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Introduction Artificial dermis is an effective therapeutic method for full-thickness dermal defects. However, the currently available artificial dermis made of porcine or bovine type I collagen has several limitations such as incomplete epithelialization and delayed migration of fibrogenic and angiogenic cells into the graft. We previously developed a composite dermal graft containing a mixture of moon jellyfish collagen and porcine type I collagen, and reported its stimulatory effect on both the re-epithelialization of the epidermis and the migration of fibrogenic and angiogenic cells into the graft. In the present study, we examined whether the same effect was observed by administering jellyfish collagen solution externally onto an artificial dermal graft made of bovine type I collagen. Methods We used a 6 mm full-thickness wound defect model. Moon jellyfish collagen was prepared as a concentrated 0.5% solution and dripped externally onto a transplanted artificial dermal graft made of bovine type I collagen. Wound repair and long-term dermal tissue remodeling were compared between mice administered jellyfish collagen solution on the bovine collagen graft and those transplanted with a composite dermal graft containing the same amounts of jellyfish and bovine collagens. The stimulatory effect of jellyfish collagen solution was also evaluated using diabetic dB/dB mice. Results External administration of jellyfish collagen solution onto the bovine collagen graft significantly accelerated wound closure compared to control saline. It also decreased the number of inflammatory cells infiltrating the wound and suppressed absorption of the transplanted graft, as well as reduced subsequent scar formation. Furthermore, external administration of jellyfish collagen solution onto the bovine collagen graft improved the delayed wound healing in diabetic model mice, and this effect was superior to that of the currently used basic fibroblast growth factor. Conclusions External administration of moon jellyfish collagen solution onto a bovine collagen graft significantly accelerated physiological wound healing and prevented excessive scar formation. It also improved wound closure in diabetic model mice, confirming its therapeutic application for intractable skin ulcers caused by impaired wound healing., Highlights • Impaired wound healing is frequently observed in elderly and diabetic patients. • Moon jellyfish collagen accelerates wound closure after full-thickness dermal defect in mice. • Jellyfish collagen used as an external medicine stimulates the elongation of regenerating epithelial cells. • Externally applied jellyfish collagen improves wound healing in diabetic model mice.

One of the most challenging problems in skin tissue engineering is the development of functional vascularized networks in wounded tissues. In this regard, improving angiogenesis is considered to be effective for the regeneration of the skin during wound healing. Therefore, different strategies have been developed to induce angiogenesis in skin tissue engineering. Using stem cells and delivery of angiogenic nanomaterials via a biocompatible scaffold are examples of these strategies. Adipose-derived stem cells could not only differentiate into endothelial and epithelial cells under appropriate conditions, but could also secrete angiogenic growth factors such as vascular endothelial growth factor, basic fibroblast growth factor and hepatocyte growth factor that induce angiogenesis in ischemia injury models. Bioglass nanoparticles are potent nanobiomaterials to improve angiogenesis in chronic wound healing. Because the wound healing process depends on the quality of newly formed blood vessels, recent interest in bioglass nanoparticles has increased dramatically. Adding some of the metallic ions (e.g., Co2+, Cu2+, Eu3+, Mg2+ and Nb5+) to the structure of bioglass nanoparticles could enhance their angiogenic properties. We purpose that delivery of ion-doped mesoporous bioactive glass nanoparticles and adipose-derived stem cells via electrospun silk fibroin nanofibrous membrane as a scaffold would improve chronic wound healing by enhancing angiogenesis.

Spermatogonial stem cells (SSCs) are crucial for maintaining spermatogenesis, studying germ line stem cell biology, and producing transgenic animals. Growth factors, including leukemia inhibitory factor (LIF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and glial cell line-derived neurotrophic factor (GDNF), are essential for in vitro culture of SSCs as well as their self-renewal and maintenance. In this study, we investigated the effects of these growth factors on porcine spermatogonial germ cell (pSGC) colony formation. We determined round alkaline phosphatase (AP)-positive pSGC colonies in the presence and absence of growth factors after 7 days of pSGC culture. EGF was found to be essential to support the formation of AP-positive pSGC colonies. The expression of epidermal growth factor receptor (EGFR) and Erb-B2 receptor tyrosine kinase 2 (ERBB2) was also altered in cultured pSGCs compared to that in feeder cells. We verified the effect of EGF signaling on pSGC colony formation using AG1478 as an EGFR inhibitor and AG879 as an ERBB2 inhibitor. pSGC colonies were observed in low dose AG1478-treated groups with EGF, whereas a high dose of AG1478 suppressed pSGC colony formation. AP-positive colonies were also observed in all AG879-treated groups. Taken together, EGFR and EGF signaling play a critical role in the initiation of colony formation of pSGCs. Our study provides insights into the mechanisms of EGF-mediated colony formation by SGCs derived from porcine testes, and will aid the development of transplantation techniques for the production of transgenic offspring.

Objective: The growth rate of pigs is related to differentiation and proliferation of muscle cells, which are regulated by growth factors and expression of growth-related genes. Thus, the objective of this study was to establish optimal culture conditions for Jeju black pig (JBP) muscle cells and determine the relationship of various factors involved in muscle growth with the proliferation of JBP muscle cells. Methods: Muscles were taken from the femur skeletal muscle of JBP embryos. After isolation of the muscle cells, cells were cultured in a 6-well plate under four different culture conditions to optimize culture conditions for JBP muscle cells. To analyze proliferation rate of JBP muscle cells, these muscle cells were seeded into 6-well plates at a density of 1.5×105 cells per well and cultured for 3 days. Western blot and quantitative real-time polymerase chain reaction were applied to verify the myogenic differentiation 1 (MyoD) expression and growth-related gene expression in JBP muscle cells, respectively. Results: We established a muscle cell line from JBP embryos and optimized its culture conditions. These muscle cells were positive for MyoD, but not for paired box 7. The proliferation rate of these muscle cells was significantly higher in a culture medium containing bFGF and epidermal growth factor + basic fibroblast growth factor (EGF+bFGF) than that without a growth factor or containing EGF alone. Treatment with EGF and bFGF significantly induced the expression of MyoD protein, an important transcription factor in muscle cells. Moreover, we checked the changes of expression of growth-related genes in JBP muscle cells by presence or absence of growth factors. Expression level of collagen type XXI alpha 1 gene was changed only when EGF and bFGF were added together to culture media for JBP muscle cells. Conclusion: Concurrent use of EGF and bFGF increased the expression of MyoD protein, thus regulating the proliferation of JBP muscle cells and the expression of growth-related genes.

Vascularization is a critical step following implantation of an engineered tissue construct in order to maintain its viability. The ability to spatially pattern or direct vascularization could be therapeutically beneficial for anastomosis and vessel in-growth. However, acellular and cell-based strategies to stimulate vascularization typically do not afford this control. We have developed an ultrasound-based method of spatially- controlling regenerative processes using acellular, composite hydrogels termed acoustically-responsive scaffolds (ARSs). An ARS consists of a fibrin matrix doped with a phase-shift double emulsion (PSDE). A therapeutic payload, which is initially contained within the PSDE, is released by an ultrasound-mediated process called acoustic droplet vaporization (ADV). During ADV, the perfluorocarbon (PFC) phase within the PSDE is vaporized into a gas bubble. In this study, we generated ex situ four different spatial patterns of ADV within ARSs containing basic fibroblast growth factor (bFGF), which were subcutaneously implanted in mice. The PFC species within the PSDE significantly affected the morphology of the ARS, based on the stability of the gas bubble generated by ADV, which impacted host cell migration. Irrespective of PFC, significantly greater cell proliferation (i.e., up to 2.9-fold) and angiogenesis (i.e., up to 3.7-fold) were observed adjacent to +ADV regions of the ARSs compared to -ADV regions. The morphology of the PSDE, macrophage infiltration, and perfusion in the implant region were also quantified. These results demonstrate that spatially-defined patterns of ADV within an ARS can elicit spatially-defined patterns of angiogenesis. Overall, these finding can be applied to improve strategies for spatially-controlling vascularization. Statement of significance Vascularization is a critical step following implantation of an engineered tissue. The ability to spatially pattern or direct vascularization could be therapeutically beneficial for inosculation and vessel in-growth. However, acellular and cell-based strategies to stimulate vascularization typically do not afford this control. We have developed an ultrasound-based method of spatially-controlling angiogenesis using acellular, composite hydrogels termed acoustically-responsive scaffolds (ARSs). An ARS consists of a fibrin matrix doped with a phase-shift double emulsion (PSDE). An ultrasound-mediated process called acoustic droplet vaporization (ADV) was used to release basic fibroblast growth factor (bFGF), which was initially contained within the PSDE. We demonstrate that spatially-defined patterns of ADV within an ARS can elicit spatially-defined patterns of angiogenesis in vivo. Overall, these finding can improve strategies for spatially-controlling vascularization.

