Mechanisms driving vestibular lamina formation and opening in the mouse

The vestibular lamina (VL) forms as an epithelial outgrowth parallel to the dental lamina (DL) in the oral cavity. During late development, it opens to create a furrow that divides the dental tissue from the cheeks and lips and is known as the vestibule. Defects in this process lead to failure in the separation of the teeth from the lips and cheeks, including the presence of multiple frenula. In this paper, the development of the VL is followed in the mouse, from epithelial placode in the embryo to postnatal opening and vestibule formation. During early outgrowth, differential proliferation controls the curvature of the VL as it extends under the forming incisors. Apoptosis plays a role in thinning the deepest part of the lamina, while terminal differentiation of the epithelium, highlighted by the expression of loricrin and flattening of the nuclei, predates the division of the VL into two to create the vestibule. Development in the mouse is compared to the human VL, with respect to the relationship of the VL to the DL, VL morphology and mechanisms of opening. Overall, this paper provides insight into an understudied part of the oral anatomy, shedding light on how defects could form in this region.     

小鼠孤雌胚胎及孤雌胚胎干细胞中印记基因的表达

小鼠孤雌胚胎体内发育最多不能超过10.5d,发育失败的主要原因是胚外组织发育缺陷和印记基因表达的异常。随着小鼠孤雌二倍体和单倍体胚胎干细胞的建系成功,不仅能作为研究印记基因的理想模型,还能够作为种子细胞应用于细胞治疗,拓宽了小鼠孤雌生殖的研究领域和再生医学应用范围。孤雌胚胎聚合作为一种简便易行的技术手段,能够显著提高孤雌胚胎干细胞的建系效率,还能促使异质胚胎间的印记基因相互补偿从而更加趋近于正常受精的胚胎干细胞。我们在文中主要阐释了小鼠孤雌胚胎、孤雌聚合胚胎、孤雌二倍体胚胎干细胞、孤雌单倍体胚胎、孤雌聚合胚胎干细胞中印记基因的表达。     

Fertilization and Empryology: The effect of epidermal growth factor on growth and differentiation of mouse preimplantation embryos in vitro

The effect of epidermal growth factor (EGF) on embryonic growth,development, attachment and spreading in vitro was studied.EGF was added to 130 embryos at the 4-cell stage; to 128 embryosat the blastocyst stage; and to 147 embryos 24 h following spreading.Development of embryos from the 4-cell to the blastocyst stage,differentiation of the inner cell mass (ICM) and trophectoderm,and the occurrence of attachment and spreading were evaluated.Embryo development was significantly inhibited in cultures supplementedwith 100 ng/ml EGF compared to the controls (P < 0.001).Development of 4-cell embryos to blastocysts occurred in 25%of the EGF group compared to 85% of controls. Spreading occurredin 20% of 4-cell embryos and 30% of blastocysts treated withEGF, compared to 80 and 90% of corresponding controls. In embryosdeveloping from the 4-cell stage, massive growth of the ICMand inhibition of the trophectoderm occurred, whereas both ICMand trophectoderm were inhibited by EGF in embryos developingfrom the blastocyst stage. Following spreading, EGF caused massivegrowth of the ICM and regression of the trophectoderm. Our preliminaryresults show that EGF may be involved in the modulation andcontrol of early embryonic growth and differentiation.     

小鼠诱导性多能干细胞的神经细胞分化与突触连接的建立及其功能分析

目的探讨小鼠诱导性多能干细胞(i PSCs)在体外某些因素的诱导下能否分化成功能性的神经细胞,以及神经细胞之间突触的发生与建立。方法首先将i PSCs悬浮培养形成拟胚体,利用维甲酸(RA)将其诱导分化成神经前体细胞,最后撤去RA贴壁培养,利用免疫荧光染色技术观察小鼠i PSCs在体外分化成神经细胞以及神经细胞之间突触发生的形态特征,利用FM1-43染色技术分析突触末端的功能活性。结果小鼠i PSCs能够在RA的诱导下向神经细胞分化,这些神经细胞可以被成熟神经元与胶质细胞标记物标记,新分化的神经元还可以观察到树突棘及突触连接的形成,在去极化刺激下突触活动增强,表现为轴突末端大量FM1-43阳性内吞颗粒。结论由小鼠i PSCs分化而来的神经细胞在体外形成突触连接的过程,提示其可以进一步分化为功能性的神经元和神经胶质细胞。     

