Mesodermal Cells Research Articles

Vitamin E and Selenium Facilitate the Osteogenesis and Adipogenesis of the Human Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells

Background and Aims: Previous studies have shown that adipose-derived mesenchymal stem/ stromal cells are one of the sources of mesenchymal stem cells (MSCs) with the capacity to differentiate into various mesodermal cell lineages. MSCs with cytokines secretion capability, which contributes to repair damaged tissues have gained wide credence for future cell-based therapeutic applications. In this study, the effect of the different dosages of vitamin E and Selenium was assessed on the stemness of the human adipose tissue-derived MSCs (AD-MSCs). Materials and Methods: Following 24 hours of cell treatments with different dosages of vitamin E and Selenium, MTT assay was used to assess the effect of them on cell proliferation. Moreover, the stemness of the AD-MSCs was assessed using osteogenic and adipogenic induction medium supplemented with the different dosages of the vitamin E and Selenium. Finally, Alizarin red and Oil-red O staining were performed to detect matrix mineralization and lipid droplet accumulation, respectively. Results: MTT data revealed that the optimal concentration for vitamin E and Selenium were 125 µM and 121 µM for the viability of the AD-MSCs. Moreover, the effect of vitamin E and Selenium were assessed by osteogenic and adipogenic differentiation by optimal dosages obtained by MTT assay, respectively. Maximum mineralization and lipid droplet aggregation of the differentiated cells were detected at IC50 in comparison with the control group. Conclusions: These results suggest that different dosages of vitamin E and Selenium could have various impacts on the proliferation and differentiation induction of human AD-MSCs.

IPSC-derived progenitor stromal cells provide new insights into aberrant musculoskeletal development and resistance to cancer in down syndrome

Down syndrome (DS) is a congenital disorder caused by trisomy 21 (T21). It is associated with cognitive impairment, muscle hypotonia, heart defects, and other clinical anomalies. At the same time, individuals with Down syndrome have lower prevalence of solid tumor formation. To gain new insights into aberrant DS development during early stages of mesoderm formation and its possible connection to lower solid tumor prevalence, we developed the first model of two types of DS iPSC-derived stromal cells. Utilizing bioinformatic and functional analyses, we identified over 100 genes with coordinated expression among mesodermal and endothelial cell types. The most significantly down-regulated processes in DS mesodermal progenitors were associated with decreased stromal progenitor performance related to connective tissue organization as well as muscle development and functionality. The differentially expressed genes included cytoskeleton-related genes (actin and myosin), ECM genes (Collagens, Galectin-1, Fibronectin, Heparan Sulfate, LOX, FAK1), cell cycle genes (USP16, S1P complexes), and DNA damage repair genes. For DS endothelial cells, our analysis revealed most down-regulated genes associated with cellular response to external stimuli, cell migration, and immune response (inflammation-based). Together with functional assays, these results suggest an impairment in mesodermal development capacity during early stages, which likely translates into connective tissue impairment in DS patients. We further determined that, despite differences in functional processes and characteristics, a significant number of differentially regulated genes involved in tumorigenesis were expressed in a highly coordinated manner across endothelial and mesodermal cells. These findings strongly suggest that microRNAs (miR-24-4, miR-21), cytoskeleton remodeling, response to stimuli, and inflammation can impact resistance to tumorigenesis in DS patients. Furthermore, we also show that endothelial cell functionality is impaired, and when combined with angiogenic inhibition, it can provide another mechanism for decreased solid tumor development. We propose that the same processes, which specify the basis of connective tissue impairment observed in DS patients, potentially impart a resistance to cancer by hindering tumor progression and metastasis. We further establish that cancer-related genes on Chromosome 21 are up-regulated, while genome-wide cancer-related genes are down-regulated. These results suggest that trisomy 21 induces a modified regulation and compensation of many biochemical pathways across the genome. Such downstream interactions may contribute toward promoting tumor resistant mechanisms.

