Development of the Goose Tongue Filiform Papillae: Could it be Tooth-Like Sense Organs? / Desarrollo de las Papilas Filiformes de la Lengua de Ganso: ¿Podrían ser Órganos Sensoriales Parecidos a los Dientes?

SUMMARY: The geese's tongue filiform papillae are particularly long, and exhibit the same morphology of a tooth, evoking the lingual teeth of several fishes. In adult animals, they contain numerous mechanical Herbst's corpuscles but no taste buds. In the embryo, they appear since stage 38 and acquire their definitive shape between stages 38 and 42. They express several proteins associated with mammalian tooth development (BMP4, β-catenin, SHH, PITX2, PAX9), also known to be linked to parrot's pseudoteeth and goose's denticulations development. Neurofilaments are early present in the papillae primordia, and appear particularly numerous in adult papillae. Our results suggest that these papillae constitute a mechanical organ with a « tooth shape » derived from ancestral odontodes, whose development is controlled by numerous genes involved in classical odontogenesis.

Las papilas filiformes de la lengua de los gansos son particularmente largas y exhiben la morfología de un diente, evocando los dientes linguales presentes en varios peces. En los animales adultos, contienen numerosos corpúsculos de Herbst mecánicos, aunque una ausencia de papilas gustativas. En el embrión, aparecen a partir del estadio 38 y adquieren su forma definitiva entre los estadios 38 y 42. Expresan varias proteínas asociadas al desarrollo dentario de los mamíferos (BMP4, β-catenina, SHH, PITX2, PAX9), también conocidas por estar asociadas al desarrollo de pseudodientes en el loro y denticulaciones en el ganso. Los neurofilamentos están presentes tempranamente en los primordios de las papilas y aparecen particularmente numerosos en las papilas adultas. Nuestros resultados sugieren que estas papilas constituyen un órgano mecánico con «forma de diente» derivado de odontoides ancestrales, cuyo desarrollo está controlado por numerosos genes implicados en la odontogénesis clásica.

The effects of altered BMP4 signaling in first branchial-arch-derived murine embryonic orofacial tissues / 国际口腔科学杂志·英文版

The first branchial arch (BA1), which is derived from cranial neural crest (CNC) cells, gives rise to various orofacial tissues. Cre mice are widely used for the determination of CNC and exploration of gene functions in orofacial development. However, there is a lack of Cre mice specifically marked BA1's cells. Pax2-Cre allele was previously generated and has been widely used in the field of inner ear development. Here, by compounding Pax2-Cre and R26R-mTmG mice, we found a specific expression pattern of Pax2

Derivation of endothelial cells from porcine induced pluripotent stem cells by optimized single layer culture system

Influence of BMP4 on Regulation of Cell Cycle and Apoptosis of Hematopoietic Stem Cells/Progenitor Cells and Its Mechanism in Chemotherapy-Induced Myelosuppression / 中国实验血液学杂志

OBJECTIVE@#To explore the effect of bone morphogenetic protein 4(BMP4) on the cell cycle and apoptosis of hemaropoictic stem and progenitor cells (HSPC) in conditions of 5-fluorouracil (5-FU)-inducing bone marrow suppression and stress hemogenesis, and its possible mechanism.@*METHODS@#The C57BL transgenic mice with BMP4 overexpression were established and were enrolled in transgenic group (BMP4 group), at the same time the wild type mice matching in age, sex and body weight were selected and were enrolled in control group (WT group). The bone marrow suppression was induced by injection with 5-FU in dose of 150 mg/kg, then the nucleated cells were isolated from bone marrow. After the HSPCs were markered with C-kit/sca-1 fluorescent antibodies, the changes of cell cycle and apoptosis of HSPC were detected by Aunexin V/PI and Ki67/DAPI double staining; the cell cycle-essociated hemotopoietic regulatory factors were detected by RT-qPCR.@*RESULTS@#Under physiologic status, there were no significant differences in cell cycle and apoptotic rate of HSPC between WT group and BMP-4 group. After the bone marrow was suppressed, the ratio of HSPC at G0 phase in BMP4 group significantly decreased(P＜0.05); the apoptosis rate of HSPC significantly increased(P＜0.05); the mRNA expression levels of hypoxia-inducing factor Hif-1α and chemotactic factor CXCL12 in stroma of BMP4 group were down-regulated significanfly(P＜0.05).@*CONCLUSION@#Under non-physiologic conditions such as stress hemogenesis or bone marrow suppression, the up-regulation of BMP4 can promote HSPC into cell cycle and apoptosis of HSPC, moreover, the BMP4 may play a regulatory role for cell cycle of HSPC through direct or indirect down-regulation of Hif-1α and CXCL-12 expressions.