Rac1对小鼠胚胎干细胞分化的调节

目的探讨Rac1对胚胎干细胞分化的影响。方法首先利用悬浮培养法制备EB建立了胚胎干细胞分化平台,Western blot方法检测Rac1在胚胎干细胞和EB中的表达;免疫荧光检测表达激活型和灭活型Rac1的胚胎干细胞的形态改变以及利用Western blot检测其对Erk的表达调控。结果活化的Rac1胚胎干细胞伸出丝状伪足,表现早期分化形态;活化的Rac1上调Erk的磷酸化水平。结论 Rac1通过调控Erk信号参与胚胎干细胞的早期分化。     

改良大鼠毛囊干细胞体外培养鉴定及诱导分化的研究

目的 探讨改良体外分离、培养、扩增、纯化SD大鼠毛囊干细胞(rHFSCs)及其免疫、超微结构的鉴定方法,观察rHFSCs的生物学特性及成脂、成骨分化能力。方法 2015年1—6月选取清洁级1周龄SD大鼠6只,在无菌条件下切取SD大鼠触须部皮肤,用 Dispase酶和Ⅳ型胶原酶混合液消化,显微镜下分离毛囊隆突部,用梯度加液组织块贴壁法培养rHFSCs,用两步酶消化法传代,用Ⅳ型胶原差速贴壁法纯化rHFSCs。收集第3代rHFSCs,采用流式细胞术、免疫荧光染色及透射电镜观察rHFSCs内部结构等方法鉴定rHFSCs。收集第1～10代rHFSCs检测细胞活力,第3、5、7、9代rHFSCs培养第1～8天时检测细胞增殖能力。收集第3代rHFSCs,按培养液的不同分为诱导组和对照组,对比两组目的基因PPAR-γ、C/EBPa、OPG、Runx2的相对表达量及染色后的光密度(OD)值变化情况,观察rHFSCs的成脂、成骨分化能力。结果 通过改良方法分离、培养、纯化的rHFSCs呈典型的铺路石状,生长曲线呈“S”形,生长增殖能力良好,克隆形成能力强。透射电镜显示细胞处于原始状态。流式细胞术检测显示Integrin β1、Integrin α6、P63呈高表达,CK15呈中等表达,符合rHFSCs标记鉴定特点。免疫荧光染色和透射电镜观察rHFSCs内部结构证明细胞的类型属于毛囊干细胞。rHFSCs成脂诱导培养7 d后RFqPCR定量结果显示,诱导组目的基因PPAR-γ、C/EBPa的相对表达量(5.598±0.168、4.757±0.416)均明显高于对照组(1.119±0.344、1.126±0.355),差异均有统计学意义(t=34.955、20.266,P值均＜0.01);诱导14 d后细胞油红O染色阳性,诱导组OD值(2.472±0.091)明显高于对照组(0.817±0.003),差异有统计学意义(t=114.641, P＜0.05)。rHFSCs成骨诱导培养14天后RFqPCR定量结果显示,诱导组目的基因OPG和Runx2的相对表达量(1.921±0.275,3.892±0.265)明显高于对照组(1.085±0.288,1.046±0.216),差异均有统计学意义(t=4.667、17.332, P值均＜0.01);诱导21 d后细胞茜素红染色阳性,诱导组OD值(0.716±0.016)明显高于对照组(0.076±0.002),差异有统计学意义(t=14.078, P＜0.01)。结论 采用改良体外分离、培养、扩增、纯化方法可成功建立rHFSCs体外培养体系,rHFSCs具有纯度大、增殖效率高、多向分化潜能强等特点,可为干细胞组织工程学相关发展提供良好的种子细胞。     

