This study explores the histological features and Bmp4 expression patterns in the replaced tooth germ of Xenopus laevis . Tooth germ formation starts from the dental placode through epithelial-mesenchymal interactions, involving various signaling pathways such as Fgf, Shh, Bmp, and Wnt. In mice, Bmp4 expression in the dental placode inhibits Pax9 expression in the dental mesenchyme. Although absent in the presumptive dental lamina of birds and toothless mammals, Bmp4 remains conserved in reptiles and fish owing to gene duplication. However, its expression in amphibian tooth germs is poorly understood. Three-month-old X. laevis were employed in this study. Initially, samples underwent paraffin embedding and were sectioned into 5 or 12 μm ribbons for H&E staining and in situ hybridization, respectively. Results revealed teeth appearing in two maxillary rows: the labial side, with prefunctional and functional teeth, and the lingual side, with replaced tooth germs behind functional teeth. Enameloid was observed between the inner dental epithelium and dental mesenchyme at the cap or early bell stages, whereas enamel and dentin formed during the late bell or mineralization stages from the replaced tooth germ. Bmp4 expression was evident in the inner dental epithelium (ameloblasts), dental papilla (odontoblasts), stellate reticulum, and Hertwig’s epithelial root sheath. Overall, these findings highlight the conservation of Bmp4 expression in X. laevis tooth development.

Gingival fibroblasts (GF) are the most abundant cell type in periodontal connective tissues, andhave distinct functional activities in the repair of periodontal tissues and in inflammatory periodontal diseases. Human gingival fibroblasts (hGF) can be used for periodontal tissue engineering. This study examined whether the alkaline phosphatase of hGF is enhanced by recombinant human BMP-4 and/or Anti human BMP-4 antibody. hGF was obtained from the excised gingival tissue of an implant patient undergoing 2nd surgery. The tissue was incubated at 37℃ in 5% CO2 and 95% humidity, and the cultivating media was changed every 2 days. The 2nd passage hGF cells were cultured in a medium containing Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 1 X antibiotic antimycotic solution. The control hGF was cultured for 7 days without rhBMP-4/Anti human BMP-4 antibody. The experimental groups were cultured for 7 with BMP-4 (10 ng/ml) and/or Anti human BMP-4 antibody. This study evaluated the differentiation of hGF to osteoblasts using alkaline phosphatase assay. In the experimental groups, the hGF showed abundant positive ALP staining. Among the experimental groups, the experimental group 3 (mixture of rhBMP-4 (20ng/㎖) and Anti human BMP-4 antibody (50000ng/㎖) showed most abundant positive ALP staining. In the control group, the hGF showed weak positive ALP staining. Overall, these results suggest that the ALP expression of hGF can enhanced by rhBMP-4 or mixture of rhBMP-4/ anti human BMP-4 antibody.

The functional cardiovascular system is comprised of distinct mesoderm-derived lineages including endothelial cells, vascular smooth muscle cells and other mesenchymal cells. Recent studies in the human embryonic stem cell differentiation model have provided evidence indicating that these cell lineages are developed from the common progenitors such as hemangioblasts and cardiovascular progenitor cells. Also, the studies have suggested that these progenitors have a common primordial progenitor, which expresses KDR (human Flk-1, also known as VEGFR2, CD309). We demonstrate here that sustained activation of BMP4 (bone morphogenetic protein 4) in hESC line, CHA15 hESC results in KDR+ mesoderm specific differentiation. To determine whether the KDR+ population derived from hESCs enhances potential to differentiate along multipotential mesodermal lineages than undifferentiated hESCs, we analyzed the development of the mesodermal cell types in human embryonic stem cell differentiation cultures. In embryoid body (EB) differentiation culture conditions, we identified an increased expression of KDR+ population from BMP4-stimulated hESC-derived EBs. After induction with additional growth factors, the KDR+ population sorted from hESCs-derived EBs displays mesenchymal, endothelial and vascular smooth muscle potential in matrix-coated monolayer culture systems. The populations plated in monolayer cultures expressed increased levels of related markers and exhibit a stable/homologous phenotype in culture terms. In conclusion, we demonstrate that the KDR+ population is stably isolated from CHA15 hESC-derived EBs using BMP4 and growth factors, and sorted KDR+ population can be utilized to generate multipotential mesodermal progenitors in vitro, which can be further differentiated into cardiovascular specific cells.

