hESCs在hEF支持下向内胚层诱导及正常与孤雌hESCs向胰岛样细胞诱导比较研究
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摘要
本研究室于2002年自主建立了人类胚胎干细胞(hESCs)系,至今已建立存有240余株hESCs系库。前期工作中成功利用人源性饲养层(hEF)体系下培养的hESCs诱导分化为限定性内胚层细胞和胰腺前体细胞,积累了诱导分化的经验。在此基础上,本课题组对hESCs向限定性内胚层细胞及胰岛素分泌细胞体外分化的条件进行了摸索和优化,首次发现hEF细胞在诱导分化过程中也发挥着辅助支持作用,对其机制进行了初步研究,显示hEF主要通过分泌可溶性因子激活ActivinA信号和Wnt信号通路发挥作用；模拟hEF细胞的辅助作用加入Wnt3a后发现Wnt3a在早期hESCs向内胚层诱导过程中发挥作用的窗口期是诱导第1天,成功建立无饲养层条件下hESCs向限定性内胚层细胞及胰岛素分泌细胞的诱导体系,在此基础上将自主建系的孤雌来源hESCs诱导分化为胰岛素分泌细胞,并对正常来源hESCs和孤雌来源hESCs在增殖能力与诱导效率等方面进行了比较,初步探讨了孤雌和正常来源hESCs向胰岛素分泌细胞体外诱导分化效率、增殖能力以及印记基因表达上的差异。本研究分为3个部分,主要研究方法和结果如下：
第一章hEF体系培养的hESCs向限定性内胚层的诱导分化
目的：1、研究在hEF细胞上培养的hESCs在高浓度ActivinA作用下向限定性内胚层分化过程中内胚层发育相关基因及蛋白表达的动态变化是否遵循体内发育模式；2、研究hEF细胞培养体系下诱导所得限定性内胚层细胞的体内外进一步分化能力；
方法：采用已有的ActivinA+FBS(AS)方法诱导hEF体系上培养的hESCs,收集O小时,6小时,12小时,1天,2天,3天,4天,5天的细胞,免疫荧光染色计数Brachyury和Sox17阳性率；real-timePCR检测原肠作用、内中外胚层、多能性标记的表达水平变化；将诱导5天所得细胞植入SCID鼠腿部肌肉,2月后对移植物进行切片HE染色,组织学分型各胚层来源比例；并将诱导5天细胞加入视黄酸,烟碱,肠促胰岛素类似物,重组人β-细胞调节素等因子,体外诱导内胚层细胞进一步分化为胰岛素分泌细胞；进行免疫荧光染色内分泌标记,PCR检测胰腺相关标记表达。
结果：PCR结果显示,采用AS法诱导hEF体系下的hESCs,在诱导第1天,原条标记GSC、Mixl1和内中胚层共同前体标记Brachyury表达明显上升,至第2天达高峰。内胚层标记Foxa2和Sox17在第2天开始上升,至第3天达高峰。染色计数结果显示Brachyury在诱导第2天达到高峰,阳性细胞率为43.7±16.6%；Sox17在诱导4天时达高峰81.7±5.4%；将诱导5天细胞移植入SCID小鼠体内后,移植物组织分型显示约75%的细胞为内胚层来源组织,外胚层来源组织比例低于1%。体外进一步诱导后,在诱导18天细胞表达胰腺前体标记pdx1, ngn3和beta2,在诱导25天细胞表达胰腺内外分泌及功能相关基因。细胞团染色显示外层为c肽和胰高血糖素双阳性细胞。
结论：在hEF体系上hESCs诱导分化为限定性内胚层细胞的过程符合体内内胚层发育规律,经过原条、内中胚层共同前体、内胚层3个阶段,获得了效率较高(约80%)的限定性内胚层细胞。该细胞具有在体内外进一步分化为更成熟内胚层组织细胞的能力。
第二章hEF细胞支持限定性内胚层分化的机制研究
目的：1、研究有饲养层及无饲养层培养体系上hESCs向内胚层细胞的诱导效率差异；2、研究hEF细胞是否分泌可溶性因子对限定性内胚层诱导产生影响；3、研究hEF细胞在限定性内胚层诱导过程中表达发育早期关键信号(TGFβ和Wnt信号)情况；4、摸索Wnt3a在限定性内胚层诱导中的作用时间窗,建立无饲养层体系下限定性内胚层诱导体系。
方法：对有饲养层体系(人饲养层H组和鼠饲养层M组)和无饲养层体系(FF组)上培养的hESCs进行AS法诱导,取诱导第5天细胞进行Soxl7染色计数和半定量PCR检测内胚层相关基因表达情况；在FF组基础上加入hEF条件培养基进行诱导(CM+A组),在诱导第5天染色计数Soxl7阳性细胞比率,PCR检测原条,内中外胚层标记的表达；取AS法诱导过程中不同时间点的hEF细胞,PCR检测胚胎早期发育关键信号TGF-β和Wnt信号通路中分泌型因子的基因表达；取FF组和CM+A组诱导1天细胞,western blot检测Wnt信号下游β-catenin的蛋白表达变化；在FF组加入25ng/ml Wnt3a作用1—4天,对不同培养时间点的细胞进行Brachyury, Sox 17染色计数,PCR检测原条,内中外胚层,胚外内胚层标记的表达。