The pattern of ciliary development in fetal mouse vestibular receptors

Summary The development of vestibular receptors in the mouse was studied by scanning electron microscopy between the 13th gestation day to birth. On the 13th gestation day, the utricle was entirely covered with microvilli, which were often grouped around small kinocilia at the center of the macula. The vertical cristae were not clearly differentiated at this stage. On the 15th gestation day, the opposite orientation of ciliary tufts in the utricle indicated the beginnings of the striola. During the whole period studied, gradients in differentiation of ciliary tufts were observed between the center and the periphery of the utricle, and the top and base of the cristae. The auxiliary structures (otolithic membranc and cupula) began to appear at the same time as the first ciliary tufts differentiated. Otoliths, still immature, were only observed as from the 16th gestation day. Differentiation of ciliary tufts on the utricle appeared to be progressive during the fetal period. However, between the 16th and 17th gestation days, a pause in the differentiation of ciliary tufts was registered. A day later, there was a pause in the increase of the utricular sensory surface, which coincided with a temporary stabilization of the decrease in the thickness of the sensory epithelium.     

激活Nodal信号通路促进小鼠胚胎干细胞向定型内胚层分化

目的:探讨激活Nodal信号通路在小鼠胚胎干细胞(embryonic stem cells,ESC)向定型内胚层(definitive endoderm,DE)分化中的作用,寻找小鼠ESC向DE分化的最佳培养体系。方法:根据培养基的不同,实验分为3组:胚胎干细胞组(基础培养+LIF)、自然分化组(基础培养基)和activin A组(基础培养基+50μg/L activin A)。细胞培养1~7 d,收集不同作用时点(1、3、5、7 d)的细胞及细胞爬片。用流式细胞术检测CXCR4阳性细胞的比例;免疫细胞化学法检测CXCR4蛋白的表达;Western blot检测OCT4及CXCR4蛋白的表达。结果:流式细胞术检测结果提示,随着培养时间的延长,CXCR4阳性细胞的比例,胚胎干细胞组在1~7 d无明显变化,自然分化组和activin A组逐渐增高,第5天达高峰(P0.05),其中activin A组最高(P0.05)。细胞免疫组化结果显示CXCR4阳性细胞呈棕褐色或棕黄色。Western blot的结果提示,随着培养时间的延长,胚胎干细胞组OCT4和CXCR4蛋白表达无明显变化;自然分化组和activin A组的CXCR4蛋白的表达逐步增高,OCT4蛋白的表达逐步下降,第5天达高峰(P0.05),其中activin A组的表达最明显(P0.05)。结论:在小鼠ESC分化过程中,在诱导培养的第5天,DE的比例最高。激活Nodal信号通路可以促进小鼠ESC向具有CXCR4分子标志的DE分化,有利于获取更多的DE细胞。     

胚胎干细胞定向分化为胰岛素分泌细胞的研究进展

胰岛移植是治疗糖尿病的有效方法之一。胚体干细胞在体外定向分化为胰岛素分泌细胞为胰岛移植提供了足够的细胞来源。概述了近年来胚胎干细胞向胰腺细胞系的诱导分化所取得的进展。     

胚胎干细胞向神经细胞诱导分化的研究

胚胎干细胞具有全能性和无限增殖的能力 ,有望成为组织工程和细胞治疗的重要种子细胞来源。如何将胚胎干细胞向特定细胞诱导分化已成为重要的课题。目前已有将胚胎干细胞诱导分化为神经细胞 ,并将其应用于动物模型治疗的报道。这些工作对今后神经系统疾病和损伤的治疗意义重大     

Cardiomyocyte differentiation from mouse embryonic stem cells using a simple and defined protocol