골 형성 단백질(Bone morphogenetic protein, BMP)은 TGF-β superfamily의 구성원 중에 하나이며, 이들은 원래 뼈 형성을 유도하는 능력에 의해 발견되었지만, 뼈 외에도 다른 세포 및 기관의 성장과 분화를 조절하는 것으로 밝혀졌다. 다기능적인 골 형성 단백질은 포유동물의 reproduction에도 매우 중요한 역할을 하는데, 예를 들어 BMP8A와 BMP8B는 생쥐의 정자 형성과 부고환에 일정한 기능을 하는 것으로 보고되었고 난소에서의 BMP15는 난포성장을 자극하고, 과립막세포(granulosa cell)의 증식시키는데 관여하는 단백질로 알려져 있다. 하지만 난관(oviduct)에서의 BMP 역할은 알려진 바가 적다. 그래서 본 연구에서는 골 형성 단백질이 착상 전 oviductal environment에 어떠한 영향을 미치는지 밝히고자 하였다. 8주령 생쥐의 Estrous cyclic oviduct을 가지고 RT-PCR과 Immunohistochemistry(IHC)를 통해 BMP2, 4의 mRNA와 단백질 발현을 확인하였다. Estrogen에 의한 BMP2, 4의 영향을 확인하고자 난소절제술을 시행한 생쥐와 Estrogen receptor alpha(ERα) Knockout 생쥐를 통해 mRNA와 단백질 발현을 확인하였다. Oviduct의 ciliated cell을 가지고 BMP2, 4의 기능을 밝히고자 siRNA실험을 진행 하였다. Estrous cyclic oviduct cDNA를 통해 RT-PCR한 결과, 이 중 Estrus 시기에 가장 높은 발현을 보인 BMP2, 4의 mRNA level은 Isthmus보다 Infundibulum과 Ampulla에 증가하였고, Immunohistochemistry (IHC)를 통해 BMP2와 BMP4 단백질은 난관 상피세포 중에서 ciliated cell에 발현되었다. 이를 ciliated cell marker인 β-tubulin과 함께 Immunofluorescence(IF)를 진행한 결과, 주로 β-tubulin-positive ciliated cell에서 발현됨을 확인하였다. 난소 절제술을 시행한 생쥐의 난관에서는 E2와 DPN (ERβ agonist)에 의해 BMP2, 4 mRNA 발현이 증가하고, ERαKO 난관의 경우 WT(Die)에 비해 BMP2, 4 mRNA, 단백질의 발현 모두 증가하였다. BMP2와 BMP4의 기능을 밝히고자 Ciliated cell line인 OA-6b를 가지고 siRNA 실험을 진행한 결과, Bmp2, Bmp4 siRNA 처리군에서 Ciliated cell marker인 FOXJ1의 발현이 줄어들고 Proliferation marker인 Ki67, Pcna의 발현이 낮아졌다. 이로써, Oviduct 내 BMP2와 BMP4 발현은 Estrogen과 양의 상관성이 있으며, Ciliated cell에서 Estrogen - ER β signaling을 통해 조절된다. Estrogen에 의해 유도되는 BMP2와 BMP4는 ciliated cell에서 Autocrine, Paracrine factor로 작용할 가능성이 있다. 더 나아가 Oviduct의 Infundibulum 및 Ampulla에 강하게 발현되는 BMP2, BMP4는 난자 및 배아에 autocrine, paracrine factor로 작용하여 oviductal cell proliferation을 조절하고, 수정을 위한 oviductal environment 조성에 중요한 역할을 하는 것으로 사료된다.