结果：采用AS法诱导有饲养层体系下hESCs在诱导第5天均获得约80%的Sox17阳性细胞率(H组82.0±8.9%和M组78.7±3.4%),而无饲养层培养体系下Sox17阳性细胞率较低(FF组22.7±5.6%)；加入hEF细胞条件培养基后Sox17阳性率上升至63.2±13.4%(CM+A组),PCR检测结果显示在诱导过程中hEF细胞表达高强度ActivinA, Nodal在使用诱导培养基后6h—12h也出现了轻微的上调表达；同时hEF细胞还表达Wnt信号通路抑制剂Dkk1和Dkk3,并且Dkk3在进入诱导过程后6h起即出现了明显的下调,至12h,24h呈逐渐减弱的趋势；Western blot结果显示CM+A组较FF组在诱导第1天Wnt下游因子β-catenin的表达更强,PCR检测Wnt下游靶基因Brachyury和c-myc的表达在CM+A组也较FF组更强,以上结果提示饲养层细胞在内胚层诱导过程中一方面通过分泌ActivinA/Nodal等因子对内胚层诱导起正面促进作用,另一方面通过减弱对Wnt信号的抑制作用,反向活化Wnt信号,对内胚层诱导发挥辅助作用。进一步在CM+A体系加入Wnt3a后发现,Wnt3a与ActivinA共同作用1-4天,均能促进限定性内胚层的分化,共同作用1天Sox17阳性率上升至85.2±3.8%,是最佳的内胚层诱导体系。
结论：hEF通过表达高强度的Activin/Nodal信号,以及减弱对Wnt信号的抑制作用对限定性内胚层分化发挥的辅助作用。在无饲养层体系上,采用Wnt3a和ActivinA联合作用1天,再单独用ActivinA培养2天能最有效促进限定性内胚层的出现。
第三章孤雌与正常来源hESCs向胰岛素分泌细胞分化比较研究
目的：探讨孤雌来源hESCs在体外诱导因素调节下向胰岛素分泌细胞体外分化的能力,以及与正常hESCs在分化效率、增殖能力以及印记基因表达上的差异。
方法：以正常来源的hESCs (chHES137),孤雌来源的hESCs (chHES32,chHES69)共三株细胞为材料,采用第二章中Wnt3a和ActivinA共同作用1天组的内胚层诱导方法,结合视黄酸、烟碱、肠促胰岛素类似物、重组人p-细胞调节素等因子,分为内胚层、原始肠管、胰腺前体、内分泌前体、胰岛素分泌细胞5个阶段诱导。在分化的d0, d3, d6,d9, d12收集细胞,通过免疫荧光染色和real-time PCR来比较胰腺发育标记蛋白(Sox17, Pdx1)阳性率,胰腺发育标记基因和印记基因的表达水平,通过Ki67染色比较增殖能力上的差异。在分化终末阶段,收集细胞进行胰岛素染色、胰腺相关功能基因检测、胰岛素和C肽释放实验检测。
结果：诱导过程中3株hESCs的胰腺发育阶段标记基因的表达遵循胰腺发育的规律,在诱导d3出现内胚层标记表达高峰,诱导d6出现原始肠管标记高峰,诱导d9为胰腺前体标记表达高峰,通过定量PCR检测显示大部分发育标记基因中chHES137和chHES69的表达明显高于chHES32, Sox17和Pdx1染色计数结果也显示chHES137和chHES69的阳性细胞率明显高于chHES32。印记基因的表达遵循着印记基因表达规律,父源性基因(PEG3,IGF2)在正常来源hESCs的表达明显强于两株孤雌来源的hESCs10倍以上。母源性印记基因(GNAS,GRB10)在正常来源hESCs的表达则明显弱于两株孤雌来源的hESCs。chHES137和chHES69诱导终末细胞团均表达胰腺功能相关基因,对不同葡萄糖浓度刺激呈现反应性。3株细胞的增殖能力没有统计学差异。
结论：在体外诱导过程中,hESCs的分化遵循着胰腺体内发育的规律,孤雌来源hESCs具有分化为胰岛素分泌细胞的能力,诱导效率和增殖能力上与正常hESCs相比未见明显差异；孤雌和正常hESCs在诱导分化过程中,印记基因的表达基本符合表观遗传学规律,并具有一定的时间依赖性。
The first human embryonic stem cell (hESC) line with our intellectual property was established in 2002. To date, we have established more than 240 hESC lines, which come into being a hESCs bank. In our previous work, the hESCs cultured on human embryonic fibroblasts (hEF) were successfully differentiated into definitive endoderm and pancreatic progenitors. Based on these work, we improved the method of differentiation and found that the hEF cells played a role in the definitive endoderm differentiation for the first time. With studying the