Down-regulation of LECT2 promotes osteogenic differentiation of MSCs via activating Wnt/β-catenin pathway

Osteoporosis is a result of the imbalance between osteoblasts and osteoclasts quantities, which is closely correlated with osteogenic differentiation (OD). Leucocyte cell-derived chemotaxin 2 (LECT2) has been reported as a regulatory factor in some chronic diseases such as hepatitis through mediating downstream target gene β-catenin. Additionally, Wnt/β-catenin is also the crucial modulatory signal pathway in OD. Mesenchymal stem cells (MSC) is a kind of mesodermal stem cells; its differentiation direction is discovered affected by Wnt/β-catenin. However, the function of LECT2 in osteoporosis still remains exploration, which encourages us to lucubrate its functional effect in regulating the OD of MSCs. In this study, we found that LECT2 was expressed at low level in MSCs with osteogenic differentiation, and knockdown of LECT2 would activate Wnt/β-catenin pathway and therefore promoting OD in MSCs. It is the first time to report that LECT2 participates in regulating OD via mediating Wnt/β-catenin. Our discovery would affirmatively help provide a novel strategy for the diagnosis and therapy methods for osteoporosis.

OFCD syndrome and extraembryonic defects are revealed by conditional mutation of the Polycomb-group repressive complex 1.1 (PRC1.1) gene BCOR

BCOR is a critical regulator of human development. Heterozygous mutations of BCOR in females cause the X-linked developmental disorder Oculofaciocardiodental syndrome (OFCD), and hemizygous mutations of BCOR in males cause gestational lethality. BCOR associates with Polycomb group proteins to form one subfamily of the diverse Polycomb repressive complex 1 (PRC1) complexes, designated PRC1.1. Currently there is limited understanding of differing developmental roles of the various PRC1 complexes. We therefore generated a conditional exon 9–10 knockout Bcor allele and a transgenic conditional Bcor expression allele and used these to define multiple roles of Bcor, and by implication PRC1.1, in mouse development. Females heterozygous for Bcor exhibiting mosaic expression due to the X-linkage of the gene showed reduced postnatal viability and had OFCD-like defects. By contrast, Bcor hemizygosity in the entire male embryo resulted in embryonic lethality by E9.5. We further dissected the roles of Bcor, focusing on some of the tissues affected in OFCD through use of cell type specific Cre alleles. Mutation of Bcor in neural crest cells caused cleft palate, shortening of the mandible and tympanic bone, ectopic salivary glands and abnormal tongue musculature. We found that defects in the mandibular region, rather than in the palate itself, led to palatal clefting. Mutation of Bcor in hindlimb progenitor cells of the lateral mesoderm resulted in 2/3 syndactyly. Mutation of Bcor in Isl1-expressing lineages that contribute to the heart caused defects including persistent truncus arteriosus, ventricular septal defect and fetal lethality. Mutation of Bcor in extraembryonic lineages resulted in placental defects and midgestation lethality. Ubiquitous over expression of transgenic Bcor isoform A during development resulted in embryonic defects and midgestation lethality. The defects we have found in Bcor mutants provide insights into the etiology of the OFCD syndrome and how BCOR-containing PRC1 complexes function in development.

Combinations of growth factors for human mesenchymal stem cell proliferation and osteogenic differentiation.

AimsHere we introduce a wide and complex study comparing effects of growth factors used alone and in combinations on human mesenchymal stem cell (hMSC) proliferation and osteogenic differentiation. Certain ways of cell behaviour can be triggered by specific peptides – growth factors, influencing cell fate through surface cellular receptors.MethodsIn our study transforming growth factor β (TGF-β), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), and vascular endothelial growth factor (VEGF) were used in order to induce osteogenesis and proliferation of hMSCs from bone marrow. These cells are naturally able to differentiate into various mesodermal cell lines. Effect of each factor itself is pretty well known. We designed experimental groups where two and more growth factors were combined. We supposed cumulative effect would appear when more growth factors with the same effect were combined. The cellular metabolism was evaluated using MTS assay and double-stranded DNA (dsDNA) amount using PicoGreen assay. Alkaline phosphatase (ALP) activity, as early osteogenesis marker, was observed. Phase contrast microscopy was used for cell morphology evaluation.ResultsTGF-β and bFGF were shown to significantly enhance cell proliferation. VEGF and IGF-1 supported ALP activity. Light microscopy showed initial extracellular matrix mineralization after VEGF/IGF-1 supply.ConclusionA combination of more than two growth factors did not support the cellular metabolism level and ALP activity even though the growth factor itself had a positive effect. This is probably caused by interplay of various messengers shared by more growth factor signalling cascades.Cite this article: Bone Joint Res 2020;9(7):412–420.