Background: Embryonic stem (ES) cells are pluripotent cells with the ability to differentiate to any cell type of the resident organism. In recent years, significant advances have been made in using these cells to obtain large numbers of cardiomyocyte (CM) ‐like cells for scientific research and clinical application. A vast number of protocols have emerged describing differentiation methods without the use of animal serum or extracts restrictive for use in a human clinical setting. These techniques follow a complicated procedure, which although successful, show a relatively varied yield among cell batches. Results: We have designed a three‐step differentiation protocol using defined reagents and a monolayer culture without feeder cells, avoiding embryoid body formation and multiple trypsin treatment, in which beating foci appeared as early as day 6 in in vitro differentiating conditions. Our results show a high yield of CM reaching approximately 60% of the differentiated cells after 13 days in vitro. Conclusions: We provide a fast, simple, reliable and reproducible protocol for inducing murine ES cells toward a CM‐like phenotype comparable to available high‐yield protocols, without the use of intermediate trypsinization/passage steps. Developmental Dynamics 245:157–165, 2016. © 2015 Wiley Periodicals, Inc.     

参麦体外诱导人胚胎生殖细胞向心肌细胞分化

目的:探讨参麦对人胚胎生殖细胞(hEGC)向心肌细胞诱导分化的作用.方法:取5～10周人胚胎生殖腺嵴,进行组织块体外培养,用直接悬浮法使hEGC形成拟胚体(EBs),用不同浓度参麦的培养基对其进行诱导分化,然后取不同时间的细胞做免疫细胞化学显色,鉴定细胞的心肌特异转录因子GATA-4和心肌肌钙蛋白-T(cTnT)表达情况.结果:参麦诱导hEGC分化为心肌细胞的最佳浓度为1g/L,诱导第3周时分化率达57 0%±3 25%,显著高于不添加任何诱导剂的对照组.诱导后细胞形态变成梭形,3周细胞突起相互连接成条索状,且排列方向趋于一致;诱导后第3天即开始出现GATA-4弱表达,第3周时表达最强;诱导后2周,细胞内开始表达cTnT,3周强阳性表达,4周表达明显增强.结论:参麦能够促进hEGC向心肌细胞分化,从而得以建立一种体外诱导hEGC分化为心肌细胞的方法.     

The allantoic and amniotic epithelia of the pig: SEM and TEM studies

Summary The epithelial layers of the allantoamnion of pig embryos and fetuses during various gestational ages were studied utilizing SEM, TEM, and light microscopic histochemistry. The allantoic endoderm exhibits a gland-like secretory activity and thereby differs greatly from that of other mammals. On the surface of this unilaminar cuboidal epithelium, the majority of the cells exhibit characteristic short vermiform ridges, while some protruding cells display larger individual microvilli. These two cell types are also distinct in thin sections. The more common granular cells with short and blunt microvillous projections and a lobated nucleus are characterized by small, Golgi-derived secretory granules in the apical cytoplasm showing a positive PAS-reaction. They contain vast glycogen deposits. Extensive regions of lateral interdigitations are found. In the younger stages, membrane thickenings of the apical plasmalemma resemble those of the urinary bladder. The cytoplasm harbors many more interwoven filaments than organelles. The second cell type, the mitochondria-rich cells, bearing longer apical microvilli in many cases, only constitutes up to 3% of the mucosal cell population. They are frequently flask-shaped, heavily reactive to oxidoreductases, and rich in lysosomes but have smaller glycogen deposits. Mitochondria-rich cells lack secretory granules but have light apical tubules, probably of endocytotic character. These cells can be found in different functional states. The amniotic epithelium is simple squamous in the younger stages and largely resembles that in other mammals exhibiting cells with few organelles but rich in filaments. Each terminal bar consists of a zonula occludens only which may open toward the end of gestation. In older fetuses, small stratified areas of cells sloughing off into the lumen appear as blisters which contain large vacuolated cells.     