mechanism of hEF induction, we added Wnt3a and ActivinA into the hESCs to differentiate the definitive endoderm by simulating the function of hEF. We got high efficiency of Sox 17 positive cells on feeder free culture system. Based on this differentiation system, we compared the efficiency of insulin-producing cells, level of imprinted genes expression and the ability of proliferation of normal derived hESCs and parthenogenetic hESCs. This research is divided into 3 parts, and the main study methods and results are as follows:
Chapter 1
Differentiation of human embryonic stem cells into definitive endoderm on hEF cells
Objective:1. To study whether the variational expression of endodermal genes and proteins of the cells on different points during the definitive endoderm induction obey the endoderm developmental rule in vivo; 2. to study the differentiation potential of the hESCs derived inducing definitive endoderm cells on hEF cells in vivo and in vitro.
Method:adopting the reported method (adding ActivinA and FBS to differentiate into definitive endoderm, AS way) to induce the hESCs on hEF cells to endoderm cells. The different points (0h,6h,12h, 1d,2d,3d,4d,5d) of cells are collected to precede the immunochemistry staining of Brachyury and Sox 17 and real-time PCR detection of the expression of gastrulation, endoderm, mesoderm, ectoderm and pluripotent markers. The 5th day differentiating cells are transplanted into the legs of SCID mice for 2 months, then the transplants are removed from the mice and sectioned to do HE staining. Meanwhile, the 5th day cells are differentiated into insulin producing cells by RA, nicotinamide, extendin-4 and betacellulin etc in vitro.
Result:the PCR results show that on 1st day of differentiation the prime streak related genes (Goosecoid, Mixl1) and mesoendoderm precursor related gene Brachyury express increasably and get the top on 2nd day. The expressions of endodermal genes (Foxa2, Sox17) increase on 2nd day and get the top on 3rd day. The counting result of Brachyury shows that the peak is on 2nd day (43.7±16.6%) and Sox17 is on 4th day (81.7±5.4%).