BMP4 Induces Cardiomyocyte Hypertrophy Through the Activation of ERK 1/2 Signaling Pathway in H9c2 Cells

Abstract Bone morphogenetic protein-4 (BMP4) is a member of the bone morphogenetic protein family which plays an important role in bone formation, inflammation and cardiac hypertrophy. The aim of this study was to investigate the underlying molecular mechanism that BMP4-induced cardiomyocyte hypertrophy. H9c2 cells were used to measure cell surface area and protein synthesis. Western blot was used to examine hypertrophic marker brain natriuretic peptide (BNP) protein expression and phosphorylation of ERK1/2. The results exhibited that cell surface area, protein synthesis and BNP protein expression were increased with BMP4 treatment. While PD98059 inhibited these effects of BMP4. In addition, BMP4 treatment increased phosphorylation of ERK1/2 in a time- and dose-dependent manner. PD98059 treatment decreased phosphorylation of ERK1/2 that was increased by BMP4. These results suggest that BMP4 induces cardiomyocyte hypertrophy through the activation of ERK1/2 cell signaling pathway.

In vitro differentiation of spermatogonial stem cells using testicular cells from Guangxi Bama mini-pig

In this study, we attempted to establish a culture system for in vitro spermatogenesis from spermatogonial stem cells (SSCs) of Bama mini-pig. Dissociated testicular cells from 1-month-old pigs were co-cultured to mimic in vivo spermatogenesis. The testicular cells were seeded in minimum essential medium alpha (α-MEM) supplemented with Knockout serum replacement (KSR). Three-dimensional colonies formed after 10 days of culture. The colonies showed positive staining for SSC-associated markers such as UCHL1, PLZF, THY1, OCT4, Dolichos biflorus agglutinin, and alkaline phosphatase. Induction of SSCs was performed in α-MEM + KSR supplemented with retinoic acid, bone morphogenetic protein 4, activin A, follicle-stimulating hormone, or testosterone. The results showed that STRA8, DMC1, PRM1, and TNP1 were upregulated significantly in the colonies after induction compared to that in testis from 1-month-old pigs, while expression levels of those genes were significantly low compared to those in 2-month-old testis. However, upregulation of ACROSIN was not significant. Replacement of α-MEM and KSR with Iscove's modified Dulbecco's medium and fetal bovine serum did not upregulate expression of these genes significantly. These results indicate that SSCs of Bama mini-pig could undergo differentiation and develop to a post-meiotic stage in α-MEM supplemented with KSR and induction factors.

Association of single nucleotide polymorphisms in AXIN2, BMP4, and IRF6 with Non-Syndromic Cleft Lip with or without Cleft Palate in a sample of the southeast Iranian population

