Specific Growth Factors Research Articles

Production of high-purity recombinant human vascular endothelial growth factor (rhVEGF165) by Pichia pastoris

Vascular endothelial growth factor (VEGF165) is a highly specific vascular endothelial growth factor that can be used to treat many cardiovascular diseases. The development of anti-tumor drugs and disease detection reagents requires highly pure VEGF165 (at least 95% purity). To date, the methods for heterologous expression and purification of VEGF165 require multiple purification steps, but the product purity remains to be low. In this study, we optimized the codons of the human VEGF165 gene (vegf165) according to the yeast codon preference. Based on the Pichia pastoris BBPB vector, we used the Biobrick method to construct a five-copy rhVEGF165 recombinant expression vector using Pgap as the promoter. In addition, a histidine tag was added to the vector. Facilitated by the His tag and the heparin-binding domain of VEGF165, we were able to obtain highly pure rhVEGF165 (purity > 98%) protein using two-step affinity chromatography. The purified rhVEGF165 was biologically active, and reached a concentration of 0.45 mg/mL. The new design of the expression vector enables production of active and highly pure rhVEGF165 ) in a simplified purification process, the purity of the biologically active natural VEGF165 reached the highest reported to date.

Forskolin-Loaded Halloysite Nanotubes as Osteoconductive Additive for the Biopolymer Tissue Engineering Scaffolds.

Here we report the use of forskolin-modified halloysite nanotubes (HNTs) as a dopant for biopolymer porous hydrogel scaffolds to impart osteoinductive properties. Forskolin is a labdane diterpenoid isolated from the Indian Coleus plant. This small molecule is widely used as a supplement in molecular biology for cell differentiation. It has been reported in some earlier publications that forskolin can activate osteodifferentiation process by cyclic adenosine monophosphate (c-AMP) signalling activation in stem cells. In presented study it was demonstrated that forskolin release from halloysite-doped scaffolds induced the osteodifferentiation of equine mesenchymal stem cells (MSCs) in vitro without addition of any specific growth factors. The reinforcement of mechanical properties of cells and intercellular space during the osteodifferentiation was demonstrated using atomic force microscopy (AFM). These clay-doped scaffolds may find applications to accelerate the regeneration of horse bone defects by inducing the processes of osteodifferentiation of endogenous MSCs.

Peripheral Blood Mononuclear Cells (PBMCs) to Dissect the Underlying Mechanisms of Bone Disease in Chronic Kidney Disease and Rare Renal Diseases

To describe the methods that can be used to obtain functional and mature osteoclasts from peripheral blood mononuclear cells (PBMCs) and report the data obtained with this model in two peculiar diseases, namely pediatric chronic kidney disease-associated mineral and bone disorders (CKD-MBD) and nephropathic cystinosis. To discuss future research possibilities in the field. Bone tissue undergoes continuous remodeling throughout life to maintain bone architecture; it involves two processes: bone formation and bone resorption with the coordinated activity of osteoblasts, osteoclasts, and osteocytes. Animal models fail to fully explain human bone pathophysiology during chronic kidney disease, mainly due to interspecies differences. The development of in vitro models has permitted to mimic human bone-related diseases as an alternative to in vivo models. Since 1997, osteoclasts have been generated in cell cultures, notably when culturing PBMCs with specific growth factors and cytokines (i.e., M-CSF and RANK-L), without the need for osteoblasts or stromal cells. These models may improve the global understanding of bone pathophysiology. They can be been used not only to evaluate the direct effects of cytokines, hormones, cells, or drugs on bone remodeling during CKD-MBD, but also in peculiar genetic renal diseases inducing specific bone impairment.

Identification of a novel iron zinc finger protein 36 (ZFP36) for predicting the overall survival of osteosarcoma based on the Gene Expression Omnibus (GEO) database.