The transplants of 5th differentiation day cells show that about 75% cells are derived from endoderm, less than 1% cells are derived from ectoderm. After differentiation in vitro, the 18th day cells express the pancreatic precursor markers pdx1, ngn3, beta2; the 25th day cells express pancreatic functional genes. The staining of clusters show the outer side of the clusters are C peptide and glucagon double positive cells.
Chapter 2
Studying the mechanism of hEF cells supporting definitive endoderm differentiation of hESCs
Objective:1. to study of whether with and without feeder cells culture system will effect the efficiency of endoderm cells; 2. to study of whether the human feeder cells secret some certain soluble factors to support the differentiation of endoderm cells; 3. to study of the activation of key signal pathways (TGFβ, Wnt) in human feeder cells during the differentiation procedure; 4. to confirm the effective time window of Wnt3a in endodermal induction and establish the feeder free inducing system into definitive endoderm cells.
Method:According to different cultivation systems, we divided the subject into three groups:(1) group H:the hES cells cultured on Human derived feeder cells; (2) group M:the hES cells cultured on Mouse derived feeder cells; (3) group FF:the hES cells cultured without feeder cells. We counted the Sox17 positive cells with immunofluorescence and detected the expression tendency of some genes correlated with gastrulating, definitive endoderm, mesoderm, and ectoderm genes using RT-PCR at 5th day in every group. Collecting the hEF cells at different time point, the expression of key signal pathways (TGFβ, Wnt) genes are detected by RT-PCR; Western blot detects theβ-catenin activation in group FF and CM+A. Based on group FF,25ng/ml Wnt3a are added from 1st to 4th day, different time cells are tested for Brachyury and Sox 17 staining and early developmental genes expression.
Result:On day 5 of induction, DE (SOX17+) cells appeared with comparable efficiency in both human and mouse feeder systems (85.0±8.9% and 78.7±3.4%, respectively). These levels were considerably superior to that obtained in the feeder-free system (22.7±5.6%). the HEF-conditioned inducing medium was used to differentiate the hESCs g rowing in group FF. The same additives were used, with the exception that the medium was cultured in HEF feeder cells for 24 h prior to the induction. Five days later, the percentage of SOX17+cells increased to 63.2±13.4%; this value was comparable to that of groups H and M. the hEF cells express strong ActivinA and the Nodal expression increases with the beginning of induction (6h-12h); hEF cells express the inhibitor of Wnt signal pathway (Dkk1, Dkk3), however, with the addition of ActivinA, the level of Dkk3 decrease obviously. With the addition of Wnt3a, we find that the different time of co-culture (1-4 days) of Wnt3a and ActivinA all can induce the sox17 positive cells, however, when the co-culture time is 1 day, the highest efficiency is gained (85.2±3.8%).
Chapter 3
Comparison of normal and parthenogentic human embryonic stem cells on inducing islet-like cells
Objective:to study of the differential potential of parthenogentic hESCs (hpESCs) into insulin producing cell clusters; to study of the difference between normal hESCs and hpESCs on differentiation efficiency, proliferating ability and imprinted-genes expression.
Method:the normal hESCs (chHES137), the hpESCs (chHES32, chHES69) are experimental materials. We adopt a modified 5-step protocol including the optimized endoderm inducing method on feeder free system with Wnt3a on 1st day. Different growth factors, such as ActivinA, retinoic acid (RA), Nicotinamide (NIC), and Exendin-4 (Ex-4), Betacellulin (BTC) etc. are added in the medium to induce the hpES cells differentiate into islet-like clusters. According to the sequence of development stages (d0, d3, d6, d9, d12), immunohistochemistry staining are proceeded to test the pancreatic developmental marker (Sox 17, Pdx1) and Real-time PCR are done to test the expression of pancreatic developmental marker and imprinted genes. Ki67 staining detects the proliferating ability.
