人胚胎生殖干细胞在体内外对卵巢癌生长抑制的研究
|
摘要
【研究背景与目的】
卵巢恶性肿瘤是妇科三大恶性肿瘤之一,发病率位居女性生殖系统恶性肿瘤第三位,因缺乏特异性症状及有效的早期诊断手段,多数患者确诊时已为晚期。卵巢恶性肿瘤易转移及广泛播散,易复发,预后差,5年生存率仅30%左右,死亡率居妇科恶性肿瘤的首位。关于卵巢癌发生发展的机制也有较多研究探讨,但目前还未有明确的解释。近年来,随着干细胞的研究进展,人们发现肿瘤细胞与干细胞有较多相似性,因而提出了“肿瘤干细胞”的假说。此假说认为:肿瘤的发生、发展及复发归因于肿瘤组织内存在具有无限增殖能力的肿瘤干细胞,而肿瘤干细胞有可能来自于干细胞的突变或融合。随着肿瘤干细胞假说的提出,人们发现许多肿瘤组织中都存在这样一群数目稀少的具有干细胞特点的细胞,这些细胞可以在免疫缺陷的小鼠体内形成完整的肿瘤组织,被称为边缘(sidepopulation,SP)细胞。在几株卵巢癌细胞系中也发现了这种细胞。肿瘤干细胞假说的提出为肿瘤的发生机制探讨及治疗学的研究提出了新的思路,从干细胞的角度来研究探讨肿瘤的各种生物学行为等,使肿瘤细胞的重塑成为可能。
干细胞治疗是当今研究的热点,其中首推全能干细胞。全能干细胞是目前所知的最具有多向分化潜能的干细胞,包括胚胎干细胞、胚胎生殖干细胞等。由于其特殊的生物学特性,使其在细胞治疗、组织修复、发育生物学、药物学等领域有着极为广阔的应用前景,成为21世纪生物医学的热点。全能干细胞的自我更新及多向分化的潜能,在多种信号通路的调节下维持在稳定的正常的平衡状态。因而,我们猜测这种具有正常发展模式的全能干细胞是否能使肿瘤细胞的生长得到控制?全能干细胞在肿瘤研究领域的应用,多是先对其进行诱导分化,再支持进一步治疗,而这种直接研究的报道较少。但已有报道鼠胚胎干细胞可以在体外抑制胰腺癌细胞的生长。近年来又有研究表明人胚胎干细胞可以逆转黑色素瘤细胞的恶性行为。胚胎生殖干细胞是全能干细胞的一种,来源于胎儿生殖嵴的原始生殖细胞,在生物学行为上与来自内细胞团的胚胎干细胞相似,且取材方便,目前还未有其可形成畸胎瘤的报道。因而,我们通过体外共培养和体内动物模型,设计研究了人胚胎生殖干细胞对卵巢癌细胞生长的作用。
第一部分人胚胎生殖干细胞的分离、培养与鉴定
【研究目的】
从废弃流产胎儿的生殖嵴分离原始生殖细胞,培养并使其转化为人胚胎生殖干细胞,并为后续实验奠定基础。
【研究方法】
1、取孕13.5天小鼠的胎鼠背部皮肤,分离培养小鼠胚胎成纤维细胞,制备小鼠胚胎成纤维细胞(mouse embryonic fibroblast,MEF)饲养层。
2、取5-7周废弃的流产胎儿的生殖嵴及其周围组织,分离出人原始生殖细胞(human primary germ cells,hPGCs)。经体外分化抑制培养,获得由hPGCs转化成的人胚胎生殖干细胞(human embryonic germ cells,hEGCs),并在体外增殖传代,简单比较胰酶消化传代法与胶原酶+机械法消化传代法的差别,初步观察探讨人白血病抑制因子(leukemia inhibitory factor,LIF)的作用。
3、取不同代数的部分hEGCs克隆,检测其碱性磷酸酶活性、胚胎阶段特异性抗原(stage-specific embryonic antigen,SSEA)-1、3、4及TRA-1-60、TRA-1-81、OCT-4的表达,并对体外自发分化进行初步的观察,以鉴定该细胞。
【结果】
1、分离并纯化培养了小鼠胚胎成纤维细胞,取其3-5代制备MEF饲养层备用。
2、将消化后的5-7周流产胎儿的生殖嵴接种于MEF饲养层上,培养2天后就出现了较明显的细胞团。经过体外抑制培养,hEGCs可以传至第11代。在传代中发现胶原酶+机械法更适合hEGCs的传代生长。在培养中发现去除LIF对细胞体外分化抑制培养无明显的影响。
3、免疫组化及免疫荧光显示培养的细胞具有较高的碱性磷酸酶活性,并表达SSEA-1、3、4,TRA-1-60,TRA-1-81,OCT-4。符合文献报道的hEGCs的特征。撤去条件培养液后,hEGCs在体外自发分化为神经球样结构及具有自动收缩节律的心肌样细胞。
【结论】
成功从废弃流产胎儿的生殖嵴及其周围组织中分离出hPGCs,并经体外分化抑制培养,获得由hPGCs转化成的hEGCs。经初步鉴定该细胞符合hEGCs的特征,为后续实验奠定了基础。
第二部分人胚胎生殖干细胞在体内外对人卵巢癌细胞SKOV3生长的作用研究
【研究目的】
通过体外共培养研究人胚胎生殖干细胞对人卵巢癌细胞SKOV3生长的作用,并进行初步的机制探讨。利用SCID小鼠建立卵巢癌的动物模型,初步研究hEGCs在体内对卵巢癌生长的作用。
【研究方法】
一、体外实验
1、实验分两组,实验组将两种细胞先后接种在六孔板中共培养,对照组只接种SKOV3细胞,共培养期间两组的培养液相同。共培养48h后,通过HE染色比较两组SKOV3细胞生长的情况,并通过细胞计数统计两组SKOV3细胞的生长变化。
2、通过免疫组化的方法检测两组SKOV3细胞的tunel和caspase-9的表达情况,以观察细胞凋亡的变化。
3、通过western blot检测两组SKOV3细胞的AKT和p-AKT蛋白的表达;并通过realtime RT-PCR检测两组SKOV3细胞的akt mRNA的表达情况,并比较其差异。
二、体内实验
1、将40只小鼠随机分成两组,利用SKOV3卵巢癌细胞在SCID小鼠皮下建立人卵巢癌的动物模型。
2、在小鼠皮下出现可触到的瘤块时(约在接种后7天,瘤块直径约0.5cm),实验组将培养的一代或二代人胚胎生殖干细胞(约2×10~5/0.1mlPBS)注入肿瘤组织内,对照组仅注射0.1mlPBS,观察并比较两组动物的肿瘤生长情况。
3、在14天、24天、35天时分别处死两组部分小鼠,将肿瘤组织固定做石蜡切片,进行tunel凋亡检测。
【结果】
一、体外实验
1、共培养48h后,HE染色显示实验组SKOV3细胞的分布较对照组稀疏,且越靠近hEGCs克隆周围的SKOV3细胞越稀疏。细胞计数显示,共培养48h后,实验组SKOV3细胞计数为(7.8±2.4)×10~4,明显少于对照组(11.5±2.3)×10~4。
2、实验组tunel染色的阳性信号明显多于对照组,且阳性信号大多聚集于干细胞克隆周围。caspase-9染色的结果与tunel染色相似的,实验组的阳性信号也多于对照组。
3、western blot显示实验组p-AKT蛋白的表达明显弱于对照组,AKT蛋白表达无明显差异。realtime RT-PCR显示实验组akt mRNA的表达都少于对照组,尤以akt2 mRNA最为明显。
二、体内实验
1、同期观察测量发现实验组动物的肿瘤生长慢于对照组。
2、Tunel凋亡染色发现,同期肿瘤组织中实验组的凋亡信号多于对照组,且随时间延长实验组凋亡信号分布逐渐弥散增多。
【结论】
HEGCs可以在体外共培养中抑制SKOV3细胞生长。其机制可能是通过降调p-AKT和akt mRNA的表达,激活caspase-9,诱导SKOV3细胞的凋亡。体内初步实验发现hEGCs也可以抑制卵巢癌的生长,此作用可能是通过诱导了癌细胞的凋亡引起的。
第三部分微流控芯片技术在体外共培养hEGCs和SKOV3细胞中的应用
【研究目的】
利用微流控芯片技术共培养hEGCs和SKOV3细胞,并与孔板共培养方法进行比较。
【研究方法】
1、根据实验要求设计微流控芯片的模型,根据常规的步骤制备微流控芯片,消毒灭菌备用。
2、实验组将hEGCs和SKOV3细胞分别接种于微流控芯片中不同的培养槽中,对照组只接种SKOV3细胞于相应的培养槽中,共培养48h,保持培养液的单向流动。
3、共培养期间利用显微镜实时观察并记录实验组与对照组SKOV3细胞的生长情况。
4、共培养结束后,检测两组SKOV3细胞的tunel的表达情况。
5、用孔板共培养作为平行对照,比较两种培养方法。
【结果】
1、设计并制备了实验所需的微流控芯片,并达到细胞培养的要求。栅栏结构的设计使培养液可以缓慢单向的流动,在12h后在进液孔与出液孔之间达到平衡。
2、在微流控芯片中,我们可以实时观察到SKOV3细胞的生长情况,并可以直观的比较实验组与对照组SKOV3细胞生长的差异。实验组SKOV3细胞生长变化较小,而对照组可以看到明显的SKOV3细胞的生长增殖,通过测量SKOV3细胞生长的外缘距出液孔外缘的距离可以比较两组的差别。
3、微流控芯片中的SKOV3细胞的tunel结果显示实验组的凋亡信号多于对照组。实验组的凋亡信号沿着液流方向逐渐减少。
4、将微流控芯片共培养与孔板共培养比较,发现微流控芯片具有较多的优点,例如可以直观实时地观察细胞的生长变化,可以控制培养液的流向和流速,可以控制微环境的变化等。
【结论】
微流控芯片技术优于传统的共培养模式:1)微流控芯片技术可以把两种贴壁细胞分离开来共培养;2)可以实时监控细胞生长变化,便于比较。3)可以控制液流的方向,研究单向的作用;4)设计方便,花费较小。微流控芯片更适合于共培养和研究细胞之间的作用。通过分泌细胞因子来抑制SKOV3细胞的生长有可能是hEGCs作用的途径之一。
【Background】
Ovarian cancer is one of the three most common malignant gynecologic tumors, whose morbidity is the third in the women's genital cancers.Because of deficiency of special symptoms and effective earlier diagnosis,it is an advanced tumor when diagnosed.Ovarian cancer is generally characterized by poor prognosis,a high rate of recurrence and early metastases.The 5-year overall survival rate is approximately 30%.The cause of ovarian cancer is unknown.In recent years,the hypothesis of cancer stem cells has been put forward because of the similarities between the stem cells and cancer cells.It is supposed that there are cancer stem cells in the tumor which contribute to the development of cancer and cancer stem cells may come from the mutation of normal stem cells.Now some kinds of side population cells which have some characteristics resemble to stem cells have been found in several kinds of cancers including ovarian cancer.The hypothesis of cancer stem cells brings new perspectives for the treatment and plasticity of tumors.
Pluripotent stem cells are the most popular one in the stem cell treatment.They demonstrate self-renewal and display the ability to differentiate into a variety of normal cell types of all three primary cell lineages based on different microenvironments.They operate normally under the control of many factors.This comparison evokes our question:do the human pluripotent stem cells,that follow this normal developmental process,have the potential to keep the growth of tumor cells under control? There is some evidence that embryonic stem(ES)cells may inhibit somatic cell growth.Lightfoot et al.reported that mouse ES cells could inhibit the growth of human pancreatic carcinoma cells in vitro.A recent study by Hendrix et al. demonstrated that metastatic melanoma cells could be reverted to a normal,skin cell-like type with the ability to form colonies similar to human embryonic stem cells (hESCs)under the microenvironment of two hESCs lines.
The human embryonic germ cell(hEGC),that has similar characteristics to embryonic stem cells coming from the inner cells,belongs to the pluripotent stem cell and comes from primordial germ cells.Our current study investigated the potential effect of hEGCs on a human ovarian cancer cell line(SKOV3)both in vitro and in vivo.
PART 1
THE ISOLATION,CULTURE AND IDENTIFICATION OF HUMAN EMBRYONIC GERM CELLS
Objective:
To isolate human primary germ cells from the gonads of abortion fetuses and turn them into human embryonic germ cells through conditioned culture.
Methods:
1、The mouse embryonic fibroblast coming from day 13 post-coitus fetuses of KM mouse was used as feeder layer cells.
2、Human primary germ cells were isolated from the gonads of abortion embryos at 5-7 weeks postconception.Human embryonic germ cells were cultured and passaged under conditioned medium.During the passage,we compared the effect of collagenase IV + mechanical digestion and trypsin.Whether LIF has effect on the culture of human embryonic germ cells was simply investigated.
3、Some hEGCs clones were taken out for characterization.Alkaline phosphatase (AKP)activity and Antibodies for stage-specific embryonic antigens(SSEA-1,3, 4),OCT-4,TRA-1-60,TRA-1-81 were detected.Spontaneous differentiation was also tested.
Results:
1、The mouse embryonic fibroblasts were cultured and passaged.Three to five passage were used as feeder layer cells.
2、After 2-3 days' primary culture,groups of tightly packed cells with distinct cell borders were recognized.HEGCs could be cultured and passaged to passage 11 under conditioned medium.Collagenase IV + mechanical digestion was fit for the passage of hEGCs.Little difference was observed after LIF was taken out of the conditioned medium.
3、Analysis was performed on some hEG clones at different passages and high level of AP activity was shown in EG clones.In addition to AKP activity,EG clones were characterized by a range of cell surface markers including:SSEA-1,SSEA-3, SSEA-4,OCT-4,TRA-1-60,TRA-1-81.When we replaced the conditioned culture medium with DMEM/F-12 and 10%heat-inactivated fetal calf serum, hEGCs could spontaneous differentiate into nerve-sphere liked cells or cells which could contract automatically.
Conclusion:
We successfully isolated and cultured hEGCs which conform to the characterization reported.
PART 2
THE EFFECT OF HUMAN EMBRYONIC GERM CELLS ON SKOV3 CELLS IN VITRO AND IN VIVO
Objective:
To investigate the effect of human embryonic germ cells on SKOV3 in vitro through coculture and in vivo using a xenograft model in SCID mouse.
Methods:
1、In vitro experiment
1)、The SKOV3 cells and hEGCs were seeded orderly onto the 6-well plates for coculture.Only SKOV3 cells were cultured in the control groups.After coculturing for 48h,HE and cell counting were performed to compare the proliferation of SKOV3 cells between the two groups.
2)、Tunel apoptosis and caspase-9 activity were detected by immtmocytochemistry.
3)、The expression of AKT and p-AKT were examined through western blot.At the same time,akt mRNA was measured by real-time PCR.
2、In vivo experiment
1)、Forty mice were randomly separated into two groups.The SKOV3 cells were subcutaneously injected into the fight flank of the SCID mice to form a xenograft model.
2)、Seven days after SKOV3 cells injection,2×10~5 EG cells in 0.1ml PBS were injected into the tumors in the experiment groups,only 0.1ml PBS was injected into the tumors in control groups.Tumor volume was assessed by measuring two axes(R1,R2)and calculated using the formula:V=1/6πR12 R2.
3)、The mice were sacrificed respectively at day 14,24,35.Tumor tissues were fixed in 10%formaldehyde,and embedded in paraffin wax,then cut into 6-μm-thick sections.The sections were also taken for tunel apoptosis assay.
Results:
1、In vitro experiment
1)、After coculturing,HE staining exhibited that SKOV3 cells near the EG clones were sparser than those far off.Cell counting showed that there was a 1.5-fold growth reduction for SKOV3 cells in the coculture group.There were(7.8±2.4)×10~4 in the coculture group,while(11.5±2.3)×10~4 in the control group.