Abstract Non-syndromic cleft lip with or without palate (NSCL/P) is a common congenital malformation worldwide, with complex etiology. It has been proposed that interaction of genes and environmental factors play a role in the predisposition to this disease. Objectives: The aim of this study was to examine the association between AXIN2 (axis inhibition protein 2) rs7224837, BMP4 (bone morphogenetic protein 4) rs17563, and IRF6 (interferon regulatory factor 6) rs861019 and 2235371 polymorphisms and NSCL/P in an Iranian population. Material and Methods: This case-control study was carried out on 132 unrelated NSCL/P patients and 156 healthy subjects. The variants were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results: The findings suggest that BMP4 rs17563 polymorphism significantly decreased the risk of NSCL/P in codominant (OR=0.36, 95%CI=0.17-0.79, p=0.012, CT vs CC and OR=0.11, 95%CI=0.01-0.88, p = 0.019, TT vs CC), dominant (OR=0.30, 95%CI=0.15-0.62, p = 0.0007, CT+TT vs CC), recessive (OR=0.12, 95%CI=0.02-0.99, p = 0.023, TT vs CC+CT), overdominant (OR=0.39, 95%CI = 0.18-0.84, p=0.021, CT vs CC+TT), and allele (OR=0.28, 95%CI=0.15-0.55, p<0.0001, T vs C) inheritance models. Our findings did not support an association between AXIN2 rs7224837 and IRF6 rs861019 polymorphism and risk/protection of NSCL/P. The IRF6 2235371 variant was not polymorphic in our population. Conclusion: The results indicate that the BMP4 rs17563 variant is likely to confer a protective effect against the occurrence of NSCL/P in a sample of the southeast Iranian population.

Hypospadias induced by maternal exposure to di-n-butyl phthalate and its mechanisms in male rat offspring / 中华男科学杂志

Objective@#To induce hypospadias in male rat offspring by maternal exposure to di-n-butyl phthalate (DBP) during late pregnancy and further investigate its mechanisms.@*METHODS@#We randomly divided 20 pregnant rats into a DBP exposure and a control group, the former treated intragastrically with DBP while the latter with soybean oil at 750 mg per kilogram of the body weight per day from gestation days (GD) 14 to 18. On postnatal day (PND) 1, we recorded the incidence rate of hypospadias and observed the histopathological changes in the genital tubercle of the hypospadiac rats. We also measured the level of serum testosterone (T) by radioimmunoassay and determined the mRNA and protein expressions of the androgen receptor (AR), sonic hedgehog (Shh), bone morphogenetic protein 4 (Bmp4) and fibroblast growth factor 8 (Fgf8) in the genital tubercle by real-time PCR and Western blot.@*RESULTS@#No hypospadiac male rats were found in the control group. The incidence rate of hypospadias in male offspring was 43.6% in the DBP-treatment group. Histological analysis confirmed hypospadiac malformation. The serum testosterone concentration was decreased in the hypospadiac male rats as compared with the controls (［0.49 ± 0.05］ vs ［1.12 ± 0.05］ ng/ml, P <0.05). The mRNA expressions of AR, Shh, Bmp4 and Fgf8 in the genital tubercle were significantly lower in the hypospadiac male rats than in the controls (AR: 0.50 ± 0.05 vs 1.00 ± 0.12, P <0.05; Shh: 0.65 ± 0.07 vs 1.00 ± 0.15, P <0.05; Bmp4: 0.42 ± 0.05 vs 1.00 ± 0.13, P <0.05; Fgf8: 0.46 ± 0.04 vs 1.00 ± 0.12, P <0.05), and so were their protein expressions (AR: 0.34 ± 0.05 vs 1.00 ± 0.09, P <0.05; Shh: 0.51 ± 0.07 vs 1.00 ± 0.12, P <0.05; Bmp4: 0.43 ± 0.05 vs 1.00 ± 0.11, P <0.05; Fgf8: 0.57 ± 0.04 vs 1.00 ± 0.13, P <0.05).@*CONCLUSIONS@#Maternal exposure to DBP during late pregnancy can induce hypospadias in the male rat offspring. DBP affects the development of the genital tubercle by reducing the serum T concentration and expressions of AR, Shh, Bmp4 and Fgf8 in the genital tubercle, which might underlie the mechanism of DBP inducing hypospadias.