Folate antagonist,methotrexate induces neuronal differentiation of human embryonic stem cells transplanted into nude mouse retina

Transplanted embryonic stem (ES) cells can be integrated into the retinas of adult mice as well-differentiated neuroretinal cells. However, the transplanted ES cells also have a tumorigenic activity as they have the ability for multipotent differentiation to various types of tissues. In the present study, human ES (hES) cells were transplanted into adult nude mouse retinas by intravitreal injections 20 h after intravitreal N-methyl-d-aspartate (NMDA) administration. After the transplantation of hES cells, the folate antagonist, methotrexate (MTX) was administrated in order to control the differentiation of the transplanted hES cells. Neuronal differentiation and teratogenic potential of hES cells were examined immunohistochemically 5 weeks after transplantation. The proliferative activity of transplanted cells was determined by both the mitotic index and the Ki-67 proliferative index. Disappearance of Oct-4-positive hES cells showing undifferentiated morphology was observed after intraperitoneal MTX treatment daily, for 15 days. Decreased mitotic and Ki-67 proliferative indices, and increased neuronal differentiation were detected in the surviving hES cells after the MTX treatment. These results suggest two important effects of intraperitoneal MTX treatment for hES cells transplanted into nude mouse retina: (1) MTX treatment following transplantation induces neuronal differentiation, and (2) MTX decreases proliferative activity and tumorigenic potential.     

胚胎干细胞向胰岛细胞诱导分化的研究

胚胎干细胞(embryonicstemcell熏ESC)因具有全能分化潜能而有望成为组织工程和细胞治疗中的重要种子细胞来源。如能将其诱导分化为能分泌胰岛素的细胞,将有助于解决胰岛素绝对或相对缺乏引起的糖尿病代谢失调状态。目前已有研究者诱导分化出能分泌胰岛素的细胞,有的将之应用于糖尿病鼠模型并取得了可喜的效果。本文对这一领域的研究现状进行综述。     

Auditory transduction in the mouse

The sensory hair cells of the mammalian cochlea transduce acoustic stimuli into auditory nerve activity. The biomechanical and molecular details of hair cell mechanotransduction are being acquired at an ever-finer level of resolution. In this review, we discuss how selected mouse mutants and transgenic models have contributed to, and will continue to shape, our understanding of the molecular basis of hair cell mechanotransduction. Functional and structural discoveries made originally in hair cells of nonmammalian vertebrates have been further pursued in the mouse inner ear, where transgenic manipulation can be applied to test molecular mechanisms. Additional insights have been obtained from mice bearing mutations in genes underlying deafness in humans. Taken together, these studies emphasize the elegance of mechanotransduction, enlarge the team of molecular players, and begin to reveal the remarkable adaptations that provide the sensitivity and temporal resolution required for mammalian hearing.     

大鼠胚胎神经干细胞黑质内移植后的存活及分化

目的：观察胚胎神经于细胞脑内移植后的存活及生长分化状况。方法：从孕11d(E11d)大鼠胚胎神经管获取神经上皮细胞，经神经巢蛋白(nestin)染色鉴定干细胞；同时植入同种大鼠黑质内。于移植后7d、14d取脑，用神经元特异烯醇化酶(NSE)、酪氨酸羟化酶(TH)免疫组化方法检测移植细胞的存活及分化状况。结果：E11d神经管上皮细胞多数呈nestin染色阳性，黑质内移植后增殖形成细胞团并随时间延长而增大。免疫组化染色显示移植细胞团内有NSE及TH免疫阳性细胞。结论：胚胎神经上皮细胞多数为神经干细胞，黑质内移植后可以存活并分化为多巴胺能神经元。     

Derivation, characterization and differentiation of human embryonic stem cells: comparing serum-containing versus serum-free media and evidence of germ cell differentiation

BACKGROUND: This study was designed to establish human embryonic stem cell (hESC) lines, to identify the differences when maintained in serum-containing versus serum-free medium and to test their potential of in vitro differentiation. METHODS: Procedures including immunosurgery were performed on 11 donated human blastocysts to establish hESC lines. The cell lines were characterized and maintained using either serum-free or serum-containing media to compare their morphology, Oct-4 expression, apoptosis and growth speed. Differentiation of these lines was evaluated by the morphology and the expression of genes belonging to the three embryonic germ layers and the germ cell lineage. RESULTS: Three hESC lines were established, and they grew at similar speed in both media (serum-containing or serum-free), but hESC cultured in serum-containing medium yielded significantly higher percentages of morphologically good colonies and cells expressing Oct-4. These cell lines differentiated spontaneously in vitro into cells expressing markers belonging to all three embryonic germ layers and germ cell markers, including c-Kit, STELLA, VASA and growth differentiation factor 9 (GDF9), in directly adherent culture. CONCLUSIONS: Three hESC lines with Taiwanese ancestry have been established, and they retain the in vitro differentiation potential with or without embryoid body (EB) formation. The data support that hESC may be capable of differentiation into germ cells although further confirmation is needed. It is also suggested that strategies such as stepwise adaptation will be needed before implementing a serum-free culture condition for hESC lines that have previously been derived in a medium containing serum.     

