecipitation assay, Nuclear‐cytoplasmic protein extraction and others were exploited to study the mechanism.Results‐We observed that PKCi could maintain naïve state of pluripotency in mESC even after prolonged culturing by reducing the expression of a critical primed transcription factor. The inhibition of phosphorylation of the PKCζ isoform is responsible for naïve state maintenance. Cell cycle analysis showed that more cells are in the G1 phase in the PKCi maintained condition than in the cells maintained in the presence of Leukemia Inhibitory Factor (LIF). Also, cell proliferation marker CDK1 expression was less in PKCi maintained cells, which probably is negatively regulated by the transcription factor KLF4. Increased nuclear expression of KLF4 was observed in PKCi maintained ESCs than that of ESCs cultured with LIF. Beside these transcription factor‐mediated regulations, PKCi exerts an epigenetic control over the ESCs absent in LIF maintained ESCs. PKCi mediated loss in phosphorylation of the epigenetic factor contributes to naïve state maintenance. Further, our data indicates towards PKCζ mediated NF‐κβ pathway activation, thereby dictating the transition of naïve to primed state of pluripotency.Conclusions‐PKCi maintained mESCs could maintain the naïve state of pluripotency. We have unraveled a multifaceted functionality of PKC signaling in the transition of naïve to primed state of pluripotency, which extends to both transcriptional and epigenetic regulation.

Transcriptome Analysis and Differential Gene Screening of Male Flower Buds in Three Age Stages of <i>Ginkgo biloba</i>

In this paper, the key genes of flowering regulation among three age stages was screened and the molecular mechanism of the flowering pathway was revealed in  Ginkgo biloba . It can provide scientific basis for promoting early flowering, molecular breeding and spreading planting of  Ginkgo biloba . High throughput sequencing technology and bioinformatics tools were used to sequence the transcriptome of male flower buds of  Ginkgo biloba  in three age stages. By analyzed the sequencing data, we screened out the key differential expression genes of age pathway. A total of 57.45 Gb of raw data was generated by transcriptome sequencing. The total number of Unigenes was 35 058 and annotated in the 8 functional databases (GO, COG, KEGG, KOG, NR, Pfam, Swiss-Prot, and eggNOG). The Unigenes were classified into 55 GO categories and 126 metabolic pathways. Analysis of differentially expressed genes revealed that 37 genes were up-regulated and 75 genes were down-regulated during the flower bud undifferentiated stage vs. initial stage of flower bud differentiation. A total of 592 genes were up-regulated and 871 genes were down-regulated during the initial stage of flower bud differentiation vs. flower bud differentiation stage. The flower bud undifferentiated stage vs. flower bud differentiation stage，961 genes were up-regulated and 1 203 genes were down-regulated. A large number of flowering related genes were discovered and finally screened out 11 key genes of flowering regulation in age pathway. including  SPL (gene.Gb_23724, gene.Gb_03922),  AP2 (gene.Gb_00766),  MADS-box (gene.Gb_01886, gene.Gb_15398, gene.Gb_28337, Gingko_newGene_2213), Gibberellin-regulated protein(gene.Gb_34467, gene.Gb_28606, gene.Gb_33214)and DELLAs protein(gene.Gb_34644).

Assessment of reproductive cycle, parasitism and biological indices of the wedge clam Donax trunculus along the Taghazout coastline (on the Moroccan Atlantic Coast)