Induced Fetal Human Muscle Stem Cells with High Therapeutic Potential in a Mouse Muscular Dystrophy Model.

SummaryDuchenne muscular dystrophy (DMD) is a progressive and fatal muscle-wasting disease caused by DYSTROPHIN deficiency. Cell therapy using muscle stem cells (MuSCs) is a potential cure. Here, we report a differentiation method to generate fetal MuSCs from human induced pluripotent stem cells (iPSCs) by monitoring MYF5 expression. Gene expression profiling indicated that MYF5-positive cells in the late stage of differentiation have fetal MuSC characteristics, while MYF5-positive cells in the early stage of differentiation have early myogenic progenitor characteristics. Moreover, late-stage MYF5-positive cells demonstrated good muscle regeneration potential and produced DYSTROPHIN in vivo after transplantation into DMD model mice, resulting in muscle function recovery. The engrafted cells also generated PAX7-positive MuSC-like cells under the basal lamina of DYSTROPHIN-positive fibers. These findings suggest that MYF5-positive fetal MuSCs induced in the late stage of iPSC differentiation have cell therapy potential for DMD.

Bone marrow-derived from the human femoral shaft as a new source of mesenchymal stem/stromal cells: an alternative cell material for banking and clinical transplantation

BackgroundMesenchymal stem/stromal cells (MSC) are commonly used in regenerative medicine. Among different tissues, iliac crest bone marrow (BM) represents the most exploited source, but its disadvantages are a painful aspiration procedure and low cell number. An alternative, readily available source of MSC for research would be beneficial for regenerative medicine development. This work aimed to propose a new source of bone marrow isolation in which the femoral shaft is taken during total hip arthroplasty (THA).MethodsIn preliminary experiments, three different gradient methods for cell separation (Ficoll-Paque 1.078 g/mL, 17% sucrose gradient, BM seeding fraction) were tested with regard to the time of primary culture, initial cell number, the phenotype, and morphology of MSC. Then human bone marrow MSC derived from two different sources, iliac crest aspirate (BM-MSCi) or femoral shaft (BM-MSCt), were analyzed in terms of cell number and colony-forming ability followed by differentiation potential of MSC into osteo-, chondro-, and adipogenic lineages as well as mRNA expression of a variety of cytokines and growth factors.ResultsOur studies showed that MSC isolated from the bone marrow of two different sources and cultured under appropriate conditions had similar characteristics and comparable propensity to differentiate into mesodermal cells. MSC derived from BM-MSCi or BM-MSCt expressed various growth factors. Interestingly, the expression of EGF, FGF, IGF, and PDGF-A was much higher in BM-MSCt than BM-MSCi.ConclusionsThe results of our study demonstrate that human MSC isolated from the BM of the femoral shaft have similar biological characteristics as MSC derived from the iliac crest, suggesting the femoral shaft as a possible alternative source for mesenchymal stem/stromal cells.

Generation of a transgene-free iPSC line and genetically modified line from a facioscapulohumeral muscular dystrophy type 2 (FSHD2) patient with SMCHD1 p.Lys607Ter mutation.

Facioscapulohumeral muscular dystrophy type2 (FSHD2), which constitutes approximately 5% of total FSHD cases and develops the same symptoms as FSHD type 1 (FSHD1), is caused by various mutations in genes including SMCHD1. We report the generation and characterization of an iPSC line derived from an FSHD2 patient carrying the SMCHD1 p.Lys607Ter mutation and its gene-corrected iPSC line which are free from transgene. These iPSC lines maintained normal karyotype, presented typical morphology, expressed endogenous pluripotency markers, and could be differentiated into ectodermal, mesodermal and endodermal cells, confirming their pluripotency.

Mini Review; Differentiation of Human Pluripotent Stem Cells into Oocytes.