Haplotypes in BMP4 and FGF Genes Increase the Risk of Peri-Implantitis

Abstract Despite the success of osseointegrated implants, failures have increased significantly, associated with development of peri-implantitis. Multiple factors influence the peri-implant bone loss, including environmental and genetic causes. BMPs (Bone morphogenetic proteins) are growth factors that induce bone formation. FGF (fibroblast growth factors) and their receptors (FGFRs) play important roles by controlling the levels of cell proliferation, differentiation and migration. BMP/FGF relationship is responsible for promoting bone regeneration and bone loss. The aim of this study was to analyze the correlation between BMP4, FGF3, FGF10 and FGFR1 genes and peri-implant bone loss. Two hundred and fifteen volunteers, with 754 dental implants, were submitted to oral examination and divided in healthy group (n=129) and peri-implantitis group (n=86). Thirteen polymorphisms in BMP4, FGF3, FGF10 and FGFR1 genes were analyzed individually and in haplotype. The chi-square test correlated genotypes, allelic and haplotype frequencies. Values of p<0.05 were considered significant. Volunteers with peri-implantitis demonstrated high incidence of total edentulism (p<0.0001) and thin peri-implant phenotype (p<0.04). Higher incidence of spontaneous bleeding, plaque and implant mobility was observed in peri-implantitis group (p<0.0001 for all). The TT polymorphic genotype for BMP4 rs2761884 was associated with healthy peri-implant (p=0.01). FGF3 rs4631909 (TT+CT genotype) also showed association with the control group (p=0.04). The frequency of C allele for FGF3 rs4631909 showed a tendency for association with peri-implantitis (p=0.08). FGF10 CCTG (p=0.03), BMP4 GAAA (p=0.05) and GGGA (p=0.02) haplotypes were associated with peri-implantitis (p=0.03). Therefore, it may be concluded that BMP4 and FGF10 haplotypes are associated with peri-implantitis.

Resumo Apesar do alto índice de sucesso em implantodontia, falhas tem aumentado drasticamente, estando associadas ao desenvolvimento de peri-implantite. A perda óssea peri-implantar é influenciada por múltiplos fatores, incluindo causas genéticas e ambientais. As BMPs (proteínas ósseas morfogenéticas) são fatores de crescimento indutores da formação óssea. Os FGFs (fatores de crescimento dos fibroblastos) e seus receptores (FGFRs) desenvolvem importante função na proliferação, diferenciação e migração celular. A relação BMP/FGF é responsável pela regeneração e perda óssea. O objetivo deste estudo foi estudar a possível correlação entre os genes BMP4, FGF3, FGF10 e FGFR1 e a perda óssea peri-implantar. Duzentos e quinze voluntários, com 754 implantes, foram submetidos ao exame oral e divididos em grupo saúde (n=129) e peri-implantite (n=86). Treze polimorfismos nos genes BMP4, FGF3, FGF10 e FGFR1 foram analisados individualmente e como haplótipos. O teste do qui-quadrado correlacionou as frequências dos genótipos, alelos e haplótipos. Valores de p<0,05 foram considerados estatisticamente significantes. Voluntários com peri-implantite mostraram alta incidência de edentulismo total (p<0,0001) e biotipo periodontal fino (p<0,04). Sangramento espontâneo, placa e mobilidade do implante foram altamente incidentes no grupo peri-implantite (p<0,0001). O genótipo polimórfico TT para BMP4 rs2761884 foi associado com saúde peri-implantar (p=0,01). FGF3 rs4631909 (genótipos TT+CT) mostraram associação com o grupo controle (p=0,04). A frequência do alelo C para FGF3 rs4631909 mostrou uma tendência de associação com peri-implantite (p=0,08). Os haplótipos FGF10 CCTG (p=0,03), BMP4 GAAA (p=0,05) e GGGA (p=0,02) foram associados com peri-implantite (p=0,03). Sendo assim, conclui-se que os haplótipos BMP4 e FGF10 estão associados com peri-implantite.

Tissue factor expression and methylation regulation in differentiation of embryonic stem cells into trophoblast

OBJECTIVE@#To explore tissue factor (TF) expression and methylation regulation in differentiation of human embryonic stem cells (hESCs) into trophoblast.@*METHODS@#Differentiation of hESCs into trophoblast was induced by bone morphogenetic protein 4 (BMP4). Expression of gene, protein of TF and DNA methylation at different time points during induction process was detected by RT-PCT, Western blot, flow cytometry and MSP-PCR method.@*RESULTS@#The expression of mRNA, protein level of TF could be detected during directional differentiation of hESCs to trophoblast cells, semi methylation-semi non methylation expression appeared at TF DNA promoter region, and it showed decreased methylation level and increased non methylation level with formation of trophoblast cell and increased expression of TF.@*CONCLUSIONS@#It shows that during differentiation of hESCs into trophoblast, the differential expression of TF is related with DNA methylation level, and it is changed with the methylation or non methylated degree. It provids new platform to furtherly explore the regulation mechanisms of specific expression of tissue factor in the process of the embryonic stem cell development.