This study was conducted to assess the physiological parameters of reproduction (sex ratio and gametogenic cycle), parasitism and biological indices (condition index and gonadal index) in Donax trunculus. Samples were collected monthly over two years (from 2016 to 2017) from three sites along the coast of Taghazout (in Southern Morocco) hosting the tourist resort Taghazout Bay. Five stages were depicted by histological examination of gonads: undifferentiated, developing, mature, spawning, and spent. The results of this histological study of the gonads of D. trunculus show that the reproductive cycle of this species has two periods: the first one is characterized by high sexual activity and extends from February to June and the second one corresponds to sexual rest and lasts from July to October. In all three stations and during both years, individuals in the undifferentiated stage (sexual rest) are encountered from July to October. Two spawning periods were observed: the first in March followed by a resumption of activity in April, and the second from May–June. Seasonal variations of the biological indices (GI and CI) were associated with the reproductive and nutritive status of bivalves. The study of parasitism by the trematode Bacciger Bacciger revealed that all the infected individuals were females in the sporocyst stage with a total prevalence between 0 and 9% in August and September respectively. Our results serve as a warning for future management measures and justify the necessity of taking into account the biology of the species in a particular area for better management of resources.

Tracking the Differentiation Status of Human Neural Stem Cells through Label-Free Raman Spectroscopy and Machine Learning-Based Analysis.

The ability to noninvasively monitor stem cells' differentiation is important to stem cell studies. Raman spectroscopy is a non-harmful imaging approach that acquires the cellular biochemical signatures. Herein, we report the first use of label-free Raman spectroscopy to characterize the gradual change during the differentiation process of live human neural stem cells (NSCs) in the in vitro cultures. Raman spectra of 600-1800 cm-1 were measured with human NSC cultures from the undifferentiated stage (NSC-predominant) to the highly differentiated one (neuron-predominant) and subsequently analyzed using various mathematical methods. Hierarchical cluster analysis distinguished two cell types (NSCs and neurons) through the spectra. The subsequently derived differentiation rate matched that measured by immunocytochemistry. The key spectral biomarkers were identified by time-dependent trend analysis and principal component analysis. Furthermore, through machine learning-based analysis, a set of eight spectral data points were found to be highly accurate in classifying cell types and predicting the differentiation rate. The predictive accuracy was the highest using the artificial neural network (ANN) and slightly lowered using the logistic regression model and linear discriminant analysis. In conclusion, label-free Raman spectroscopy with the aid of machine learning analysis can provide the noninvasive classification of cell types at the single-cell level and thus accurately track the human NSC differentiation. A set of eight spectral data points combined with the ANN method were found to be the most efficient and accurate. Establishing this non-harmful and efficient strategy will shed light on the in vivo and clinical studies of NSCs.

Neuron Replating, a Powerful and Versatile Approach to Study Early Aspects of Neuron Differentiation.

Neuron differentiation includes formation and outgrowth of neurites that differentiate into axons or dendrites. Directed neurite outgrowth is controlled by growth cones that protrude and retract actin-rich structures to sense environmental cues. These cues control local actin filament dynamics, steer growth cones toward attractants and away from repellents, and navigate neurites through the developing brain. Rodent hippocampal neurons are widely used to study the mechanisms underlying neuron differentiation. Genetic manipulation of isolated neurons including gene inactivation or reporter gene expression can be achieved by classical transfections methods, but these methods are restricted to neurons cultured for several days, after neurite formation or outgrowth. Instead, electroporation allows gene manipulation before seeding. However, reporter gene expression usually takes up to 24 h, and time course of gene inactivation depends on the half live of the targeted mRNA and gene product. Hence, these methods do not allow to study early aspects of neuron differentiation. In the present study, we provide a detailed protocol in which we combined electroporation-based gene manipulation of mouse hippocampal neurons before initial seeding with a replating step after 2 d in vitro (DIV) that resets neurons into an undifferentiated stage. By categorizing neurons according to their differentiation stage, thorough morphometric analyses, live imaging of actin dynamics in growth cones as well as guidance cue-mediated growth cone morphologic changes, we demonstrate that differentiation and function of replated neurons did not differ from non-replated neurons. In summary, we provide a protocol that allows to thoroughly characterize differentiation of mouse primary hippocampal neurons.

The effects of denatured major bovine whey proteins on the digestive tract, assessed by Caco-2 cell differentiation and on viability of suckling mice.