Primary Ovarian Insufficiency (POI) is one of the main diseases causing female infertility that occurs in about 1% of women between 30-40 years of age. There are few effective methods for the treatment of women with POI. In the past few years, stem cell-based therapy as one of the most highly investigated new therapies has emerged as a promising strategy for the treatment of POI. Human pluripotent stem cells (hPSCs) can self-renew indefinitely and differentiate into any type of cell. Human Embryonic Stem Cells (hESCs) as a type of pluripotent stem cells are the most powerful candidate for the treatment of POI. Human-induced Pluripotent Stem Cells (hiPSCs) are derived from adult somatic cells by the treatment with exogenous defined factors to create an embryonic-like pluripotent state. Both hiPSCs and hESCs can proliferate and give rise to ectodermal, mesodermal, endodermal, and germ cell lineages. After ovarian stimulation, the number of available oocytes is limited and the yield of total oocytes with high quality is low. Therefore, a robust and reproducible in-vitro culture system that supports the differentiation of human oocytes from PSCs is necessary. Very few studies have focused on the derivation of oocyte-like cells from hiPSCs and the details of hPSCs differentiation into oocytes have not been fully investigated. Therefore, in this review, we focus on the differentiation potential of hPSCs into human oocyte-like cells.

Pcdh18a regulates endocytosis of E-cadherin during axial mesoderm development in zebrafish

The notochord defines the axial structure of all vertebrates during development. Notogenesis is a result of major cell reorganization in the mesoderm, the convergence and the extension of the axial cells. However, it is currently not fully understood how these processes act together in a coordinated way during notochord formation. The prechordal plate is an actively migrating cell population in the central mesoderm anterior to the trailing notochordal plate cells. We show that prechordal plate cells express Protocadherin 18a (Pcdh18a), a member of the cadherin superfamily. We find that Pcdh18a-mediated recycling of E-cadherin adhesion complexes transforms prechordal plate cells into a cohesive and fast migrating cell group. In turn, the prechordal plate cells subsequently instruct the trailing mesoderm. We simulated cell migration during early mesoderm formation using a lattice-based mathematical framework and predicted that the requirement for an anterior, local motile cell cluster could guide the intercalation and extension of the posterior, axial cells. Indeed, a grafting experiment validated the prediction and local Pcdh18a expression induced an ectopic prechordal plate-like cell group migrating towards the animal pole. Our findings indicate that the Pcdh18a is important for prechordal plate formation, which influences the trailing mesodermal cell sheet by orchestrating the morphogenesis of the notochord.

Embryogenesis of an Invertebrate Lymphoid Organ: The Asterina Gibbosa Axial Organ (Asterids, Echinodermata)

The metamerization of the coelom leads to the axial complex genesis. Mesodermic cells appear besides the stone canal (larva 12 days old): they constitute the axial organ (AO) which develop, in the ventral side, the oral part (PO). This last part corresponds (sensu stricto) to the ancestral lymphoïd organ.

NO placental inflammation.

NO placental inflammation.

Morphogenetic mechanisms forming the notochord rod: The turgor pressure-sheath strength model.

The notochord is a defining feature of chordates. During notochord formation in vertebrates and tunicates, notochord cells display dynamic morphogenetic movement, called convergent extension, in which cells intercalate and align at the dorsal midline. However, in cephalochordates, the most basal group of chordates, the notochord is formed without convergent extension. It is simply developed from mesodermal cells at the dorsal midline. This suggests that convergent extension movement of notochord cells is a secondarily acquired developmental attribute in the common ancestor of olfactores (vertebrates+tunicates), and that the chordate ancestor innovated the notochord upon a foundation of morphogenetic mechanisms independent of cell movement. Therefore, this review focuses on biological features specific to notochord cells, which have been well studied using clawed frogs, zebrafish, and tunicates. Attributes of notochord cells, such as vacuolation, membrane trafficking, extracellular matrix formation, and apoptosis, can be understood in terms of two properties: turgor pressure of vacuoles and strength of the notochord sheath. To maintain the straight rod-like structure of the notochord, these parameters must be counterbalanced. In the future, the turgor pressure-sheath strength model, proposed in this review, will be examined in light of quantitative molecular data and mathematical simulations, illuminating the evolutionary origin of the notochord.