BackgroundThe purpose of this study is to explore the relationship between the ferroptosis-related gene zinc finger protein 36 (ZFP36) and the prognosis of osteosarcoma patients after surgery.MethodsDifferential expression genes (DEGs) between osteosarcoma and normal tissues were screened using osteosarcoma chip data in GEO database. Based on the median expression quantity, ferroptosis DEGs were divided into high and low expression groups. Combined with its corresponding clinical survival data, the survival analysis of ferroptosis DEGs was carried out using the Survival package, and ferroptosis-related genes related to prognosis were identified. Next, the clinical data of 60 osteosarcoma patients treated in Jiangyin Hospital Affiliated to Nanjing University of Chinese Medicine, Zhongda Hospital and Nanjing Drum Tower Hospital from January 2011 to January 2016 were retrospectively analyzed. Immunohistochemistry and reverse transcription quantitative polymerase chain reaction (qRT-PCR) were used to detect gene expression in osteosarcoma. The Kaplan-Meier method and log rank test were used for univariate survival analysis, the Cox regression method was used for multivariate analysis, and the nomogram was constructed for internal verification on this basis.ResultsImmunohistochemical and reverse transcription quantitative PCR results showed that the expression of ZFP36 was mainly higher in cancer-adjacent tissues than in tumor tissues. There were significant differences in age, tumor location, Enneking stage, and tumor specific growth factor (TSGF) between the high and low expression groups of ZFP36 (P<0.05). The final study included 60 patients, of whom 23 patients died (mortality rate: 38.33%), and 37 patients survived (survival rate: 61.67%), with a median progression-free survival (PFS) of 32.5 months and a median overall survival (OS) of 77 months. The Cox multivariate analysis showed that distant metastasis and ZFP36 were independent risk factors affecting tumor progression (P=0.021 and P=0.006, respectively). Elevated ZFP36 can significantly prolong the OS and PFS of osteosarcoma patients. In internal verification, the Concordance index (C-index) of the nomogram was 0.7211 [95% confidence interval (CI): 0.6308–0.8115], and the prediction model had certain accuracy.ConclusionsElevated ZFP36 can significantly prolong the OS and PFS in osteosarcoma patients. At the same time, ZFP36 could be used as a new predictive biomarker and novel therapeutic target for osteosarcoma patients.

DNCP induces the differentiation of induced pluripotent stem cells into odontoblasts by activating the Smad/p-Smad and p38/p-p38 signaling pathways.

In recent years, stem cells have been studied for treating tooth loss. The present study aimed to investigate the roles of dentin non-collagen protein (DNCP)-associated microenvironments in the differentiation of induced pluripotent stem cells (iPSCs) into dentin cells. iPSCs were cultured and identified by examining octamer-binding transcription-factor-4 (Oct-4) and sex-determining region-Y-2 (Sox-2) expression. iPSCs were differentiated by culturing DNCP-associated microenvironments (containing specific growth factors), and they were divided into control, DNCP, DNCP+bone morphogenetic proteins (BMPs) and DNCP+Noggin (a BMP inhibitor) groups. Msh homeobox 1 (Msx-1), dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP-1) mRNA expression was evaluated using reverse transcription-quantitative PCR. The levels of p38, phosphorylated (p)-p38, Smad and p-Smad were determined by western blotting. Upon treatment with mouse embryonic fibroblasts, iPSCs-dependent embryoid bodies (EBs) were successfully generated. iPSCs exhibited increased Oct-4 and Sox-2 expression. Differentiated iPSCs had higher expression levels of DSPP, DMP-1 and Msx-1 in the DNCP group compared with those in the control group (P<0.05). Noggin treatment significantly downregulated, while BMPs administration significantly increased the expression levels of DSPP, DMP-1 and Msx-1 compared with those of the DNCP group (P<0.05). The ratios of p-p38/p38 and p-Smad/Smad were significantly higher in the DNCP group compared with those in the control group (P<0.05). Noggin and BMPs significantly decreased ratios of p-p38/p38, compared with those of the DNCP group (P<0.05). In conclusion, DNCP induced the differentiation of iPSCs into odontoblasts by activating the Smad/p-Smad and p38/p-p38 signaling pathways.