Alpha-lactalbumin (α-LA) and β-lactoglobulin (β-LG) are contained in bovine milk whey. Chemical and physical treatments are known to alter the conformation of these proteins and we have previously reported that α-LA denatured with trifluoroethanol (TFE) and isolated from sterilized market milk inhibited the growth of rat crypt IEC-6 cells. In the present study, we aimed to evaluate the effects of TFE-treated α-LA and β-LG on cell growth using cultured intestinal cells and on their safety using a suckling mouse model. First, we investigated the effect of the TFE-treated whey proteins on human colonic Caco-2 cells at various differentiation stages. In the undifferentiated stage, we assessed cell growth by a water-soluble tetrazolium-1 method. The native whey proteins enhanced cell proliferation, whereas the TFE-treated whey proteins strongly inhibited cell growth. We investigated cell barrier function in the post-differentiated stage by measuring transepithelial electrical resistance (TER). Not only native but also the TFE-treated whey proteins increased TER. Next, we evaluated whether the TFE-treated α-LA and β-LG have adverse effects on healthy suckling mice. No mice given by the TFE-treated samples showed any adverse symptoms. We also performed a safety test using a human rotavirus infected gastrointestinal disease suckling mice model. Even the TFE-treated whey proteins appeared to prevent the development of diarrheal symptoms without any adverse effects. Although we cannot know the effect of long-term ingestion of denatured whey proteins, these results suggest that they have no adverse effects on differentiated intestinal cells and digestive tract, at least in short-term ingestion.

Extracellular laminin regulates hematopoietic potential of pluripotent stem cells through integrin β1-ILK-β-catenin-JUN axis

Recombinant matrices have enabled feeder cell-free maintenance cultures of human pluripotent stem cells (hPSCs), with laminin 511-E8 fragment (LM511-E8) being widely used. However, we herein report that hPSCs maintained on LM511-E8 resist differentiating to multipotent hematopoietic progenitor cells (HPCs), unlike hPSCs maintained on LM421-E8 or LM121-E8. The latter two LM-E8s bound weakly to hPSCs compared with LM511-E8 and activated the canonical Wnt/β-catenin signaling pathway. Moreover, the extracellular LM-E8-dependent preferential hematopoiesis was associated with a higher expression of integrin β1 (ITGB1) and downstream integrin-linked protein kinase (ILK), β-catenin and phosphorylated JUN. Accordingly, the lower coating concentration of LM511-E8 or addition of a Wnt/β-catenin signaling activator, CHIR99021, facilitated higher HPC yield. In contrast, the inhibition of ILK, Wnt or JNK by inhibitors or mRNA knockdown suppressed the HPC yield. These findings suggest that extracellular laminin scaffolds modulate the hematopoietic differentiation potential of hPSCs by activating the ITGB1-ILK-β-catenin-JUN axis at the undifferentiated stage. Finally, the combination of low-concentrated LM511-E8 and a revised hPSC-sac method, which adds bFGF, SB431542 and heparin to the conventional method, enabled a higher yield of HPCs and higher rate for definitive hematopoiesis, suggesting a useful protocol for obtaining differentiated hematopoietic cells from hPSCs in general.

Phenotypic switch of smooth muscle cells in paediatric chronic intestinal pseudo-obstruction syndrome.

Smooth Muscle Cells (SMC) are unique amongst all muscle cells in their capacity to modulate their phenotype. Indeed, SMCs do not terminally differentiate but instead harbour a remarkable capacity to dedifferentiate, switching between a quiescent contractile state and a highly proliferative and migratory phenotype, a quality often associated to SMC dysfunction. However, phenotypic plasticity remains poorly examined in the field of gastroenterology in particular in pathologies in which gut motor activity is impaired. Here, we assessed SMC status in biopsies of infants with chronic intestinal pseudo‐obstruction (CIPO) syndrome, a life‐threatening intestinal motility disorder. We showed that CIPO‐SMCs harbour a decreased level of contractile markers. This phenotype is accompanied by an increase in Platelet‐Derived Growth Factor Receptor‐alpha (PDGFRA) expression. We showed that this modulation occurs without origin‐related differences in CIPO circular and longitudinal‐derived SMCs. As we characterized PDGFRA as a marker of digestive mesenchymal progenitors during embryogenesis, our results suggest a phenotypic switch of the CIPO‐SMC towards an undifferentiated stage. The development of CIPO‐SMC culture and the characterization of SMC phenotypic switch should enable us to design therapeutic approaches to promote SMC differentiation in CIPO.