Differentiation of human umbilical cord matrix-derived mesenchymal stem cells into germ-like cells

Mesenchymal Stem Cells [MSCs] are multipotent cells that can be collected from different sources. Under specific conditions, MSCs can be differentiated to tissue specific cells in vitro. Human Umbilical Cord Mesenchymal Stem Cells [hUCMSCs] can easily be harvested and cultured in in vitro conditions. Production of germ cells from mesenchymal stem cells is a very interesting and promising area in the field of reproductive medicine. In the present study, the possible trans-differentiation of hUCMSCs into Primordial like Germ Cell [PGC] was performed in vitro under specific condition. Human umbilical cord mesenchymal stem cells were cultured and expanded in DMEM medium containing 10% FBS. The cultured cells were studied for differentiation ability to adipocytes and osteocytes. Furthermore, MSCs related markers were identified by flow cytometry method. For PGC differentiation, hUCMS cells were cultured in differentiation medium containing Bone Morphogenetic Protein 4 [BMP4] and it was followed by retinoic acid [RA]. Real time PCR and immunocytochemistry analysis were performed to evaluate the expression of PGC specific genes and proteins, respectively. Our results showed that hUCMSCs cultured in the presence of BMP4 and RA are able to trans differentiate in to PGC like cells in vitro. Real time PCR and immunocytochemistry results showed that differentiated cells expressed PGC specific markers after 14 days of culture. Based on these results, it was concluded that hUCMSC may be considered as a promising alternative cell source in reproductive medicine. More studies including laboratory and also animal models are needed to evaluate the functionality of differentiated PGCs before introducing them to clinical applications

The similarity between human embryonic stem cell-derived epithelial cells and ameloblast-lineage cells / 国际口腔科学杂志·英文版

This study aimed to compare epithelial cells derived from human embryonic stem cells (hESCs) to human ameloblast-lineage cells (ALCs), as a way to determine their potential use as a cell source for ameloblast regeneration. Induced by various concentrations of bone morphogenetic protein 4 (BMP4), retinoic acid (RA) and lithium chloride (LiCl) for 7 days, hESCs adopted cobble-stone epithelial phenotype (hESC-derived epithelial cells (ES-ECs)) and expressed cytokeratin 14. Compared with ALCs and oral epithelial cells (OE), ES-ECs expressed amelogenesis-associated genes similar to ALCs. ES-ECs were compared with human fetal skin epithelium, human fetal oral buccal mucosal epithelial cells and human ALCs for their expression pattern of cytokeratins as well. ALCs had relatively high expression levels of cytokeratin 76, which was also found to be upregulated in ES-ECs. Based on the present study, with the similarity of gene expression with ALCs, ES-ECs are a promising potential cell source for regeneration, which are not available in erupted human teeth for regeneration of enamel.

KDM6B epigenetically regulates odontogenic differentiation of dental mesenchymal stem cells / 国际口腔科学杂志·英文版

