A Human Embryonic Stem Cell Model for Trophoblast Differentiation and Placental..

用于滋养层分化和胎盘的人类胚胎干细胞模型。

• 批准号: 7633376
• 负责人: THADDEUS G GOLOS
• 金额: $ 14.69万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7633376
• 关键词: 17p; 3-Dimensional; Achievement; Adherent Culture; Adhesions; Age; Apoptosis; Appearance; Appendix; Area; Baltimore; Biological Assay; Biological Markers; Boston; Burn injury; Canada; Cell Count; Cell Line; Cell model; Cells; Characteristics; Choriocarcinoma; Chorionic villi; Coculture Techniques; Collaborations; Collagen Type IV; Commit; Communication; Complement; Condition; Cues; Culture Media; Cytokeratin; Daily; Data; Development; Digestion; District of Columbia; Drops; Embryo; Embryonic Development; Endometrial; Endometrium; Endothelial Cells; Endothelium; Environment; Estradiol; Event; Exhibits; Exposure to; Extracellular Matrix; Fetal Growth; Fibroblasts; Figs - dietary; Funding; Gel; Gene Expression; Genes; Genetic Transcription; Glucose; Growth Factor; Gynecologic; HLA G antigen; Hormones; Human; Human Chorionic Gonadotropin; Hyperglycemia; Immunohistochemistry; In Vitro; Indium; Individual; Integrins; International; Invasive; Investigation; Laboratories; Laminin; Lead; Ligands; Lysine; Maintenance; Maternal-Fetal Exchange; Mediating; Membrane Proteins; Microarray Analysis; Modeling; Modification; Molecular; Mononuclear; Morphogenesis; Morphology; Mus; Ontario; Organogenesis; Oxidative Stress; PTK2 gene; Pathway interactions; Personal Satisfaction; Phenotype; Phosphorylation; Physiological; Pilot Projects; Placenta; Placentation; Plasmids; Plastics; Play; Population; Pregnancy; Preparation; Primates; Principal Investigator; Progesterone; Proliferating; Publications; Rate; Regulation; Reproduction; Research Personnel; Role; San Francisco; Signal Transduction; Signal Transduction Pathway; Site; Small Interfering RNA; Societies; Staging; Stem Cell Research; Stem cells; Stromal Cells; Structure; Study models; Surface; Suspension Culture; Suspension substance; Suspensions; System; Thick; Time; Transcript; Trypsin; Undifferentiated; Villous; Week; Western Blotting; Woman; abstracting; base; cell growth; cellular imaging; cytotrophoblast; day; diabetic; embryo stage 2; enhanced green fluorescent protein; fetal; human embryonic stem cell; implantation; in vivo; interest; mRNA Expression; matrigel; migration; natural Blastocyst Implantation; nonhuman primate; novel; paxillin; pluripotency; prevent; progenitor; programs; research study; response; shear stress; stem; success; tool; trophoblast; two-dimensional

Principal Investigator/Program Director (Last, First, Middle): MagneSS, R.R. Pjt. IV PI: GolOS, T.G. lines express a markedly different repertoire of genes, suggesting that these cell lines may respond differently to some experimental conditions, such as oxidative stress or adhesion and migration. Publications acknowledging support of HD38843 (collaborating P01P.l.s are in bold) Burleigh, D.W., K. Stewart, K.M. Grindle, R.R.Magness, H.H. Kay,and T.G.Golos. 2004 Proliferation and Apoptosis in Placenta from Diabetic Women. J. Soc. Gynecol. Invest. 11:36-41. Liu, Y.P.,O.V.Dovzhenko, M.A. Garthwaite, S.V.Dambaeva, M. Burning, LM. Pollastrini, and T.G. Golos. 