Palindromic-assisted self-annealing transcription amplification for reliable genotyping of epidermal growth factor receptor exon mutations

Reliable discrimination of specific epidermal growth factor receptor (EGFR) gene mutations plays a critical role in guiding lung cancer therapeutics. Until now, convenient and accurate recognition of the specific deletion of EGFR exons has remained particularly challenging. Herein, we propose a palindromic-assisted self-annealing transcription amplification (PASTA) strategy for the reliable detection of circulating EGFR exon mutations. We designed a palindromic DNA hairpin nanorobot consisting of a palindromic tail, a T7 promoter, a target recognition region, and a transcription template. The nanorobot enabled prompt self-assembly into a target-hairpin/hairpin-target dimer in the presence of single-stranded DNA target and further triggered in vitro transcription. In a proof-of-concept experiment for detecting circulating 15n-del EGFR mutation, a detection limit of 0.8 fM and a linear detection range of 1 fM to 100 pM was achieved, and an accuracy of 100% was reached in clinical validation by analyzing 20 samples from clinical lung cancer patients. Empowered by the intrinsic sensitivity and selectivity, the proposed PASTA approach will lead to the development of a universal platform for reliable molecular subtyping.

Фактор диференціації росту 11: загальнобіологічні властивості, метаболічні ефекти та можлива патофізіологічна роль при артеріальній гіпертензії, ожирінні, цукровому діабеті та залежній від віку патології (огляд літератури)

В огляді наведені сучасні літературні дані щодо загальнобіологічних властивостей фактора диференціації росту 11 (GDF11), його участі в ембріогенезі, онкогенезі, ангіогенезі, старінні й апоптозі, відмінностей між GDF11 та міостатином, перспектив призначення рекомбінантного GDF11, експериментальних досліджень ефектів GDF11 на тваринах, можливостей клінічного застосування GDF11, його різнобічної дії при серцево-судинних захворюваннях, участі в тромбогенезі, використання в дієтології, спортивній медицині і трансфузіології, можливостей генної терапії гіпертензивного серця та її потенційних мішеней.

Cholenic acid derivative UniPR1331 impairs tumor angiogenesis via blockade of VEGF/VEGFR2 in addition to Eph/ephrin

Angiogenesis, the formation of new blood vessels from preexisting ones, is crucial for tumor growth and metastatization, and is considered a promising therapeutic target. Unfortunately, drugs directed against a specific proangiogenic growth factor or receptor turned out to be of limited benefit for oncology patients, likely due to the high biochemical redundancy of the neovascularization process. In this scenario, multitarget compounds that are able to simultaneously tackle different proangiogenic pathways are eagerly awaited. UniPR1331 is a 3β-hydroxy-Δ5-cholenic acid derivative, which is already known to inhibit Eph–ephrin interaction. Here, we employed an analysis pipeline consisting of molecular modeling and simulation, surface plasmon resonance spectrometry, biochemical assays, and endothelial cell models to demonstrate that UniPR1331 directly interacts with the vascular endothelial growth factor receptor 2 (VEGFR2) too. The binding of UniPR1331 to VEGFR2 prevents its interaction with the natural ligand vascular endothelial growth factor and subsequent autophosphorylation, signal transduction, and in vitro proangiogenic activation of endothelial cells. In vivo, UniPR1331 inhibits tumor cell-driven angiogenesis in zebrafish. Taken together, these data shed light on the pleiotropic pharmacological effect of UniPR1331, and point to Δ5-cholenic acid as a promising molecular scaffold for the development of multitarget antiangiogenic compounds.

Mesenchymal Stromal Cell Differentiation for Generating Cartilage and Bone-Like Tissues In Vitro.

In the field of tissue engineering, progress has been made towards the development of new treatments for cartilage and bone defects. However, in vitro culture conditions for human bone marrow mesenchymal stromal cells (hBMSCs) have not yet been fully defined. To improve our understanding of cartilage and bone in vitro differentiation, we investigated the effect of culture conditions on hBMSC differentiation. We hypothesized that the use of two different culture media including specific growth factors, TGFβ1 or BMP2, as well as low (2% O2) or high (20% O2) oxygen tension, would improve the chondrogenic and osteogenic potential, respectively. Chondrogenic and osteogenic differentiation of hBMSCs isolated from multiple donors and expanded under the same conditions were directly compared. Chondrogenic groups showed a notable upregulation of chondrogenic markers compared with osteogenic groups. Greater sGAG production and deposition, and collagen type II and I accumulation occurred for chondrogenic groups. Chondrogenesis at 2% O2 significantly reduced ALP gene expression and reduced type I collagen deposition, producing a more stable and less hypertrophic chondrogenic phenotype. An O2 tension of 2% did not inhibit osteogenic differentiation at the protein level but reduced ALP and OC gene expression. An upregulation of ALP and OC occurred during osteogenesis in BMP2 containing media under 20% O2; BMP2 free osteogenic media downregulated ALP and also led to higher sGAG release. A higher mineralization was observed in the presence of BMP2 during osteogenesis. This study demonstrates how the modulation of O2 tension, combined with tissue-specific growth factors and media composition can be tailored in vitro to promote chondral or endochondral differentiation while using the same donor cell population.