Background: Pelvic organ prolapse (POP) is a common debilitating condition affecting approximately 30-40% of women. The FDA issued a warning about polypropylene mesh used for pelvic floor repair due to erosion, exposure and other complications and banned it in 2019. The application of stem cell therapy and growth factors has strongly promoted the development of pelvic tissue engineering.Purpose: we intend to address the issues of direct application of growth factors, the side effects of long-term exogenous treatment, and the directional differentiation of stem cells. Methods: we evaluated the paracrine effects and directional differentiation of adipose mesenchymal stem cells through stable overexpression of basic fibroblast growth factor (bFGF).Results: we found that the modified stem cells could continuously and stably release bFGF in the initial stage and could spontaneously differentiate into fibroblasts with a high differentiation efficiency in the later stage.Conclusion: following ADSCs are designed to continuously release controllable levels of growth factors during the control period of repair, taking advantage of the paracrine function of stem cells to accelerate cell growth and extracellular matrix (ECM) reconstruction during the early stage of stem cell implantation, and then stem cells are differentiated into target tissues-fibroblasts to accelerate the reconstruction of pelvic floor tissues, this study demonstrated the strong therapeutic potential of this approach for pelvic tissue engineering.Abbreviations POP: Pelvic organ prolapse; ADSCs: Adipose-derived stem cells; bFGF: Basic fibroblast growth factor; BMSCs: Bone marrow-derived mesenchymal stem cells; HUVECs: Human umbilical vein endothelial cells; EMSCs: Endometrial mesenchymal stem cells; VEGF: Vascular endothelial growth factor; PDGF: Platelet-derived growth factor ECM: Extracellular matrix; IGF: Insulin-like growth factor; HGF: Hepatocyte growth factor; EGF: Epidermal growth factor; BMP-2: Bone morphogenetic protein 2; FBR: Foreign body reaction.

Ovaries are the primary physiological source of female sex hormones, which play a crucial role in maintaining ovarian cycle, determining secondary sexual characteristics and preparing the endometrium for implantation. In vitro follicle engineering has been used to investigate follicle development, including ovarian hormone production and gamete maturation. To engineer functional follicles, culture and expansion of the primary ovarian cells are essential. However, the phenotypic and functional characteristics of primary ovarian cells are often lost during culture. The objective of this study is to develop an optimized culture system for maintaining ovarian cell growth and functionality. Granulosa cells (GCs) and theca cells (TCs) were isolated from female rats. The addition of follicle-stimulating hormone (FSH) or luteinizing hormone (LH) to the basal culture media significantly enhanced the secretion of estradiol from GCs and androstenedione from TCs. Serum concentrations of 5% and 10% had a similar role in promoting ovarian cell expansion and secretion of estradiol and androstenedione hormones from both types of cells. Growth differentiation factor 9 (GDF9), bone morphogenic protein 15 (BMP15), BMP7 and basic fibroblast growth factor (bFGF) enhanced GC proliferation and estradiol production, respectively. Among them, the effect of bFGF was most significant. bFGF also enhanced TC proliferation. When GCs and TCs were cultured in 5% serum, gonadotropin and bFGF-containing medium, they proliferated exponentially throughout the culture period of up to 40 days while maintaining their functional characteristics. Taken together, these results indicate that our medium formula is optimal for maximizing proliferation of functionally differentiated ovarian cells.

The molecular signaling cascades that regulate angiogenesis and microvascular remodeling are fundamental to normal development, healthy physiology, and pathologies such as inflammation and cancer. Yet quantifying such complex, fractally branching vascular patterns remains difficult. We review application of NASA’s globally available, freely downloadable VESsel GENeration (VESGEN) Analysis software to numerous examples of 2D vascular trees, networks, and tree-network composites. Upon input of a binary vascular image, automated output includes informative vascular maps and quantification of parameters such as tortuosity, fractal dimension, vessel diameter, area, length, number, and branch point. Previous research has demonstrated that cytokines and therapeutics such as vascular endothelial growth factor, basic fibroblast growth factor (fibroblast growth factor-2), transforming growth factor-beta-1, and steroid triamcinolone acetonide specify unique “fingerprint” or “biomarker” vascular patterns that integrate dominant signaling with physiological response. In vivo experimental examples described here include vascular response to keratinocyte growth factor, a novel vessel tortuosity factor; angiogenic inhibition in humanized tumor xenografts by the anti-angiogenesis drug leronlimab; intestinal vascular inflammation with probiotic protection by Saccharomyces boulardii, and a workflow programming of vascular architecture for 3D bioprinting of regenerative tissues from 2D images. Microvascular remodeling in the human retina is described for astronaut risks in microgravity, vessel tortuosity in diabetic retinopathy, and venous occlusive disease.

Estrogen is essential for the skin to maintain its physiological function. The binding of estrogen to the estrogen receptor (ER) activates gene transcription, which has biological effects on the target tissue. Estrogen levels and ER expression are known to decrease with aging and exposure to ultraviolet light (UV); therefore, increased estrogen levels and ER expression may improve age-related changes in the skin. Rehmannia root has been reported to have blood circulation-promoting and anti-inflammatory effects; however, few studies have reported the effects of Rehmannia root on skin. In this study, we examined the effects of Rehmannia glutinosa Libosch. var. purpurea Makino root extract (RE) on ER expression, and estrogen, RE, or their related ingredients increased ER expression in human epidermal keratinocytes, human dermal fibroblasts, and skin models. Moreover, RE increased the production of basic fibroblast growth factor, transforming growth factor β1, and epidermal growth factor. The mixture of estrogen and RE improved extracellular matrix (ECM) production to a greater degree than estrogen and RE independently. Although high population doubling levels (PDL) and UV irradiation downregulated ER expression, RE upregulated ER expression in high PDL cells and UV irradiated cells. In addition, RE increased the expression of epidermal differentiation marker proteins compared to their expression levels in the absence of RE. The collective findings suggest that RE aids in the prevention of skin aging by upregulating the ER expression that has been decreased by aging and UV and promoting estrogen activity, ECM production, and epidermal differentiation.

Background: In vitro obtaining oocytes can be an appropriate alternative for patients with gonadal insufficiency or cancer survivors. The purpose of the current research was isolating stem cells from ovarian cortical tissue as well as evaluating the effectiveness of follicle stimulating hormone (FSH), basic fibroblast growth factor (bFGF), and neurotrophin 3 (NT3) in differentiating to oocyte-like cells. Methods: A human ovary was dissected and cortical tissue pieces were cultured for cell isolation. Isolated cells were divided into 8 groups (3 cases in each group) of control, FSH, NT3, bFGF, FSH+NT3, FSH+bFGF, NT3+bFGF, and FSH+NT3+ bFGF. Pluripotency specific gene (OCT4-A and Nanog), initial germ cells (c-KIT and VASA) and PF growth initiators (GDF-9 and Lhx-8) were evaluated by qRT-PCR. Experiments were performed in triplicate and there were 3 samples in each group. The results were analyzed using one-way ANOVA and p-value less than 0.05 was considered statistically significant. Results: Flow cytometry results showed that cells isolated from the ovarian cortex expressed markers of pluripotency. The results showed that the expression of Nanog, OCT4, GDF-9 and VASA was significantly increased in FSH+NT3 group, while treatment with bFGF caused significant expression of c-KIT and Lhx-8 (p

Most characterized angiogenic modulators are proteolytic fragments of structural plasma and/or matrix components. Herein, we have identified a novel anti-angiogenic peptide generated by the in vitro hydrolysis of the C-terminal moiety of the fibrinogen alpha chain, produced by the snake venom metalloprotease bothropasin (SVMP), a hemorrhagic proteinase in Bothrops jararaca venom. The 14-amino acids peptide (alphastatin-C) is a potent antagonist of basic fibroblast growth factor, induced endothelial cell (HUVEC-CS) proliferation, migration and capillary tube formation in matrigel. It also inhibits cell adhesion to fibronectin. The basis of the antagonism between bFGF and alphastatin-C is elucidated by the inhibition of various bFGF induced signaling pathways and their molecular components modification, whenever the combination of the stimuli is provided, in comparison to the treatment with bFGF only. To corroborate to the potential therapeutic use of alphastatin-C, we have chosen to perform in vivo assays in two distinct angiogenic settings. In chick model, alphastatin-C inhibits chorioallantoic membrane angiogenesis. In mouse, it efficiently reduces tumor number and volume in a melanoma model, due to the impairment of tumor neovascularization in treated mice. In contrast, we show that the alphastatin-C peptide induces arteriogenesis, increasing pial collateral density in neonate mice. alphastatin-C is an efficient new antiangiogenic FGF-associated agent in vitro, it is an inhibitor of embryonic and tumor vascularization in vivo while, it is an arteriogenic agent. The results also suggest that SVMPs can be used as in vitro biochemical tools to process plasma and/or matrix macromolecular components unraveling new angiostatic peptides.