2)、There were more positive signals of tunel and caspase-9 in the coculture group, especially near the clones,than the control group.
3)、SKOV3 cells in the coculture group showed less p-AKT and akt mRNA than those in the control group.
2、In vivo experiment
1)、The time-course of tumor volume change was detected.It is noteworthy that the rumors in the EG group grew dramatically slower than those in the control group.
2)、More positive signals of the tunel apoptosis were detected in the experiment group.
Conclusion:
The hEGCs could inhibit the growth of SKOV3 cells in vitro by inducing apoptosis by inhibiting AKT pathway and activating caspase-9.The transplantation of EG cells suppressed the growth of tumor which may be caused by inducing apoptosis of SKOV3 cells.
PART 3
COCULTURE HUMAN EMBRYONIC GERM CELLS WITH SKOV3 CELLS ON A MICROFLUIDIC CHIP
Objective:
A well-designed microfluidic device with unidirectional-perfusion has been developed to observe the effect of human embryonic germ(hEG)cells on SKOV3 cells.
Methods:
1、The microfluidic chip was designed according our experiment and fabricated as reported.
2、The hEGCs and SKOV3 cells in the experiment group were seeded in the inlet reserviors and the outlet reserviors separately,and were cocultured for two days. In the control group,just SKOV3 cells were seeded in the outlet reservoirs.The medium was perfused unidirectionally from the inlet to the outlet reserviors.
3、The growth of SKOV3 cells were observed online.
4、Tunel apoptosis was detected after coculturing.
5、Coculture on 6-well plate was used as a parallel group to compare the differences between the two cell culture methods.
Results:
1、A microfluidic chip suitable for cell culturing was designed according to the experiment.The barrier makes it possible that medium can flow slowly and come to the balance between the inlet reserviors and the outlet reservoirs after 12h.
2、Using our device,the growth of SKOV3 cells could be observed online.The growth inhibition of SKOV3 cells by hEG cells was monitored intuitionally.
3、More apoptosis signals in SKOV3 cells culture area were detected in the coculture group,which decreased along flowing of the medium.
4、Compared with the traditional coculture,microfluidic chip showed many advantages in the experiment.
Conclusion:
In conclusion,the results demonstrated that microfluidic chip might be a potential tool to invest the effect of stem cells on cancer cells with intuitionistic cell-based screens.The perfused microfluidic system could control the change of culture microenvironment better and assure uniform perfusion of the cell culture media throughout the cell culture chamber.
卵巢恶性肿瘤是妇科三大恶性肿瘤之一,发病率位居女性生殖系统恶性肿瘤第三位,因缺乏特异性症状及有效的早期诊断手段,多数患者确诊时已为晚期。卵巢恶性肿瘤易转移及广泛播散,易复发,预后差,5年生存率仅30%左右,死亡率居妇科恶性肿瘤的首位。关于卵巢癌发生发展的机制也有较多研究探讨,但目前还未有明确的解释。近年来,随着干细胞的研究进展,人们发现肿瘤细胞与干细胞有较多相似性,因而提出了“肿瘤干细胞”的假说。此假说认为:肿瘤的发生、发展及复发归因于肿瘤组织内存在具有无限增殖能力的肿瘤干细胞,而肿瘤干细胞有可能来自于干细胞的突变或融合。随着肿瘤干细胞假说的提出,人们发现许多肿瘤组织中都存在这样一群数目稀少的具有干细胞特点的细胞,这些细胞可以在免疫缺陷的小鼠体内形成完整的肿瘤组织,被称为边缘(sidepopulation,SP)细胞。在几株卵巢癌细胞系中也发现了这种细胞。肿瘤干细胞假说的提出为肿瘤的发生机制探讨及治疗学的研究提出了新的思路,从干细胞的角度来研究探讨肿瘤的各种生物学行为等,使肿瘤细胞的重塑成为可能。
干细胞治疗是当今研究的热点,其中首推全能干细胞。全能干细胞是目前所知的最具有多向分化潜能的干细胞,包括胚胎干细胞、胚胎生殖干细胞等。由于其特殊的生物学特性,使其在细胞治疗、组织修复、发育生物学、药物学等领域有着极为广阔的应用前景,成为21世纪生物医学的热点。全能干细胞的自我更新及多向分化的潜能,在多种信号通路的调节下维持在稳定的正常的平衡状态。因而,我们猜测这种具有正常发展模式的全能干细胞是否能使肿瘤细胞的生长得到控制?全能干细胞在肿瘤研究领域的应用,多是先对其进行诱导分化,再支持进一步治疗,而这种直接研究的报道较少。但已有报道鼠胚胎干细胞可以在体外抑制胰腺癌细胞的生长。近年来又有研究表明人胚胎干细胞可以逆转黑色素瘤细胞的恶性行为。胚胎生殖干细胞是全能干细胞的一种,来源于胎儿生殖嵴的原始生殖细胞,在生物学行为上与来自内细胞团的胚胎干细胞相似,且取材方便,目前还未有其可形成畸胎瘤的报道。因而,我们通过体外共培养和体内动物模型,设计研究了人胚胎生殖干细胞对卵巢癌细胞生长的作用。
第一部分人胚胎生殖干细胞的分离、培养与鉴定
【研究目的】
从废弃流产胎儿的生殖嵴分离原始生殖细胞,培养并使其转化为人胚胎生殖干细胞,并为后续实验奠定基础。
【研究方法】
1、取孕13.5天小鼠的胎鼠背部皮肤,分离培养小鼠胚胎成纤维细胞,制备小鼠胚胎成纤维细胞(mouse embryonic fibroblast,MEF)饲养层。
2、取5-7周废弃的流产胎儿的生殖嵴及其周围组织,分离出人原始生殖细胞(human primary germ cells,hPGCs)。经体外分化抑制培养,获得由hPGCs转化成的人胚胎生殖干细胞(human embryonic germ cells,hEGCs),并在体外增殖传代,简单比较胰酶消化传代法与胶原酶+机械法消化传代法的差别,初步观察探讨人白血病抑制因子(leukemia inhibitory factor,LIF)的作用。
3、取不同代数的部分hEGCs克隆,检测其碱性磷酸酶活性、胚胎阶段特异性抗原(stage-specific embryonic antigen,SSEA)-1、3、4及TRA-1-60、TRA-1-81、OCT-4的表达,并对体外自发分化进行初步的观察,以鉴定该细胞。
【结果】
1、分离并纯化培养了小鼠胚胎成纤维细胞,取其3-5代制备MEF饲养层备用。
2、将消化后的5-7周流产胎儿的生殖嵴接种于MEF饲养层上,培养2天后就出现了较明显的细胞团。经过体外抑制培养,hEGCs可以传至第11代。在传代中发现胶原酶+机械法更适合hEGCs的传代生长。在培养中发现去除LIF对细胞体外分化抑制培养无明显的影响。
3、免疫组化及免疫荧光显示培养的细胞具有较高的碱性磷酸酶活性,并表达SSEA-1、3、4,TRA-1-60,TRA-1-81,OCT-4。符合文献报道的hEGCs的特征。撤去条件培养液后,hEGCs在体外自发分化为神经球样结构及具有自动收缩节律的心肌样细胞。
【结论】
成功从废弃流产胎儿的生殖嵴及其周围组织中分离出hPGCs,并经体外分化抑制培养,获得由hPGCs转化成的hEGCs。经初步鉴定该细胞符合hEGCs的特征,为后续实验奠定了基础。
第二部分人胚胎生殖干细胞在体内外对人卵巢癌细胞SKOV3生长的作用研究
【研究目的】
通过体外共培养研究人胚胎生殖干细胞对人卵巢癌细胞SKOV3生长的作用,并进行初步的机制探讨。利用SCID小鼠建立卵巢癌的动物模型,初步研究hEGCs在体内对卵巢癌生长的作用。
【研究方法】
一、体外实验
1、实验分两组,实验组将两种细胞先后接种在六孔板中共培养,对照组只接种SKOV3细胞,共培养期间两组的培养液相同。共培养48h后,通过HE染色比较两组SKOV3细胞生长的情况,并通过细胞计数统计两组SKOV3细胞的生长变化。
2、通过免疫组化的方法检测两组SKOV3细胞的tunel和caspase-9的表达情况,以观察细胞凋亡的变化。
3、通过western blot检测两组SKOV3细胞的AKT和p-AKT蛋白的表达;并通过realtime RT-PCR检测两组SKOV3细胞的akt mRNA的表达情况,并比较其差异。
二、体内实验
1、将40只小鼠随机分成两组,利用SKOV3卵巢癌细胞在SCID小鼠皮下建立人卵巢癌的动物模型。
2、在小鼠皮下出现可触到的瘤块时(约在接种后7天,瘤块直径约0.5cm),实验组将培养的一代或二代人胚胎生殖干细胞(约2×10~5/0.1mlPBS)注入肿瘤组织内,对照组仅注射0.1mlPBS,观察并比较两组动物的肿瘤生长情况。
3、在14天、24天、35天时分别处死两组部分小鼠,将肿瘤组织固定做石蜡切片,进行tunel凋亡检测。
【结果】
一、体外实验
1、共培养48h后,HE染色显示实验组SKOV3细胞的分布较对照组稀疏,且越靠近hEGCs克隆周围的SKOV3细胞越稀疏。细胞计数显示,共培养48h后,实验组SKOV3细胞计数为(7.8±2.4)×10~4,明显少于对照组(11.5±2.3)×10~4。
2、实验组tunel染色的阳性信号明显多于对照组,且阳性信号大多聚集于干细胞克隆周围。caspase-9染色的结果与tunel染色相似的,实验组的阳性信号也多于对照组。
3、western blot显示实验组p-AKT蛋白的表达明显弱于对照组,AKT蛋白表达无明显差异。realtime RT-PCR显示实验组akt mRNA的表达都少于对照组,尤以akt2 mRNA最为明显。
二、体内实验
1、同期观察测量发现实验组动物的肿瘤生长慢于对照组。
2、Tunel凋亡染色发现,同期肿瘤组织中实验组的凋亡信号多于对照组,且随时间延长实验组凋亡信号分布逐渐弥散增多。
【结论】
HEGCs可以在体外共培养中抑制SKOV3细胞生长。其机制可能是通过降调p-AKT和akt mRNA的表达,激活caspase-9,诱导SKOV3细胞的凋亡。体内初步实验发现hEGCs也可以抑制卵巢癌的生长,此作用可能是通过诱导了癌细胞的凋亡引起的。
第三部分微流控芯片技术在体外共培养hEGCs和SKOV3细胞中的应用
【研究目的】
利用微流控芯片技术共培养hEGCs和SKOV3细胞,并与孔板共培养方法进行比较。
【研究方法】
1、根据实验要求设计微流控芯片的模型,根据常规的步骤制备微流控芯片,消毒灭菌备用。
2、实验组将hEGCs和SKOV3细胞分别接种于微流控芯片中不同的培养槽中,对照组只接种SKOV3细胞于相应的培养槽中,共培养48h,保持培养液的单向流动。
3、共培养期间利用显微镜实时观察并记录实验组与对照组SKOV3细胞的生长情况。
4、共培养结束后,检测两组SKOV3细胞的tunel的表达情况。
5、用孔板共培养作为平行对照,比较两种培养方法。
【结果】
1、设计并制备了实验所需的微流控芯片,并达到细胞培养的要求。栅栏结构的设计使培养液可以缓慢单向的流动,在12h后在进液孔与出液孔之间达到平衡。
2、在微流控芯片中,我们可以实时观察到SKOV3细胞的生长情况,并可以直观的比较实验组与对照组SKOV3细胞生长的差异。实验组SKOV3细胞生长变化较小,而对照组可以看到明显的SKOV3细胞的生长增殖,通过测量SKOV3细胞生长的外缘距出液孔外缘的距离可以比较两组的差别。
3、微流控芯片中的SKOV3细胞的tunel结果显示实验组的凋亡信号多于对照组。实验组的凋亡信号沿着液流方向逐渐减少。
4、将微流控芯片共培养与孔板共培养比较,发现微流控芯片具有较多的优点,例如可以直观实时地观察细胞的生长变化,可以控制培养液的流向和流速,可以控制微环境的变化等。
【结论】
微流控芯片技术优于传统的共培养模式:1)微流控芯片技术可以把两种贴壁细胞分离开来共培养;2)可以实时监控细胞生长变化,便于比较。3)可以控制液流的方向,研究单向的作用;4)设计方便,花费较小。微流控芯片更适合于共培养和研究细胞之间的作用。通过分泌细胞因子来抑制SKOV3细胞的生长有可能是hEGCs作用的途径之一。
【Background】
Ovarian cancer is one of the three most common malignant gynecologic tumors, whose morbidity is the third in the women's genital cancers.Because of deficiency of special symptoms and effective earlier diagnosis,it is an advanced tumor when diagnosed.Ovarian cancer is generally characterized by poor prognosis,a high rate of recurrence and early metastases.The 5-year overall survival rate is approximately 30%.The cause of ovarian cancer is unknown.In recent years,the hypothesis of cancer stem cells has been put forward because of the similarities between the stem cells and cancer cells.It is supposed that there are cancer stem cells in the tumor which contribute to the development of cancer and cancer stem cells may come from the mutation of normal stem cells.Now some kinds of side population cells which have some characteristics resemble to stem cells have been found in several kinds of cancers including ovarian cancer.The hypothesis of cancer stem cells brings new perspectives for the treatment and plasticity of tumors.
Pluripotent stem cells are the most popular one in the stem cell treatment.They demonstrate self-renewal and display the ability to differentiate into a variety of normal cell types of all three primary cell lineages based on different microenvironments.They operate normally under the control of many factors.This comparison evokes our question:do the human pluripotent stem cells,that follow this normal developmental process,have the potential to keep the growth of tumor cells under control? There is some evidence that embryonic stem(ES)cells may inhibit somatic cell growth.Lightfoot et al.reported that mouse ES cells could inhibit the growth of human pancreatic carcinoma cells in vitro.A recent study by Hendrix et al. demonstrated that metastatic melanoma cells could be reverted to a normal,skin cell-like type with the ability to form colonies similar to human embryonic stem cells (hESCs)under the microenvironment of two hESCs lines.
The human embryonic germ cell(hEGC),that has similar characteristics to embryonic stem cells coming from the inner cells,belongs to the pluripotent stem cell and comes from primordial germ cells.Our current study investigated the potential effect of hEGCs on a human ovarian cancer cell line(SKOV3)both in vitro and in vivo.
PART 1
THE ISOLATION,CULTURE AND IDENTIFICATION OF HUMAN EMBRYONIC GERM CELLS
Objective:
To isolate human primary germ cells from the gonads of abortion fetuses and turn them into human embryonic germ cells through conditioned culture.
Methods:
1、The mouse embryonic fibroblast coming from day 13 post-coitus fetuses of KM mouse was used as feeder layer cells.
2、Human primary germ cells were isolated from the gonads of abortion embryos at 5-7 weeks postconception.Human embryonic germ cells were cultured and passaged under conditioned medium.During the passage,we compared the effect of collagenase IV + mechanical digestion and trypsin.Whether LIF has effect on the culture of human embryonic germ cells was simply investigated.
3、Some hEGCs clones were taken out for characterization.Alkaline phosphatase (AKP)activity and Antibodies for stage-specific embryonic antigens(SSEA-1,3, 4),OCT-4,TRA-1-60,TRA-1-81 were detected.Spontaneous differentiation was also tested.
Results:
1、The mouse embryonic fibroblasts were cultured and passaged.Three to five passage were used as feeder layer cells.
2、After 2-3 days' primary culture,groups of tightly packed cells with distinct cell borders were recognized.HEGCs could be cultured and passaged to passage 11 under conditioned medium.Collagenase IV + mechanical digestion was fit for the passage of hEGCs.Little difference was observed after LIF was taken out of the conditioned medium.