An in situ tissue engineering scaffold with growth factors combining angiogenesis and osteoimmunomodulatory functions for advanced periodontal bone regeneration

BackgroundThe regeneration of periodontal bone defect remains a vital clinical challenge. To date, numerous biomaterials have been applied in this field. However, the immune response and vascularity in defect areas may be key factors that are overlooked when assessing the bone regeneration outcomes of biomaterials. Among various regenerative therapies, the up-to-date strategy of in situ tissue engineering stands out, which combined scaffold with specific growth factors that could mimic endogenous regenerative processes.ResultsHerein, we fabricated a core/shell fibrous scaffold releasing basic fibroblast growth factor (bFGF) and bone morphogenetic protein-2 (BMP-2) in a sequential manner and investigated its immunomodulatory and angiogenic properties during periodontal bone defect restoration. The in situ tissue engineering scaffold (iTE-scaffold) effectively promoted the angiogenesis of periodontal ligament stem cells (PDLSCs) and induced macrophage polarization into pro-healing M2 phenotype to modulate inflammation. The immunomodulatory effect of macrophages could further promote osteogenic differentiation of PDLSCs in vitro. After being implanted into the periodontal bone defect model, the iTE-scaffold presented an anti-inflammatory response, provided adequate blood supply, and eventually facilitated satisfactory periodontal bone regeneration.ConclusionsOur results suggested that the iTE-scaffold exerted admirable effects on periodontal bone repair by modulating osteoimmune environment and angiogenic activity. This multifunctional scaffold holds considerable promise for periodontal regenerative medicine and offers guidance on designing functional biomaterials.Graphic

P–803 Novel culture conditions for the improvement of the in vitro expansion of human male fetal germ cells

Abstract Study question Do different ECMs/substrates and growth media culture conditions improve in vitro male human primordial germ cell (hPGC) expansion? Summary answer We achieved in vitro expansion improvement of male hPGCs with specific growth factors such as LIF, EGF, FGF2 and GDNF on gelatin- and vitronectin-coating cultures. What is known already PGCs are the precursors of male and female gametes, which are specified during early mammalian post-implantation embryonic development. PGCs undergo sequential cell divisions to differentiate into pro-spermatogonia (pSPG). In vitro propagation of pSPG could be important to understand the transition to spermatogonial stem cells (SSCs), important for fertility preservation in patients with infertility. Here, we aimed at performing a comparative analysis on in vitro feeder-free culture systems, based on different extracellular matrix (ECM) and growth media culture conditions, to support the expansion of the male germline stem cell populations using second trimester human male gonads as primary material. Study design, size, duration We collected human 2nd trimester male fetal gonads from elective abortions. Male gonads were dissected in saline solution (0.9% NaCl) and were either fixed overnight in 4% paraformaldehyde (PFA) for immunohistochemistry or disaggregated by enzymatic digestion for in vitro culture. Participants/materials, setting, methods After differential plating, fetal cells were cultured for 6 days. Disaggregated gonads were cultured with two different growing media (Medium 1 supplemented with LIF, EGF, FGF–2 and GDNF and Medium 2 supplemented with RA, BMP 4 and Activin A) on gelatin, laminin, vitronectin or matrigel coated plates. Cultured cells were immunostained, quantified for the expression of DDX4 and POU5F1 after 3 days (D3) and 6 days (D6) of culture. Main results and the role of chance We pursued to evaluate whether germ cells dissociated from a pool of male fetal gonads could propagate in vitro when cultured for D6 in different conditions. We observed that expansion of POU5F1-positive early PGCs and DDX4-positive late PGCs was only observed when cells were plated on gelatin or vitronectin and cultured with Medium 1, containing the growth factors LIF, EGF, FGF2 and GDNF. However, a reduced percentage of PGCs was observed in all four different coatings when grown with Medium 2, containing RA, BMP4 and Activin A. We analyzed the relative expression of the PGC markers POU5F1, DDX4 and MAGEA4 in histological sections of gonads from embryos at 18.5 weeks of gestation. Two populations of hPGCs were observed: ∼10–30% of the gonadal cells expressed solely DDX4 (late PGCs), whereas less than 10% of gonadal cells expressed POU5F1 (early PGCs). SOX9 and STARD1 expression was evaluated, confirming the presence of Sertoli cells and Leydig cells, respectively. Limitations, reasons for caution Due to the limited and difficulty to obtain human fetal tissue, a limited number of samples were used. Wider implications of the findings: We expanded human male fetal germ cells in vitro for D6 on gelatin and vitronectin coated plates with Medium 1, containing growth factors LIF, EGF, FGF2 and GDNF. Our findings provide a 2D culture system to expand hPGCs that could be useful to study propagation to pSPGs and eventually SSCs. Trial registration number Not applicable