Result:the expression pattern of pancreatic developmental genes obey the pancreas development rule in 3 hESCs. The level of endodermal markers achieves the peak at 3rd day, primary gut markers achieve the peak at 6th day, at 9th day the pancreatic precursor markers appear. Most of developmental genes are stronger in chHES137and chHES69 than chHES32. the imprinted genes'expression obey the rule too. Paternal genes (PEG3,IGF2) mainly express in normal hESCs.
The results from immunohistochemistry and RT-PCR showed that the islet-like clusters expressed the islet specific hormones and functional markers. Insulin release test and electron microscopy indicated that the clusters had the biochemical function of islet. The islet-like clusters derived from phES cells had the basic characteristics of islet. There is no significant difference between 3 hESCs in proliferating ability.
第一章hEF体系培养的hESCs向限定性内胚层的诱导分化
目的：1、研究在hEF细胞上培养的hESCs在高浓度ActivinA作用下向限定性内胚层分化过程中内胚层发育相关基因及蛋白表达的动态变化是否遵循体内发育模式；2、研究hEF细胞培养体系下诱导所得限定性内胚层细胞的体内外进一步分化能力；
方法：采用已有的ActivinA+FBS(AS)方法诱导hEF体系上培养的hESCs,收集O小时,6小时,12小时,1天,2天,3天,4天,5天的细胞,免疫荧光染色计数Brachyury和Sox17阳性率；real-timePCR检测原肠作用、内中外胚层、多能性标记的表达水平变化；将诱导5天所得细胞植入SCID鼠腿部肌肉,2月后对移植物进行切片HE染色,组织学分型各胚层来源比例；并将诱导5天细胞加入视黄酸,烟碱,肠促胰岛素类似物,重组人β-细胞调节素等因子,体外诱导内胚层细胞进一步分化为胰岛素分泌细胞；进行免疫荧光染色内分泌标记,PCR检测胰腺相关标记表达。
结果：PCR结果显示,采用AS法诱导hEF体系下的hESCs,在诱导第1天,原条标记GSC、Mixl1和内中胚层共同前体标记Brachyury表达明显上升,至第2天达高峰。内胚层标记Foxa2和Sox17在第2天开始上升,至第3天达高峰。染色计数结果显示Brachyury在诱导第2天达到高峰,阳性细胞率为43.7±16.6%；Sox17在诱导4天时达高峰81.7±5.4%；将诱导5天细胞移植入SCID小鼠体内后,移植物组织分型显示约75%的细胞为内胚层来源组织,外胚层来源组织比例低于1%。体外进一步诱导后,在诱导18天细胞表达胰腺前体标记pdx1, ngn3和beta2,在诱导25天细胞表达胰腺内外分泌及功能相关基因。细胞团染色显示外层为c肽和胰高血糖素双阳性细胞。
结论：在hEF体系上hESCs诱导分化为限定性内胚层细胞的过程符合体内内胚层发育规律,经过原条、内中胚层共同前体、内胚层3个阶段,获得了效率较高(约80%)的限定性内胚层细胞。该细胞具有在体内外进一步分化为更成熟内胚层组织细胞的能力。
第二章hEF细胞支持限定性内胚层分化的机制研究
目的：1、研究有饲养层及无饲养层培养体系上hESCs向内胚层细胞的诱导效率差异；2、研究hEF细胞是否分泌可溶性因子对限定性内胚层诱导产生影响；3、研究hEF细胞在限定性内胚层诱导过程中表达发育早期关键信号(TGFβ和Wnt信号)情况；4、摸索Wnt3a在限定性内胚层诱导中的作用时间窗,建立无饲养层体系下限定性内胚层诱导体系。
方法：对有饲养层体系(人饲养层H组和鼠饲养层M组)和无饲养层体系(FF组)上培养的hESCs进行AS法诱导,取诱导第5天细胞进行Soxl7染色计数和半定量PCR检测内胚层相关基因表达情况；在FF组基础上加入hEF条件培养基进行诱导(CM+A组),在诱导第5天染色计数Soxl7阳性细胞比率,PCR检测原条,内中外胚层标记的表达；取AS法诱导过程中不同时间点的hEF细胞,PCR检测胚胎早期发育关键信号TGF-β和Wnt信号通路中分泌型因子的基因表达；取FF组和CM+A组诱导1天细胞,western blot检测Wnt信号下游β-catenin的蛋白表达变化；在FF组加入25ng/ml Wnt3a作用1—4天,对不同培养时间点的细胞进行Brachyury, Sox 17染色计数,PCR检测原条,内中外胚层,胚外内胚层标记的表达。