3、Analysis was performed on some hEG clones at different passages and high level of AP activity was shown in EG clones.In addition to AKP activity,EG clones were characterized by a range of cell surface markers including:SSEA-1,SSEA-3, SSEA-4,OCT-4,TRA-1-60,TRA-1-81.When we replaced the conditioned culture medium with DMEM/F-12 and 10%heat-inactivated fetal calf serum, hEGCs could spontaneous differentiate into nerve-sphere liked cells or cells which could contract automatically.
Conclusion:
We successfully isolated and cultured hEGCs which conform to the characterization reported.
PART 2
THE EFFECT OF HUMAN EMBRYONIC GERM CELLS ON SKOV3 CELLS IN VITRO AND IN VIVO
Objective:
To investigate the effect of human embryonic germ cells on SKOV3 in vitro through coculture and in vivo using a xenograft model in SCID mouse.
Methods:
1、In vitro experiment
1)、The SKOV3 cells and hEGCs were seeded orderly onto the 6-well plates for coculture.Only SKOV3 cells were cultured in the control groups.After coculturing for 48h,HE and cell counting were performed to compare the proliferation of SKOV3 cells between the two groups.
2)、Tunel apoptosis and caspase-9 activity were detected by immtmocytochemistry.
3)、The expression of AKT and p-AKT were examined through western blot.At the same time,akt mRNA was measured by real-time PCR.
2、In vivo experiment
1)、Forty mice were randomly separated into two groups.The SKOV3 cells were subcutaneously injected into the fight flank of the SCID mice to form a xenograft model.
2)、Seven days after SKOV3 cells injection,2×10~5 EG cells in 0.1ml PBS were injected into the tumors in the experiment groups,only 0.1ml PBS was injected into the tumors in control groups.Tumor volume was assessed by measuring two axes(R1,R2)and calculated using the formula:V=1/6πR12 R2.
3)、The mice were sacrificed respectively at day 14,24,35.Tumor tissues were fixed in 10%formaldehyde,and embedded in paraffin wax,then cut into 6-μm-thick sections.The sections were also taken for tunel apoptosis assay.
Results:
1、In vitro experiment
1)、After coculturing,HE staining exhibited that SKOV3 cells near the EG clones were sparser than those far off.Cell counting showed that there was a 1.5-fold growth reduction for SKOV3 cells in the coculture group.There were(7.8±2.4)×10~4 in the coculture group,while(11.5±2.3)×10~4 in the control group.
2)、There were more positive signals of tunel and caspase-9 in the coculture group, especially near the clones,than the control group.
3)、SKOV3 cells in the coculture group showed less p-AKT and akt mRNA than those in the control group.
2、In vivo experiment
1)、The time-course of tumor volume change was detected.It is noteworthy that the rumors in the EG group grew dramatically slower than those in the control group.
2)、More positive signals of the tunel apoptosis were detected in the experiment group.
Conclusion:
The hEGCs could inhibit the growth of SKOV3 cells in vitro by inducing apoptosis by inhibiting AKT pathway and activating caspase-9.The transplantation of EG cells suppressed the growth of tumor which may be caused by inducing apoptosis of SKOV3 cells.
PART 3
COCULTURE HUMAN EMBRYONIC GERM CELLS WITH SKOV3 CELLS ON A MICROFLUIDIC CHIP
Objective:
A well-designed microfluidic device with unidirectional-perfusion has been developed to observe the effect of human embryonic germ(hEG)cells on SKOV3 cells.
Methods:
1、The microfluidic chip was designed according our experiment and fabricated as reported.
2、The hEGCs and SKOV3 cells in the experiment group were seeded in the inlet reserviors and the outlet reserviors separately,and were cocultured for two days. In the control group,just SKOV3 cells were seeded in the outlet reservoirs.The medium was perfused unidirectionally from the inlet to the outlet reserviors.
3、The growth of SKOV3 cells were observed online.
4、Tunel apoptosis was detected after coculturing.
5、Coculture on 6-well plate was used as a parallel group to compare the differences between the two cell culture methods.
Results:
1、A microfluidic chip suitable for cell culturing was designed according to the experiment.The barrier makes it possible that medium can flow slowly and come to the balance between the inlet reserviors and the outlet reservoirs after 12h.
2、Using our device,the growth of SKOV3 cells could be observed online.The growth inhibition of SKOV3 cells by hEG cells was monitored intuitionally.
3、More apoptosis signals in SKOV3 cells culture area were detected in the coculture group,which decreased along flowing of the medium.
4、Compared with the traditional coculture,microfluidic chip showed many advantages in the experiment.
Conclusion:
In conclusion,the results demonstrated that microfluidic chip might be a potential tool to invest the effect of stem cells on cancer cells with intuitionistic cell-based screens.The perfused microfluidic system could control the change of culture microenvironment better and assure uniform perfusion of the cell culture media throughout the cell culture chamber.
引文
1、裴雪涛.干细胞生物学.科学出版社.2003.7.6.
2、Zhang SC,Wemig M,Duncan ID,Brustle O,Thomson JA.In vitro differentiation of transplantable neural precursors from human embryonic stem cells.Nat Biotechnol.2001;19:1129-1133.
3、Kaufman DS,Hanson ET,Lewis RL,Auerbach R,Thomson JA.Hematopoietic colony-forming cells derived from human embryonic stem cells.Proc Natl Acad Sci U S A.2001;98:10716-10721.
4、Xu C,Police S,Rao N,Carpenter MK.Characterization and enrichment of cardiomyocytes derived from human embryonic stem cells.Circ Res.2002;91:501-508.
5、He JQ,Ma Y,Lee Y,Thomson JA,Kamp TJ.Human embryonic stem cells develop into multiple types of cardiac myocytes:action potential characterization.Circ Res.2003;93:32-39.
6、Assady S,Maor G,Amit M,Itskovitz-Eldor J,Skorecki KL,Tzukerman M.Insulin production by human embryonic stem cells.Diabetes.2001;50:1691-1697.
7、Zhan X,Dravid G,Ye Z,Hammond H,Shamblott M,Gearhart J,Cheng L.Functional antigen-presenting leucocytes derived from human embryonic stem cells in vitro.Lancet.2004;364:163-171.
8、Evans MJ,Kaufman MH.Establishment in culture of pluripotential cells from mouse embryos.Nature.1981;292:154-156.
9、Thomson JA,Kalishman J,Golos TG,Durning M,Harris CP,Becker RA,Hearn JP. Isolation of a primate embryonic stem cell line. Proc Natl Acad Sci U S A. 1995;92:7844-7848.
10、 Thomson JA, Kalishman J, Golos TG, Durning M, Harris CP, Hearn JP. Pluripotent cell lines derived from common marmoset (Callithrix jacchus) blastocysts. Biol Reprod. 1996;55:254-259.
11、 Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282:1145-1147.
12、 Shamblott MJ, Axelman J, Wang S, Bugg EM, Littlefield JW, Donovan PJ, Blumenthal PD, Huggins GR, Gearhart JD. Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc Natl Acad Sci U S A. 1998;95:13726-13731.
13、 Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T, Okita K, Mochiduki Y, Takizawa N, Yamanaka S. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol. 2008;26:101-6.
14、 Chung Y, Klimanskaya I, Becker S, Li T, Maserati M, Lu SJ , Zdravkovic T, Ilic D, Genbacev O, Fisher S, Krtolica A, Lanzal R. Human Embryonic Stem Cell Lines Generated without Embryo Destruction. Cell Stem Cell. 2008; 2:113-117.
15、 Cowan CA, Klimanskaya I, McMahon J, Atienza J, Witmyer J, Zucker JP, Wang S, Morton CC, McMahon AP, Powers D, Melton DA. Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med. 2004;350:1353-1356.
16、 Zhang QX, Li Y, Huang SL, He ZX. Human embryonic stem cells preliminarily established in China. Chin Med J. 2002;82:1314-1318.
17、 Li F, Liu Y, Chen D, Lin X, Li J, Wang J, Peng Y, Wang S, Wang Y. Leukemia inhibitory factor-expressing human embryonic lung fibroblasts as feeder cells for human embryonic germ cells. Cells Tissues Organs. 2007;186:221-8.
18、 Saxena S, Hanwate M, Deb K, Sharma V, Totey S. FGF2 secreting human fibroblast feeder cells: A novel culture system for human embryonic stem cells. Mol Reprod Dev. 2008 Mar 3.
19、 Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, Carpenter MK. Feeder free growth of undifferentiated human embryonic stem cells. Nat biotechnol. 2001;19:971-975.
20、 Amit M, Shariki C, Margulets V, Itskovitz-Eldor J. Feeder layer- and serum-free culture of human embryonic stem cells. Biol Reprod. 2004;70:837-45.
21、 Ginis I, Luo Y, Miura T, Thies S, Brandenberger R, Gerecht-Nir S, Amit M, Hoke A, Carpenter MK, Itskovitz-Eldor J, Rao MS. Differences between human and mouse embryonic stem cells. 2004;269:360-80.
22、 Xu RH, Peck RM, Li DS, Feng X, Ludwig T, Thomson JA. Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nat Methods. 2005;2:185-190.
23、 Xu C, Rosier E, Jiang J, Lebkowski JS, Gold JD, O'Sullivan C, Delavan-Boorsma K, Mok M, Bronstein A, Carpenter MK. Basic fibroblast growth factor supports undifferentiated human embryonic stem cell growth without conditioned medium. Stem Cells. 2005;23:315-23.
24、 Levenstein ME, Ludwig TE, Xu RH, Lianas RA, VanDenHeuvel-Kramer K, Manning D, Thomson JA. Basic Fibroblast Growth Factor Support of Human Embryonic Stem Cell. Stem Cells. 2006;24:568-574.
25、 James D, Levine AJ, Besser D, Hemmati-Brivanlou A. TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells. Development. 2005; 132:1273-82.
26、 Dravid G, Ye Z, Hammond H, Chen G, Pyle A, Donovan P, Yu X, Cheng L. Defining the role of Wnt/beta-catenin signaling in the survival, proliferation, and selfrenewal of human embryonic stem cells. Stem Cells. 2005;23:1489-501.
27、 Metallo CM, Azarin SM, Ji L, de Pablo JJ, Palecek SP. Engineering tissue from human embryonic stem cells. J Cell Mol Med. 2008 Jan 11.
28、 Stewart R, Stojkovic M, Lako M. Mechanisms of self-renewal in human embryonic stem cells. Eur J Cancer. 2006;42:1257-72.
29、 Park JH, Kim SJ, Lee JB, Song JM, Kim CG, Roh S 2nd, Yoon HS. Establishment of a human embryonic germ cell line and comparison with mouse and human embryonic stem cells.Mol Cells. 2004; 17:309-15.
30、 Byrne JA, Mitalipov SM, Clepper L, Wolf DP. Transcriptional profiling of rhesus monkey embryonic stem cells. Biol Reprod. 2006;75:908-15.
31、 Buaas FW, Kirsh AL, Sharma M, McLean DJ, Morris JL, Griswold MD, de Rooij DG, Braun RE. Plzf is required in adult male germ cells for stem cell self-renewal. Nat Genet. 2004;36:647-652.
32、 Perrier AL, Tabar V, Barberi T, Rubio ME, Bruses J, Topf N, Harrison NL, Studer L. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci U S A. 2004,101: 12543-12548.
33、 Khanna A, Swistowska AM, Rao MS, Zeng X. Generation and transplantation of dopaminergic neurons derived from embryonic stem cells. Curr Stem Cell Res Ther. 2007;2:139-47.
34、 Rolletschek A, Chang H, Guan K, Czyz J, Meyer M, Wobus AM. Differentiation of embryonic stem cell-derived dopaminergic neurons is enhanced by survival-promoting factors. Mech Dev. 2001; 105:93-104.
35、 Cho MS, Lee YE, Kim JY, Chung S, Cho YH, Kim DS, Kang SM, Lee H, Kim MH, Kim JH, Leem JW, Oh SK, Choi YM, Hwang DY, Chang JW, Kim DW. Highly efficient and large-scale generation of functional dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci U S A. 2008; 105:3392-7.
36、 Lamba DA, Karl MO, Ware CB, Reh TA. Efficient generation of retinal progenitor cells from human embryonic stem cells. Proc Natl Acad Sci U S A. 2006; 103:12769-74.
37、 Coraux C, Hilmi C, Rouleau M, Spadafora A, Hinnrasky J, Ortonne JP, Dani C, Aberdam D. Reconstituted skin from murine embryonic stem cells. Curr Biol. 2003;13:849-853.
38、 Metallo CM, Vodyanik MA, de Pablo JJ, Slukvin II, Palecek SP. The response of human embryonic stem cell-derived endothelial cells to shear stress. Biotechnol Bioeng. 2008 Jan 30.
39、 Chen Y, Amende I, Hampton TG, Yang Y, Ke Q, Min JY, Xiao YF, Morgan JP. Vascular endothelial growth factor promotes cardiomyocyte differentiation of embryonic stem cells. Am J Physiol Heart Circ Physiol. 2006;291:H1653-8.
40、 Mummery C, van der Heyden MA, de Boer TP, Passier R, Ward D, van den Brink S, van Rooijen M, van de Stolpe A. Cardiomyocytes from human and mouse embryonic stem cells. Methods Mol Med. 2007; 140:249-72.
41、 Xiao Q, Zeng L, Zhang Z, Margariti A, Ali ZA, Channon KM, Xu Q, Hu Y. Sca-1~+ progenitors derived from embryonic stem cells differentiate into endothelial cells capable of vascular repair after arterial injury. Arterioscler Thromb Vasc Biol. 2006;26:2244-51.
42、 Yamahara K, Sone M, Itoh H, Yamashita JK, Yurugi-Kobayashi T, Homma K, Chao TH, Miyashita K, Park K, Oyamada N, Sawada N, Taura D, Fukunaga Y, Tamura N, Nakao K. Augmentation of neovascularizaiton in hindlimb ischemia by combined transplantation of human embryonic stem cells-derived endothelial and mural cells. LoS ONE. 2008;3:e1666.
43、 Kurita R, Sasaki E, Yokoo T, Hiroyama T, Takasugi K, Imoto H, Izawa K, Dong Y, Hashiguchi T, Soda Y, Maeda T, Suehiro Y, Tanioka Y, Nakazaki Y, Tani K. Tall/Scl Gene Transduction Using a Lentiviral Vector Stimulates Highly Efficient Hematopoietic Cell Differentiation from Common Marmoset (Callithrix jacchus) Embryonic Stem Cells. Stem Cells. 2006;24:2014-2022.
44、 Chang KH, Nelson AM, Cao H, Wang L1, Nakamoto B, Ware CB, Papayannopoulou T. Definitive-like erythroid cells derived from human embryonic stem cells coexpress high levels of embryonic and fetal globins with little or no adult globin. Blood. 2006; 108:1515-23.
45、 Vodyanik MA, Bork JA, Thomson JA, Slukvin II. Human embryonic stem cell-derived CD34+ cells: efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential. Blood. 2005; 105:617-26.
46、 Woll PS, Martin CH, Miller JS, Kaufman DS. Human embryonic stem cell-derived NK cells acquire functional receptors and cytolytic activity. J Immunol. 2005;175:5095-103.
47、 Slukvin II, Vodyanik MA, Thomson JA, Gumenyuk ME, Choi KD. Directed differentiation of human embryonic stem cells into functional dendritic cells through the myeloid pathway. J Immunol. 2006; 176:2924-32.
48、 Galic Z, Kitchen SG, Kacena A, Subramanian A, Burke B, Cortado R, Zack JA. T lineage differentiation from human embryonic stem cells. Proc Natl Acad Sci U S A. 2006;103:11742-7.
49、 Anderson JS, Bandi S, Kaufman DS, Akkina R. Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy. Retrovirology. 2006;3:24.
50、 Trivedi P, Hematti P. Derivation and immunological characterization of mesenchymal stromal cells from human embryonic stem cells. Exp Hematol. 2008;36:350-9.
51、 Tremoleda JL, Forsyth NR, Khan NS, Wojtacha D, Christodoulou I, Tye BJ, Racey SN, Collishaw S, Sottile V, Thomson AJ, Simpson AH, Noble BS, McWhir J. Bone tissue formation from human embryonic stem cells in vivo. Cloning Stem Cells. 2008;10:119-32.