Background: Glioblastoma multiforme is the most aggressive astrocytoma in adults. Glioblastoma is a heterogenous tumor formed from various cells including astrocyte and cancer stem cells. Here, we explain the isolation, culture, morphology and specific markers of human glioblastoma astrocyte and stem cells. Methods: We isolated astrocyte and cancer stem cells from human glioblastoma tissue. The obtained glioblastoma tissues were digested and cultured in DMEM12, B27 supplemented with basic fibroblast growth factor and epidermal growth factor. The morphology and specific markers were assessed in astrocyte and cancer stem cell of human glioblastoma through immunocytochemistry. Results: Results indicated that there were two morphology types in cell culture including epithelioid morphology and fibroblastic morphology. The astrocyte confirmed via expression of the Glial fibrillary acidic protein (GFAP) protein. Cancer stem cells were round and floating in the culture medium. Immunocytochemical staining indicated that nestin and SRY-box 2 (SOX2) antigens were positively expressed in primary neurospheres. Conclusion: The expression of glial and stem cell markers shows that both cells are in the human glioblastoma.

The present study was undertaken to evaluate the effect of different concentration of FGF2 viz. 5 ng (T1), 10 ng (T2), and 20 ng/mL (T3) on cumulus cell expansion, oocyte maturation, in vitro embryo production, total cell number (TCN) of the blastocyst, and expression of the FGF2 and FGFR2 transcripts in buffalo oocytes and the embryos. Results showed that the effect of FGF2 on the diameter of buffalo COC was significantly higher (P0.05) in the T1 group than the other groups at 24h of maturation. The maturation and cleavage rate of oocytes was significantly higher (P0.05) in the T3 group than the control, however, the values did not different (P0.05) from other groups. The effect of FGF2 on morula and blastocyst yield did not different (P0.05) between treatment groups. However, the TCN of the blastocyst was slightly higher (P0.05) in the T3 group than the control and other groups. In subsequent trials, the expression of the FGF2 transcript was higher (P0.05) in A-grade of oocytes than the C- and D-grade of oocytes, but the expression was not different (P0.05) from the B-grade of oocytes. While the FGFR2 expression was higher (P0.05) in cumulus cells than any grades of oocytes. The relative abundance of FGF2 and FGFR2 transcripts was significantly higher (P0.05) in the 2-cell stage of the embryo than the other stages of embryos. This study was further extended to characterize the FGF2 ligand-binding site in the D3 domain of the buffalo FGF2 receptor. Bioinformatics analysis showed that the bovine FGF2 ligand-binding site in the D3 domain of buffalo was different from the D3 domain of the cattle.

The scarcity of chondrocytes is a major challenge for cartilage tissue engineering. Monolayer expansion is necessary to amplify the limited number of chondrocytes needed for clinical application. Growth factors are often added to improve monolayer culture conditions, promoting proliferation, and enhancing chondrogenesis. Limited knowledge on the biosafety of the cell products manipulated with growth factors in culture has driven this study to evaluate the impact of growth factor cocktail supplements in chondrocyte culture medium on chondrocyte genetic stability and tumorigenicity. The growth factors were basic fibroblast growth factor (b-FGF), transforming growth factor β2 (TGF β2), insulin-like growth factor 1 (IGF-1), insulin-transferrin-selenium (ITS), and platelet-derived growth factor (PD-GF). Nasal septal chondrocytes cultured in growth factor cocktail exhibited a significantly high proliferative capacity. Comet assay revealed no significant DNA damage. Flow cytometry showed chondrocytes were mostly at G0-G1 phase, exhibiting normal cell cycle profile with no aneuploidy. We observed a decreased tumour suppressor genes’ expression (p53, p21, pRB) and no TP53 mutations or tumour formation after 6 months of implantation in nude mice. Our data suggest growth factor cocktail has a low risk of inducing genotoxic and tumorigenic effects on chondrocytes up to passage 6 with 16.6 population doublings. This preclinical tumorigenicity and genetic instability evaluation is crucial for further clinical works.

Lymphangiogenesis, or formation of new lymphatic vessels, is a tightly regulated process that is controlled by growth factor signaling and biomechanical cues. Lymphatic endothelial cells (LECs) undergo remodeling, migration, and proliferation to invade the surrounding extracellular matrix (ECM) during both physiological and pathological lymphangiogenesis. This study optimized conditions for an in vitro three-dimensional (3-D) collagen-based model that induced LEC invasion and recapitulated physiological formation of lymphatic capillaries with lumens. Invasion of LECs was enhanced in the presence of sphingosine 1-phosphate (S1P). Effects of various known lymphangiogenic factors, vascular endothelial growth factor (VEGF)-A, basic fibroblast growth factor (bFGF), interleukin (IL)-8, and hepatocyte growth factor (HGF), were tested on LEC sprout formation synergistically with VEGF-C. Several of these growth factors significantly enhanced LEC invasion, and synergistic effects of some of these further enhanced the sprouting density and lumen volume. To determine the contribution of specific ECM components, we analyzed the expression of different integrin subunits. Basal expressions of the integrin α5- and integrin β1-subunits were high in LECs. The addition of fibronectin, which mediates cellular responses through these integrins, enhanced LEC sprouting density and sprout length dose-dependently. siRNA-mediated knockdown of the integrin β1-subunit suppressed LEC invasion and also inhibited VEGF receptor (VEGFR)3 and ERK activation. Furthermore, exposing LECs to the inflammatory mediator lipopolysaccharide (LPS) inhibited sprouting. This optimized model for LEC invasion includes S1P, VEGF-C, and fibronectin within a 3-D collagen matrix, along with VEGF-C, VEGF-A, bFGF, and HGF in the culture medium, and provides a useful tool to investigate the functional effect of various lymphangiogenic factors and inhibitors.

Golden snub-nosed monkeys are endangered animals in China, and their cells have been demonstrated to be important as genetic resources and in applications for advancing biological research. Moreover, in primary research, basic fibroblast growth factor (bFGF) is used to promote the proliferation of fibroblasts to create abundant cells for cryopreservation. To further investigate the effect of bFGF on the efficiency of preservation of fibroblasts obtained from an endangered species, a fibroblast cell line was isolated from a dead golden snub-nosed monkey. Cell viability and mitochondrial membrane potential were assessed using CCK8 and JC-1 assay kits. The karyotype was analyzed by chromosomal microarray analysis, while RNA sequencing and gene expression analyses were performed to assess molecular changes in response to bFGF. Flow cytometry was used to characterize changes in cell surface markers in response to bFGF treatment. The results showed that cells maintained typical fibroblast morphology, while cell viability and mitochondrial membrane potential were not significantly affected between three and eight passages (p > 0.05). We also observed that the addition of bFGF promoted fibroblast proliferation and increased mitochondrial membrane potential. In addition, the bFGF treatment did not alter the normal karyotype of cells, downregulating fibroblast-associated genes and upregulating those associated with cell regulation, including those of the WNT, PI3K and MAPK pathways. The addition of bFGF also increased CD29, CD90, CD105, CD34 and CD44 expression while decreasing that of CD14 and HLA-DR at the protein level. Taken together, these results demonstrate that bFGF may upregulate the WNT, PI3K and MAPK pathways to promote cell proliferation while also increasing the expression of genes and surface markers associated with mesenchymal and hematopoietic cell linages.

Objective: Bone morphogenetic protein 4 (BMP4) and basic fibroblast growth factor (bFGF) play important roles in embryonic heart development. Also, two epigenetic modifying molecules, 5ˊ-azacytidine (5ˊ-Aza) and valproic acid (VPA) induce cardiomyogenesis in the infarcted heart. In this study, we first evaluated the role of BMP4 and bFGF in cardiac trans-differentiation and then the effectiveness of 5´-Aza and VPA in reprogramming and cardiac differentiation of human adipose tissue-derived stem cells (ADSCs). Materials and Methods: In this experimental study, human ADSCs were isolated by collagenase I digestion. For cardiac differentiation, third to fifth-passaged ADSCs were treated with BMP4 alone or a combination of BMP4 and bFGF with or without 5ˊ-Aza and VPA pre-treatment. After 21 days, the expression of cardiac-specific markers was evaluated by reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR, immunocytochemistry, flow cytometry and western blot analyses. Results: BMP4 and more prominently a combination of BMP4 and bFGF induced cardiac differentiation of human ADSCs. Epigenetic modification of the ADSCs by 5ˊ-Aza and VPA significantly upregulated the expression of OCT4A, SOX2, NANOG, Brachyury/T and GATA4 but downregulated GSC and NES mRNAs. Furthermore, pre-treatment with 5ˊ-Aza and VPA upregulated the expression of TBX5, ANF, CX43 and CXCR4 mRNAs in three-week differentiated ADSCs but downregulated the expression of some cardiac-specific genes and decreased the population of cardiac troponin I-expressing cells. Conclusion: Our findings demonstrated the inductive role of BMP4 and especially BMP4 and bFGF combination in cardiac trans-differentiation of human ADSCs. Treatment with 5ˊ-Aza and VPA reprogrammed ADSCs toward a more pluripotent state and increased tendency of the ADSCs for mesodermal differentiation. Although pre-treatment with 5ˊ-Aza and VPA counteracted the cardiogenic effects of BMP4 and bFGF, it may be in favor of migration, engraftment and survival of the ADSCs after transplantation.