结果：采用AS法诱导有饲养层体系下hESCs在诱导第5天均获得约80%的Sox17阳性细胞率(H组82.0±8.9%和M组78.7±3.4%),而无饲养层培养体系下Sox17阳性细胞率较低(FF组22.7±5.6%)；加入hEF细胞条件培养基后Sox17阳性率上升至63.2±13.4%(CM+A组),PCR检测结果显示在诱导过程中hEF细胞表达高强度ActivinA, Nodal在使用诱导培养基后6h—12h也出现了轻微的上调表达；同时hEF细胞还表达Wnt信号通路抑制剂Dkk1和Dkk3,并且Dkk3在进入诱导过程后6h起即出现了明显的下调,至12h,24h呈逐渐减弱的趋势；Western blot结果显示CM+A组较FF组在诱导第1天Wnt下游因子β-catenin的表达更强,PCR检测Wnt下游靶基因Brachyury和c-myc的表达在CM+A组也较FF组更强,以上结果提示饲养层细胞在内胚层诱导过程中一方面通过分泌ActivinA/Nodal等因子对内胚层诱导起正面促进作用,另一方面通过减弱对Wnt信号的抑制作用,反向活化Wnt信号,对内胚层诱导发挥辅助作用。进一步在CM+A体系加入Wnt3a后发现,Wnt3a与ActivinA共同作用1-4天,均能促进限定性内胚层的分化,共同作用1天Sox17阳性率上升至85.2±3.8%,是最佳的内胚层诱导体系。
结论：hEF通过表达高强度的Activin/Nodal信号,以及减弱对Wnt信号的抑制作用对限定性内胚层分化发挥的辅助作用。在无饲养层体系上,采用Wnt3a和ActivinA联合作用1天,再单独用ActivinA培养2天能最有效促进限定性内胚层的出现。
第三章孤雌与正常来源hESCs向胰岛素分泌细胞分化比较研究
目的：探讨孤雌来源hESCs在体外诱导因素调节下向胰岛素分泌细胞体外分化的能力,以及与正常hESCs在分化效率、增殖能力以及印记基因表达上的差异。
方法：以正常来源的hESCs (chHES137),孤雌来源的hESCs (chHES32,chHES69)共三株细胞为材料,采用第二章中Wnt3a和ActivinA共同作用1天组的内胚层诱导方法,结合视黄酸、烟碱、肠促胰岛素类似物、重组人p-细胞调节素等因子,分为内胚层、原始肠管、胰腺前体、内分泌前体、胰岛素分泌细胞5个阶段诱导。在分化的d0, d3, d6,d9, d12收集细胞,通过免疫荧光染色和real-time PCR来比较胰腺发育标记蛋白(Sox17, Pdx1)阳性率,胰腺发育标记基因和印记基因的表达水平,通过Ki67染色比较增殖能力上的差异。在分化终末阶段,收集细胞进行胰岛素染色、胰腺相关功能基因检测、胰岛素和C肽释放实验检测。
结果：诱导过程中3株hESCs的胰腺发育阶段标记基因的表达遵循胰腺发育的规律,在诱导d3出现内胚层标记表达高峰,诱导d6出现原始肠管标记高峰,诱导d9为胰腺前体标记表达高峰,通过定量PCR检测显示大部分发育标记基因中chHES137和chHES69的表达明显高于chHES32, Sox17和Pdx1染色计数结果也显示chHES137和chHES69的阳性细胞率明显高于chHES32。印记基因的表达遵循着印记基因表达规律,父源性基因(PEG3,IGF2)在正常来源hESCs的表达明显强于两株孤雌来源的hESCs10倍以上。母源性印记基因(GNAS,GRB10)在正常来源hESCs的表达则明显弱于两株孤雌来源的hESCs。chHES137和chHES69诱导终末细胞团均表达胰腺功能相关基因,对不同葡萄糖浓度刺激呈现反应性。3株细胞的增殖能力没有统计学差异。
结论：在体外诱导过程中,hESCs的分化遵循着胰腺体内发育的规律,孤雌来源hESCs具有分化为胰岛素分泌细胞的能力,诱导效率和增殖能力上与正常hESCs相比未见明显差异；孤雌和正常hESCs在诱导分化过程中,印记基因的表达基本符合表观遗传学规律,并具有一定的时间依赖性。
The first human embryonic stem cell (hESC) line with our intellectual property was established in 2002. To date, we have established more than 240 hESC lines, which come into being a hESCs bank. In our previous work, the hESCs cultured on human embryonic fibroblasts (hEF) were successfully differentiated into definitive endoderm and pancreatic progenitors. Based on these work, we improved the method of differentiation and found that the hEF cells played a role in the definitive endoderm differentiation for the first time. With studying the mechanism of hEF induction, we added Wnt3a and ActivinA into the hESCs to differentiate the definitive endoderm by simulating the function of hEF. We got high efficiency of Sox 17 positive cells on feeder free culture system. Based on this differentiation system, we compared the efficiency of insulin-producing cells, level of imprinted genes expression and the ability of proliferation of normal derived hESCs and parthenogenetic hESCs. This research is divided into 3 parts, and the main study methods and results are as follows:
Chapter 1
Differentiation of human embryonic stem cells into definitive endoderm on hEF cells
Objective:1. To study whether the variational expression of endodermal genes and proteins of the cells on different points during the definitive endoderm induction obey the endoderm developmental rule in vivo; 2. to study the differentiation potential of the hESCs derived inducing definitive endoderm cells on hEF cells in vivo and in vitro.
Method:adopting the reported method (adding ActivinA and FBS to differentiate into definitive endoderm, AS way) to induce the hESCs on hEF cells to endoderm cells. The different points (0h,6h,12h, 1d,2d,3d,4d,5d) of cells are collected to precede the immunochemistry staining of Brachyury and Sox 17 and real-time PCR detection of the expression of gastrulation, endoderm, mesoderm, ectoderm and pluripotent markers. The 5th day differentiating cells are transplanted into the legs of SCID mice for 2 months, then the transplants are removed from the mice and sectioned to do HE staining. Meanwhile, the 5th day cells are differentiated into insulin producing cells by RA, nicotinamide, extendin-4 and betacellulin etc in vitro.
Result:the PCR results show that on 1st day of differentiation the prime streak related genes (Goosecoid, Mixl1) and mesoendoderm precursor related gene Brachyury express increasably and get the top on 2nd day. The expressions of endodermal genes (Foxa2, Sox17) increase on 2nd day and get the top on 3rd day. The counting result of Brachyury shows that the peak is on 2nd day (43.7±16.6%) and Sox17 is on 4th day (81.7±5.4%).
The transplants of 5th differentiation day cells show that about 75% cells are derived from endoderm, less than 1% cells are derived from ectoderm. After differentiation in vitro, the 18th day cells express the pancreatic precursor markers pdx1, ngn3, beta2; the 25th day cells express pancreatic functional genes. The staining of clusters show the outer side of the clusters are C peptide and glucagon double positive cells.