52、 Agarwal S, Holton KL, Lanza R. Efficient Differentiation of Functional Hepatocytes from Human Embryonic Stem Cells. Stem Cells. 2008 Feb 21.
53、 Hay DC, Zhao D, Fletcher J, Hewitt ZA, McLean D, Urruticoechea-Uriguen A, Black JR, Elcombe C, Ross JA, Wolf R, Cui W. Efficient Differentiation of Hepatocytes from Human Embryonic Stem Cells Exhibiting Markers Recapitulating Liver Development In Vivo. Stem Cells. 2008 Jan 31.
54、 Lavon N, Yanuka O, Benvenisty N. The Effect of Overexpression of Pdx1 and Foxa2 on the Differentiation of Human Embryonic Stem Cells into Pancreatic Cells. Stem Cells. 2006;24:1923-1930.
55、 Liew CG, Shah NN, Briston SJ, Shepherd RM, Khoo CP, Dunne MJ, Moore HD, Cosgrove KE, Andrews PW. PAX4 enhances beta-cell differentiation of human embryonic stem cells. PLoS ONE. 2008; 3:el783.
56、 Mueller D, Shamblott MJ, Fox HE, Gearhart JD, Martin LJ. Transplanted human embryonic germ cell-derived neural stem cells replace neurons and oligodendrocytes in the forebrain of neonatal mice with excitotoxic brain damage. J Neurosci Res. 2005;82:592-608.
57、 Ohtaka T, Matsui Y, Obinata M. Hematopoietic development of primordial germ cell-derived mouse embryonic germ cells in culture. Biochem Biophys Res Commun. 1999;260:475-482.
58、 Kim MS, Hwang NS, Lee J, Kim TK, Leong K, Shamblott MJ, Gearhart J, Elisseeff J. Musculoskeletal differentiation of cells derived from human embryonic germ cells. Stem Cells. 2005;23:113-123.
59、 Chen B, Shi J, Zheng J, Chen Y, Wang K, Yang Q, Chen X, Yang Z, Zhou X, Zhu Y, Chu J, Liu A, Sheng HZ. Differentiation of liver cells from human primordial germ cell-derived progenitors. Differentiation. 2007;75:350-9.
60、 Guillaume DJ, Zhang SC. Human embryonic stem cells: a potential source of transplantable neural progenitor cells. Neurosurg Focus. 2008;24:E3.
61、 Khanna A, Swistowska AM, Rao MS, Zeng X. Generation and transplantation of dopaminergic neurons derived from embryonic stem cells. Curr Stem Cell Res Ther. 2007;2:139-47.
62、 Daadi MM, Maag AL, Steinberg GK. Adherent self-renewable human embryonic stem cell-derived neural stem cell line: functional engraftment in experimental stroke model. PLoS ONE. 2008;3:e1644.
63、 http://diabetes.about.com/b/2008/02/25/human-embryonic-stem-cells-generate-in sulin-producing-cells-in-mice.htm.
64、 Xu C, Lebkowski J, Gold JD. Growth and differentiation of human embryonic stem cells for cardiac cell replacement therapy. Curr Stem Cell Res Ther. 2006;1:173-87.
65、 Asahina K, Teramoto K, Teraoka H. Embryonic stem cells: hepatic differentiation and regenerative medicine for the treatment of liver disease. Curr Stem Cell Res Ther. 2006;1:139-56.
66、 Wang Z, Lu H, Wang YC, Cong XQ. Human embryonic stem cells and liver diseases: From basic research to future clinical application. J Dig Dis. 2008;9:14-9.
67、 Bandi S, Akkina R. Human embryonic stem cell (hES) derived dendritic cells are functionally normal and are susceptible to HIV-1 infection. AIDS Res Ther. 2008;5:1.
68、 Cao T, Lu K, Fu X, Heng BC. Differentiated fibroblastic progenies of human embryonic stem cells for toxicology screening. Cloning Stem Cells. 2008;10:1-10.
69、 Lightfoot HM, Cairns BA, Lapaglia M, Pleasant A, Meyer A, Fair J. Murine Embryonic Stem (ES) cells inhibit human pancreatic carcinoma cell growth in vitro. J Sur Res. 2004; 121:342.
70、 Woll PS, Martin CH, Miller JS, Kaufman DS. Human embryonic stem cell-derived NK cells acquire functional receptors and cytolytic activity. J Immunol. 2005;175:5095-103.
71、 http://www.nature.com/news/2006/061106/full/061106-17.html.
72、 Postovit LM, Seftor EA, Seftor RE, Hendrix MJ. A three-dimensional model to study the epigenetic effects induced by the microenvironment of human embryonic stem cells. Stem Cells. 2006;24:501-505.
73、 Postovit LM, Margaryan NV, Seftor EA, Kirschmann DA, Lipavsky A, Wheaton WW, Abbott DE, Seftor RE, Hendrix MJ. Human embryonic stem cell microenvironment suppresses the tumorigenic phenotype of aggressive cancer cells. Proc Natl Acad Sci U S A. 2008; 105:4329-34.
1、薛清彩,吴绍华.人原始生殖细胞的研究现状.四川解剖学杂志.2007:15:37-39。
2、Shamblott MJ,Axelman J,Wang S,Bugg EM,Littlefield JW,Donovan PJ,Blumenthal PD,Huggins GR,Gearhart JD.Derivation of pluripotent stem cells from cultured human primordial germ cells.Proc.Natl.Acad.Sci.U.S.A.1998;95:13726-13731.
3、Aflatoonian B,Moore H.Human primordial germ cells and embryonic germ cells,and their use in cell therapy.Curr Opin Biotechnol.2005;16:530-5.
4、姜景岩,孔北华,沈柏均.人胚骨髓基质细胞饲养层对人胚胎生殖细胞生长的作用生殖与避孕.2005;25:131-136.
5、Li F,Liu Y,Chen D,Lin X,Li J,Wang J,Peng Y,Wang S,Wang Y.Leukemia inhibitory factor-expressing human embryonic lung fibroblasts as feeder cells for human embryonic germ cells.Cells Tissues Organs.2007;186:221-8.
6、Halberstadt C,Emerich DF,Gores P.Use of Sertoli cell transplants to provide local immunoprotection for tissue grafts.Expert Opin Biol Ther.2004;4:813- 25.
7、He J,Wang Y,Li YL.Fibroblast-like cells derived from the gonadal ridges and dorsal mesenteries of human embryos as feeder cells for the culture of human embryonic germ cells.J Biomed Sci.2007;14:617-28.
8、Kleinman HK,McGarvey ML,Hassell JR,Star VL,Cannon FB,Laurie GW,Martin GR.Basement membrane complexes with biological activity.Biochemistry.1986;25:312-8.
9、Klimanskaya I,Chung Y,Meisner L,Johnson J,West MD,Lanza R.Human embryonic stem cells derived without feeder cells.Lancet.2005;365:1636-1641.
10、Stojkovic P,Lako M,Stewart R,Przyborski S,Armstrong L,Evans J,Murdoch A,Strachan T,Stojkovic M.An autogeneic feeder cell system that efficiently supports growth of undifferentiated human embryonic stem cells. Stem Cells. 2005;23:306-314.
11、 Stojkovic P, Lako M, Przyborski S, Stewart R, Armstrong L, Evans J, Zhang X, Stojkovic M. Human-serum matrix supports undifferentiated growth of human embryonic stem cells. Stem Cells. 2005;23:895-902.
12、 Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, Carpenter MK. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol. 2001; 19:971-4.
13、 Chen HF, Kuo HC, Chien CL, Shun CT, Yao YL, Ip PL, Chuang CY, Wang CC, Yang YS, Ho HN. Derivation, characterization and differentiation of human embryonic stem cells: comparing serum-containing versus serum-free media and evidence of germ cell differentiation. Hum Reprod. 2007;22:567-77.
14、Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, Carpenter MK. Basic fibroblast growth supports undifferentiated human embryonic stem cell growth without conditioned medium. Stem Cells. 2005; 23:315-23.
15、 Xu C, Rosier E, Jiang J, Lebkowski JS, Gold JD, O'Sullivan C, Delavan-Boorsma K, Mok M, Bronstein A, Carpenter MK. Basic Fibroblast Growth Factor Support of Human Embryonic Stem Cell. Stem Cells. 2006;24:568-574.
16、 Ginis I, Luo Y, Miura T, Thies S, Brandenberger R, Gerecht-Nir S, Amit M, Hoke A, Carpenter MK, Itskovitz-Eldor J, Rao MS. Differences between human and mouse embryonic stem cells. 2004;269:360-80.
17、 Turnpenny L, Spalluto CM, Perrett RM, Shea MO, Hanley KP, Cameron IT, Wilson DI, Hanley NA. Evaluating Human Embryonic Germ Cells: Concord and Conflict as Pluripotent Stem Cells. Stem Cells. 2006;24:212-220.
18、 Buaas FW, Kirsh AL, Sharma M, McLean DJ, Morris JL, Griswold MD, de Rooij DG, Braun RE. Plzf is required in adult male germ cells for stem cell self-renewal. Nat Genet. 2004;36:647-652.
19、 Perrett RM, Turnpenny L, Eckert JJ, O'Shea M, Sonne SB, Cameron IT, Wilson DI, Rajpert-De Meyts E, Hanley NA. The Early Human Germ Cell Lineage Does Not Express SOX2 During In Vivo Development or Upon In Vitro Culture. Biol Reprod. 2008 Jan 16.
20、 Turnpenny L, Brickwood S, Spalluto CM, Piper K, Cameron IT, Wilson DI, Hanley NA. Derivation of human embryonic germ cells: an alternative source of pluripotent stem cells. Stem Cells. 2003; 21:598-609.
21、 Perrier AL, Tabar V, Barberi T, Rubio ME, Bruses J, Topf N, Harrison NL, Studer L. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci U S A. 2004;101:12543-12548.
22、 Kurita R, Sasaki E, Yokoo T, Hiroyama T, Takasugi K, Imoto H, Izawa K, Dong Y, Hashiguchi T, Soda Y, Maeda T, Suehiro Y, Tanioka Y, Nakazaki Y, Tani K. Tall/Scl Gene Transduction Using a Lentiviral Vector Stimulates Highly Efficient Hematopoietic Cell Differentiation from Common Marmoset (Callithrix jacchus) Embryonic Stem Cells. Stem Cells. 2006;24:2014-2022.
23、 Anderson JS, Bandi S, Kaufman DS, Akkina R. Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy. Retrovirology. 2006;3:24.
24、 Galic Z, Kitchen SG, Kacena A, Subramanian A, Burke B, Cortado R, Zack JA. T lineage differentiation from human embryonic stem cells. Proc Natl Acad Sci U S A. 2006; 103:11742-7.
25、 Slukvin II, Vodyanik MA, Thomson JA, Gumenyuk ME, Choi KD. Directed differentiation of human embryonic stem cells into functional dendritic cells through the myeloid pathway. J Immunol. 2006; 176:2924-32.
26、 Lavon N, Yanuka O, Benvenisty N. The Effect of Overexpression of Pdx1 and Foxa2 on the Differentiation of Human Embryonic Stem Cells into Pancreatic Cells. Stem Cells. 2006;24:1923-1930.
27、 Shamblott MJ, Axelman J, Littlefield JW, Blumenthal PD, Huggins GR, Cui Y, Cheng L, Gearhart JD. Human embryonic germ cell derivatives express a broad range of developmentally distinct markers and proliferate extensively in vitro. Proc Natl Acad Sci U S A.2001;98:113-118.
28、 Mueller D, Shamblott MJ, Fox HE, Gearhart JD, Martin LJ. Transplanted human embryonic germ cell-derived neural stem cells replace neurons and oligodendrocytes in the forebrain of neonatal mice with excitotoxic brain damage. J Neurosci Res. 2005;82:592-608.
29、 Ohtaka T, Matsui Y, Obinata M. Hematopoietic development of primordial germ cell-derived mouse embryonic germ cells in culture. Biochem Biophys Res Commun. 1999;260:475-482.
30、 Kim MS, Hwang NS, Lee J, Kim TK, Leong K, Shamblott MJ, Gearhart J, Elisseeff J. Musculoskeletal differentiation of cells derived from human embryonic germ cells. Stem Cells. 2005;23:113-123.
31、 Varghese S, Theprungsirikul P, Ferran A, Hwang N, Canver A, Elisseeff J. Chondrogenic differentiation of human embryonic germ cell derived cells in hydrogels. Conf Proc IEEE Eng Med Biol Soc. 2006; 1:2643-6.
32、 Chen B, Shi J, Zheng J, Chen Y, Wang K, Yang Q, Chen X, Yang Z, Zhou X, Zhu Y, Chu J, Liu A, Sheng HZ. Differentiation of liver cells from human primordial germ cell-derived progenitors. Differentiation. 2007;75:350-9.
33、 Turnpenny L, Cameron IT, Spalluto CM, Hanley KP, Wilson DI, Hanley NA Human embryonic germ cells for future neuronal replacement therapy. Brain Res Bull. 2005;68:76-82.
34、 Mueller D, Shamblott MJ, Fox HE, Gearhart JD, Martin LJ. Transplanted human embryonic germ cell-derived neural stem cells replace neurons and oligodendrocytes in the forebrain of neonatal mice with excitotoxic brain damage. J Neurosci Res. 2005;82:592-608.
属性不符
1、 Stewart R, Stojkovic M, Lako M. Mechanisms of self-renewal in human embryonic stem cells. Eur J Cancer. 2006;42:1257-72.
2、 Bibikova M, Chudin E, Wu B, Zhou L, Garcia EW, Liu Y, Shin S, Plaia TW, Auerbach JM, Arking DE, Gonzalez R, Crook J, Davidson B, Schulz TC, Robins A, Khanna A, Sartipy P, Hyllner J, Vanguri P, Savant-Bhonsale S, Smith AK, Chakravarti A, Maitra A, Rao M, Barker DL, Loring JF, Fan JB. Human embryonic stem cells have a unique epigenetic signature. Genome Res. 2006; 16:1075-83.
3、 Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature. 2001;414:105-111.
4、 Postovit LM, Costa FF, Bischof JM, Seftor EA, Wen B, Seftor RE, Feinberg AP, Soares MB, Hendrix MJ. The commonality of plasticity underlying multipotent tumor cells and embryonic stem cells. J Cell Biochem. 2007;9999:1-10.
5、 Bjerkvig R, Tysnes BB, Aboody KS, Najbauer J, Terzis AJ. Opinion: the origin of the cancer stem cell: current controversies and new insights. Nat Rev Cancer. 2005;5:899-904.
6、 Yilmaz OH, Valdez R, Theisen BK, Guo W, Ferguson DO, Wu H, Morrison SJ. Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells. Nature. 2006;441:475-82.
7、 Postovit LM, Seftor EA, Seftor RE, Hendrix MJ. Influence of the microenvironment on melanoma cell fate determination and phenotype. Cancer Res. 2006;66:7833-7836.
8、 Hendrix MJ, Seftor EA, Seftor RE, Kasemeier-Kulesa J, Kulesa PM, Postovit LM. Reprogramming metastatic tumour cells with embryonic microenvironments. Nat Rev Cancer. 2007;7:246-255.
9、 Szotek PP, Pieretti-Vanmarcke R, Masiakos PT, Dinulescu DM, Connolly D, Foster R,Dombkowski D,Preffer F,MacLaughlin DT,Donahoe PK.Ovarian cancer side population defines cells with stem cell-like characteristics and Mullerian Inhibiting Substance responsiveness.Proc Natl Acad Sci U S A.2006;103:11154-9.
10、Bukovsky A,Virant-Klun I,Svetlikova M,Willson I.Ovarian germ cells.Methods Enzymol.2006;419:208-58.
11、Rahimi G,Isachenko E,Sauer H,Wartenberg M,Isachenko V,Hescheler J,Mallmann P,Nawroth F.Measurement of apoptosis in long-term cultures of human ovarian tissue.Reproduction.2001;122:657-663.