2004 Maintenance of Pluripotency in Human Embryonic Stem Cells Stably Over-Expressing Enhanced Green Fluorescent Protein. Stem Cells Dev. 13:636-45. Liu, Y.P.,S.V.Dambaeva, Oksana V. Dovzhenko, M.A. Garthwaite, and T.G.Golos. 2005 Stable plasmid- based siRNA silencing of gene expression in human embryonic stem cells. Stem Cells Dev. 14:487-92. Burleigh, D.W., K. M. Grindle, R. L. Grendell, C.M. Kendziorski, R.R. Magness and T.G. Golos. 2006 Microarray Analysis of BeWo and JEG3 Trophoblast Cell Lines: Identification of Differentially Expressed Transcripts. In press. Golos, T.G., LM. Pollastrini, and B. Gerami-Naini. 2006. Human embryonic stem cells as a model for trophoblast differentiation. Sem. Reproof. Med. In press. Burleigh, D.W., K. M. Grindle, R. L. Grendell, C.M.Kendziorski, and T.G. Golos. Differential regulation of gene transcription by hyperglycemia in BeWo and JEG3 Trophoblast Cell Lines. In Preparation. Abstracts presented: D.W. Burleigh, K. Stewart, K.M. Grindle, R.R. Magness, H.H. Kay, and T.G. Golos. Proliferation and Apoptosis in Placenta from Diabetic Women. Presented at the 48th Annual Meeting of the Society for Gynecologic Investigation, Toronto, Ontario, Canada, March 14-17, 2001. D.W. Burleigh, M. Durning and T.G. Golos. Glucose-regulated placental and trophoblast (gene) mRNA expression. Presented at the 35th Annual Meeting of the Society for the Study of Reproduction, Baltimore, MD, July 28-31, 2002. Y. Song, Y.X. Wen, B. Gerami-Naini, M. Durning, T. G. Golos, and J. Zheng. Differentiation of Endothelial Cells from Human Embryonic Stem (ES)Cells. Presented at the 50th Annual Meeting of the Society for Gynecologic Investigation, Washington, D.C., March 26-29, 2003. Burleigh, D.W., R.L.Grindell, K.M. Grindle, R.R.Magness and T.G.Golos. Microarray analysis of JEG3 and BeWo cell lines: comparison of differentially expressed transcripts. Presented at the 51st Annual Meeting of the Society for Gynecologic Investigation, Houston, TX, March 2004. Liu, Y.P., O.V. Dovzhenko, M.A. Garthwaite, S.V. Dambaeva, M. Durning, L.M. Pollastrini, and T.G. Golos. Maintenance of pluripotency in human embryonic stem cells stably over-expressing enhanced green fluorescent protein. Presented at the 2nd Annual Meeting of the International Society for Stem Cell Research, Boston MA, June 2004. Gerami-Naini, B., O.V. Dovzhenko, M. Durning, M.A. Garthwaite, and T.G.Golos. Detecting expression of proliferation, differentiation and apoptosis markers in trophoblast cells derived from embryoid bodies grown in three-dimensional matrix structure and in suspension culture. Presented at the 3rd Annual Meeting of the International Society of Stem Cell Research, San Francisco, CA, June 23-25, 2005. Shift of focus to an ESC model for placental development. The control of trophoblast differentiation was a longstanding area of both interest and activity in our lab,in which we had used primary cultures as well as established human choriocarcinoma cell lines. In collaboration with James Thomson, we had previously shown that nonhuman primate ESC have an enhanced ability to differentiate into trophoblasts in comparison to mouse ESC (14, 15), an observation subsequently also determined for human ESC (13). However, this differentiation was still found to be of low efficiency in hESC. Thus, during this time period, we initiated independent