Homologous feeder culture system can efficiently promote the proliferation of embryonic germ (EG) cells or embryonic stem (ES) cells while avoiding contamination by exogenous proteins and pathogens. In this study, we compared the potency of using homologous porcine embryonic fibroblasts (PEFs), gonadal stromal cells (GSCs), porcine adipose-derived stem cells (PASCs), or porcine amniotic fluid stem (PAFS) cells as feeder cells for porcine EG growth, with the commonly used mouse embryonic fibroblasts (MEFs). We compared the feeder cell growth rates; secretion of growth factors including stem cell factor (SCF), basic fibroblast growth factor (bFGF), and leukemia inhibitory factor (LIF); the effects of growth factors on porcine PGC growth; and EG growth rates when individual cells were used as feeders. Our results showed that feeder cells secreted limited amounts of growth factors, and supplementation of growth factors can significantly improve the formation of EG colonies and number of passages (P < 0.05). GSC and PEF were more suitable for EG growth because of their faster growth rate and their support on EG growth. In conclusion, this study identified novel homologous cells that can be used for EG production.

1. This study examined whether in vitro proliferation and maintenance of cockerel spermatogonial stem cells (SSCs) could be improved by adding different combinations of growth factors (GFs), including glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF) or leukaemia inhibitory factor (LIF) into the culture medium. 2. The SSCs were isolated from the testes of immature cockerels. For short-term cultures, a medium supplemented with different combinations of GFs for 7 d in 5 replicates was used. The groups were classified as follows: without GF (control group); with GDNF (G group); with GDNF and bFGF (GF group); and with GDNF, bFGF and LIF (GFL group). The number of colonies and cells per colony, as well as the transcript abundance of STRA8 and OCT4 genes, was determined 7 d after the initial culturing. Immunofluorescence staining of SSEA-1, SSEA-3 and VASA protein markers, besides periodic acid-Schiff (PAS) staining, was carried out. 3. The number of colonies and cells per colony increased in the G, GF and GFL groups, compared to the control group (P < 0.01); however, the highest proliferation and colony formation were observed in the GFL group. The positive immunofluorescence staining of SSEA-1, SSEA-3 and VASA protein markers, as well as PAS staining, confirmed the self-renewal and colonisation of cockerel SSCs. The proliferation results were supported by the increased STRA8 and OCT4 transcript abundance in the treated groups (G, GF and GLF), compared to the control group. The SSC proliferation was associated with the higher transcript abundance of STAR8 and OCT4 genes in the GFL group, compared to the G and GF groups (P < 0.01). 4. The results showed that proliferation and colony-forming capacity of cockerel SSCs were positively improved by GDNF, bFGF and LIF. However, the most significant effect was observed when the medium was supplemented with LIF in combination with GDNF and bFGF.

Gholamreza Farnoosh,1 Mohammad-Reza Mahmoudian-Sani2 1Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran; 2Thalassemia and Hemoglobinopathy Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, IranCorrespondence: Mohammad-Reza Mahmoudian-Sani Tel +98 61-33750410Fax +98 61-33750427Email mohamadsani495@gmail.comIntroduction: Hearing Loss (HL) is known as the most common sensory processing disorder across the world. An effective treatment which has been currently used for patients suffering from this condition is cochlear implant (CI). The major limitation of this treatment is the need for a healthy auditory neuron (AN). Accordingly, mesenchymal cells (MCs) are regarded as good candidates for cell-based therapeutic approaches. The present study aimed to investigate the potentials of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) for differentiation towards ANs along with using treatments with growth factors and microRNA (miRNA) transfection in vitro.Methods: To this end, neurospheres derived from hBM-MSCs were treated via basic fibroblast growth factor (bFGF), neurotrophin-3 (NT-3), and brain-derived neurotrophic factor (BDNF) as growth factors N2 and B27 supplements, as well as miRNA-96, -182, -183 transfected into hBM-MSCs in order to evaluate the differentiation of such cells into ANs.Results: Treatments with growth factors demonstrated a significant increase in neurogenin 1 (Ngn1) and sex determining region Y-box 2 (SOX2) markers; but tubulin, microtubule-associated protein 2 (MAP2), and GATA binding protein 3 (GATA3) markers were not statistically significant. The findings also revealed that miRNA-182 expression in miRNA-183 family could boost the expressions of some AN marker (ie, Ngn1, SOX2, peripherin, and nestin) in vitro.Discussion: It can be concluded that miRNA is probably a good substitute for growth factors used in differentiating into ANs. Transdifferentiation of hBM-MSCs into ANs, which does not occur under normal conditions, may be thus facilitated by miRNAs, especially miRNA-182, or via a combination of miRNA and growth factors.Keywords: microRNA-183 family, auditory neuron, human bone marrow-derived mesenchymal stem cell, growth factor

Background Collagen is the most abundant structural protein in the mammalian connective tissue and represents approximately 30% of animal protein. The current study evaluated the potential capacity of collagen extract derived from Nile tilapia skin in improving the cutaneous wound healing in rats and investigated the underlying possible mechanisms. A rat model was used, and the experimental design included a control group (CG) and the tilapia collagen treated group (TCG). Full-thickness wounds were conducted on the back of all the rats under general anesthesia, then the tilapia collagen extract was applied topically on the wound area of TCG. Wound areas of the two experimental groups were measured on days 0, 3, 6, 9, 12, and 15 post-wounding. The stages of the wound granulation tissues were detected by histopathologic examination and the expression of vascular endothelial growth factor (VEGF), and transforming growth factor (TGF-ß1) were investigated using immunohistochemistry. Moreover, relative gene expression analysis of transforming growth factor-beta (TGF-ß1), basic fibroblast growth factor (bFGF), and alpha-smooth muscle actin (α-SMA) were quantified by real-time qPCR. Results The histopathological assessment showed noticeable signs of skin healing in TCG compared to CG. Immunohistochemistry results revealed remarkable enhancement in the expression levels of VEGF and TGF-β1 in TCG. Furthermore, TCG exhibited marked upregulation in the VEGF, bFGF, and α-SMA genes expression. These findings suggested that the topical application of Nile tilapia collagen extract can promote the cutaneous wound healing process in rats, which could be attributed to its stimulating effect on recruiting and activating macrophages to produce chemotactic growth factors, fibroblast proliferation, and angiogenesis. Conclusions The collagen extract could, therefore, be a potential biomaterial for cutaneous wound healing therapeutics.

Basic fibroblast growth factor (bFGF) promotes epithelial cell proliferation and angiogenesis but its clinical applications are limited by its short half-life and low retention. Recently developed gelatin hydrogel sheets able to release physiologically active substances in a controlled manner have the potential to overcome these issues. In this study, the effects of gelatin hydrogel sheets impregnated with bFGF on flap survival and angiogenesis were examined in a murine skin flap model. A flap of 1 × 3 cm was generated on the backs of 60 C57BL/6 mice. The mice were divided into five groups (n = 12/group): Group I, untreated; Group II, treated with a gelatin hydrogel sheet impregnated with saline; Group III, treated with bFGF (50 µg) without sheets; Groups IV and V, treated with gelatin hydrogel sheets impregnated with 50 and 100 µg of bFGF, respectively. On the seventh day after surgery, the flap survival area and vascular network were examined and hematoxylin and eosin and von Willebrand factor staining were used for histological examinations. The flap survival areas were significantly larger in Groups IV and V than in other groups. The area of new vessels was significantly larger in Group IV than in the other groups. In the murine skin flap model, gelatin hydrogel sheets impregnated with bFGF promoted angiogenesis and improved flap survival. These findings support the use of bFGF-impregnated gelatin hydrogel sheets for improving ischemic flap survival in clinical settings.