Isolation and characterization of mesenchymal stem cells derived from amniotic fluid: A prospective study

Objectives: The study aims to isolate, expand, and check the feasibility and differentiation potential of amniotic fluid mesenchymal stem cell (AF-MSC) from the minimum amount of amniotic fluid. Materials and Methods: Amniotic fluid samples were collected from women undergoing 2nd trimester amniocentesis between 16 and 24 weeks of period of gestation. MSCs were isolated and characterized by MSCs surface marker profiling and were expanded in specific growth media to assess their differentiation capability into osteocytes, chondrocytes, and adipocytes. The differentiation was confirmed using specific staining. Results: The isolated AF-MSCs showed successful stem cell population for 18 samples out of 23. All the isolated AF-MSCs showed positivity for MSCs surface markers. For osteocyte differentiation, cells were cultured in osteogenic induction media for 4 weeks, and the differentiation was confirmed by staining with Alizarin Red S stain, which showed extracellular matrix mineralization. For adipocytes differentiation, the induction media exhibited lipid droplets and positive staining with Oil Red O stain. Similarly, cells cultured in chondrocytes differentiation media, showed positive staining with Alcian Blue. Conclusion: AF-MSCs have the capacity to differentiate into common mesodermal cell types. Considering their easy accessibility, amniotic fluid could be a good source for MSCs with a greater potential for cellular therapy in various chronic disabling diseases, for example, spinal cord injuries, massive bone and cartilage damage, and demyelinating diseases.

Modeling early stages of endoderm development in epiblast stem cell aggregates with supply of extracellular matrices.

Endoderm precursors expressing FoxA2 and Sox17 develop from the epiblast through the gastrulation process. In this study, we developed an experimental system to model the endoderm‐generating gastrulation process using epiblast stem cells (EpiSCs). To this end, we established an EpiSC line i22, in which enhanced green fluorescent protein is coexpressed with Foxa2. Culturing i22 EpiSCs as aggregates for a few days was sufficient to initiate Foxa2 expression, and further culturing of the aggregates in Matrigel promoted the sequential activation of transcription factor genes involved in endoderm precursor development, e.g., Eomes, Gsc, and Sox17. In aggregation culture of i22 cells for 3 days, all cells expressed POU5F1, SOX2, and E‐cadherin, a signature of the epiblast, whereas expression of GATA4 and SOX17 was also activated moderately in dispersed cells, suggesting priming of these cells to endodermal development. Embedding the aggregates in Matrigel for further 3 days elicited migration of the cells into the lumen of laminin‐rich matrices covering the aggregates, in which FOXA2 and SOX17 were expressed at a high level with the concomitant loss of E‐cadherin, indicating the migratory phase of endodermal precursors. Prolonged culturing of the aggregates generated three segregating cell populations found in post‐gastrulation stage embryos: (1) definitive endoderm co‐expressing high SOX17, GATA4, and E‐cadherin, (2) mesodermal cells expressing a low level of GATA4 and lacking E‐cadherin, and (3) primed epiblast cells expressing POU5F1, SOX2 without E‐cadherin. Thus, aggregation of EpiSCs followed by embedding of aggregates in the laminin‐rich matrix models the gastrulation‐dependent endoderm precursor development.

Evaluation of the therapeutic potential of mesenchymal stromal cells during in vitro production: search for markers related to immunomodulatory properties

