| 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 1Differentiation of human embryonic stem cells into definitive endoderm on hEF cellsObjective: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 2Studying the mechanism of hEF cells supporting definitive endoderm differentiation of hESCsObjective: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 3Comparison of normal and parthenogentic human embryonic stem cells on inducing islet-like cellsObjective: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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