Transcriptomics-Based Phenotypic Screening Supports Drug Discovery in Human Glioblastoma Cells.

Simple SummaryGlioblastoma (GBM) remains a particularly challenging cancer, with an aggressive phenotype and few promising treatment options. Future therapy will rely heavily on diagnosing and targeting aggressive GBM cellular phenotypes, both before and after drug treatment, as part of personalized therapy programs. Here, we use a genome-wide drug-induced gene expression (DIGEX) approach to define the cellular drug response phenotypes associated with two clinical drug candidates, the phosphodiesterase 10A inhibitor Mardepodect and the multi-kinase inhibitor Regorafenib. We identify genes encoding specific drug targets, some of which we validate as effective antiproliferative agents and combination therapies in human GBM cell models, including HMGCoA reductase (HMGCR), salt-inducible kinase 1 (SIK1), bradykinin receptor subtype B2 (BDKRB2), and Janus kinase isoform 2 (JAK2). Individual, personalized treatments will be essential if we are to address and overcome the pharmacological plasticity that GBM exhibits, and DIGEX will play a central role in validating future drugs, diagnostics, and possibly vaccine candidates for this challenging cancer.We have used three established human glioblastoma (GBM) cell lines—U87MG, A172, and T98G—as cellular systems to examine the plasticity of the drug-induced GBM cell phenotype, focusing on two clinical drugs, the phosphodiesterase PDE10A inhibitor Mardepodect and the multi-kinase inhibitor Regorafenib, using genome-wide drug-induced gene expression (DIGEX) to examine the drug response. Both drugs upregulate genes encoding specific growth factors, transcription factors, cellular signaling molecules, and cell surface proteins, while downregulating a broad range of targetable cell cycle and apoptosis-associated genes. A few upregulated genes encode therapeutic targets already addressed by FDA approved drugs, but the majority encode targets for which there are no approved drugs. Amongst the latter, we identify many novel druggable targets that could qualify for chemistry-led drug discovery campaigns. We also observe several highly upregulated transmembrane proteins suitable for combined drug, immunotherapy, and RNA vaccine approaches. DIGEX is a powerful way of visualizing the complex drug response networks emerging during GBM drug treatment, defining a phenotypic landscape which offers many new diagnostic and therapeutic opportunities. Nevertheless, the extreme heterogeneity we observe within drug-treated cells using this technique suggests that effective pan-GBM drug treatment will remain a significant challenge for many years to come.

Continuous expression of reprogramming factors induces and maintains mouse pluripotency without specific growth factors and signaling inhibitors.