12、Stahelin BJ,Marti U,Solioz M,Zimmermann H,Reichen J.False positive staining in the TUNEL assay to detect apoptosis in liver and intestine is caused by endogenous nucleases and inhibited by diethyl pyrocarbonate.Mol Pathol.1998;51:204-208.
13、Li P,Nijhawan D,Budihardjo I,Srinivasula SM,Ahrnad M,Alnemri ES,Wang X.Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade.Cell.1997;91:479-89.
14、Park JY,Park JM,Jang JS,Choi JE,Kim KM,Cha SI,Kim CH,Kang YM,Lee WK,Kam S,Park RW,Kim IS,Lee JT,Jung TH.Caspase 9 promoter polymorphisms and risk of primary lung cancer.Hum Mol Genet.2006;15:1963-1971.
15、Liu JR,Opipari AW,Tan L,Jiang Y,Zhang Y,Tang H,Nunez G Dysfunctional apoptosome activation in ovarian cancer:implications for chemoresistance.Cancer Res.2002;62:924-931.
16、Altieri DC.Survivin and apoptosis control.Adv Cancer Res.2003;88:31252.
17、Huang HM,Huang CJ,Yen JJ.Mc121 is a common target of stem cell factor and interleukin25 for apoptosis prevention activity via MEKPMAPK and PI23KPAkt pathways.Blood.2000;96:1764-71.
18、姚鹏,杨晓明,胡大荣.PI3KPAKT途径在HGF介导肝干细胞抗凋亡调控中的作 用.中华微生物学和免疫学杂志.2005:25:89-93.
19、Hendrix MJ,Seflor EA,Seflor RE,Kasemeier-Kulesa J,Kulesa PM,Postovit LM Reprogramming metastatic tumour cells with embryonic microenvironments.Nat Rev Cancer.2007;7:246-255.
20、孙晓杰,黄常志.P13K-Akt信号通路与肿瘤.世界华人消化杂志.2006:14:306-311.
21、Conus NM,Hannan KM,Cristiano BE,Hemmings BA,Pearson RB.Direct identification of tyrosine 474 as a regulatory phosphorylation site for the Akt protein kinase.J Biol Chem.2002;277:38021-38028.
22、Noske A,Kaszubiak A,Weichert W,Sers C,Niesporek S,Koch I,Schaefer B,Sehouli J,Dietel M,Lage H,Denkert C.Specific inhibition of AKT2 by RNA interference results in reduction of ovarian cancer cell proliferation:increased expression of AKT in advanced ovarian cancer.Cancer Lett.2007;246:190-200.
23、Sun M,Wang G,Paciga JE,Feldman RI,Yuan ZQ,Ma XL,Shelley SA,Jove R,Tsichlis PN,Nicosia SV,Cheng JQ.AKT1/PKBalpha kinase is frequently elevated in human cancers and its constitutive activation is required for oncogenic transformation in NIH3T3 cells.Am J Pathol.2001;159:431-7.
24、Page C,Lin HJ,Jin Y,Castle VP,Nunez G,Huang M,Lin J.Overexpression of Akt/AKT can modulate chemotherapy-induced apoptosis.Anticancer Res.2000;20:407-16.
25、Cheng JQ,Godwin AK,Bellacosa A,Taguchi T,Franke TF,Hamilton TC,Tsichlis PN,Testa JR.AKT2,a putative oncogene encoding a member of a subfamily of proteinserine/threonine kinases,is amplified in human ovarian carcinomas.Proc Natl Acad Sci U S A.1992;89:9267-71.
26、Yuan ZQ,Sun M,Feldman RI,Wang G,Ma X,Jiang C,Coppola D,Nicosia SV,Cheng JQ.Frequent activation of AKT2 and induction of apoptosis by inhibition of phosphoinositide-3-OH kinase/Akt pathway in human ovarian cancer.Oncogene. 2000;19:2324-30.
27、 Bellacosa A, de Feo D, Godwin AK, Bell DW, Cheng JQ, Altomare DA, Wan M, Dubeau L, Scambia G, Masciullo V, Ferrandina G, Benedetti Panici P, Mancuso S, Neri G, Testa JR. Molecular alterations of the AKT2 oncogene in ovarian and breast carcinomas. Int J Cancer. 1995;64:280-5.
28、 Armstrong L, Hughes O, Yung S, Hyslop L, Stewart R, Wappler I, Peters H, Walter T, Stojkovic P, Evans J, Stojkovic M, Lako M. The role of PI3K/AKT, MAPK/ERK and NFkappabeta signalling in the maintenance of human embryonic stem cell pluripotency and viability highlighted by transcriptional profiling and functional analysis. Hum Mol Genet. 2006; 15:1894-1913.
29、 Miura T, Mattson MP, Rao MS. Cellular lifespan and senescence signaling in embryonic stem cells. Aging Cell. 2004;3:333-343.
30、 Prince ME, Sivanandan R, Kaczorowski A, Wolf GT, Kaplan MJ, Dalerba P, Weissman IL, Clarke MF, Ailles LE .Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. PNAS. 2007 104:973-978.
31、 http://www.eurekalert.org/pub_releases/2007-02/uomh-uri012907.php
32、 O'Brien CA, Pollett A, Gallinger S, Dick JE. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature. 2007;445:106-10.
33、 Woll PS, Martin CH, Miller JS, Kaufman DS. Human embryonic stem cell-derived NK cells acquire functional receptors and cytolytic activity. J Immunol. 2005;175:5095-103.
34、 Germano IM, Uzzaman M, Benveniste RJ, Zaurova M, Keller G. Apoptosis in human glioblastoma cells produced using embryonic stem cell-derived astrocytes expressing tumor necrosis factor-related apoptosis-inducing ligand. J Neurosurg. 2006;105:88-95.
35、 Lightfoot HM, Cairns BA, Lapaglia M, Pleasant A, Meyer A, Fair J. Murine Embryonic Stem (ES) cells inhibit human pancreatic carcinoma cell growth in vitro. J Sur Res. 2004;121:342.
36、 http://www.nature.com/news/2006/061106/full/061106-17.html
37、 Postovit LM, Seftor EA, Seftor RE, Hendrix MJ. A three-dimensional model to study the epigenetic effects induced by the microenvironment of human embryonic stem cells. Stem Cells. 2006;24:501-505.
38、 Postovit LM, Margaryan NV, Seftor EA, Kirschmann DA, Lipavsky A, Wheaton WW, Abbott DE, Seftor RE, Hendrix MJ. Human embryonic stem cell microenvironment suppresses the tumorigenic phenotype of aggressive cancer cells. Proc Natl Acad Sci U S A. 2008;105:4329-34.
1、Beebe D J,Mensing GA,Walker GM.Physics and Applications of Microfluidics in Biology.Annu Rev Biomed Eng.2002;4:261-286.
2、Sia SK,Whitesides GM.Microfluidic devices based on poly(dimethylsiloxane)for biological studies.Electrophoresis.2003;24:3563-3576.
3、Ju XL,Li D,Gao N,Shi Q,Hou HS.Hepatogenic Differentiation of Mesenchymal Stem Cells using Microfluidic Chips.Biotechnol J.2008;doi:10,1002/biot.200707152.
4、岳志红,张正.微流控芯片技术及其在检验医学中的应用.现代仪器.2004;5:14-16.
5、Chiu DT,Jeon NL,Huang S,Kane RS,Wargo CJ,Choi IS,Ingber DE,Whitesides GM.Patterned deposition of cells and proteins onto surfaces by using three-dimensional microfluidic systems.Proc Natl Acad Sci U S A.2000;97:2408-2413.
6、Glasgow IK,Zeringue HC,Beebe DJ,Choi SJ,Lyman JT,Chan NG,Wheeler MB.Handling individual mammalian embryos using microfluidics.IEEE Trans Biomed Eng.2001;48:570-578.
7、Hatch A,Kamholz AE,Hawkins KR,Munson MS,Schilling EA,Weigl BH,Yager P.A rapid diffusion immunoassay in a T-sensor.Nat Biotechnol.2001;19:461-465.
8、Yang J,Huang Y,Wang XB,Becker FF,Gascoyne PR.Cell separation on micro fabricated electrodes using dielectrophoretic/gravitational field flow fractionation.Anal Chem.1999;71:911-918.
9、Heasley LE.Autocrine and paracrine signaling through neuropeptide receptors in human cancer.Oncogene.2001;20:1563-9.
10、Tsai RYL,Mckay RDG.Cell contact regulates fate choice by cortical stem cells.J Neurosci.2000;20:3725-35.
11、 Purpura KA, Aubin JE, Zandstra PW. Sustained in vitro expansion of bone progenitors is cell density dependent. Stem Cells. 2003;22:39-50.
12、 Javazon EH, Colter DC, Schwarz EJ, Prockop DJ. Rat marrow stromal cells are more sensitive to plating density and expand more rapidly from single-cell-derived colonies than human marrow stromal cells. Stem Cells. 2001; 19:219-25.
13、 Zandstra PW, Le HV, Daley GQ, Griffith LG, Lauffenburger DA. Leukemia inhibitory factor (LIF) concentration modulates embryonic stem cell self-renewal and differentiation independently of proliferation. Biotechnol Bioeng. 2000;69:607-17.
14、 Kim L, Vahey MD, Lee HY, Voldman J. Microfluidic arrays for logarithmically perfused embryonic stem cell culture. Lab Chip. 2006;3:394-406.
15、 Rosenthal A, Macdonald A, Voldman J. Cell patterning chip for controlling the stem cell microenvironment. Biomaterials. 2007;21:3208-16.
16、 Chung BG, Flanagan LA, Rhee SW, Schwartz PH, Lee AP, Monuki ES, Jeon NL. Human neural stem cell growth and differentiation in a gradient-generating microfluidec device. Lab chip. 2005;5:401-406.
17、 Nagrath S, Sequist LV, Maheswaran S, Bell DW, Irimia D, Ulkus L, Smith MR, Kwak EL, Digumarthy S, Muzikansky A, Ryan P, Balis UJ, Tompkins RG, Haber DA, Toner M. Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature. 2007;450:1235-39.
18、 Nie FQ, Yamada M, Kobayashi J, Yamato M, Kikuchi A, Okano T. On-chip cell migration assay using microfluidic channels. Biomaterials. 2007;27:4017-22.
19、 Zhong JF, Chen Y, Marcus JS, Scherer A, Quake SR, Taylor CR, Weiner LP. A microfluidic processor for gene expression profiling of single human embryonic stem cells. Lab Chip. 2008;8:68-74.
Chung BG, Flanagan LA, Rhee SW, Schwartz PH, Lee AP, Monuki ES, Jeon NL (2005) Human neural stem cell growth and differentiation in a gradient-generating microfluidec device. Lab chip 5:401-406
Hatch A, Kamholz AE, Hawkins KR, Munson MS, Schilling EA, Weigl BH, Yager P (2001) A rapid diffusion immunoassay in a T-sensor. Nat Biotechnol 19:461-465
Ju XL, Li D, Gao N, Shi Q, Hou HS (2008) Hepatogenic Differentiation of Mesenchymal Stem Cells using Microfluidic Chips. Biotechnol J. doi:10,1002/biot.200707152
Lightfoot HM, Cairns BA, Lapaglia M B, Pleasant BA, Meyer AA, Fair J (2004) Murine Embryonic Stem (ES) cells inhibit human pancreatic carcinoma cell growth in vitro. J Sur Res 121:342
Mueller D, Shamblott MJ, Fox HE, Gearhart JD, Martin LJ (2005) Transplanted human embryonic germ cell-derived neural stem cells replace neurons and oligodendrocytes in the forebrain of neonatal mice with excitotoxic brain damage. J Neurosci Res 82:592-608
Nie FQ, Yamada M, Kobayashi J, Yamato M, Kikuchi A, Okano T (2007) On-chip cell migration assay using microfluidic channels. Biomaterials 27:4017-22
Postovit LM, Seftor EA, Seftor RE Hendrix MJ (2006) A three-dimensional model to study the epigenetic effects induced by the microenvironment of human embryonic stem cells. Stem Cells 24:501-505
Postovit LM, Costa FF, Bischof JM Seftor EA, Wen B, Seftor RE, Feinberg AP, Soares MB, Hendrix MJ (2007) The commonality of plasticity underlying multipotent tumor cells and embryonic stem cells. J Cell Biochem 9999:1-10
Rosenthal A, Macdonald A, Voldman J (2007) Cell patterning chip for controlling the stem cell microenvironment. Biomaterials 21:3208-16
Shamblott MJ, Axelman J, Wang S Bugg EM, Littlefield JW, Donovan PJ,
Blumenthal PD, Huggins GR, Gearhart JD (1998) Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc Natl Acad Sci U S A 95:13726-13731
Shamblott MJ, Axelman J, Littlefield JW, Blumenthal PD, Huggins GR, Cui Y,
Cheng L, Gearhart JD (2001) Human embryonic germ cell derivatives express a broad range of developmentally distinct markers and proliferate extensively in vitro. Proc Natl Acad Sci U S A 98:113-118
Sia SK, Whitesides GM (2003) Microfluidic devices based on poly (dimethylsiloxane) for biological studies. Electrophoresis 24:3563-3576
Turnpenny L, Brickwood S, Spalluto CM, Piper K, Cameron IT, Wilson DI, Hanley NA (2003) Derivation of human embryonic germ cells: an alternative source of pluripotent stem cells. Stem Cells 21:598-609
1 Postovit LM, Costa FF, Bischof JM et al. The commonality of plasticity underlying multipotent tumor cells and embryonic stem cells. J Cell Biochem 2007; 9999: 1-10.
2 Bjerkvig R, Tysnes BB, Aboody KS et al. Opinion: the origin of the cancer stem cell: current controversies and new insights. Nat Rev Cancer 2005; 5: 899-904.
3 Galmozzi E, Facchetti F, LaPorta CA. Cancer stem cells and therapeutic perspectives. Curr Med Chem 2006; 13: 603-607.
4 Postovit LM, Seftor EA, Seftor RE et al. Influence of the microenvironment on melanoma cell fate determination and phenotype. Cancer Res 2006; 66: 7833-7836.
5 Lightfoot HM, Cairns BA, Lapaglia M et al. Murine Embryonic Stem (ES) cells inhibit human pancreatic carcinoma cell growth in vitro. J Sur Res 2004; 121: 342.
6 Postovit LM, Seftor EA, Seftor RE et al. A three-dimensional model to study the epigenetic effects induced by the microenvironment of human embryonic stem cells. Stem Cells 2006; 24: 501-505.
7 Shamblott MJ, Axelman J, Wang S et al. Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc Natl Acad Sci U S A 1998; 95: 13726-13731.
8 Noske A, Kaszubiak A, Weichert W et al. Specific inhibition of AKT2 by RNA interference results in reduction of ovarian cancer cell proliferation: increased expression of AKT in advanced ovarian cancer. Cancer Lett 2007; 246: 190-200.
9 Stewart R, Stojkovic M, Lako M. Mechanisms of self-renewal in human embryonic stem cells. Eur J Cancer 2006; 42: 1257-1272.
10 Turnpenny L, Brickwood S, Spalluto CM et al. Derivation of human embryonic germ cells: an alternative source of pluripotent stem cells. Stem Cells 2003; 21: 598-609.
11 Park JH, Kim SJ, Lee JB et al. Establishment of a human embryonic germ cell line and comparison with mouse and human embryonic stem cells. Mol Cells 2004; 17: 309-315.
12 Shamblott MJ, Axelman J, Littlefield JW et al. Human embryonic germ cell derivatives express a broad range of developmentally distinct markers and proliferate extensively in vitro. Proc Natl Acad Sci U S A 2001; 98: 113-118.
13 Mueller D, Shamblott MJ, Fox HE et al. Transplanted human embryonic germ cell-derived neural stem cells replace neurons and oligodendrocytes in the forebrain of neonatal mice with excitotoxic brain damage. J Neurosci Res 2005; 82: 592-608.