Expression profile of GnRH-like peptide during gonadal sex differentiation in the cephalopod kisslip cuttlefish, Sepia lycidas

Gonadotropin-releasing hormone (GnRH) is one of the most important neuroendocrine regulators for animal reproduction. GnRH-like peptide (GnRH-like) has recently been shown to play a critical reproductive role mainly in gametogenesis or steroidogenesis in the gonads of some molluscs, including cephalopods. However, its involvement in gonadal sex differentiation remains unknown. Here, we show the expression profile of GnRH-like in the brain of the cephalopod kisslip cuttlefish, Sepia lycidas, throughout gonadal sex differentiation, by quantitative real time RT-PCR and immunohistochemistry. We found that GnRH-like could be detected in the brain at a sexually undifferentiated stage, and its expression level significantly increased upon initiation of gonadal sex differentiation. However, no significant difference in GnRH-like expression levels was observed between sexes during gonadal sex differentiation. Additionally, we demonstrated immunoreactivity of GnRH-like in glial cells or immature neurons, which are mainly distributed in the non-reproductive related area of the cephalopod brain, suggesting the immature function of the reproductive endocrine axis during early ontogenesis. Our results demonstrate for the first time, the expression profile of GnRH-like during early ontogenesis in cephalopods.

Cloning and Sequence Analysis of <i>GBM5</i> Gene from <i>Ginkgo biloba</i>

AGAMOUS (AG), a member of MADS-box gene family, is a key gene related to regulation of flower development in plants. The AG homologous gene GBM5 (Accession No.: AY114304.1) was obtained from the transcriptome of Ginkgo biloba . Primers were designed according to the obtained GBM5 gene sequence, and the full-length cDNA sequence of GBM5 gene with 732 bp in size and 243 amino acids in coding product can be synthetized by designing primers. The comparative analysis of evolutionary tree and amino acid sequence showed that ginkgo GBM5 was closely related to cycads AG and black spruce AG . qPCR analysis showed that the expression of GBM5 in different tissues of Ginkgo biloba was different at different periods, the expression levels of different tissues are as follows: Female flower>Male flower>Primordia>Foliage; Its expression is high in the undifferentiated stage of flower bud, the expression patterns were: April leaf>May leaf>September leaf, April primordia>May primordia>September primordia.

Production of Cardiomyocyte-Like Cells by Fibroblast Reprogramming with Defined Factors.

Over the last decade, great achievements have been made in the field of direct epigenetic reprogramming, which converts one type of adult somatic cells into another type of differentiated cells, such as direct reprogramming of fibroblasts into cardiomyocytes, without passage through an undifferentiated pluripotent stage. Discovery of direct cardiac reprogramming offers a promising therapeutic strategy to prevent/attenuate cardiac fibrotic remodeling in a diseased heart. Furthermore, in vitro reprogramming of fibroblasts into cardiomyocyte-like cells provides new avenues to conduct basic mechanistic studies, to test drugs, and to model cardiac diseases in a dish. Here, we describe a detailed step-by-step protocol for in vitro production of induced cardiomyocyte-like cells (iCMs) from fibroblasts. The related procedures include high-quality fibroblast isolation of different origins (neonatal cardiac, tail-tip, and adult cardiac fibroblasts), retroviral preparation of reprogramming factors, and iCM generation by fibroblast reprogramming via retroviral transduction of Gata4, Mef2c, and Tbx5. A detailed written protocol will help many other laboratories, inexperienced in this area, to use and further improve this technology in their studies of cardiac regenerative medicine.