Background & Aim Mesenchymal stromal/stem cells (MSCs) are a heterogeneous population characterized by self-renewing and ability to differentiate into mesodermal cell origin. Wharton Jelly MSC (WJ-MSC) are a promising candidate for cell therapy due to their ease of isolation and their tissue regeneration capabilities. Moreover, they can regulate the immune response by secreting a set of soluble factors and by establishing direct interactions with the cells of immune system. These immunomodulation properties are used in the treatment of different pathologies such as graft versus host disease. In order to use WJ-MSCs for clinical applications, an in vitro expansion step is critical to obtain the sufficient dose. This step imposes several physicochemical and hydrodynamic constraints that can influence the quality and functional properties of cells. The objective of this work was to identify and validate predictive markers related to immunomodulatory properties of WJ-MSCs. For this purpose, CD119, CD200 and Notch1 were chosen and studied. Methods, Results & Conclusion At first, markers distribution was studied by flow cytometry. Secondly, immunomodulatory potential of WJ-MSC was evaluated by a coculture of these cells with CD4, CD8 lymphocytes, followed by the study of lymphocytes proliferation by flow cytometry. These experimentss were performed under different conditions: WJ-MSCs cultivated in normoxia (N) and hypoxia (H), early passage and senescent cells, and under stimulation with gamma interferon (IFN-γ). Our results showed a heterogeneity of distribution of the markers studied. In addition, our study revealed that WJ-MSC cultivated in H was more immunosuppressive than those cultivated in N. Moreover, we showed that the cellular senescence induced a reduction of the immunosuppressive properties, and that IFN-γ stimulation increased WJ-MSC immunosuppression potential. Finally, our study highlighted a trend towards a correlation between the expression of CD119 and Notch1 by WJ-CSM and their immunomodulatory properties. To conclude, the culture conditions and cell senescence affect the immunomodulatory properties of WJ-CSM hence the interest of verifying these properties after the cell expansion procedures. Furthermore CD119 and Notch1 receptors may be predictive markers of the immunomodulatory properties of WJ -CSM to be checked during in vitro amplification.

Bovine tongue epithelium-derived cells: A new source of bovine mesenchymal stem cells.

Mesenchymal stem cells (MSCs) possess the ability to differentiate into multiple cell lineages, and thus, confer great potential for use in regenerative medicine and biotechnology. In the present study, we attempted to isolate and characterize bovine tongue tissue epithelium-derived MSCs (boT-MSCs) and investigate the culture conditions required for long-term culturing of boT-MSCs. boT-MSCs were successfully isolated by the collagenase digestion method and their proliferative capacity was maintained for up to 20 or more passages. We observed a significant increase in the proliferation of boT-MSCs during the 20 consecutive passages under low-glucose Dulbecco’s modified Eagle’s medium culture condition among the three culture conditions. These boT-MSCs presented pluripotency markers (octamer-binding transcription factor 3/4 (Oct3/4) and sex determining region Y-box2 (Sox2)) and cell surface markers, which included CD13, CD29, CD44, CD73, CD90, CD105, CD166, and major histocompatibility complex (MHC) class I (MHC-I) but not CD11b, CD14, CD31, CD34, CD45, CD80, CD86, CD106, CD117, and MHC-II at third passage. Moreover, these boT-MSCs could differentiate into mesodermal (adipocyte, osteocyte, and chondrocyte) cell lineages. Thus, the present study suggests that the tongue of bovines could be used as a source of bovine MSCs.

Reconstruction of Ewing Sarcoma Developmental Context from Mass-Scale Transcriptomics Reveals Characteristics of EWSR1-FLI1 Permissibility.

Ewing sarcoma is an aggressive pediatric cancer of enigmatic cellular origins typically resulting from a single translocation event t (11; 22) (q24; q12). The resulting fusion gene, EWSR1-FLI1, is toxic or unstable in most primary tissues. Consequently, attempts to model Ewing sarcomagenesis have proven unsuccessful thus far, highlighting the need to identify the cellular features which permit stable EWSR1-FLI1 expression. By re-analyzing publicly available RNA-Sequencing data with manifold learning techniques, we uncovered a group of Ewing-like tissues belonging to a developmental trajectory between pluripotent, neuroectodermal, and mesodermal cell states. Furthermore, we demonstrated that EWSR1-FLI1 expression levels control the activation of these developmental trajectories within Ewing sarcoma cells. Subsequent analysis and experimental validation demonstrated that the capability to resolve R-loops and mitigate replication stress are probable prerequisites for stable EWSR1-FLI1 expression in primary tissues. Taken together, our results demonstrate how EWSR1-FLI1 hijacks developmental gene programs and advances our understanding of Ewing sarcomagenesis.