14 Ohtaka T, Matsui Y, Obinata M. Hematopoietic development of primordial germ cell-derived mouse embryonic germ cells in culture. Biochem Biophys Res Commun 1999; 260: 475-482.
15 Kim MS, Hwang NS, Lee J et al. Musculoskeletal differentiation of cells derived from human embryonic germ cells. Stem Cells 2005; 23: 113-123.
16 Rahimi G, Isachenko E, Sauer H et al. Measurement of apoptosis in long-term cultures of human ovarian tissue. Reproduction 2001; 122: 657-663.
17 Stahelin BJ, Marti U, Solioz M et al. False positive staining in the TUNEL assay to detect apoptosis in liver and intestine is caused by endogenous nucleases and inhibited by diethyl pyrocarbonate. Mol Pathol 1998; 51: 204-208.
18 Liu JR, Opipari AW, Tan L et al. Dysfunctional apoptosome activation in ovarian cancer: implications for chemoresistance. Cancer Res 2002; 62: 924-931.
19 Park JY, Park JM, Jang JS et al. Caspase 9 promoter polymorphisms and risk of primary lung cancer. Hum Mol Genet 2006; 15: 1963-1971.
20 Hendrix MJ, Seftor EA, Seftor RE et al. Reprogramming metastatic tumour cells with embryonic microenvironments. Nat Rev Cancer 2007; 7: 246-255.
21 Reya T, Morrison SJ, Clarke MF et al. Stem cells, cancer, and cancer stem cells. Nature 2001; 414: 105-111.
22 Penninger JM, Woodgett J. Stem cells. PTEN—coupling tumor suppression to stem cells? Science 2001; 294: 2116-2118.
23 Conus NM, Hannan KM, Cristiano BE et al. Direct identification of tyrosine 474 as a regulatory phosphorylation site for the Akt protein kinase. J Biol Chem 2002; 277: 38021-38028.
24 Armstrong L, Hughes O, Yung S et al. The role of PI3K/AKT, MAPK/ERK and NFkappabeta signalling in the maintenance of human embryonic stem cell pluripotency and viability highlighted by transcriptional profiling and functional analysis. Hum Mol Genet 2006; 15: 1894-1913.
25 Miura T, Mattson MP, Rao MS. Cellular lifespan and senescence signaling in embryonic stem cells. Aging Cell 2004; 3: 333-343.
2、Zhang SC,Wemig M,Duncan ID,Brustle O,Thomson JA.In vitro differentiation of transplantable neural precursors from human embryonic stem cells.Nat Biotechnol.2001;19:1129-1133.
3、Kaufman DS,Hanson ET,Lewis RL,Auerbach R,Thomson JA.Hematopoietic colony-forming cells derived from human embryonic stem cells.Proc Natl Acad Sci U S A.2001;98:10716-10721.
4、Xu C,Police S,Rao N,Carpenter MK.Characterization and enrichment of cardiomyocytes derived from human embryonic stem cells.Circ Res.2002;91:501-508.
5、He JQ,Ma Y,Lee Y,Thomson JA,Kamp TJ.Human embryonic stem cells develop into multiple types of cardiac myocytes:action potential characterization.Circ Res.2003;93:32-39.
6、Assady S,Maor G,Amit M,Itskovitz-Eldor J,Skorecki KL,Tzukerman M.Insulin production by human embryonic stem cells.Diabetes.2001;50:1691-1697.
7、Zhan X,Dravid G,Ye Z,Hammond H,Shamblott M,Gearhart J,Cheng L.Functional antigen-presenting leucocytes derived from human embryonic stem cells in vitro.Lancet.2004;364:163-171.
8、Evans MJ,Kaufman MH.Establishment in culture of pluripotential cells from mouse embryos.Nature.1981;292:154-156.
9、Thomson JA,Kalishman J,Golos TG,Durning M,Harris CP,Becker RA,Hearn JP. Isolation of a primate embryonic stem cell line. Proc Natl Acad Sci U S A. 1995;92:7844-7848.
10、 Thomson JA, Kalishman J, Golos TG, Durning M, Harris CP, Hearn JP. Pluripotent cell lines derived from common marmoset (Callithrix jacchus) blastocysts. Biol Reprod. 1996;55:254-259.
11、 Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282:1145-1147.
12、 Shamblott MJ, Axelman J, Wang S, Bugg EM, Littlefield JW, Donovan PJ, Blumenthal PD, Huggins GR, Gearhart JD. Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc Natl Acad Sci U S A. 1998;95:13726-13731.
13、 Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T, Okita K, Mochiduki Y, Takizawa N, Yamanaka S. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol. 2008;26:101-6.
14、 Chung Y, Klimanskaya I, Becker S, Li T, Maserati M, Lu SJ , Zdravkovic T, Ilic D, Genbacev O, Fisher S, Krtolica A, Lanzal R. Human Embryonic Stem Cell Lines Generated without Embryo Destruction. Cell Stem Cell. 2008; 2:113-117.
15、 Cowan CA, Klimanskaya I, McMahon J, Atienza J, Witmyer J, Zucker JP, Wang S, Morton CC, McMahon AP, Powers D, Melton DA. Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med. 2004;350:1353-1356.
16、 Zhang QX, Li Y, Huang SL, He ZX. Human embryonic stem cells preliminarily established in China. Chin Med J. 2002;82:1314-1318.
17、 Li F, Liu Y, Chen D, Lin X, Li J, Wang J, Peng Y, Wang S, Wang Y. Leukemia inhibitory factor-expressing human embryonic lung fibroblasts as feeder cells for human embryonic germ cells. Cells Tissues Organs. 2007;186:221-8.
18、 Saxena S, Hanwate M, Deb K, Sharma V, Totey S. FGF2 secreting human fibroblast feeder cells: A novel culture system for human embryonic stem cells. Mol Reprod Dev. 2008 Mar 3.
19、 Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, Carpenter MK. Feeder free growth of undifferentiated human embryonic stem cells. Nat biotechnol. 2001;19:971-975.
20、 Amit M, Shariki C, Margulets V, Itskovitz-Eldor J. Feeder layer- and serum-free culture of human embryonic stem cells. Biol Reprod. 2004;70:837-45.
21、 Ginis I, Luo Y, Miura T, Thies S, Brandenberger R, Gerecht-Nir S, Amit M, Hoke A, Carpenter MK, Itskovitz-Eldor J, Rao MS. Differences between human and mouse embryonic stem cells. 2004;269:360-80.
22、 Xu RH, Peck RM, Li DS, Feng X, Ludwig T, Thomson JA. Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nat Methods. 2005;2:185-190.
23、 Xu C, Rosier E, Jiang J, Lebkowski JS, Gold JD, O'Sullivan C, Delavan-Boorsma K, Mok M, Bronstein A, Carpenter MK. Basic fibroblast growth factor supports undifferentiated human embryonic stem cell growth without conditioned medium. Stem Cells. 2005;23:315-23.
24、 Levenstein ME, Ludwig TE, Xu RH, Lianas RA, VanDenHeuvel-Kramer K, Manning D, Thomson JA. Basic Fibroblast Growth Factor Support of Human Embryonic Stem Cell. Stem Cells. 2006;24:568-574.
25、 James D, Levine AJ, Besser D, Hemmati-Brivanlou A. TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells. Development. 2005; 132:1273-82.
26、 Dravid G, Ye Z, Hammond H, Chen G, Pyle A, Donovan P, Yu X, Cheng L. Defining the role of Wnt/beta-catenin signaling in the survival, proliferation, and selfrenewal of human embryonic stem cells. Stem Cells. 2005;23:1489-501.
27、 Metallo CM, Azarin SM, Ji L, de Pablo JJ, Palecek SP. Engineering tissue from human embryonic stem cells. J Cell Mol Med. 2008 Jan 11.
28、 Stewart R, Stojkovic M, Lako M. Mechanisms of self-renewal in human embryonic stem cells. Eur J Cancer. 2006;42:1257-72.
29、 Park JH, Kim SJ, Lee JB, Song JM, Kim CG, Roh S 2nd, Yoon HS. Establishment of a human embryonic germ cell line and comparison with mouse and human embryonic stem cells.Mol Cells. 2004; 17:309-15.
30、 Byrne JA, Mitalipov SM, Clepper L, Wolf DP. Transcriptional profiling of rhesus monkey embryonic stem cells. Biol Reprod. 2006;75:908-15.
31、 Buaas FW, Kirsh AL, Sharma M, McLean DJ, Morris JL, Griswold MD, de Rooij DG, Braun RE. Plzf is required in adult male germ cells for stem cell self-renewal. Nat Genet. 2004;36:647-652.
32、 Perrier AL, Tabar V, Barberi T, Rubio ME, Bruses J, Topf N, Harrison NL, Studer L. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci U S A. 2004,101: 12543-12548.
33、 Khanna A, Swistowska AM, Rao MS, Zeng X. Generation and transplantation of dopaminergic neurons derived from embryonic stem cells. Curr Stem Cell Res Ther. 2007;2:139-47.
34、 Rolletschek A, Chang H, Guan K, Czyz J, Meyer M, Wobus AM. Differentiation of embryonic stem cell-derived dopaminergic neurons is enhanced by survival-promoting factors. Mech Dev. 2001; 105:93-104.
35、 Cho MS, Lee YE, Kim JY, Chung S, Cho YH, Kim DS, Kang SM, Lee H, Kim MH, Kim JH, Leem JW, Oh SK, Choi YM, Hwang DY, Chang JW, Kim DW. Highly efficient and large-scale generation of functional dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci U S A. 2008; 105:3392-7.
36、 Lamba DA, Karl MO, Ware CB, Reh TA. Efficient generation of retinal progenitor cells from human embryonic stem cells. Proc Natl Acad Sci U S A. 2006; 103:12769-74.
37、 Coraux C, Hilmi C, Rouleau M, Spadafora A, Hinnrasky J, Ortonne JP, Dani C, Aberdam D. Reconstituted skin from murine embryonic stem cells. Curr Biol. 2003;13:849-853.
38、 Metallo CM, Vodyanik MA, de Pablo JJ, Slukvin II, Palecek SP. The response of human embryonic stem cell-derived endothelial cells to shear stress. Biotechnol Bioeng. 2008 Jan 30.
39、 Chen Y, Amende I, Hampton TG, Yang Y, Ke Q, Min JY, Xiao YF, Morgan JP. Vascular endothelial growth factor promotes cardiomyocyte differentiation of embryonic stem cells. Am J Physiol Heart Circ Physiol. 2006;291:H1653-8.
40、 Mummery C, van der Heyden MA, de Boer TP, Passier R, Ward D, van den Brink S, van Rooijen M, van de Stolpe A. Cardiomyocytes from human and mouse embryonic stem cells. Methods Mol Med. 2007; 140:249-72.
41、 Xiao Q, Zeng L, Zhang Z, Margariti A, Ali ZA, Channon KM, Xu Q, Hu Y. Sca-1~+ progenitors derived from embryonic stem cells differentiate into endothelial cells capable of vascular repair after arterial injury. Arterioscler Thromb Vasc Biol. 2006;26:2244-51.
42、 Yamahara K, Sone M, Itoh H, Yamashita JK, Yurugi-Kobayashi T, Homma K, Chao TH, Miyashita K, Park K, Oyamada N, Sawada N, Taura D, Fukunaga Y, Tamura N, Nakao K. Augmentation of neovascularizaiton in hindlimb ischemia by combined transplantation of human embryonic stem cells-derived endothelial and mural cells. LoS ONE. 2008;3:e1666.
43、 Kurita R, Sasaki E, Yokoo T, Hiroyama T, Takasugi K, Imoto H, Izawa K, Dong Y, Hashiguchi T, Soda Y, Maeda T, Suehiro Y, Tanioka Y, Nakazaki Y, Tani K. Tall/Scl Gene Transduction Using a Lentiviral Vector Stimulates Highly Efficient Hematopoietic Cell Differentiation from Common Marmoset (Callithrix jacchus) Embryonic Stem Cells. Stem Cells. 2006;24:2014-2022.
44、 Chang KH, Nelson AM, Cao H, Wang L1, Nakamoto B, Ware CB, Papayannopoulou T. Definitive-like erythroid cells derived from human embryonic stem cells coexpress high levels of embryonic and fetal globins with little or no adult globin. Blood. 2006; 108:1515-23.
45、 Vodyanik MA, Bork JA, Thomson JA, Slukvin II. Human embryonic stem cell-derived CD34+ cells: efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential. Blood. 2005; 105:617-26.
46、 Woll PS, Martin CH, Miller JS, Kaufman DS. Human embryonic stem cell-derived NK cells acquire functional receptors and cytolytic activity. J Immunol. 2005;175:5095-103.
47、 Slukvin II, Vodyanik MA, Thomson JA, Gumenyuk ME, Choi KD. Directed differentiation of human embryonic stem cells into functional dendritic cells through the myeloid pathway. J Immunol. 2006; 176:2924-32.
48、 Galic Z, Kitchen SG, Kacena A, Subramanian A, Burke B, Cortado R, Zack JA. T lineage differentiation from human embryonic stem cells. Proc Natl Acad Sci U S A. 2006;103:11742-7.
49、 Anderson JS, Bandi S, Kaufman DS, Akkina R. Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy. Retrovirology. 2006;3:24.
50、 Trivedi P, Hematti P. Derivation and immunological characterization of mesenchymal stromal cells from human embryonic stem cells. Exp Hematol. 2008;36:350-9.
51、 Tremoleda JL, Forsyth NR, Khan NS, Wojtacha D, Christodoulou I, Tye BJ, Racey SN, Collishaw S, Sottile V, Thomson AJ, Simpson AH, Noble BS, McWhir J. Bone tissue formation from human embryonic stem cells in vivo. Cloning Stem Cells. 2008;10:119-32.
52、 Agarwal S, Holton KL, Lanza R. Efficient Differentiation of Functional Hepatocytes from Human Embryonic Stem Cells. Stem Cells. 2008 Feb 21.
53、 Hay DC, Zhao D, Fletcher J, Hewitt ZA, McLean D, Urruticoechea-Uriguen A, Black JR, Elcombe C, Ross JA, Wolf R, Cui W. Efficient Differentiation of Hepatocytes from Human Embryonic Stem Cells Exhibiting Markers Recapitulating Liver Development In Vivo. Stem Cells. 2008 Jan 31.
54、 Lavon N, Yanuka O, Benvenisty N. The Effect of Overexpression of Pdx1 and Foxa2 on the Differentiation of Human Embryonic Stem Cells into Pancreatic Cells. Stem Cells. 2006;24:1923-1930.
55、 Liew CG, Shah NN, Briston SJ, Shepherd RM, Khoo CP, Dunne MJ, Moore HD, Cosgrove KE, Andrews PW. PAX4 enhances beta-cell differentiation of human embryonic stem cells. PLoS ONE. 2008; 3:el783.
56、 Mueller D, Shamblott MJ, Fox HE, Gearhart JD, Martin LJ. Transplanted human embryonic germ cell-derived neural stem cells replace neurons and oligodendrocytes in the forebrain of neonatal mice with excitotoxic brain damage. J Neurosci Res. 2005;82:592-608.
57、 Ohtaka T, Matsui Y, Obinata M. Hematopoietic development of primordial germ cell-derived mouse embryonic germ cells in culture. Biochem Biophys Res Commun. 1999;260:475-482.
58、 Kim MS, Hwang NS, Lee J, Kim TK, Leong K, Shamblott MJ, Gearhart J, Elisseeff J. Musculoskeletal differentiation of cells derived from human embryonic germ cells. Stem Cells. 2005;23:113-123.
59、 Chen B, Shi J, Zheng J, Chen Y, Wang K, Yang Q, Chen X, Yang Z, Zhou X, Zhu Y, Chu J, Liu A, Sheng HZ. Differentiation of liver cells from human primordial germ cell-derived progenitors. Differentiation. 2007;75:350-9.