studies in the laboratory to determine whether we could devise alternative experimental systems in which the ability to differentiate into trophoblasts was enhanced. Soon after we obtained results demonstrating the instructive effects of extracellular matrix on trophoblast differentiation (below), we recognized that the embryoid body paradigm represented an opportunity to develop a unique model for studying early events in placental morphogenesis, and we began to collaborate with Dr. Zheng in the P01 to develop the background data and the tools for the studies proposed in Projects //, III and IV. Thus, we are now focusing our efforts on the hESC paradigms we have developed for modeling placental morphogenesis and PHS 398/2590 (Rev.09/04) IV-21 Continuation Format Page Principal Investigator/Program Director(Last, First, Middle): MagneSS, R.R. Pjt. IV PI: GoloS, T.G. this will be the focus of the competitive renewal of this Project. It should be recognized, thus, that Project IV in the preceding funding period supported in part experiments that collected the preliminary data for Core B, Project IV, andProject III, andnewdirections undertaken by Project II in this A1submission. MG3 Every day Every 3 days Every 5 days Every day Every 3 days Every Sdays Time of EB Culture o 10 20 so Day of Culture Fig. 3. Hormone secretion by EBs during 144 hrs of Fig. 4. Secretionof hCG by Matrigel-embedded EBs. The suspension culture, in comparison with unconditioned results of three representative experiments with ~ 50 medium. EBs/3ml dish are shown. Embryoid body formation. Although hCG secretion is detectable in spontaneously differentiating ESC cultures (18,and Gerami and Golos, unpublished), the typically modest levels of hCG secretion indicate that spontaneous trophoblast differentiation, while consistently detectable, is not extensive. We considered embryoid bodies (EBs) as an alternative model to evaluate trophoblast differentiation from hESC. EBs were prepared generally as previously described (32). Within 2 days after the initiation of suspension EB culture, the secretion of hCG, progesterone and estradiol was detectable in the culture medium, in comparison with unconditioned medium (Fig. 3). Reasoning that the EBs may be a good model for not only trophoblast differentiation, but also perhaps placenta! morphogenesis, we initiated pilot studies to evaluate EB cell growth in an ECMenvironment as a first approximation of implantation in the uterine endometrium. We cultured EBs in suspension for 9 days, and transferred them individually to 50-100 ul volume drops of growth factor reduced (BD Biosciences) Matrigel which had been allowed to gel at 37¿C. The Matrigel "rafts" containing the EBs were then transferred to fresh PHS 398/2590 (Rev. 09/04) IV-22 Continuation Format Page Principal Investigator/Program Director (Last, First, Middle): Magness, R.R. Pjt. IV PI: GolOS, T.G. EB culture medium. Culture was then continued, with gentle rocking,to prevent adhesion to the plastic. Please see Core B for details on embryoid body formation and culture in Matrigel. Fig. 5. Trophoblast-like outgrowths projecting from the surfaces of representative EBs explanted to Matrigel rafts. With these cultures, we noted a frequent appearance of projections from the surface of the EBs (Fig. 5). Although the appearance of the outgrowths was heterogeneous, virtually all EBs exhibited outgrowths into the Matrigel environment. These Matrigel-embedded EBs were maintained in culture for up to 50 days, and we determined hCG secretion into the culture medium (Fig. 4). Replicate cultures had medium replaced every 1, 3 or 5 days. While hCG secretion was detectable but relatively modest for the first 20 days in culture, there was a dramatic increase in hCG secretion that occurred reproducibly at approximately day 20 (three independent experiments are shown in Fig. 4). High levels of hCG (and progesterone and estradiol-17p) secretion were sustained for at least 4 weeks, and daily medium change promoted the highest level of hCG secretion. Additional experiments with delayed transfer to Matrigel demonstrated that exposure to matrix and not age of the EB was the critical element in the initiation of trophoblast differentiation (Appendix). Comparing secretion rates in suspension vs. Matrigel culture, an increase of > 10,000-fold induction is typical. Thus, the dramatic increase in hormone secretion following Matrigel explantation suggests that in suspension EBs, trophoblasts are relatively undifferentiated but that Matrigel exposure promotes cells from a progenitor/stem trophoblast, to a committed cell undergoing terminal differentiation. IHC studies demonstrate readily discerned populations of cytokeratin/hCG-positive cells. We conclude that trophoblast-like cells differentiate in EB cultures. Given these results, we wished to determine if association of EBs with Matrigel under "two-dimensional" conditions was sufficient to promote long-term hormone secretion. This is particularly important with regards to our ability to evaluate differentiation on diverse matrix surfaces. We conducted experiments with suspension EB cultures transferred to culture dishes and allowed to adhere to a thin coating of Matrigel on the culture surface (Fig. 6, also Appendix). . EB plated on Matrigel coated plates 1200 1000 E 800 [ I O 600 [¿ 400 . 200 ' T J A n n n fi H III 1 5 10 11 12 13 14 15 16 17 18 19 20 21 22 25 Day of Culture Fig. 6. Secretion of hCG in EBs allowed to Matrigel-coated dishes in adherent culture. PHS 398/2590 (Rev. 09/04) L f - 30 35 40 43 attach to Fig. 7. Representative images of cells derived from embryoid bodies cultured on Matrigel. Syncytial cells were seen 6 days after passage, whereas freshly passaged cells were all mononuclear(not shown). IV-23 Continuation Format Page Principal Investigator/ProgramDirector (Last, First, Middle): MagneSS, R.R. Pjt.IV PI: GolOS, T.G. The results demonstratethat hCG, progesteroneand estradiol-17p secretion beganto increaseapproximately 10 days after attachmentto the culture surface and was sustainedfor 2-4 weeks, depending on the thickness ofthe Matrigel. For several of the replicate wells in this experiment, we wished to determine if the outgrowths could be passaged and replated and would sustain proliferation. Cellular outgrowths were resuspended following trypsin treatment and were replated on Matrigel-coated plates. Cells were readily adherent and continued to proliferate, and passagedcells in all wells exhibited at least some syncytiumformation (Fig. 7), characteristic of both primary human villous