ObjectivesDerivation and maintenance of pluripotent stem cells (PSCs) generally require optimized and complex culture media, which hinders the derivation of PSCs from various species. Expression of Oct4, Sox2, Klf4, and c‐Myc (OSKM) can reprogram somatic cells into induced PSCs (iPSCs), even for species possessing no optimal culture condition. Herein, we explored whether expression of OSKM could induce and maintain pluripotency without PSC‐specific growth factors and signaling inhibitors.MethodsThe culture medium of Tet‐On‐OSKM/Oct4‐GFP mouse embryonic stem cells (ESCs) was switched from N2B27 with MEK inhibitor, GSK3β inhibitor, and leukemia inhibitory factor (LIF) (2iL) to N2B27 with doxycycline. Tet‐On‐OSKM mouse embryonic fibroblast (MEF) cells were reprogrammed in N2B27 with doxycycline. Cell proliferation was traced. Pluripotency was assessed by expression of ESC marker genes, teratoma, and chimera formation. RNA‐Seq was conducted to analyze gene expression.ResultsVia continuous expression of OSKM, mouse ESCs (OSKM‐ESCs) and the resulting iPSCs (OSKM‐iPSCs) reprogrammed from MEF cells propagated stably, expressed pluripotency marker genes, and formed three germ layers in teratomas. Transcriptional landscapes of OSKM‐iPSCs resembled those of ESCs cultured in 2iL and were more similar to those of ESCs cultured in serum/LIF. Furthermore, OSKM‐iPSCs contributed to germline transmission.ConclusionsExpression of OSKM could induce and maintain mouse pluripotency without specific culturing factors. Importantly, OSKM‐iPSCs could produce gene‐modified animals through germline transmission, with potential applications in other species.

Characterisation of the serum cytokine profile in feline atopic skin syndrome.

Feline atopic skin syndrome (FASS) is a pruritic and inflammatory skin disease commonly encountered in cats. Three previous reports evaluated cytokine immune activation in cats diagnosed with feline allergic dermatitis. However, no significant upregulations were observed in allergic cats compared to healthy controls. To evaluate differences in the serum cytokine profile of cats diagnosed with FASS compared to healthy cats, and correlate serum markers with the extent of FASS skin disease using clinical scoring systems. Thirteen client-owned FASS cats and 12 healthy control cats. Thirteen cytokine and chemokines from the serum of FASS cats and healthy controls were analysed using a commercially available feline-specific multiplex assay. Patients with FASS had a significant increase in serum concentrations of interferon-gamma (IFN-γ), interleukin (IL)-2, IL-13 and IL-18. In addition, cytokine/chemokines involved in inflammation and chemotaxis [IL-8, C-C Motif Chemokine Ligand (CCL)5, CCL2 and CXCL12], as well as growth factors, stem cell factor and Fms-related tyrosine kinase 3 ligand (Flt3L), also were significantly elevated. A significant positive correlation (r=0.64) between the serum levels of Flt3L and Scoring Feline Allergic Dermatitis (SCORFAD) score was observed. These results demonstrate the activation of a broad array of immune secretory cytokines in the serum of cats with FASS, which are largely associated with a mixed Th1 and Th2 inflammatory response along with specific growth factors. Further larger-sample studies are needed to assess the modulation of serum biomarkers in FASS by pharmacological/therapeutic interventions.

Gene Therapy: A Review

The goal of gene-enhanced tissue engineering is to regenerate lost tissue by the local delivery of cells that have been genetically-enhanced to deliver physiologic levels of specific growth factors. The basis for this approach lies in the presence of a population of progenitor cells that can be induced, under the influence of these growth factors, to differentiate into the specific cells required for tissue regeneration, with guidance from local clues in the wound environment. Keywords: Gene therapy, Growth factor, Tissue engineering, Regeneration.

Shared Genetic Etiology between Alzheimer's Disease and Blood Levels of Specific Cytokines and Growth Factors.

Late-onset Alzheimer’s disease (AD) has a significant genetic and immunological component, but the molecular mechanisms through which genetic and immunity-related risk factors and their interplay contribute to AD pathogenesis are unclear. Therefore, we screened for genetic sharing between AD and the blood levels of a set of cytokines and growth factors to elucidate how the polygenic architecture of AD affects immune marker profiles. For this, we retrieved summary statistics from Finnish genome-wide association studies of AD and 41 immune marker blood levels and assessed for shared genetic etiology, using a polygenic risk score-based approach. For the blood levels of 15 cytokines and growth factors, we identified genetic sharing with AD. We also found positive and negative genetic concordances—implying that genetic risk factors for AD are associated with higher and lower blood levels—for several immune markers and were able to relate some of these results to the literature. Our results imply that genetic risk factors for AD also affect specific immune marker levels, which may be leveraged to develop novel treatment strategies for AD.