60、 Guillaume DJ, Zhang SC. Human embryonic stem cells: a potential source of transplantable neural progenitor cells. Neurosurg Focus. 2008;24:E3.
61、 Khanna A, Swistowska AM, Rao MS, Zeng X. Generation and transplantation of dopaminergic neurons derived from embryonic stem cells. Curr Stem Cell Res Ther. 2007;2:139-47.
62、 Daadi MM, Maag AL, Steinberg GK. Adherent self-renewable human embryonic stem cell-derived neural stem cell line: functional engraftment in experimental stroke model. PLoS ONE. 2008;3:e1644.
63、 http://diabetes.about.com/b/2008/02/25/human-embryonic-stem-cells-generate-in sulin-producing-cells-in-mice.htm.
64、 Xu C, Lebkowski J, Gold JD. Growth and differentiation of human embryonic stem cells for cardiac cell replacement therapy. Curr Stem Cell Res Ther. 2006;1:173-87.
65、 Asahina K, Teramoto K, Teraoka H. Embryonic stem cells: hepatic differentiation and regenerative medicine for the treatment of liver disease. Curr Stem Cell Res Ther. 2006;1:139-56.
66、 Wang Z, Lu H, Wang YC, Cong XQ. Human embryonic stem cells and liver diseases: From basic research to future clinical application. J Dig Dis. 2008;9:14-9.
67、 Bandi S, Akkina R. Human embryonic stem cell (hES) derived dendritic cells are functionally normal and are susceptible to HIV-1 infection. AIDS Res Ther. 2008;5:1.
68、 Cao T, Lu K, Fu X, Heng BC. Differentiated fibroblastic progenies of human embryonic stem cells for toxicology screening. Cloning Stem Cells. 2008;10:1-10.
69、 Lightfoot HM, Cairns BA, Lapaglia M, Pleasant A, Meyer A, Fair J. Murine Embryonic Stem (ES) cells inhibit human pancreatic carcinoma cell growth in vitro. J Sur Res. 2004; 121:342.
70、 Woll PS, Martin CH, Miller JS, Kaufman DS. Human embryonic stem cell-derived NK cells acquire functional receptors and cytolytic activity. J Immunol. 2005;175:5095-103.
71、 http://www.nature.com/news/2006/061106/full/061106-17.html.
72、 Postovit LM, Seftor EA, Seftor RE, Hendrix MJ. A three-dimensional model to study the epigenetic effects induced by the microenvironment of human embryonic stem cells. Stem Cells. 2006;24:501-505.
73、 Postovit LM, Margaryan NV, Seftor EA, Kirschmann DA, Lipavsky A, Wheaton WW, Abbott DE, Seftor RE, Hendrix MJ. Human embryonic stem cell microenvironment suppresses the tumorigenic phenotype of aggressive cancer cells. Proc Natl Acad Sci U S A. 2008; 105:4329-34.
1、薛清彩,吴绍华.人原始生殖细胞的研究现状.四川解剖学杂志.2007:15:37-39。
2、Shamblott MJ,Axelman J,Wang S,Bugg EM,Littlefield JW,Donovan PJ,Blumenthal PD,Huggins GR,Gearhart JD.Derivation of pluripotent stem cells from cultured human primordial germ cells.Proc.Natl.Acad.Sci.U.S.A.1998;95:13726-13731.
3、Aflatoonian B,Moore H.Human primordial germ cells and embryonic germ cells,and their use in cell therapy.Curr Opin Biotechnol.2005;16:530-5.
4、姜景岩,孔北华,沈柏均.人胚骨髓基质细胞饲养层对人胚胎生殖细胞生长的作用生殖与避孕.2005;25:131-136.
5、Li F,Liu Y,Chen D,Lin X,Li J,Wang J,Peng Y,Wang S,Wang Y.Leukemia inhibitory factor-expressing human embryonic lung fibroblasts as feeder cells for human embryonic germ cells.Cells Tissues Organs.2007;186:221-8.
6、Halberstadt C,Emerich DF,Gores P.Use of Sertoli cell transplants to provide local immunoprotection for tissue grafts.Expert Opin Biol Ther.2004;4:813- 25.
7、He J,Wang Y,Li YL.Fibroblast-like cells derived from the gonadal ridges and dorsal mesenteries of human embryos as feeder cells for the culture of human embryonic germ cells.J Biomed Sci.2007;14:617-28.
8、Kleinman HK,McGarvey ML,Hassell JR,Star VL,Cannon FB,Laurie GW,Martin GR.Basement membrane complexes with biological activity.Biochemistry.1986;25:312-8.
9、Klimanskaya I,Chung Y,Meisner L,Johnson J,West MD,Lanza R.Human embryonic stem cells derived without feeder cells.Lancet.2005;365:1636-1641.
10、Stojkovic P,Lako M,Stewart R,Przyborski S,Armstrong L,Evans J,Murdoch A,Strachan T,Stojkovic M.An autogeneic feeder cell system that efficiently supports growth of undifferentiated human embryonic stem cells. Stem Cells. 2005;23:306-314.
11、 Stojkovic P, Lako M, Przyborski S, Stewart R, Armstrong L, Evans J, Zhang X, Stojkovic M. Human-serum matrix supports undifferentiated growth of human embryonic stem cells. Stem Cells. 2005;23:895-902.
12、 Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, Carpenter MK. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol. 2001; 19:971-4.
13、 Chen HF, Kuo HC, Chien CL, Shun CT, Yao YL, Ip PL, Chuang CY, Wang CC, Yang YS, Ho HN. Derivation, characterization and differentiation of human embryonic stem cells: comparing serum-containing versus serum-free media and evidence of germ cell differentiation. Hum Reprod. 2007;22:567-77.
14、Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, Carpenter MK. Basic fibroblast growth supports undifferentiated human embryonic stem cell growth without conditioned medium. Stem Cells. 2005; 23:315-23.
15、 Xu C, Rosier E, Jiang J, Lebkowski JS, Gold JD, O'Sullivan C, Delavan-Boorsma K, Mok M, Bronstein A, Carpenter MK. Basic Fibroblast Growth Factor Support of Human Embryonic Stem Cell. Stem Cells. 2006;24:568-574.
16、 Ginis I, Luo Y, Miura T, Thies S, Brandenberger R, Gerecht-Nir S, Amit M, Hoke A, Carpenter MK, Itskovitz-Eldor J, Rao MS. Differences between human and mouse embryonic stem cells. 2004;269:360-80.
17、 Turnpenny L, Spalluto CM, Perrett RM, Shea MO, Hanley KP, Cameron IT, Wilson DI, Hanley NA. Evaluating Human Embryonic Germ Cells: Concord and Conflict as Pluripotent Stem Cells. Stem Cells. 2006;24:212-220.
18、 Buaas FW, Kirsh AL, Sharma M, McLean DJ, Morris JL, Griswold MD, de Rooij DG, Braun RE. Plzf is required in adult male germ cells for stem cell self-renewal. Nat Genet. 2004;36:647-652.
19、 Perrett RM, Turnpenny L, Eckert JJ, O'Shea M, Sonne SB, Cameron IT, Wilson DI, Rajpert-De Meyts E, Hanley NA. The Early Human Germ Cell Lineage Does Not Express SOX2 During In Vivo Development or Upon In Vitro Culture. Biol Reprod. 2008 Jan 16.
20、 Turnpenny L, Brickwood S, Spalluto CM, Piper K, Cameron IT, Wilson DI, Hanley NA. Derivation of human embryonic germ cells: an alternative source of pluripotent stem cells. Stem Cells. 2003; 21:598-609.
21、 Perrier AL, Tabar V, Barberi T, Rubio ME, Bruses J, Topf N, Harrison NL, Studer L. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci U S A. 2004;101:12543-12548.
22、 Kurita R, Sasaki E, Yokoo T, Hiroyama T, Takasugi K, Imoto H, Izawa K, Dong Y, Hashiguchi T, Soda Y, Maeda T, Suehiro Y, Tanioka Y, Nakazaki Y, Tani K. Tall/Scl Gene Transduction Using a Lentiviral Vector Stimulates Highly Efficient Hematopoietic Cell Differentiation from Common Marmoset (Callithrix jacchus) Embryonic Stem Cells. Stem Cells. 2006;24:2014-2022.
23、 Anderson JS, Bandi S, Kaufman DS, Akkina R. Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy. Retrovirology. 2006;3:24.
24、 Galic Z, Kitchen SG, Kacena A, Subramanian A, Burke B, Cortado R, Zack JA. T lineage differentiation from human embryonic stem cells. Proc Natl Acad Sci U S A. 2006; 103:11742-7.
25、 Slukvin II, Vodyanik MA, Thomson JA, Gumenyuk ME, Choi KD. Directed differentiation of human embryonic stem cells into functional dendritic cells through the myeloid pathway. J Immunol. 2006; 176:2924-32.
26、 Lavon N, Yanuka O, Benvenisty N. The Effect of Overexpression of Pdx1 and Foxa2 on the Differentiation of Human Embryonic Stem Cells into Pancreatic Cells. Stem Cells. 2006;24:1923-1930.
27、 Shamblott MJ, Axelman J, Littlefield JW, Blumenthal PD, Huggins GR, Cui Y, Cheng L, Gearhart JD. Human embryonic germ cell derivatives express a broad range of developmentally distinct markers and proliferate extensively in vitro. Proc Natl Acad Sci U S A.2001;98:113-118.
28、 Mueller D, Shamblott MJ, Fox HE, Gearhart JD, Martin LJ. Transplanted human embryonic germ cell-derived neural stem cells replace neurons and oligodendrocytes in the forebrain of neonatal mice with excitotoxic brain damage. J Neurosci Res. 2005;82:592-608.
29、 Ohtaka T, Matsui Y, Obinata M. Hematopoietic development of primordial germ cell-derived mouse embryonic germ cells in culture. Biochem Biophys Res Commun. 1999;260:475-482.
30、 Kim MS, Hwang NS, Lee J, Kim TK, Leong K, Shamblott MJ, Gearhart J, Elisseeff J. Musculoskeletal differentiation of cells derived from human embryonic germ cells. Stem Cells. 2005;23:113-123.
31、 Varghese S, Theprungsirikul P, Ferran A, Hwang N, Canver A, Elisseeff J. Chondrogenic differentiation of human embryonic germ cell derived cells in hydrogels. Conf Proc IEEE Eng Med Biol Soc. 2006; 1:2643-6.
32、 Chen B, Shi J, Zheng J, Chen Y, Wang K, Yang Q, Chen X, Yang Z, Zhou X, Zhu Y, Chu J, Liu A, Sheng HZ. Differentiation of liver cells from human primordial germ cell-derived progenitors. Differentiation. 2007;75:350-9.
33、 Turnpenny L, Cameron IT, Spalluto CM, Hanley KP, Wilson DI, Hanley NA Human embryonic germ cells for future neuronal replacement therapy. Brain Res Bull. 2005;68:76-82.
34、 Mueller D, Shamblott MJ, Fox HE, Gearhart JD, Martin LJ. Transplanted human embryonic germ cell-derived neural stem cells replace neurons and oligodendrocytes in the forebrain of neonatal mice with excitotoxic brain damage. J Neurosci Res. 2005;82:592-608.
属性不符
1、 Stewart R, Stojkovic M, Lako M. Mechanisms of self-renewal in human embryonic stem cells. Eur J Cancer. 2006;42:1257-72.
2、 Bibikova M, Chudin E, Wu B, Zhou L, Garcia EW, Liu Y, Shin S, Plaia TW, Auerbach JM, Arking DE, Gonzalez R, Crook J, Davidson B, Schulz TC, Robins A, Khanna A, Sartipy P, Hyllner J, Vanguri P, Savant-Bhonsale S, Smith AK, Chakravarti A, Maitra A, Rao M, Barker DL, Loring JF, Fan JB. Human embryonic stem cells have a unique epigenetic signature. Genome Res. 2006; 16:1075-83.
3、 Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature. 2001;414:105-111.
4、 Postovit LM, Costa FF, Bischof JM, Seftor EA, Wen B, Seftor RE, Feinberg AP, Soares MB, Hendrix MJ. The commonality of plasticity underlying multipotent tumor cells and embryonic stem cells. J Cell Biochem. 2007;9999:1-10.
5、 Bjerkvig R, Tysnes BB, Aboody KS, Najbauer J, Terzis AJ. Opinion: the origin of the cancer stem cell: current controversies and new insights. Nat Rev Cancer. 2005;5:899-904.
6、 Yilmaz OH, Valdez R, Theisen BK, Guo W, Ferguson DO, Wu H, Morrison SJ. Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells. Nature. 2006;441:475-82.
7、 Postovit LM, Seftor EA, Seftor RE, Hendrix MJ. Influence of the microenvironment on melanoma cell fate determination and phenotype. Cancer Res. 2006;66:7833-7836.
8、 Hendrix MJ, Seftor EA, Seftor RE, Kasemeier-Kulesa J, Kulesa PM, Postovit LM. Reprogramming metastatic tumour cells with embryonic microenvironments. Nat Rev Cancer. 2007;7:246-255.
9、 Szotek PP, Pieretti-Vanmarcke R, Masiakos PT, Dinulescu DM, Connolly D, Foster R,Dombkowski D,Preffer F,MacLaughlin DT,Donahoe PK.Ovarian cancer side population defines cells with stem cell-like characteristics and Mullerian Inhibiting Substance responsiveness.Proc Natl Acad Sci U S A.2006;103:11154-9.
10、Bukovsky A,Virant-Klun I,Svetlikova M,Willson I.Ovarian germ cells.Methods Enzymol.2006;419:208-58.
11、Rahimi G,Isachenko E,Sauer H,Wartenberg M,Isachenko V,Hescheler J,Mallmann P,Nawroth F.Measurement of apoptosis in long-term cultures of human ovarian tissue.Reproduction.2001;122:657-663.
12、Stahelin BJ,Marti U,Solioz M,Zimmermann H,Reichen J.False positive staining in the TUNEL assay to detect apoptosis in liver and intestine is caused by endogenous nucleases and inhibited by diethyl pyrocarbonate.Mol Pathol.1998;51:204-208.
13、Li P,Nijhawan D,Budihardjo I,Srinivasula SM,Ahrnad M,Alnemri ES,Wang X.Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade.Cell.1997;91:479-89.
14、Park JY,Park JM,Jang JS,Choi JE,Kim KM,Cha SI,Kim CH,Kang YM,Lee WK,Kam S,Park RW,Kim IS,Lee JT,Jung TH.Caspase 9 promoter polymorphisms and risk of primary lung cancer.Hum Mol Genet.2006;15:1963-1971.
15、Liu JR,Opipari AW,Tan L,Jiang Y,Zhang Y,Tang H,Nunez G Dysfunctional apoptosome activation in ovarian cancer:implications for chemoresistance.Cancer Res.2002;62:924-931.
16、Altieri DC.Survivin and apoptosis control.Adv Cancer Res.2003;88:31252.
17、Huang HM,Huang CJ,Yen JJ.Mc121 is a common target of stem cell factor and interleukin25 for apoptosis prevention activity via MEKPMAPK and PI23KPAkt pathways.Blood.2000;96:1764-71.
18、姚鹏,杨晓明,胡大荣.PI3KPAKT途径在HGF介导肝干细胞抗凋亡调控中的作 用.中华微生物学和免疫学杂志.2005:25:89-93.
19、Hendrix MJ,Seflor EA,Seflor RE,Kasemeier-Kulesa J,Kulesa PM,Postovit LM Reprogramming metastatic tumour cells with embryonic microenvironments.Nat Rev Cancer.2007;7:246-255.
20、孙晓杰,黄常志.P13K-Akt信号通路与肿瘤.世界华人消化杂志.2006:14:306-311.
21、Conus NM,Hannan KM,Cristiano BE,Hemmings BA,Pearson RB.Direct identification of tyrosine 474 as a regulatory phosphorylation site for the Akt protein kinase.J Biol Chem.2002;277:38021-38028.