cytotrophoblast cultures as well as BMP4-treated hES cells (16,33). In addition, hCG, progesterone and estradiol secretion was maintained through at least two passageswith these cells (not shown). Similar results have been obtained in experiments with cytokeratin-positive cells dispersed from suspension EBs with limitedtrypsin digestion. EB outgrowths on individual ECMcomponents. Wehave begun to conduct preliminary experiments onthe differentiation of trophoblastic outgrowths from hESC-derived embryoid bodies, as assayed by hCG secretion. Initial results are shown in Fig. 8, which presents hCG levels in EBs plated in 2-dimensional culture on Matrigel, laminin, and poly-l-lysine-coated dishes. While culture on Matrigel promotes the highest secretion of hCG, laminin (Fig. 8) and collagen IV (not shown) also promote substantially higher levels than other surfaces, includingpoly-l-lysine. Hormone secretion is not due to substantially different EB adhesion (cell number, day2) or proliferation (cell number, day 24) (Fig.9). Further 2-D model studies, extended to signaling and microarray analysis, are proposed in Aim 1. Fig. 8. Secretion ofhCGin 2-DEBcultures on varied Fig. 9. Cellnumber in 2-DEBculture experiments on matrix protein surfaces. various matrix surfaces. Integrin signaling components. Relevant to Specific Aim 1,we have conducted preliminary western blot and immunohistochemistry analysis of undifferentiated hESC and suspension and 2-D Matrigel cultured EBs from hESC. Western blots were optimized by the Molecular Core. Preliminary data in Fig. 10 demonstrates expression of several of these molecules by western blot of extracts from suspension EBs, including FAK, ROCK, and paxillin. While not unexpectedly, all are expressed in differentiated as well as undifferentiated cells, defining their activation/phosphorylation states is the critical experiment, which is proposed in

首席研究员/项目总监（最后、第一、中间）：MagneSS，R.R. Pjt IV PI：GolOS，T.G. 表达细胞系具有明显不同的基因库，表明这些细胞系可能有不同的反应 一些实验条件，例如氧化应激或粘附和迁移。 HD38843 的出版物兼容支持（合作 P01P.l.s 以粗体显示） Burleigh, D.W.、K. Stewart、K.M. Grindle、R.R.Magness、H.H. Kay 和 T.G.Golos 2004 年 糖尿病女性的胎盘细胞凋亡。11：36-41。 刘，Y.P.，O.V. Dovzhenko，M.A. Garthwaite，S.V. Dambaeva，M. Burning，LM Pollastrini，和 T.G. 2004年维持人类胚胎干细胞的多能性稳定过度表达增强型绿色 荧光蛋白。13：636-45。 Liu，Y.P.，S.V.Dambaeva，Oksana V. Dovzhenko，M.A. Garthwaite，和 T.G.Golos 2005 年稳定质粒- 基于 siRNA 的人类胚胎干细胞基因表达沉默。 14：487-92。 Burleigh，D.W.，K.M. Grindle，R.L. Grendell，C.M. Kendziorski，R.R. Magness 和 T.G. BeWo 和 JEG3 滋养层细胞系的微阵列分析：差异表达的鉴定 文字记录正在出版。 Golos, T.G.、LM. Pollastrini 和 B. Gerami-Naini，2006 年。人类胚胎干细胞作为模型。 滋养细胞分化。 Burleigh，D.W.，K. M. Grindle，R. L. Grendell，C.M. Kendziorski，和 T.G. 准备中的 BeWo 和 JEG3 滋养层细胞系中高血糖的基因转录。 提出的摘要： D.W. Burleigh、K.M. Grindle、R.R. Magness、H.H. Golos 和 T.G. 糖尿病妇女胎盘细胞凋亡在第 48 届糖尿病学会年会上发表。 妇科调查，加拿大安大略省多伦多，2001 年 3 月 14-17 日。 D.W. Burleigh，M. Durning 和 T.G. Golos。 在马里兰州巴尔的摩生殖研究学会第 35 届年会上提出。 2002 年 7 月 28 日至 31 日。 Y. Song、Y.X. Wen、B. Gerami-Naini、M. Durning、T. G. Golos 和 J. Cheng。 人类胚胎干 (ES) 细胞在第 50 届学会年会上发表。 妇科调查，华盛顿特区，2003 年 3 月 26-29 日。 Burleigh, D.W.、R.L.Grindell、K.M. Grindle、R.R.Magness 和 T.G.Golos 对 JEG3 和的微阵列分析。 BeWo 细胞系：差异表达转录本的比较，在第 51 届年会上发表。 妇科调查协会，德克萨斯州休斯顿，2004 年 3 月。 刘，Y.P.，O.V. Dovzhenko，M.A. Garthwaite，S.V. Dambaeva，M. Durning，L.M. Pollastrini，和 T.G. 