Mesenchymal stem cells (MSCs) have been identified and isolated from dental tissues, including stem cells from apical papilla, which demonstrated the ability to differentiate into dentin-forming odontoblasts. The histone demethylase KDM6B (also known as JMJD3) was shown to play a key role in promoting osteogenic commitment by removing epigenetic marks H3K27me3 from the promoters of osteogenic genes. Whether KDM6B is involved in odontogenic differentiation of dental MSCs, however, is not known. Here, we explored the role of KDM6B in dental MSC fate determination into the odontogenic lineage. Using shRNA-expressing lentivirus, we performed KDM6B knockdown in dental MSCs and observed that KDM6B depletion leads to a significant reduction in alkaline phosphate (ALP) activity and in formation of mineralized nodules assessed by Alizarin Red staining. Additionally, mRNA expression of odontogenic marker gene SP7 (osterix, OSX), as well as extracellular matrix genes BGLAP (osteoclacin, OCN) and SPP1 (osteopontin, OPN), was suppressed by KDM6B depletion. When KDM6B was overexpressed in KDM6B-knockdown MSCs, odontogenic differentiation was restored, further confirming the facilitating role of KDM6B in odontogenic commitment. Mechanistically, KDM6B was recruited to bone morphogenic protein 2 (BMP2) promoters and the subsequent removal of silencing H3K27me3 marks led to the activation of this odontogenic master transcription gene. Taken together, our results demonstrated the critical role of a histone demethylase in the epigenetic regulation of odontogenic differentiation of dental MSCs. KDM6B may present as a potential therapeutic target in the regeneration of tooth structures and the repair of craniofacial defects.

Differentiation potential of CD41⁺ cells derived from the mouse aorta-gonad-mesonephros region, yolk sac and embryonic circulating blood / 中华血液学杂志

<p><b>OBJECTIVE</b>To compare the differentiation ability difference of hematopoietic, mesenchymal and endothelial potential between CD41⁺ cells derived from the mouse aorta-gonadmesonephros (AGM) region, yolk sac (YS) and embryonic circulating blood (CB).</p><p><b>METHODS</b>CD41⁺ cells were sorted from AGM, YS and CB. The CD45 and c-kit expression were studied in CD41⁺ cells by flow cytometry. IL-3 and bone morphogenetic protein 4 (BMP-4) treatment together with semi solid culture were used to assess hematopoietic potential difference of CD41⁺ cells. Immunofluorescence staining of α-SMA was used to assess mesenchymal potential difference. The endothelial cell induction system was used to assess endothelial potential difference.</p><p><b>RESULTS</b>The proportions of CD45+ cells in CD41⁺ population were 51.9% (AGM), 45.8% (YS) and 22.2% (CB), respectively, while those of c-kit⁺ cells were 40.0% (AGM), 39.6% (YS) and 36.2% (CB), respectively. After stimulated by IL-3 factor, the number of total colonies increased in all three groups-derived CD41⁺ cells compared to that of unstimulated group[(14.1±1.9) vs (1.2±0.2), (32.4±1.1) vs (18.4±2.2) and (41.8±0.9) vs (10.4±1.8)], (P<0.01). After stimulated by BMP-4 factor, compared to unstimulated group, CFU-Mix colony number in CD41⁺ cells from AGM region and YS were significantly decreased[(0.5±0.6) vs (3.2±0.8), (1.3±0.7) vs (7.4±1.7)](P<0.01), but there was no difference in CB group[(2.5±0.5) vs (3.9±1.5)](P>0.01). The mesenchymal marker α-SMA was highly expressed in CD41⁺ cells from AGM region and YS, but lowly expressed in CD41⁺ cells from CB.</p><p><b>CONCLUSION</b>There are some differences between CD41⁺ cells in AGM region, YS and CB on hematopoietic cell surface marker expression, hematopoietic colony formation with IL-3 and BMP-4 stimulation.</p>