Isolation, Characterization, and Differentiation of Stem Cells From Various Dental Sources: An In Vitro Study

Aim: The aim of the current study was to isolate stem cells from various dental sources such as dental pulp, periodontal ligament (PDL), and apical papilla, and to characterize stem cells by staining for the presence/absence of specific surface markers and also to differentiate stem cells into osteogenic, chondrogenic, and adipogenic cell lineages by exposing them to specific growth factors under the ideal conditions. Materials and Methods: A total of 117 samples were included in the study, consisting of 30 pulp, 50 gingival, 35 PDL, and 2 apical papilla samples. The pulp was extirpated and transported to the Central Research Laboratory. Gingival connective tissue was collected from the participants undergoing any crown lengthening procedure or any gingivectomy procedure from the Department of Periodontology. A similar procedure was also followed for apical papilla and PDL. Isolation was done followed by the identification of the cells by immunocytochemistry using different markers. Once the identity of cells was confirmed, these cells were treated with different culture media to attain 70% to 100% confluency. Then the medium was replaced with a conditioning medium containing specific growth factors for differentiation into osteogenic, chondrogenic, and adipogenic cell lineages. Result: In our study, the number of samples collected and processed was 117. The isolation rate of stem cells from the above-collected samples was 70%. Statistical analysis—no statistical analysis was done as there was no variability expected. Conclusion: Our study showed that stem cells could be isolated, differentiated, and characterized from different dental sources.

Canine Pituitary Organoids as 3D In Vitro Model for Cushing Disease

Cushing disease (CD) is a serious endocrine disorder that is most often caused by an ACTH-secreting pituitary adenoma. Patients can be treated medically when surgery is not an option or was unsuccessful. However, currently used pituitary-targeting drugs are effective in only 40% of patients. To efficiently identify new pituitary-targeting treatment options, we need an in vitro system that closely mimics in vivo conditions. We therefore aimed to establish organoid cultures of normal anterior pituitary and corticotroph adenomas. Organoids or tumoroids are miniature three-dimensional (3D) structures grown from stem cells, that closely resemble the organ or tumor they originate from. Because CD is a thousand times more prevalent in dogs than in humans, and hypophysectomy is the treatment of choice, we used canine tissues. Normal anterior pituitary glands were collected from three healthy dogs that were euthanized for reasons unrelated to the current study. Corticotroph adenomas were collected from six dogs that underwent transsphenoidal hypophysectomy at our University Clinic. The dogs were diagnosed with CD based on clinical signs, endocrine testing, and CT scan imaging. Normal anterior pituitary and corticotroph adenoma cells were cultured in a 3D matrix (basement membrane extract) with anterior pituitary organoid medium containing specific growth factors and ligands, which was refreshed twice a week. The organoids and tumoroids were characterized with histopathology and RT-qPCR. Structures resembling organoids or tumoroids grew from all nine samples (3 normal, 6 adenoma) that were put in culture. Both cystic and dense structures were observed. The organoids and tumoroids expanded rapidly, and could be passaged once every week. The organoids and tumoroids were successfully cultured up until passage number 10, and were then frozen down. Histopathology showed that the organoid or tumoroid cells morphologically resembled healthy anterior pituitary or corticotroph adenoma cells. All organoids cultures expressed mRNA of pituitary stem cell markers SOX2 and SOX9. This study shows that corticotroph adenomas can be cultured as tumoroids in vitro, something not previously published in any species. Based on the many opportunities in organoid culture (e.g., high-throughput drug screenings, gene editing, studying developmental processes), we expect that this in vitro model will pave the way to efficiently and reliably identify new treatment options for CD. Not only for humans, but also for our best friends: dogs.