Chapter 2
Studying the mechanism of hEF cells supporting definitive endoderm differentiation of hESCs
Objective:1. to study of whether with and without feeder cells culture system will effect the efficiency of endoderm cells; 2. to study of whether the human feeder cells secret some certain soluble factors to support the differentiation of endoderm cells; 3. to study of the activation of key signal pathways (TGFβ, Wnt) in human feeder cells during the differentiation procedure; 4. to confirm the effective time window of Wnt3a in endodermal induction and establish the feeder free inducing system into definitive endoderm cells.
Method:According to different cultivation systems, we divided the subject into three groups:(1) group H:the hES cells cultured on Human derived feeder cells; (2) group M:the hES cells cultured on Mouse derived feeder cells; (3) group FF:the hES cells cultured without feeder cells. We counted the Sox17 positive cells with immunofluorescence and detected the expression tendency of some genes correlated with gastrulating, definitive endoderm, mesoderm, and ectoderm genes using RT-PCR at 5th day in every group. Collecting the hEF cells at different time point, the expression of key signal pathways (TGFβ, Wnt) genes are detected by RT-PCR; Western blot detects theβ-catenin activation in group FF and CM+A. Based on group FF,25ng/ml Wnt3a are added from 1st to 4th day, different time cells are tested for Brachyury and Sox 17 staining and early developmental genes expression.
Result:On day 5 of induction, DE (SOX17+) cells appeared with comparable efficiency in both human and mouse feeder systems (85.0±8.9% and 78.7±3.4%, respectively). These levels were considerably superior to that obtained in the feeder-free system (22.7±5.6%). the HEF-conditioned inducing medium was used to differentiate the hESCs g rowing in group FF. The same additives were used, with the exception that the medium was cultured in HEF feeder cells for 24 h prior to the induction. Five days later, the percentage of SOX17+cells increased to 63.2±13.4%; this value was comparable to that of groups H and M. the hEF cells express strong ActivinA and the Nodal expression increases with the beginning of induction (6h-12h); hEF cells express the inhibitor of Wnt signal pathway (Dkk1, Dkk3), however, with the addition of ActivinA, the level of Dkk3 decrease obviously. With the addition of Wnt3a, we find that the different time of co-culture (1-4 days) of Wnt3a and ActivinA all can induce the sox17 positive cells, however, when the co-culture time is 1 day, the highest efficiency is gained (85.2±3.8%).
Chapter 3
Comparison of normal and parthenogentic human embryonic stem cells on inducing islet-like cells
Objective:to study of the differential potential of parthenogentic hESCs (hpESCs) into insulin producing cell clusters; to study of the difference between normal hESCs and hpESCs on differentiation efficiency, proliferating ability and imprinted-genes expression.
Method:the normal hESCs (chHES137), the hpESCs (chHES32, chHES69) are experimental materials. We adopt a modified 5-step protocol including the optimized endoderm inducing method on feeder free system with Wnt3a on 1st day. Different growth factors, such as ActivinA, retinoic acid (RA), Nicotinamide (NIC), and Exendin-4 (Ex-4), Betacellulin (BTC) etc. are added in the medium to induce the hpES cells differentiate into islet-like clusters. According to the sequence of development stages (d0, d3, d6, d9, d12), immunohistochemistry staining are proceeded to test the pancreatic developmental marker (Sox 17, Pdx1) and Real-time PCR are done to test the expression of pancreatic developmental marker and imprinted genes. Ki67 staining detects the proliferating ability.
Result:the expression pattern of pancreatic developmental genes obey the pancreas development rule in 3 hESCs. The level of endodermal markers achieves the peak at 3rd day, primary gut markers achieve the peak at 6th day, at 9th day the pancreatic precursor markers appear. Most of developmental genes are stronger in chHES137and chHES69 than chHES32. the imprinted genes'expression obey the rule too. Paternal genes (PEG3,IGF2) mainly express in normal hESCs.
The results from immunohistochemistry and RT-PCR showed that the islet-like clusters expressed the islet specific hormones and functional markers. Insulin release test and electron microscopy indicated that the clusters had the biochemical function of islet. The islet-like clusters derived from phES cells had the basic characteristics of islet. There is no significant difference between 3 hESCs in proliferating ability.
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