A preliminary study on the culture of single hematopoietic stem cell / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the biological behavior including survival and proliferation of CD34 + CD38--Lin--cells when they are cultured at single cell level.</p><p><b>METHODS</b>Purified umbilical cord blood CD34 + CD38--Lin--cells were separated at single cell level in 96-well plates using flow cytometry for four groups: control group (CD34 + CD38--Lin--cell plus stem cell medium) , Shh group (CD34 + CD38--Lin--cell plus stem cell medium and Shh), BMP-4 group (CD34 + CD38--Lin--cell plus stem cell medium and BMP-4), Jagged-1 group (CD34 + CD38--Lin--cell plus stem cell medium and Jagged-1). Methylcellulose medium was used in the colony-forming experiment which was also in four groups as previously. The number of cells and colony-forming units in each well for the four groups was evaluated at different time points (day 1, 3, 7) with fluorescence microscopy counting method.</p><p><b>RESULTS</b>Division of single cell was observed to be amplified in all of these groups from day 3. And meanwhile, after 1-week culture, the survival rates for the treated groups were all higher than the control group (Jagged-1 group > BMP-4 group > Shh group > control), while the cell number in each well was also highest in the Jagged-1 group (Jagged-1 group > BMP-4 group > control). The number of wells with a cell number of zero was significantly fewer in all treated groups (especially the Jagged-1 group) than in the control group; meanwhile, the number of wells with a cell number higher than 17 was evidently higher in all the treated groups (especially the BMP-4 group) more than controls. Colony-forming units for erythroid (BFU-E), granulocyte (CFU-G), macrophage (CFU-M), and granulocyte macrophage (CFU-GM) were observed for all of these experimental groups, and there was no significant difference between the four experimental groups.</p><p><b>CONCLUSIONS</b>CD34 + CD38 - Lin - cell can achieve the survival, self-renewal and proliferation when cultured at single cell level, and the adding of Shh, BMP-4, and Jagged-1 can enhance such capabilities. However, CD34 + CD38 - Lin - cell can only maintain cell totipotency in its niche.</p>

Overexpression of bone morphogenetic protein 4 in STO fibroblast feeder cells represses the proliferation of mouse embryonic stem cells in vitro

Embryonic stem cells (ESCs) can be propagated in vitro on feeder layers of mouse STO fibroblast cells. The STO cells secrete several cytokines that are essential for ESCs to maintain their undifferentiated state. In this study, we found significant growth inhibition of mouse ESCs (mESCs) cultured on STO cells infected with adenovirus containing a dominant-negative mutant form of IkappaB (rAd-dnIkappaB). This blockage of the NF-kappaB signal pathway in STO cells led to a significant decrease in [3H]thymidine incorporation and colony formation of mESCs. Expression profile of cytokines secreted from the STO cells revealed an increase in the bone morphogenetic protein4 (BMP4) transcript level in the STO cells infected with adenoviral vector encoding dominant negative IkappaB (rAd-dnIkappaB). These results suggested that the NF-kappaB signaling pathway represses expression of BMP4 in STO feeder cells. Conditioned medium from the rAd-dnIkappaB-infected STO cells also significantly reduced the colony size of mESCs. Addition of BMP4 prevented colony formation of mESCs cultured in the conditioned medium. Our finding suggested that an excess of BMP4 in the conditioned medium also inhibits proliferation of mESCs.

Expression of BMP4 mature peptide in eukaryotic cells and its differentiation-inhibiting effect in culturing induced pluripotent stem cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the role of bone morphogenetic protein 4 (BMP4) in culturing induced pluripotent stem cells (iPSCs) and the related signal pathways.</p><p><b>METHODS</b>We amplified the mature peptide of BMP4 from the placenta through RT-PCR, and IgK secretion peptide was ligated to the N-terminal of BMP4 mature peptide. The recombinant plasmid pPYCAG-IgK-BMP4 was transfected into 293T cells and screened with puromycin, and the positive clones for expressing BMP4 were verified by cell immunofluorescence and Western blotting. To test the bioactivity of BMP4, iPSCs were cultured in the medium supplemented with leukemia inhibitory factor (LIF) plus the supernatant containing BMP4, and the cell phenotype, cell differentiation capacity into lineages of the 3 germ layers and expression levels of pluripotency-associated genes were investigated.</p><p><b>RESULTS</b>Smad1 was phosphorylated by BMP4 from the culture medium. iPSCs cultured in the medium supplemented with LIF plus the supernatant containing BMP4 for 3 passages maintained the phenotype of stem cells with the expression levels of pluripotency-associated genes not affected. These iPSCs also maintained the capacity to differentiate into cell lineages of the 3 germ layers.</p><p><b>CONCLUSION</b>BMP4 can be efficiently expressed in mammalian cells to maintain the multipotent differentiation capacity of the iPSCs in in vitro culture.</p>

Effect of bone morphogenetic protein-4 on the proliferation and differentiation of rat hepatic precursor cells / 中南大学学报(医学版)