In tumor tissue, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor A (VEGFA) promote tumorigenesis by activating angiogenesis, but targeting single factor may produce drug resistance and compensatory angiogenesis. The Peptibody with bFGF/VEGFA was designed to simultaneously blockade these two factors. We were aiming to produce this Fc fusion protein in a large scale. The biological characterizations of Peptibody strains were identified as Escherichia coli and the fermentation mode was optimized in the shake flasks and 10‐L bioreactor. The fermentation was scaled up to 100 L, with wet cell weight (WCW) 126 g/L, production 1.41 g/L, and productivity 0.35 g/(L·h) of IPTG induction. The target protein was isolated by cation‐exchange, hydrophobic and Protein A chromatography, with total recovery of 60.28% and HPLC purity of 86.71%. The host cells protein, DNA, and endotoxin residues were within the threshold. In mouse model, immunization of Peptibody vaccine could significantly suppressed the tumor growth and angiogenesis, with inhibition rate of 57.73 and 39.34%. The Peptibody vaccine could elicit high‐titer anti‐bFGF and anti‐VEGFA antibodies, which inhibited the proliferation and migration of Lewis lung cancer cell cells by decreasing the Akt/MAPK signal pathways. Therefore, the Peptibody with bFGF/VEGFA might be used as a therapeutic tumor vaccine. The large‐scale process we developed could support its industrial production and pre‐clinical study in the future.

Background Some recent studies on insulin receptor tyrosine kinase substrate (IRTKS) have focused more on its functions in diseases. However, there is a lack of research on the role of IRTKS in carcinomas and its mechanism remains ambiguous. In this study, we aimed to clarify the role and mechanism of IRTKS in the carcinogenesis of colorectal cancer (CRC). Methods We analysed the expression of IRTKS in CRC tissues and normal tissues by researching public databases. Cancer tissues and adjacent tissues of 67 CRC patients who had undergone radical resection were collected from our center. Quantitative real-time polymerase chain reaction and immunohistochemistry were performed in 52 and 15 pairs of samples, respectively. In vitro and in vivo experiments were conducted to observe the effect of IRTKS on CRC cells. Gene Set Enrichment Analysis and Metascape platforms were used for functional annotation and enrichment analysis. We detected the protein kinase B (AKT) phosphorylation and cell viability of SW480 transfected with small interfering RNAs (siRNAs) with or without basic fibroblast growth factor (bFGF) through immunoblotting and proliferation assays. Results The expression of IRTKS in CRC tissues was higher than that in adjacent tissues and normal tissues (all P Conclusion IRTKS mediated the bFGF-induced cell proliferation through the phosphorylation of AKT in CRC cells, which may contribute to tumorigenicity in vivo.

Hydroxyapatite/collagen (HAP/Col) composite has a nanostructure and composition similar to that of natural bone. Herein, we have evaluated the beneficial effects of acid-electrolyzed functional water (FW) in combination with HAP/Col composite as an irrigation material in a rat calvarium defect model. The rats were divided into four groups: control, PBS irrigation; FW, FW irrigation; HAP/Col, filled with HAP/Col; FW + HAP/Col, FW irrigation prior to HAP/Col filling. Bone volume (BV) and bone mineral density (BMD) of the newly formed bone were analyzed by microcomputed tomography. The results indicated that the combined use of FW and HAP/Col significantly augmented both BV (12.25 ± 1.93 mm3 , control: 3.22 ± 0.55 mm3 , 6 weeks) and BMD (120.09 ± 14.76 cm3 /mg vs. control: 54.67 ± 7.20 cm3 /mg, 6 weeks) in a time-dependent manner, which might be attributed to the soluble factor-inducing ability of FW. Based on this assumption, bFGF concentration in peripheral blood was measured. bFGF concentration was significantly increased in the FW + HAP/Col group (68.25 ± 9.2 pg/ml vs. control: 21.70 ± 8.18 pg/ml, 6 hr). Real-time PCR demonstrated significant augmentation of MCSF (2.82 ± 0.59-fold), RANKL (2.51 ± 0.33-fold) and BMP7 (1.66 ± 0.25-fold) (bone regeneration-related genes) and PDGF (1.31 ± 0.15-fold), VEGF (3.27 ± 0.42-ld) and IL-8 (6.77 ± 2.02-fold) (angiogenic genes) mRNAs in the FW + HAP/Col group. Taken together, these results suggest that the combined use of FW and HAP/Col induces bone regeneration, presumably by inducing the factors contributing to bone regeneration and angiogenesis.

In this study, a new continuous muscle cell line, LYCMS (large yellow croaker muscle cell line), derived from the muscle tissue of larva of large yellow croaker (Larimichthys crocea) was developed with modified DMEM/F12 medium at 27 °C. The muscle cell line could be passaged at different ratios for different growth rates. Karyotype analysis showed that a large proportion of LYCMS cells had 48 chromosomes. The proliferation of LYCMS cell line could be affected by mammalian growth factors such as human basic fibroblast growth factor (b-FGF), epidermal growth factor (EGF), and hepatocyte growth factor (HGF). GFP expression experiments indicated that the LYCMS cell line could be used for exogenous genes’ expression. Different virus response-related genes tested in this study showed diverse change types in expression before and after (0–24 h) polycytidylic acid (poly I: C) challenge of LYCMS cells. This is the first study of virus response signaling pathways of large yellow croaker based on the muscle cell line. The results showed that compared with the in vivo experiments, the use of the LYCMS cell line for immune research is more convenient, efficient, and rapid. By using this model, we demonstrated that MDA5-IPS1-TRAF6-NFκB-cytokines, MDA5-IPS1-TRAF3-IRF3-interferon or TLR22-TRIF-IRF3-interferon, TLR8-MyD88-NFκB-cytokines, and TLR3-TRIF-IRF3-interferon pathways were able to response to poly I: C challenge in the muscle cell line of large yellow croaker.

Liraglutide is a glucagon-like peptide-1 receptor (GLP-1R) agonist and incretin mimetic used for the treatment of Type 2 diabetes mellitus. It has also been shown to have a beneficial role in the cardiovascular system. Here, we investigated the mechanism by which liraglutide promotes angiogenesis using human umbilical vein endothelial cells (HUVECs). HUVECs were treated with various concentrations of liraglutide, and assessed by wound healing assay and tube formation assay as measures of angiogenesis. We found that liraglutide at 10 and 100 nmol/L greatly promoted the angiogenic ability of HUVECs. Next, we examined the JAK2/STAT3 signaling pathway and found that liraglutide treatment led to JAK2/STAT3 activation and significant increase in the angiogenic mediator expressions, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and endothelial nitric oxide synthase (eNOS) in HUVECs. Treatment with JAK2 inhibitor, AG490, in HUVECs successfully reduced the observed effects of liraglutide. We conclude that liraglutide promotes the angiogenic ability of HUVECs by activating the JAK2/STAT3 signaling pathway and upregulating its downstream factors, VEGF, bFGF and eNOS. Thus, liraglutide may provide ischemic relief for diabetic patients with cardiovascular diseases in addition to glycemic control.

Objectives Mesenchymal stem cells (MSCs) have been intensively investigated as to their therapeutic potentials. However, the full chemical-defined medium supporting the isolation and expansion of human MSCs has not been developed yet. Materials and methods Here, we developed the full chemical-defined medium, NBVbe medium, via RNA sequencing, bioinformatic analysis, and growth factor screening. Results The NBVbe medium contains N2B27 medium with the BSA (bovine serum albumin) replaced by the recombinant human albumin, bFGF (basic fibroblast growth factor), vitamin C, and EGF (epidermal growth factor). The NBVbe medium could support the isolation and expansion of human MSCs from the umbilical cords. Conclusions The full chemical-defined medium supporting the isolation and expansion of human MSCs has been developed. This would be helpful for further optimization of the MSC medium, their clinical applications, and molecular characterization.

Dental pulp stem cells (DPSCs) have a high capacity for differentiation and the ability to regenerate a dentin/pulp-like complex. Numerous studies have provided evidence of DPSCs' differentiation capacity, such as in neurogenesis, adipogenesis, osteogenesis, chondrogenesis, angiogenesis, and dentinogenesis. The molecular mechanisms and functions of DPSCs' differentiation process are affected by growth factors and scaffolds. For example, growth factors such as basic fibroblast growth factor (bFGF), transforming growth factor-β (TGF-β), nerve growth factor (NGF), platelet-derived growth factor (PDGF), and bone morphogenic proteins (BMPs) influence DPSC fate, including in differentiation, cell proliferation, and wound healing. In addition, several types of scaffolds, such as collagen, hydrogel, decellularized bioscaffold, and nanofibrous spongy microspheres, have been used to characterize DPSC cellular attachment, migration, proliferation, differentiation, and functions. An appropriate combination of growth factors and scaffolds can enhance the differentiation capacity of DPSCs, in terms of optimizing not only dental-related expression but also dental pulp morphology. For a cell-based clinical approach, focus has been placed on the tissue engineering triad [cells/bioactive molecules (growth factors)/scaffolds] to characterize DPSCs. It is clear that a deep understanding of the mechanisms of stem cells, including their aging, self-renewal, microenvironmental homeostasis, and differentiation correlated with cell activity, the energy for which is provided from mitochondria, should provide new approaches for DPSC research and therapeutics. Mitochondrial functions and dynamics are related to the direction of stem cell differentiation, including glycolysis, oxidative phosphorylation, mitochondrial metabolism, mitochondrial transcription factor A (TFAM), mitochondrial elongation, and mitochondrial fusion and fission proteins. This review summarizes the effects of major growth factors and scaffolds for regenerating dentin/pulp-like complexes, as well as elucidating mitochondrial properties of DPSCs for the development of advanced applications research.