22、Noske A,Kaszubiak A,Weichert W,Sers C,Niesporek S,Koch I,Schaefer B,Sehouli J,Dietel M,Lage H,Denkert C.Specific inhibition of AKT2 by RNA interference results in reduction of ovarian cancer cell proliferation:increased expression of AKT in advanced ovarian cancer.Cancer Lett.2007;246:190-200.
23、Sun M,Wang G,Paciga JE,Feldman RI,Yuan ZQ,Ma XL,Shelley SA,Jove R,Tsichlis PN,Nicosia SV,Cheng JQ.AKT1/PKBalpha kinase is frequently elevated in human cancers and its constitutive activation is required for oncogenic transformation in NIH3T3 cells.Am J Pathol.2001;159:431-7.
24、Page C,Lin HJ,Jin Y,Castle VP,Nunez G,Huang M,Lin J.Overexpression of Akt/AKT can modulate chemotherapy-induced apoptosis.Anticancer Res.2000;20:407-16.
25、Cheng JQ,Godwin AK,Bellacosa A,Taguchi T,Franke TF,Hamilton TC,Tsichlis PN,Testa JR.AKT2,a putative oncogene encoding a member of a subfamily of proteinserine/threonine kinases,is amplified in human ovarian carcinomas.Proc Natl Acad Sci U S A.1992;89:9267-71.
26、Yuan ZQ,Sun M,Feldman RI,Wang G,Ma X,Jiang C,Coppola D,Nicosia SV,Cheng JQ.Frequent activation of AKT2 and induction of apoptosis by inhibition of phosphoinositide-3-OH kinase/Akt pathway in human ovarian cancer.Oncogene. 2000;19:2324-30.
27、 Bellacosa A, de Feo D, Godwin AK, Bell DW, Cheng JQ, Altomare DA, Wan M, Dubeau L, Scambia G, Masciullo V, Ferrandina G, Benedetti Panici P, Mancuso S, Neri G, Testa JR. Molecular alterations of the AKT2 oncogene in ovarian and breast carcinomas. Int J Cancer. 1995;64:280-5.
28、 Armstrong L, Hughes O, Yung S, Hyslop L, Stewart R, Wappler I, Peters H, Walter T, Stojkovic P, Evans J, Stojkovic M, Lako M. The role of PI3K/AKT, MAPK/ERK and NFkappabeta signalling in the maintenance of human embryonic stem cell pluripotency and viability highlighted by transcriptional profiling and functional analysis. Hum Mol Genet. 2006; 15:1894-1913.
29、 Miura T, Mattson MP, Rao MS. Cellular lifespan and senescence signaling in embryonic stem cells. Aging Cell. 2004;3:333-343.
30、 Prince ME, Sivanandan R, Kaczorowski A, Wolf GT, Kaplan MJ, Dalerba P, Weissman IL, Clarke MF, Ailles LE .Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. PNAS. 2007 104:973-978.
31、 http://www.eurekalert.org/pub_releases/2007-02/uomh-uri012907.php
32、 O'Brien CA, Pollett A, Gallinger S, Dick JE. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature. 2007;445:106-10.
33、 Woll PS, Martin CH, Miller JS, Kaufman DS. Human embryonic stem cell-derived NK cells acquire functional receptors and cytolytic activity. J Immunol. 2005;175:5095-103.
34、 Germano IM, Uzzaman M, Benveniste RJ, Zaurova M, Keller G. Apoptosis in human glioblastoma cells produced using embryonic stem cell-derived astrocytes expressing tumor necrosis factor-related apoptosis-inducing ligand. J Neurosurg. 2006;105:88-95.
35、 Lightfoot HM, Cairns BA, Lapaglia M, Pleasant A, Meyer A, Fair J. Murine Embryonic Stem (ES) cells inhibit human pancreatic carcinoma cell growth in vitro. J Sur Res. 2004;121:342.
36、 http://www.nature.com/news/2006/061106/full/061106-17.html
37、 Postovit LM, Seftor EA, Seftor RE, Hendrix MJ. A three-dimensional model to study the epigenetic effects induced by the microenvironment of human embryonic stem cells. Stem Cells. 2006;24:501-505.
38、 Postovit LM, Margaryan NV, Seftor EA, Kirschmann DA, Lipavsky A, Wheaton WW, Abbott DE, Seftor RE, Hendrix MJ. Human embryonic stem cell microenvironment suppresses the tumorigenic phenotype of aggressive cancer cells. Proc Natl Acad Sci U S A. 2008;105:4329-34.
1、Beebe D J,Mensing GA,Walker GM.Physics and Applications of Microfluidics in Biology.Annu Rev Biomed Eng.2002;4:261-286.
2、Sia SK,Whitesides GM.Microfluidic devices based on poly(dimethylsiloxane)for biological studies.Electrophoresis.2003;24:3563-3576.
3、Ju XL,Li D,Gao N,Shi Q,Hou HS.Hepatogenic Differentiation of Mesenchymal Stem Cells using Microfluidic Chips.Biotechnol J.2008;doi:10,1002/biot.200707152.
4、岳志红,张正.微流控芯片技术及其在检验医学中的应用.现代仪器.2004;5:14-16.
5、Chiu DT,Jeon NL,Huang S,Kane RS,Wargo CJ,Choi IS,Ingber DE,Whitesides GM.Patterned deposition of cells and proteins onto surfaces by using three-dimensional microfluidic systems.Proc Natl Acad Sci U S A.2000;97:2408-2413.
6、Glasgow IK,Zeringue HC,Beebe DJ,Choi SJ,Lyman JT,Chan NG,Wheeler MB.Handling individual mammalian embryos using microfluidics.IEEE Trans Biomed Eng.2001;48:570-578.
7、Hatch A,Kamholz AE,Hawkins KR,Munson MS,Schilling EA,Weigl BH,Yager P.A rapid diffusion immunoassay in a T-sensor.Nat Biotechnol.2001;19:461-465.
8、Yang J,Huang Y,Wang XB,Becker FF,Gascoyne PR.Cell separation on micro fabricated electrodes using dielectrophoretic/gravitational field flow fractionation.Anal Chem.1999;71:911-918.
9、Heasley LE.Autocrine and paracrine signaling through neuropeptide receptors in human cancer.Oncogene.2001;20:1563-9.
10、Tsai RYL,Mckay RDG.Cell contact regulates fate choice by cortical stem cells.J Neurosci.2000;20:3725-35.
11、 Purpura KA, Aubin JE, Zandstra PW. Sustained in vitro expansion of bone progenitors is cell density dependent. Stem Cells. 2003;22:39-50.
12、 Javazon EH, Colter DC, Schwarz EJ, Prockop DJ. Rat marrow stromal cells are more sensitive to plating density and expand more rapidly from single-cell-derived colonies than human marrow stromal cells. Stem Cells. 2001; 19:219-25.
13、 Zandstra PW, Le HV, Daley GQ, Griffith LG, Lauffenburger DA. Leukemia inhibitory factor (LIF) concentration modulates embryonic stem cell self-renewal and differentiation independently of proliferation. Biotechnol Bioeng. 2000;69:607-17.
14、 Kim L, Vahey MD, Lee HY, Voldman J. Microfluidic arrays for logarithmically perfused embryonic stem cell culture. Lab Chip. 2006;3:394-406.
15、 Rosenthal A, Macdonald A, Voldman J. Cell patterning chip for controlling the stem cell microenvironment. Biomaterials. 2007;21:3208-16.
16、 Chung BG, Flanagan LA, Rhee SW, Schwartz PH, Lee AP, Monuki ES, Jeon NL. Human neural stem cell growth and differentiation in a gradient-generating microfluidec device. Lab chip. 2005;5:401-406.
17、 Nagrath S, Sequist LV, Maheswaran S, Bell DW, Irimia D, Ulkus L, Smith MR, Kwak EL, Digumarthy S, Muzikansky A, Ryan P, Balis UJ, Tompkins RG, Haber DA, Toner M. Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature. 2007;450:1235-39.
18、 Nie FQ, Yamada M, Kobayashi J, Yamato M, Kikuchi A, Okano T. On-chip cell migration assay using microfluidic channels. Biomaterials. 2007;27:4017-22.
19、 Zhong JF, Chen Y, Marcus JS, Scherer A, Quake SR, Taylor CR, Weiner LP. A microfluidic processor for gene expression profiling of single human embryonic stem cells. Lab Chip. 2008;8:68-74.
Chung BG, Flanagan LA, Rhee SW, Schwartz PH, Lee AP, Monuki ES, Jeon NL (2005) Human neural stem cell growth and differentiation in a gradient-generating microfluidec device. Lab chip 5:401-406
Hatch A, Kamholz AE, Hawkins KR, Munson MS, Schilling EA, Weigl BH, Yager P (2001) A rapid diffusion immunoassay in a T-sensor. Nat Biotechnol 19:461-465
Ju XL, Li D, Gao N, Shi Q, Hou HS (2008) Hepatogenic Differentiation of Mesenchymal Stem Cells using Microfluidic Chips. Biotechnol J. doi:10,1002/biot.200707152
Lightfoot HM, Cairns BA, Lapaglia M B, Pleasant BA, Meyer AA, Fair J (2004) Murine Embryonic Stem (ES) cells inhibit human pancreatic carcinoma cell growth in vitro. J Sur Res 121:342
Mueller D, Shamblott MJ, Fox HE, Gearhart JD, Martin LJ (2005) Transplanted human embryonic germ cell-derived neural stem cells replace neurons and oligodendrocytes in the forebrain of neonatal mice with excitotoxic brain damage. J Neurosci Res 82:592-608
Nie FQ, Yamada M, Kobayashi J, Yamato M, Kikuchi A, Okano T (2007) On-chip cell migration assay using microfluidic channels. Biomaterials 27:4017-22
Postovit LM, Seftor EA, Seftor RE Hendrix MJ (2006) A three-dimensional model to study the epigenetic effects induced by the microenvironment of human embryonic stem cells. Stem Cells 24:501-505
Postovit LM, Costa FF, Bischof JM Seftor EA, Wen B, Seftor RE, Feinberg AP, Soares MB, Hendrix MJ (2007) The commonality of plasticity underlying multipotent tumor cells and embryonic stem cells. J Cell Biochem 9999:1-10
Rosenthal A, Macdonald A, Voldman J (2007) Cell patterning chip for controlling the stem cell microenvironment. Biomaterials 21:3208-16
Shamblott MJ, Axelman J, Wang S Bugg EM, Littlefield JW, Donovan PJ,
Blumenthal PD, Huggins GR, Gearhart JD (1998) Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc Natl Acad Sci U S A 95:13726-13731
Shamblott MJ, Axelman J, Littlefield JW, Blumenthal PD, Huggins GR, Cui Y,
Cheng L, Gearhart JD (2001) Human embryonic germ cell derivatives express a broad range of developmentally distinct markers and proliferate extensively in vitro. Proc Natl Acad Sci U S A 98:113-118
Sia SK, Whitesides GM (2003) Microfluidic devices based on poly (dimethylsiloxane) for biological studies. Electrophoresis 24:3563-3576
Turnpenny L, Brickwood S, Spalluto CM, Piper K, Cameron IT, Wilson DI, Hanley NA (2003) Derivation of human embryonic germ cells: an alternative source of pluripotent stem cells. Stem Cells 21:598-609
1 Postovit LM, Costa FF, Bischof JM et al. The commonality of plasticity underlying multipotent tumor cells and embryonic stem cells. J Cell Biochem 2007; 9999: 1-10.
2 Bjerkvig R, Tysnes BB, Aboody KS et al. Opinion: the origin of the cancer stem cell: current controversies and new insights. Nat Rev Cancer 2005; 5: 899-904.
3 Galmozzi E, Facchetti F, LaPorta CA. Cancer stem cells and therapeutic perspectives. Curr Med Chem 2006; 13: 603-607.
4 Postovit LM, Seftor EA, Seftor RE et al. Influence of the microenvironment on melanoma cell fate determination and phenotype. Cancer Res 2006; 66: 7833-7836.
5 Lightfoot HM, Cairns BA, Lapaglia M et al. Murine Embryonic Stem (ES) cells inhibit human pancreatic carcinoma cell growth in vitro. J Sur Res 2004; 121: 342.
6 Postovit LM, Seftor EA, Seftor RE et al. A three-dimensional model to study the epigenetic effects induced by the microenvironment of human embryonic stem cells. Stem Cells 2006; 24: 501-505.
7 Shamblott MJ, Axelman J, Wang S et al. Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc Natl Acad Sci U S A 1998; 95: 13726-13731.
8 Noske A, Kaszubiak A, Weichert W et al. Specific inhibition of AKT2 by RNA interference results in reduction of ovarian cancer cell proliferation: increased expression of AKT in advanced ovarian cancer. Cancer Lett 2007; 246: 190-200.
9 Stewart R, Stojkovic M, Lako M. Mechanisms of self-renewal in human embryonic stem cells. Eur J Cancer 2006; 42: 1257-1272.
10 Turnpenny L, Brickwood S, Spalluto CM et al. Derivation of human embryonic germ cells: an alternative source of pluripotent stem cells. Stem Cells 2003; 21: 598-609.
11 Park JH, Kim SJ, Lee JB et al. Establishment of a human embryonic germ cell line and comparison with mouse and human embryonic stem cells. Mol Cells 2004; 17: 309-315.
12 Shamblott MJ, Axelman J, Littlefield JW et al. Human embryonic germ cell derivatives express a broad range of developmentally distinct markers and proliferate extensively in vitro. Proc Natl Acad Sci U S A 2001; 98: 113-118.
13 Mueller D, Shamblott MJ, Fox HE et al. Transplanted human embryonic germ cell-derived neural stem cells replace neurons and oligodendrocytes in the forebrain of neonatal mice with excitotoxic brain damage. J Neurosci Res 2005; 82: 592-608.
14 Ohtaka T, Matsui Y, Obinata M. Hematopoietic development of primordial germ cell-derived mouse embryonic germ cells in culture. Biochem Biophys Res Commun 1999; 260: 475-482.
15 Kim MS, Hwang NS, Lee J et al. Musculoskeletal differentiation of cells derived from human embryonic germ cells. Stem Cells 2005; 23: 113-123.
16 Rahimi G, Isachenko E, Sauer H et al. Measurement of apoptosis in long-term cultures of human ovarian tissue. Reproduction 2001; 122: 657-663.
17 Stahelin BJ, Marti U, Solioz M et al. False positive staining in the TUNEL assay to detect apoptosis in liver and intestine is caused by endogenous nucleases and inhibited by diethyl pyrocarbonate. Mol Pathol 1998; 51: 204-208.
18 Liu JR, Opipari AW, Tan L et al. Dysfunctional apoptosome activation in ovarian cancer: implications for chemoresistance. Cancer Res 2002; 62: 924-931.
19 Park JY, Park JM, Jang JS et al. Caspase 9 promoter polymorphisms and risk of primary lung cancer. Hum Mol Genet 2006; 15: 1963-1971.
20 Hendrix MJ, Seftor EA, Seftor RE et al. Reprogramming metastatic tumour cells with embryonic microenvironments. Nat Rev Cancer 2007; 7: 246-255.
21 Reya T, Morrison SJ, Clarke MF et al. Stem cells, cancer, and cancer stem cells. Nature 2001; 414: 105-111.
22 Penninger JM, Woodgett J. Stem cells. PTEN—coupling tumor suppression to stem cells? Science 2001; 294: 2116-2118.
23 Conus NM, Hannan KM, Cristiano BE et al. Direct identification of tyrosine 474 as a regulatory phosphorylation site for the Akt protein kinase. J Biol Chem 2002; 277: 38021-38028.
24 Armstrong L, Hughes O, Yung S et al. The role of PI3K/AKT, MAPK/ERK and NFkappabeta signalling in the maintenance of human embryonic stem cell pluripotency and viability highlighted by transcriptional profiling and functional analysis. Hum Mol Genet 2006; 15: 1894-1913.
25 Miura T, Mattson MP, Rao MS. Cellular lifespan and senescence signaling in embryonic stem cells. Aging Cell 2004; 3: 333-343.