OBJECTIVE@#To determine the regulation effect of bone morphogenetic protein-4 (BMP-4) on the proliferation and differentiation of rat hepatic precursor cells.@*METHODS@#We used Noggin (200 ng/mL) as the function blocking control of BMP-4, and the hepatic precursor cells of WB-F344 were treated with recombinant BMP-4 at 50 ng/mL at different time points. The proliferation of WB-F344 cells were tested by methyl thiazolyl tetrazolium (MTT) colorimetric assay. The ultrastructural characters of differentiated WB-F344 cells regulated by BMP-4 were observed under a transmission electron microscope. RT-PCR was used to examine mRNA expression of specific molecular markers for different cellular phenotypes potentially differentiated from the WB-F344 cells.@*RESULTS@#At different time points, the absorbance values in the BMP-4 treatment groups were higher than those in the control groups of Noggin and blank treatment (P<0.01). The WB-F344 cells treated with BMP-4 exhibited typical ultrastructural characters of well-differentiated epithelial cells. The hepatocyte mRNA markers were more significantly promoted in the differentiated WB-F344 cells in the BMP-4 treatment group than those in the other 2 control groups.@*CONCLUSION@#BMP-4 can promote the proliferation and directional differentiation towards hepatocytes of rat hepatic precursor cells of WB-F344.

The Immunohistochemical Patterns of Calcification-related Molecules in the Epidermis and Dermis of the Zebrafish (Danio rerio)

BACKGROUND: The scales of bony fish represent a significant reservoir of calcium and calcification of the elasmoid scale is known to be associated with deposition of mineral crystals from the epidermis to dermis. However, little is known about the exact mechanisms of calcium deposition, mobilization and regeneration occurring in the zebrafish skin. OBJECTIVE: The purpose of this study was to investigate the expression of calcification-related molecular mediators in both the epidermis and dermis of the zebrafish (Danio rerio), using immunohistochemical study. METHODS: We examined the skin of zebrafish in four populations of different ages (i.e. 20 days post-fertilization (dpf), 35 dpf, 50 dpf, and the adult zebrafish), using several immunohistochemical markers, including bone morphogenetic protein 4 (BMP-4), beta-catenin, osteocalcin, osteopontin and osteonectin. RESULTS: BMP-4, osteopontin and osteonectin were moderately expressed in the epidermis of zebrafish after 35 dpf. Also, some of the cells in the upper dermis showed strong positivity for BMP-4, osteocalcin, osteopontin and osteonetin. CONCLUSION: Our results suggest that BMP-4, osteocalcin, osteopontin and osteonectin may play a role in the process of calcification of the elasmoid scale.

The Role of Microtubule in the Regulation of Endochondral Bone Formation in the Developing Mouse Cranial Base / 체질인류학회지

Endochondral bone formation of the developing cranial base is a complex process. This mechanism requires precise orchestration of many cellular events and cartilage matrix metabolism, such as proliferation, becoming round in shape, termination of proliferation, hypertrophic size-increase, and finally programmed cell death. Active formation and degradation of cartilage matrix take place, in which microtubules are involved for intracellular events; bone apposition follows these events. However, the involvement of microtubules during these changes in the developing cranial base has not been identified yet. Thus, we investigated the involvement of microtubules in the regulation of endochondral bone formation during cranial base development. Using tubulin-binding drug nocodazole, we examined the effects of altering the structure and function of microtubules during in vivo organ culture of the mouse cranial base. Cultured specimens were analyzed with HE staining, immunohistochemistry, and cell counting in order to study the morphological and molecular changes that occurred in the tissues. Disruption of the microtubular array by nocodazole reduced cells expressing proliferation marker Ki67, osteogenic marker BSP, and BMP4 within the sphenooccipital synchondrosis region; chondrocyte hypertrophy was ceased in the hypertrophic zone; degeneration of cartilage matrix and bone matrix apposition was inhibited in the ossification center of the basooccipital cranial base. Our data demonstrated that disruption of microtubules by nocodazole have multiple inhibitory effects on the sequential changes that occur during endochondral bone formation, suggesting the importance of normal microtubule-polymerization in cranial base development.