Acupuncture is widely used in the treatment of cerebral hemorrhage, and it improves outcomes in experimental animal models and patients. However, the mechanisms underlying the effectiveness of acupuncture treatment for cerebral hemorrhage are still unclear. In this study, a model of intracerebral hemorrhage was produced by injecting 50 μL autologous blood into the caudate nucleus in Wistar rats. Acupuncture at Baihui (DU20) and Qubin (GB7) acupoints was performed at a depth of 1.0 inch, 12 hours after blood injection, once every 24 hours. The needle was rotated at 200 r/min for 5 minutes, For each 30-minute session, needling at 200 r/min was performed for three sessions, each lasting 5 minutes. For the positive control group, at 6 hours, and 1, 2, 3 and 7 days after induction of hemorrhage, the rats were intraperitoneally injected with 1 mL aniracetam (0.75 mg/mL), three times a day. The Bederson behavioral test was used to assess palsy in the contralateral limbs. Western blot assay was used to examine the expression levels of Nestin and basic fibroblast growth factor in the basal ganglia. Immunohistochemistry was performed to count the number of Nestin- and glial cell line-derived neurotrophic factor-positive cells in the basal ganglia. Acupuncture effectively reduced hemorrhage and brain edema, elevated the expression levels of Nestin and basic fibroblast growth factor in the basal ganglia, and increased the number of Nestin- and glial cell line-derived neurotrophic factor-positive cells in the basal ganglia. Together, these findings suggest that acupuncture promotes functional recovery after cerebral hemorrhage by increasing the expression of neurotrophic factors. The study was approved by the Committee for Experimental Animals of Heilongjiang Medical Laboratory Animal Center (approval No. 2017061001) on June 10, 2017.

The translation of growth factors (GFs) into clinical applications is limited by their low stability in physiological environments. Controlled GF delivery through biomaterial vehicles provides protection from proteases, targeted delivery, and longer term release profiles. However, current methods used to incorporate GFs into biomaterials still present limitations. While direct adsorption and encapsulation result in burst release, covalent incorporation provides a tailorable release profile but generally requires more complicated processes and chemical modification of the GFs. Bioaffinity methods provide long-term release profiles but fail in their specificity, resulting in GF-dependent applicability and release profiles. In the present study, we introduce tyraminated poly-vinyl-alcohol (PVA-Tyr) as a GF-delivery vehicle that can covalently incorporate native GFs through a photo-initiated cross-linking process via formation of bi-phenol bonds. Mass loss and release studies revealed that protein-loaded PVA-Tyr hydrogels had highly tailorable degradation times from 7 to 92 days, during which the covalently incorporated proteins were released in a linear fashion. The incorporation of bovine serum albumin (BSA), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), or brain-derived growth factor (BDNF) resulted in similar incorporation efficiencies and release profiles, demonstrating the low specificity and versatility of the system. Furthermore, functional studies demonstrated that VEGF, bFGF and BDNF released from the PVA-Tyr hydrogels retained the ability to increase the metabolic activity, migration, and 3D vessel formation of endothelial cells and mesenchymal stem cells. Taken together, this demonstrates that PVA-Tyr shows high potential as a highly tailorable GF delivery tool for a range of different regenerative medicine applications.

In this study, we established a new cell line (Cynoglossus semilaevis kidney cells, CSK) from kidney of the half-smooth tongue sole (Cynoglossus semilaevis). The cells were subcultured over 1 000 days and passaged for more than 100 times. Additionally, CSK cells were optimally maintained in Dulbecco’s modified Eagle medium nutrient mixture F-12 supplemented with HEPES, antibiotics, fetal bovine serum, 2-mercaptoethanol, and basic fibroblast growth factor. The optimum growth temperature for CSK cells was 25°C, and the cells showed a fibroblast-like phenotype. Chromosome analysis revealed that CSK cells had a normal diploid karyotype with 2n=42. CSK cells were susceptible to Grouper nervous necrosis virus (NNV), and cytopathic effects were observed at 3–5 days postinfection. The NNV sensitivity of CSK cells was related to the high abundance of virions in the cytoplasm, as observed by electron microscopy. Additionally, CSK cells could be successfully transfected with a green fluorescent protein reporter plasmid, and fluorescent signals were easily observed. Finally, immunocytochemistry analysis showed that CSK cells were supporting cells. Overall, we established this new cell line, which may have potential applications in the identification of viral pathogens affecting the half-smooth tongue sole.

Tumorigenic cancer stem cells (CSCs) exist in various tumors including the cutaneous squamous cell carcinoma (cSCC) as a minor subpopulation and are tightly associated with metastasis and therapeutic resistance. Better understanding of CSCs properties is essential for the novel therapeutic strategy targeted toward these cancers. The cSCC stem cells (cSCCSCs) were enriched from a cSCC cell line A431 by repeated sphere culture, and identified via the expression analysis of stemness marker genes and CD44 proteolysis. MiR-199a-5p was previously reported to be related with the proteolysis modulation of CD44, so the specific regulation mechanisms were verified by overexpression in vitro and in vivo. MiR-199a-5p is under-expressed in cSCCSCs and functions as a tumor suppressive molecule. Overexpression of miR-199a-5p reduced the stemness of cSCCSCs and inhibited cell proliferation. By targeting the deacetylase Sirt1, miR-199a-5p inhibited cellular proteolysis of CD44 and reduced the CD44 intracellular domain (CD44ICD) release and nuclear translocation. Overexpression of CD44ICD reversed the effects of miR-199a-5p overexpression or Sirt1 silencing, and increased the transcriptional expression of stemness genes. Our results revealed that the miR-199a-5p/Sirt1/CD44ICD signaling pathway regulates cSCCSCs progression by affecting its migration ability and tumorigenicity, therefore can be utilized to develop a curative approach for cSCC. Abbreviations: CSCs: cancer stem cells; cSCC cutaneous squamous cell carcinoma; cSCCSCs: cSCC stem cells; CD44ICD: CD44 intracellular domain; HA: hyaluronic acid; HNSCC: hand and neck squamous cell carcinoma; ESCC: esophageal squamous cell carcinoma;MMPs: matrix metalloproteinases; SFM: sphere formation medium; EGF: epidermal growth factor; bFGF: basic fibroblast growth factor; BSA: bovine serum albumin; CCK-8: cell counting kit-8

Human adipose-derived stem cells are used in regenerative medicine for treating various diseases including osteoarthritis, degenerative arthritis, cartilage or tendon injury, etc. However, their use in neurological disorders is limited, probably due to the lack of a quick and efficient induction method of transforming these cells into neural stem or progenitor cells. In this study, we reported a highly efficient and simple method to induce adipose-derived stem cells into neural progenitor cells within 12 hours, using serum-free culture combined with a well-defined induction medium (epidermal growth factor 20 ng/ml and basic fibroblast growth factor, both at 20 ng/ml, with N2 and B27 supplements). These adipose-derived stem cell-derived neural progenitor cells grow as neurospheres, can self-renew to form secondary neurospheres, and can be induced to become neurons and glial cells. Real-time polymerase chain reaction showed significantly upregulated expression of neurogenic genes Sox2 and Nestin with a moderate increase in stemness gene expression. Raybio human growth factor analysis showed a significantly upregulated expression of multiple neurogenic and angiogenic cytokines such as brain-derived neurotrophic factor, glial cell line-derived neurotrophic growth factor, nerve growth factor, basic fibroblast growth factor and vascular endothelial growth factor etc. Therefore, adipose-derived stem cell-derived neurospheres can be a new source of neural progenitor cells and hold great potential for future cell replacement therapy for treatment of various refractory neurological diseases.