稳定过度表达增强型绿色以维持人胚胎干细胞的多能性 荧光蛋白在国际干细胞研究学会第二届年会上提出， 马萨诸塞州波士顿，2004 年 6 月。 Gerami-Naini, B.、O.V. Dovzhenko、M. Durning、M.A. Garthwaite 和 T.G.Golos 的表达。 来源于生长在拟胚体中的滋养层细胞的增殖、分化和凋亡标记 三维矩阵结构和悬浮培养在第三届年会上提出。 国际干细胞研究学会，加利福尼亚州旧金山，2005 年 6 月 23-25 日。 将焦点转向胎盘发育的 ESC 模型 滋养层分化的控制是一个因素。 我们实验室长期感兴趣和活动的领域，我们在其中使用了原代培养物以及 我们之前与 James Thomson 合作建立了人类绒毛膜癌细胞系。 结果表明，与人类相比，非人灵长类 ESC 分化为滋养层细胞的能力增强 小鼠 ESC (14, 15)，随后的观察结果也确定了人类 ESC (13)。 仍然发现 hESC 的分化效率较低，因此，在这段时间内，我们开始了。 在实验室进行独立研究，以确定我们是否可以设计替代实验系统 在我们获得结果后不久，分化为滋养层细胞的能力就得到了增强。 为了证明细胞外基质对滋养层分化的指导作用（如下），我们 认识到类胚体范式代表了开发独特模型的机会 研究胎盘形态发生的早期事件，我们开始与郑博士合作P01 为项目 //、III 和 IV 中提出的研究开发背景数据和工具。 我们将精力集中在我们为胎盘形态发生建模而开发的 hESC 范式上 PHS 398/2590 (Rev.09/04) IV-21 延续格式页 首席研究员/项目总监（最后，第一，中间）：MagneSS，R.R. Pjt IV PI：GoloS，T.G. 这将是该项目竞争性更新的重点，因此，应该认识到，项目IV在。 之前的资助期支持了部分实验，收集了核心 B 的初步数据， 项目 IV、项目 III 以及项目 II 在本 A1 提交中采取的新方向。 MG3 每天 每3天一次 每 5 天一次 每天 每3天一次 每天 EB 培养时间 o 10 20 左右 文化日 图3.图4的144小时内EB分泌的激素。基质胶包埋的EB分泌hCG。 悬浮培养，与三个代表性实验的无条件结果进行比较，约 50 显示了 EB/3ml 培养皿。 尽管在自发分化的 ESC 中可检测到 hCG 分泌。 培养物（18，以及 Gerami 和 Golos，未发表），通常适度的 hCG 分泌水平表明 我们认为自发的滋养层分化虽然可以持续检测到，但并不广泛。 拟胚体（EB）作为评估 hESC 滋养层分化的替代模型。 一般如前所述(32)制备，在开始悬浮EB培养后2天内， 与对照组相比，培养基中可检测到 hCG、孕酮和雌二醇的分泌 无条件培养基（图3）。 推断 EB 不仅可能是滋养层分化的良好模型，而且可能是 胎盘！形态发生，我们启动了初步研究以评估 ECM 环境中的 EB 细胞生长 我们将 EB 悬浮培养 9 天，并近似植入子宫内膜。 将它们分别转移至 50-100 ul 体积的生长因子还原滴 (BD Biosciences) Matrigel 已在 37° 下胶凝C. 然后将含有 EB 的基质胶“筏”转移至新鲜的 PHS 398/2590（修订版 09/04） IV-22 延续格式页 首席研究员/项目总监（最后、第一、中间）：Magnes, R.R. Pjt IV PI：GolOS, T.G. 然后继续培养 EB 培养基，轻轻摇动，以防止粘附到塑料上。 有关拟胚体形成和基质胶培养的详细信息，请参阅核心 B。 图 5. 从滋养层突出的滋养层样生长物 移植到基质胶筏上的代表性 EB 的表面。 在这些文化中，我们注意到经常出现 EB 表面的投影（图 5）。 产物的外观是异质的， 几乎所有 EB 都会生长到基质胶中 这些基质胶嵌入的 EB 是 在培养物中维持长达 50 天，并且我们 测定 hCG 分泌到培养基中（图 1）。 4) 重复培养物每 1、3 或 1 次更换培养基。 5天后虽然可检测到hCG分泌。 前 20 天的文化相对温和， hCG 分泌急剧增加 大约在第 20 天可重复（三个独立的 实验如图4所示）。 孕酮和雌二醇-17p) 分泌持续至少 4 周，并且每天更换培养基 促进最高水平的 hCG 分泌 延迟转移至基质胶的附加实验。 证明暴露于基质而不是 EB 的年龄是启动的关键因素 滋养层分化（附录）比较悬浮液与基质胶培养物的分泌率。 诱导后增加> 10,000 倍是典型的，因此，激素分泌随后急剧增加。 基质胶外植表明，在悬浮 EB 中，滋养层细胞相对未分化，但 基质胶暴露促进细胞从祖细胞/干滋养层转变为经历终末期的定型细胞 IHC 研究表明细胞角蛋白/hCG 阳性细胞群体易于识别。 结论是滋养层样细胞在 EB 培养物中分化。 鉴于这些结果，我们希望确定 EB 与基质胶在“二维”下是否存在关联 条件足以促进长期激素分泌，这对于激素分泌而言尤其重要。 我们评估不同基质表面差异的能力我们用悬浮液进行了实验。 EB 培养物转移至培养皿中并使其粘附在培养物上的薄层基质胶上 表面（图 6，也是附录）。 EB 镀在基质胶涂层板上 1200 1000 E 800 [ 我 600 欧 [?? 400。 200' 蒂亚戈 n n n fi H III 1 5 10 11 12 13 14 15 16 17 18 19 20 21 22 25 文化日 图 6. 允许 EB 中 hCG 的分泌 贴壁培养中的基质胶包被培养皿。 PHS 398/2590（修订版 09/04） L f - 30 35 40 43 附于图7。来自细胞的代表性图像 在基质胶上培养的拟胚体。 传代后 6 天可见，而新鲜的 传代细胞均为单核细胞（未显示）。 IV-23 延续格式页 首席研究员/项目总监（最后、第一、中间）：MagneSS, R.R. Pjt.IV PI：GolOS, T.G. 结果表明，hCG、孕酮和雌二醇-17p 分泌开始增加约 附着到培养表面后 10 天，持续 2-4 周，具体取决于厚度 基质胶。 对于本实验中的几个重复孔，我们希望确定产物是否可以 传代并重新铺板并将维持增殖，然后将细胞产物重悬。 胰蛋白酶处理并重新铺板到基质胶包被的板上，细胞很容易贴壁并继续。 增殖，并且所有孔中的传代细胞至少表现出一些合胞体形成（图7），特征 原代人绒毛细胞滋养层培养物以及 BMP4 处理的 hES 细胞 (16,33)。 此外，hCG、孕酮和雌二醇的分泌通过至少两次传代得以维持 细胞（未显示）在使用分散的细胞角蛋白阳性细胞的实验中获得了类似的结果。 来自胰蛋白酶消化有限的悬浮 EB。 EB 在各个 ECM 组件上的生长 我们已经开始进行初步实验。 通过 hCG 分泌测定，滋养层细胞从 hESC 衍生的胚状体分化。 初步结果如图 8 所示，显示了铺在 2 维培养物中的 EB 中的 hCG 水平。 基质胶、层粘连蛋白和聚-L-赖氨酸包被的培养皿中，基质胶上的培养物可促进最高水平的分泌。 hCG、层粘连蛋白（图 8）和胶原蛋白 IV（未显示）也比其他表面促进显着更高的水平， 包括聚-L-赖氨酸的激素分泌并非由于 EB 粘附的显着差异（细胞数，第 2 天）。 或增殖（细胞数量，第 24 天）（图 9）。 分析，在目标 1 中提出。 图 8. 不同的 2-DEB 培养物中 hCG 的分泌 图 9. 2-DEB 培养物实验中的细胞数 基质蛋白质表面。 与特定目标1相关的整合素信号成分，我们进行了初步的蛋白质印迹和 对未分化 hESC 以及悬浮液和 2-D Matrigel 培养的 EB 进行免疫组织化学分析 图 10 中的初步数据显示了 hESC 的蛋白质印迹。 通过对悬浮 EB 提取物进行蛋白质印迹分析其中几种分子的表达，包括 FAK、 ROCK 和桩蛋白虽然并不意外，但均以分化和未分化形式表达。 细胞，定义其激活/磷酸化状态是关键的实验，这是在