Adverse effects of nicotine on cardiogenic differentiation from human embryonic stem cells detected by single-cell RNA sequencing

Tobacco smoking was one of the important adverse factors for congenital heart disease. The effects of nicotine, the main component of tobacco, on human embryonic cardiogenesis and related mechanisms remain poorly understood. This work used single-cell RNA sequencing to investigate the effects of nicotine on human embryonic stem cell (hESC) line H9 and its underlying mechanisms during cardiac differentiation. H9 was cultured in feeder-free medium and differentiated in cardiac condition medium when cells reached 90% confluent. Cell viability was detected by MTT after different concentration of nicotine treatment. Different expressed genes during cardiac differentiation was analyzed by single-cell RNA sequencing (scRNA-seq). Key gene expressions were confirmed by qPCR and Western blot. Results showed that 0.1μM–10μM nicotine did not affect H9 cell proliferation. Nicotine 1 μM down-regulated cardiac progenitor cell, mesoderm cell, smooth muscle cell and neural crest cell relatively. Snail1/2 regulating endocardial cushion development were downregulated apparently at differention day 6. Nicotine didn’t affect bry-1 and mesp-1 but inhibited cardiac transcript factors. Consequently, the expression of cTnI, a marker of cardiomyocytes was decreased significantly. The data suggest direct adverse effects of nicotine on heart development at the single-cell level and offer a new approach for estimate drug and environmental toxicity on the pathogenesis of the embryonic cardiovascular system development.

P63 Mutation Impacts the Structure of Cardiopharyngeal Field Derived Muscles in Mice

The cardiopharyngeal field (CPF) includes anterior lateral mesoderm of the first heart field and the contiguous pharyngeal mesoderm. The latter gives rise to second‐heart‐field‐derived heart regions and branchiomeric head muscles. Neural crest cells (NCCs) surround and infiltrate mesoderm cells, forming connective tissue of head and heart musculature. NCCs are also involved in the septation of the cardiac outflow tract, the patterning of pharyngeal arch arteries, and in the proper development of attachments of branchiomeric muscles to skull, jaw, hyoid bone, and laryngeal cartilages.The p63 (aka Trp63) transcription factor is essential for the differentiation of epithelial tissue (ectoderm and endoderm) during embryogenesis. To form the correct amount of mature ectodermal, mesodermal, and NCC populations and to control the differentiation of the CPF, p63 interacts with downstream transcription factors, such as Tbx1, Isl1, and nkx2.5. We analyzed how the loss of p63 expression in p63−/− mice affected the mesodermal derivatives of the CPF. We hypothesized that the loss of p63 decreases the number of mesodermal cells differentiating from the CPF, causing a smaller mesodermal cell population in the CPF. We expected to see smaller CPF derived muscles in the p63 mutant mice, as well as deficient connective tissue in head and heart musculature as well as weakened attachments of craniofacial muscles.To determine if and when certain muscular defects appear, we performed muscle antibody staining and dissected wildtype littermates and p63−/− mice (B6.129S7‐Trp63tm2Brd/J strain) aged embryonic day (E) 12–18. We have collected minimum of five specimens for each age. We studied craniofacial, laryngeal, as well as cardiac musculature by analyzing the antibody stained specimens under the microscope and by performing microdissections under a stereomicroscope. We used 3D computed tomography scan images (taken with a uCT scanner, Skyscan 1172) to compare the craniofacial skeletons between wildtype and mutant mice.In p63−/− mice, branchiomeric muscles such as the masseter, buccinator, and nasolabialis were properly attached to bony elements but were smaller than what was seen in the wildtype mice. The p63−/− head is flexed and the chin is closer to the chest than in the wildtype specimens: this posture could be caused by the shortened and less dense sternocleidomastoid muscle in the p63−/− mice. Corresponding with previous reports, our p63 −/− mice also showed several malformations (e.g., poorly developed limbs, cleft palate, cardiac abnormalities) stereotypical of this mouse mutant and which are also seen in humans with p63 mutations such as in ADULT (acro‐dermatoungual‐lacrimal‐tooth) and EEC (Ectrodactyly‐Ectodermal Dysplasia‐Cleft Lip /Palate) syndromes. Despite that p63 expression is restricted to epithelium, the described defects reveal that p63 expression is important to much earlier, and shared developmental processes, notably NCC proliferation and differentiation, that do impact tissues derived from mesoderm as well as from epithelium. Analyzing the influence of p63 on CPF differentiation will contribute to a better understanding of regulatory mechanisms during head and heart development including the role of these mechanisms in human congenital syndromes.