A simple, rapid, efficient, and specialized culture system was successfully developed in this study to induce human embryonic stem cells into dopaminergic neurons in vitro. It only took 5 days to generate quickly and directly a large number of homogeneous neural stem cell (NSC) spheres by the introduction of small molecules LDN (inhibitor of BMP [bone morphogenetic protein] pathway that inhibits BMP type I receptors ALK2 and ALK3), SB431542 (inhibitor of TGF-β/Activin/Dodal pathway that inhibits ALK4, ALK5, and ALK7), CHIR99021 (inhibitors of GSK-3 [glycogen synthase kinase 3]), and basic fibroblast growth factor (bFGF). The dopaminergic neurons were successfully induced at day 25 (tyrosine hydroxylase [TH] expressed) and at day 32 (TH highly expressed) with high purity (TH/Tuj1: 84.14% and 93.15%, respectively) by the addition of FGF8 (fibroblast growth factor 8), sonic hedgehog (SHH), and Purmorphamine after the generation of NSC at day 5. And, the dopaminergic neurons induced by this system successfully survived and integrated into the striatum of cynomolgus monkey brain after transplantation, which verified the efficiency of the induction system developed in this study, suggesting the potential clinical application in cell therapy for neurological diseases.

A human hepatoma cell line synthesizes, as evidenced by metabolic labeling, an endothelial cell mitogen that is found to be mostly cell associated. The hepatoma-derived growth factor (HDGF) has been purified to homogeneity by a combination of Bio-Rex 70, heparin-Sepharose, and reverse-phase chromatography; it is a cationic polypeptide with a molecular weight of about 18,500-19,000. HDGF is structurally related to basic fibroblast growth factor (FGF). Immunological analysis demonstrates that antiserum prepared against a synthetic peptide corresponding to the amino-terminal sequence of basic FGF cross-reacts with HDGF when analyzed by electrophoretic blotting and by immunoprecipitation. Sequence analysis of tryptic fragments demonstrates that HDGF contains sequences that are homologous to both amino-terminal and carboxyl-terminal sequences of basic FGF.

Synovial mesenchymal stem cell (SMSC) is the promising cell source of cartilage regeneration but has several issues to overcome such as limited cell proliferation and heterogeneity of cartilage regeneration ability. Previous reports demonstrated that basic fibroblast growth factor (bFGF) can promote proliferation and cartilage differentiation potential of MSCs in vitro, although no reports show its beneficial effect in vivo. The purpose of this study is to investigate the promoting effect of bFGF on cartilage regeneration using human SMSC in vivo. SMSCs were cultured with or without bFGF in a growth medium, and 2 &times, 105 cells were aggregated to form a synovial pellet. Synovial pellets were implanted into osteochondral defects induced in the femoral trochlea of severe combined immunodeficient mice, and histological evaluation was performed after eight weeks. The presence of implanted SMSCs was confirmed by the observation of human vimentin immunostaining-positive cells. Interestingly, broad lacunae structures and cartilage substrate stained by Safranin-O were observed only in the bFGF (+) group. The bFGF (+) group had significantly higher O&rsquo, Driscoll scores in the cartilage repair than the bFGF (&minus, ) group. The addition of bFGF to SMSC growth culture may be a useful treatment option to promote cartilage regeneration in vivo.

Vasculature plays a vital role in human biology as blood vessels transport nutrients and oxygen throughout the body. Endothelial cells (ECs), specifically, are key as they maintain barrier functions between the circulating blood and the surrounding tissues. ECs derived from human pluripotent stem cells (hPSCs) are utilized to study vascular development and disease mechanisms within in vitro models. Additionally, ECs derived from induced pluripotent stem cells (iPSCs) hold great promise for advancing personalized medicine, cell therapies, and tissue-engineered constructs by creating patient-specific cell populations. Here, we describe a xeno-free, serum-free differentiation protocol for deriving ECs from hPSCs. In brief, mesoderm progenitor cells are derived via WNT pathway activation. Following this, EC maturation is achieved with exogenous vascular endothelial growth factor A (VEGFA) and basic fibroblast growth factor 2 (bFGF2). We have characterized these cells as expressing mature EC markers and have illustrated their functionality in vitro.

Lactic acid bacteria (LAB) are known to have beneficial effects on immune responses when they are orally administered as bacterial products. Although the beneficial effects of LAB have been reported for the genera Lactobacillus and Lactococcus, little has been uncovered on the effects of the genus Enterococcus on skin wound-healing. In this study, we aimed to clarify the effect of heat-killed Enterococcus faecalis KH2 (heat-killed KH2) strain on the wound-healing process and to evaluate the therapeutic potential in chronic skin wounds. We analyzed percent wound closure, re-epithelialization, and granulation area, and cytokine and growth factor production. We found that heat-killed KH2 contributed to the acceleration of re-epithelialization and the formation of granulation tissue by inducing tumor necrosis factor-α, interleukin-6, basic fibroblast growth factor, transforming growth factor (TGF)-β1, and vascular endothelial growth factor production. In addition, heat-killed KH2 also improved wound closure, which was accompanied by the increased production of TGF-β1 in diabetic mice. Topical administration of heat-killed KH2 might have therapeutic potential for the treatment of chronic skin wounds in diabetes mellitus. In the present study, we concluded that heat-killed KH2 promoted skin wound-healing through the formation of granulation tissues and the production of inflammatory cytokines and growth factors.

Angiogenesis is a hallmark of fibrocalcific aortic valve disease (CAVD). An imbalance of pro- and anti-angiogenic factors is thought to play a role in driving this disease process, and valvular interstitial cells (VICs) may act as a significant source of these factors. CAVD is also known to exhibit sexual dimorphism in its presentation, and previous work suggested that VICs may exhibit cellular-scale sex differences in the context of angiogenesis. The current study sought to investigate the production of angiogenesis-related factors by male and female VICs possessing quiescent (qVIC) or activated (aVIC) phenotypes. Production of several pro-angiogenic growth factors was elevated in porcine aVICs relative to qVICs, with sex differences found in both the total amounts secreted and their distribution across media vs. lysate. Porcine valvular endothelial cells (VECs) were also sex-separated in culture and found to behave similarly with respect to metabolic activity, viability, and tubulogenesis, but male VECs exhibited higher proliferation rates than female VECs. VECs responded to sex-matched media conditioned by VICs with increased tubulogenesis, but decreased proliferation, particularly upon treatment with aVIC-derived media. It is likely that this attenuation of proliferation resulted from a combination of decreased basic fibroblast growth factor and increased thrombospondin-2 (TSP2) secreted by aVICs. Overall, this study indicates that VICs regulate angiogenic VEC behavior via an array of paracrine molecules, whose secretion and sequestration are affected by both VIC phenotype and sex. Moreover, strong sex differences in TSP2 secretion by VICs may have implications for understanding sexual dimorphism in valve fibrosis, as TSP2 is also a powerful regulator of fibrosis.

RNA-binding proteins (RBPs) are essential regulators controlling both the cellular transcriptome and translatome. These processes enable cellular plasticity, an important prerequisite for growth. Cellular growth is a complex, tightly controlled process. Using cancer cells as model, we looked for RBPs displaying strong expression in published transcriptome datasets. Interestingly, we found the Pumilio (Pum) protein family to be highly expressed in all these cells. Moreover, we observed that Pum2 is regulated by basic fibroblast growth factor (bFGF). bFGF selectively enhances protein levels of Pum2 and the eukaryotic initiation factor 4E (eIF4E). Exploiting atomic force microscopy and in vitro pulldown assays, we show that Pum2 selects for eIF4E mRNA binding. Loss of Pum2 reduces eIF4E translation. Accordingly, depletion of Pum2 led to decreased soma size and dendritic branching of mature neurons, which was accompanied by a reduction in essential growth factors. In conclusion, we identify Pum2 as an important growth factor for mature neurons. Consequently, it is tempting to speculate that Pum2 may promote cancer growth.

Endothelial cells (ECs), the primary component of the vasculature, play a crucial role in neovascularization. However, the number of endogenous ECs is inadequate for both experimental purposes and clinical applications. Porcine ovarian putative stem cells (poPSCs), although not pluripotent, are characterized by great plasticity. Therefore, this study aimed to investigate whether poPSCs have the potential to differentiate into cells of endothelial lineage. poPSCs were immunomagnetically isolated from postnatal pig ovaries based on the presence of SSEA-4 protein. Expression of mesenchymal stem cells (MSCs) markers after pre-culture, both at the level of mRNA: ITGB1, THY, and ENG and corresponding protein: CD29, CD90, and CD105 were significantly higher compared to the control ovarian cortex cells. To differentiate poPSCs into ECs, inducing medium containing vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), ascorbic acid, and heparin was applied. After 14 days, poPSC differentiation into ECs was confirmed by immunofluorescence staining for vascular endothelial cadherin (VECad) and vascular endothelial growth factor receptor-2 (VEGFR-2). Semi-quantitative WB analysis of these proteins confirmed their high abundance. Additionally, qRT-PCR showed that mRNA expression of corresponding marker genes: CDH5, KDR was significantly higher compared with undifferentiated poPSCs. Finally, EC functional status was confirmed by the migration test that revealed that they were capable of positive chemotaxis, while tube formation assay demonstrated their ability to develop capillary networks. In conclusion, our results provided evidence that poPSCs may constitute the MSC population in the ovary and confirmed that they might be a potential source of ECs for tissue engineering.