Role of oxygen exposure on the differentiation of human induced pluripotent stem cells in 2D and 3D cardiac organoids

Human induced pluripotent stem cells (hiPSC) have the ability to differentiate theoritically into any cell type. The development of organoid systems exhibiting the essential features of human organ such as liver and heart is of high interest. Optimizing the culture conditions to obtain the highest cardiac organoids efficacy is crucial. In fact, cardiac differentiation protocols have been established by essentially focusing on specific growth factors on hiPSC differentiation efficiency. However, the optimal environmental factors such as the optimal oxygen exposure to obtain cardiac myocytes in network are still unclear. The mesoderm germ layer differentiation is known to be enhanced by low oxygen exposure. Yet, the effect of low oxygen exposure on the molecular and functional maturity of the hiPSC-derived cardiomyocytes remains unexplored. We aimed here at comparing the molecular and functional consequences of low (5% O 2 or LOE) and high oxygen exposure (21% O 2 or HOE) on cardiac differentiation of hiPSCs in 2D monolayer and 3D organoids protocols. hiPSC-CMs were differentiated through both the 2D (monolayer) and 3D (embryoid body) protocols using several lines. Cardiac marker expression and cell morphology were assessed using qRT-PCR and immunofluorescence. The mitochondrial localization and metabolic properties were evaluated by high-resolution respirometry and mitochondrial staining. The intracellular Ca 2+ handling and contractile properties were also monitored using confocal fluorescent microscopy and atomic force microscopy. Our results indicated that the 2D cardiac monolayer can only be differentiated in HOE. The 3D cardiac organoids containing hiPSC-CMs in LOE exhibited higher cardiac markers expression such as troponin T (TnTc), RyR2, Serca2a, alpha and beta heavy myosin chains. Moreover, we found enhanced contractile force, hypertrophy and steadier SR Ca 2+ release reflected by a more regular spontaneous Ca 2+ transients associated with a higher maximal amplitude and lower spontaneous Ca 2+ events revealing a better SR Ca 2+ handling in LOE. Similar beat rate, preserved distribution of mitochondria and similar oxygen consumption by the mitochondrial respiratory chain complexes were also observed. Our results brought evidences that LOE is moderately beneficial for the 3D cardiac organoids with hPSC-CMs exhibiting further maturity. In contrast, the 2D cardiac monolayers strictly require HOE.

Serotonin Induces Inflammatory Cytokine Production and Regulates Lymphatic Endothelial Cell Function

Serotonin or 5-hydroxytryptamine (5-HT) is a neuromodulator, with both neuroendocrine and neurotransmitter functions, which is synthesized in serotonergic neurons in the central nervous system and enterochromaffin cells throughout the gastrointestinal tract. The lymphatics play a central role in inflammatory and immune response and lymphatic endothelial cells (LECs) are active participants in these processes. Several immune cells store or respond to 5-HT thereby implicating it in inflammatory pathways and it is also known as a growth factor for several types of non-tumoral cells. Lymphatic muscle has been shown to be significantly responsive to 5-HT however the effects of 5-HT on modulation of lymphatic endothelial phenotype or inflammatory function is not well understood. In this study, we hypothesized that increased levels of 5-HT modulate LEC morphological and phenotypic behavior and also induces cytokine and chemokine production that contribute to inflammation. LECs were initially evaluated for the expression of specific receptors by real time PCR. HDLECs were treated with 5-HT and its receptor antagonist and the effects of 5-HT on LEC proliferation, migration and tube formation was evaluated. Real time PCR was carried out to evaluate the alterations in specific cytokines, chemokines and growth factors produced by LECs in response to 5-HT treatment. In addition, conditioned medium was collected from LECs treated with 5-HT and/or its receptor inhibitor and subjected to cytokine and chemokine array analysis. Our real time PCR analysis show that LECs show varying basal level expression of the 5-HT receptors (5-HTR1-7) with 5-HTR7 showing the highest level of expression. Serotonin treatment of the LECs significantly induces LEC proliferation and migration shown by XTT and Boyden chamber assays. Further, LECs treated with 5-HT for 24hrs show significant induction of several growth factors, inflammatory and lymphangiogenic molecules including angiogenin, endoglin and IL23 indicating the activation of key inflammatory or lymphangiogenic pathways. In keeping with these findings, our results also show that 5-HT treatment enhances lymphatic tube formation that is abrogated by 5-HTR inhibition. Further, 5-HT also activates the PI3K/AKT and ERK MAPK key lymphangiogenic and growth promoting pathways in the LECs. Taken together, we show that 5-HT alters phenotypic and morphological behavior of LECs and activate several growth factors and signaling molecules that are pro-lymphangiogenic and thus can be a viable therapeutic strategy for modulation of lymphangiogenesis and LEC function during inflammation.