Neural differentiation in the OTT-6050 mouse teratoma: Effects of intracerebral environment on the neural differentiation of embryoid bodies

Summary Small embryoid bodies (EB's) from the OTT-6050 transplantable mouse teratoma, obtained by gravity filtration through a 74 mesh, were injected into the right cerebral hemisphere of syngeneic newborn or adult mice of both sexes in order to produce differentiating teratomas after a single passage. In subsequent experiments, two solid tumors resulting from two different EB-implants into the brains of adult hosts were used to initiate sequential tumors and were carried intracerebrally in adult mice for 12 and 18 passages respectively. The animals were sacrificed when signs of increased intracranial pressure developed. Survival times were as follows: single passages in adult mice: mean, 35 days; single passages in neonatal mice: mean, 19 days; sequential passages in adult mice: mean, 25 days. Multipotential stem cells accounted for l/2 to 3/4 of the cens in all tumors. Primitive neural cells, ependymoblastic rosettes, neuroblasts and glia were present in all; stem cells, primitive neural cells and rosettes decreased proportionately as the more differentiated neural populations became prominent. Mature ganglion cells were found only in the sequentially passaged tumors and in tumors maintained for more than one month after a single passage in adult mice. Synapses were noted in the most differentiated areas. Neuroblasts were infrequent in tumors developing in neonatal hosts, and mature ganglion cells were absent. Glial fibrillary acidic protein was present by the 24th day in tumors obtained in adult hosts after single passage and in sequential passages.Both in the OTT-6050-derived tumor fractions IB-9 and IB-21, previously reported, and in the EB-derived tumors described in the present study the cerebral microenvironment did not appear to have unique properties favoring neural differentiation and maturation, since similar neural features were found in their subcutaneous counterparts. The findings reported suggest that any accentuation of neuroepithelial differentiation elicited by injecting EB's either intracerebrally or subcutaneously is apparently directly related to the total time of in vivo maintenance of the tumor and therefore presumably to the length of time necessary for such maturation to occur.Supported by Research Grant CA 11689 of the National Cancer Institute and by Neuropathology Research Training Grant NS 5 T32 NS 7111 of the National Institute of Neurological and Communicative Diseases and Stroke, USPHS     

昆明小鼠胚胎干细胞向心肌细胞的分化

目的饲养层制作及复苏培养昆明小鼠胚胎干细胞,探索体外诱导胚胎干细胞向心肌细胞分化。方法饲养层制作,复苏及体外培养胚胎干细胞,采用一步法消化贴壁胚胎干细胞,悬浮培养5d,形成拟胚体(EB),待EB贴壁后,加入淫羊藿苷(Icraiin,ICA)诱导液对其诱导并每天观察,免疫荧光检测心肌细胞特异性肌钙蛋白T(cTnT)和心室肌球蛋白轻链(MLC-2v)的表达。结果胚胎干细胞悬浮聚集5d,形成类似球状的拟胚体,将拟胚体贴壁诱导8d,发现拟胚体中出现跳动,诱导后10d拟胚体跳动率达65%,显著高于空白对照组和阴性对照组,一个分化拟胚体中一般出现1-3个跳动点,节律约为50-80times/min,在诱导10d时跳动的合胞体,免疫荧光表明cTnT和MLC-2v表达为阳性。结论胚胎干细胞经悬浮聚集培养5d后经10-7mol/L淫羊藿苷诱导,得到了可以跳动的心肌细胞团。