Human-induced pluripotent stem cell-derived-vascular smooth muscle cells (hiPSC-VSMC) and their secretome have been shown to promote angiogenesis and wound healing. However, there is a paucity of research on how the extracellular matrix (ECM) microenvironment may impact the hiPSC-VSMC's functions. In this study, we investigated the effect of specific ECM ligand-integrin interaction on hiPSC-VSMC's paracrine secretion, cell viability, and morphology. Here, we show precise modulation of hiPSC-VSMC in a fibronectin functionalized fibrillar collagen scaffold by targeting their integrin β3. The secretion of proangiogenic growth factor, basic fibroblast growth factor (bFGF) was found to be fibronectin-dependent via αvβ3 integrin interactions. In addition, our data show the possible role of a positive feedback loop between integrin β3, bFGF, and matrix metalloproteinase-2 in regulating hiPSC-VSMC's morphology and cell viability. Finally, the secretome with enhanced bFGF shows potential for future wound healing applications.

The pathophysiology of multiple sclerosis involves an autoimmune and a neurodegenerative mechanism. Central nervous system-infiltrating immune cells in multiple sclerosis also possess a neuroprotective activity through secretion of neurotrophins, such as brain-derived neurotrophic factor. Fingolimod was shown to slow the progression of disability and loss of brain volume. The objective of this study was to explore whether fingolimod induces secretion of neurotrophins by immune cells. Blood was drawn from 21 patients before the initiation of treatment with fingolimod and at 6 and 12 months of follow-up. The levels of the neurotrophic factors brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, β-nerve growth factor, neurotrophin-3, neurotrophin-4, basic fibroblast growth factor, epidermal growth factor, and vascular endothelial growth factor were screened in the supernatants of separated T cells and monocyte cultures using a customized, multiplex enzyme-linked immunosorbent assay. Brain-derived neurotrophic factor levels were further validated by a specific enzyme-linked immunosorbent assay. Treatment with fingolimod significantly increased brain-derived neurotrophic factor secretion from T cells. A specific enzyme-linked immunosorbent assay confirmed these results in the supernatant of T cells after 6 and 12 months of therapy. T cells that reach the bloodstream of fingolimod-treated patients with multiple sclerosis may contribute to the neuroprotective effect of this therapy by increased secretion of brain-derived neurotrophic factor. This mechanism of action of fingolimod in patients with multiple sclerosis has not been previously reported.

Olfactory bulb and nasal mucosa are one of the sources for neural stem cell, including the superior and middle turbinates (MT). The middle and inferior turbinates (IT) provides the largest area of nasal mucosa which is technically easier to harvest the stem cell for future transplantation. The ability of nasal respiratory epithelial cells (RECs) and nasal fibroblasts (NFs) from both middle and inferior turbinates to differentiate into neural lineage (NL) cells were compared in this study. Six redundant human MT and IT from post-sinus surgery were digested and cultured. The RECs and NFs were separated and induced with neurotrophic factors of forskolin, human basic fibroblast growth factor (bFGF), platelet-derived growth factor-AA (PDGF-AA) and heregulin-β1-EGF-domain. Based on immunocytochemistry and quantitative PCR, the NL induced NFs of MT expressed GFAP, Nestin and P75 receptor. NL induced RECs from MT and IT expressed GFAP and Nestin but did not express the P75 receptor protein. Regarding the control, the non-induced RECs and fibroblasts expressed Nestin only. This study demonstrated that nasal mucosa cells from both IT and MT have the potential to differentiate into neural lineage cells even though the fibroblasts of MT are superior in term of quality. Hopefully, these tissues will provide better donor area with less morbidity for autologous or allograft transplantation in future neural regenerative medicine.

The objective of this study was to investigate the effects of different growth factors on the proliferation of Bama mini-pig spermatogonial stem cells (SSCs) in vitro. The growth factors glial cell line-derived neurotrophic factor (GDNF), leukaemia inhibitory factor (LIF), GDNF family receptor alpha-1 (GFRα1) and basic fibroblast growth factor (bFGF) were investigated. The SSCs were seeded on SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) feeder layers. Cultivation of the cells were subjected to a factorial design of the growth factors GDNF + bFGF, GDNF + bFGF + GFRα1, LIF + bFGF and LIF + bFGF + GFRα1. The SSCs could propagate for 25 passages in the medium adding GDNF + bFGF + GFRα1, 22 passages in the medium adding GDNF + bFGF, 6 passages in the medium adding LIF + bFGF, or LIF + bFGF + GFRα1. qRT-PCR analysis showed that the highest mRNA expression levels of NANOG, POU5F, DDX4, GFRα1 and UCHL1 were detected in the group adding GDNF + bFGF + GFRα1. The SSCs from the group adding GDNF + bFGF + GFRα1 also showed UCHL1-, DBA- and CDH1-positive staining. Moreover, Stra8 and Scp3 expression, and haploid peak were detected after induction of the SSCs from the group adding GDNF + bFGF + GFRα1. In conclusion, pig SSCs could be maintained for long term in the presence of GDNF, bFGF, and GFRα1.

Background: Meningioma and glioma are common central nervous system tumors. Hypoxic tumor cells secrete angiogenic cytokines, such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) that stimulate neovascular formation and inflammatory cytokine, such as TNF-α and IL-1β. We measured these serum levels in patients with glial cell tumors and meningioma. Materials and Methods: This was a case-control study in 2014-2015 on patients diagnosed with meningioma/glioma. All demographic and clinical data were registered. The tumor volume and intraoperative bleeding were recorded. Serum levels of VEGF, PDGF, FGF, TNF-α and IL-1β were measured by ELISA methods. Results: Ninety-six patients were enrolled in this study, 32 in each group. Patients VEGF level with cranial tumor, glioma/meningioma had increased. VEGF level was highest among grade IV tumors, larger tumors, and in glioblastoma multiform. There was an upsurge in VEGF serum level as glioma grade increased. The highest VEGF levels were seen in parasagittal meningioma. In contrast to VEGF, PDGF was slightly elevated in glial cell tumors, which was significantly elevated in meningioma. Higher PDGF correlated with increased intraoperative bleeding, especially in meningioma cases. Oligodendroglial tumors expressed higher PDGF levels in contrast to other glial tumors. FGF level was not statistically significant. TNF-α and IL-1β expressions were significantly higher in the meningioma and glioma group in comparison to control group. Conclusion: We found increased VEGF and PDGF serum levels in CNS patient’s tumor. A different role for PDGF was found in the pathogenesis of neovascularization of meningioma, as well as oligodendroglioma. No significant result was found for FGF. TNF-α and IL-1β can serve as key prognostic biomarker in high-grade glioma and meningioma patients.

The endothelial cells play a crucial role in the progression of angiogenesis, which causes cell re-modulation, proliferation, adhesion, migration, invasion and survival. Angiogenic factors like cytokines, cell adhesion molecules, growth factors, vasoactive peptides, proteolytic enzymes (metalloproteinases) and plasminogen activators bind to their receptors on endothelial cells and activate the signal transduction pathways like epidermal growth factor receptor (EGFR phosphatidylinositol 3-kinase and (PI3K)/AKT/mammalian target of rapamycin (mTOR) which initiate the process of angiogenesis. Cytokines that stimulate angiogenesis include direct and indirect proangiogenic markers. The direct proangiogenic group of markers consists of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF-2) and hepatocyte growth factor (HGF) whereas the indirect proangiogenic markers include transforming growth factor-beta (TGF-β), interleukin 6 (IL-6), interleukin 8 (IL-8) and platelet-derived growth factor (PDGF). VEGF and FGF-2 are the strongest activators of angiogenesis which stimulate migration and proliferation of endothelial cells in existing vessels to generate and stabilize new blood vessels. VEGF is released in hypoxic conditions as an effect of the hypoxia-inducible factor (HIF-1α) and causes re-modulation and inflammation of bronchi cell. Cell re-modulation and inflammation leads to the development of various lung disorders like pulmonary hypertension, chronic obstructive pulmonary disease, asthma, fibrosis and lung cancer. This indicates that there is a firm link between overexpression of VEGF and FGF-2 with lung disorders. Various natural and synthetic drugs are available for reducing the overexpression of VEGF and FGF-2 which can be helpful in treating lung disorders. Researchers are still searching for new angiogenic inhibitors which can be helpful in the treatment of lung disorders. The present review emphasizes on molecular mechanisms and new drug discovery focused on VEGF and FGF-2 inhibitors and their role as anti-angiogenetic agents in lung disorders.