2011-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/657103摘要：人類胚胎幹細胞(Humanembryonicstemcells,hESCs)是具有持續自我更新，及分化成各種細胞的多功能幹細胞。過去的研究指出把人類胚胎幹細胞注射至免疫系統缺乏的實驗鼠，會產生包含許多種類的組織細胞形成的畸胎瘤。在細胞培養實驗時人類胚胎幹細胞會聚集成類胚胎體(embryoidbodies,EBs)，並且自動分化成各種組織細胞，其中包括造血性幹細胞(hematopoieticstemcells,HSCs)，若在培養過程中加入細胞激素以及第四型骨型態發生蛋白(bonemorphogeneticprotein-4,BMP-4)可促進增加造血幹細胞的分化。而除了透過類胚胎體培養的方式外，將人類胚胎幹細胞與老鼠骨髓基質細胞株OP9、MS5或是主動脈-性腺-中腎產生的基質細胞(aorta-gonad-mesonephros-derivedstromalcells)共同培養則是通常被用來從人類胚胎幹細胞產生CD34+CD43+之細胞群，此細胞群具有B淋巴球、自然殺手細胞或是骨髓細胞前驅特性；目前針對人類胚胎幹細胞產生造血性幹細胞的淋巴組織細胞生成方面的研究文獻並不多。近來Dr.Timmermans發現T淋巴球細胞可由人類胚胎幹細胞與OP9基質細胞共同培養時出現的造血性區域(hematopoieticzones,HZs)產生。若在有生長激素的狀態下，如FLT3-L(fms-liketyrosinekinase3receptorligand)、IL-7、SCF(stemcellfactor)，將造血性區域移至表現DLL的OP9細胞株(OP9-Delta-like-l)上會促進T淋巴球的大量生成。相較於T淋巴球，B淋巴球生成的研究較不成功。B淋巴球的發育可被分為幾個階段，由proB前驅細胞、proB細胞、preB細胞、未成熟B細胞、成熟B細胞到漿細胞，而這些B細胞的發育是透過一些只會作用在B細胞之轉錄因子包括第一型早期B細胞因子(EarlyBcellfactor1,EBF1)及Pax5等等。進行細胞培養時，於mECS/OP9細胞共同培養的系統中加入Flt-3L可以產生大量具功能的B淋巴球，但相同的系統仍無法有效的在人類胚胎幹細胞培養系統產生B淋巴球。經由過去的文獻顯示在老鼠骨髓造血幹細胞中表現第一型早期B細胞因子(EBF1)會造成多功能幹細胞分化成B淋巴球，因此在本研究計畫中我們將嘗試兩步驟的方式來從人類胚胎幹細胞分化產生B淋巴球。首先，我們將從人類胚胎幹細胞中產生造血性幹細胞，再進一步將造血性幹細胞分化出B淋巴球。這樣的分化不僅是使我們了解B淋巴球發育的真實過程也提供了適於研究控制B淋巴球功能因子的系統。這樣的系統可以做為日後治療B淋巴癌或是B淋巴球缺乏疾病的參考。<br> Abstract: Human embryonic stem cells (hESCs) are pluripotent stem cells that are able to maintain theirself-renewal for a prolonged period and are capable of differentiating into all the cell types in vitro andin vivo. These cells therefore are potentially good targets for stem cell-based transplantation therapyand there have been growing interest in deriving cells from hESCs for this purpose. Previous studyindicated that hESCs injected into immunedeficient mice produced teratomas comprised of cells ofmultiple tissue types, suggesting their pluripotent potential. To achieve application of clinical andexperimental medicine, in vitro generating cells of hematopoietic system is needed. In vitroaggregation of hESCs into clusters of cells termed “embryoid bodies” (EBs) allows for thespontaneous and random differentiation of hESCs into multiple tissue types, including hematopoieticstem cells (HSCs) and combination with cytokines and bone morphogenetic protein-4 (BMP-4)promotes HSCs differentiation. Beside EB formation, coculture system of hESC with mouse bonemarrow stromal cell lines, OP9, MS5 or aorta-gonad-mesonephros-derived stromal cells is often usedto generate CD34+CD43+ population with precursor activity for B, NK, and myeloid cells fromhESC. So far the studies of lymphopoiesis from hESC-derived HSCs are limited. However, onlynature killer (NK) cells generation from hESC-derived HSCs is well characterized. Recently,Timmermans et al. showed that T cells could be generated from hESC-derived HPCs present in aspecial structure “hematopoietic zones” (HZs). The HZs are formed of endothelium-lined cellclumps, reminiscent of blood islands during embryonic development when hESCs cultured on OP-9stromal cells. T cells largely generates from this HZs when HZs transferred on OP9-DLL (Delta-like1) cells in the presence of growth factors (fms-like tyrosine kinase 3 receptor ligand (FLT3-L), IL-7,stem cell factor (SCF)). Compared to T cells generation, B cells generated from hESC have been lesssuccessful. The development of B cells can be divided into several stages, from pre-pro B cell, pro-Bcell, pre-B cell, immature B cell, mature B cell to plasma cells and the developmental stages areunder control of B-lineage specific transcriptional factors, such as early B cell factor 1 (EBF) andPax5 etc. The addition of Flt-3L to mESC/OP-9 coculture system efficiently generates functional Blymphocytes in vitro, in contrast, the same coculture system does not generate hESC-derived Blymphocytes efficiently. Moreover, enforced expression of EBF1 in mouse bone marrow(BM)-derived HSCs restricted pluripotent cells differentiate into B lymphocytes. In this study, wewill generate B lymphocytes from human embryonic stem cells (hESCs) by two-step way. First,hematopoietic progenitors cells (HPCs) will generate from hESC and further differentiate into Blymphocytes. The study of B lymphopoiesis from hESC not only reveals the real biology of B celldevelopment but also provides an useful system to study the factors controlling B cell function.Moreover, this system may provide future therapeutic strategies to manipulate B leukemia and B celldeficient diseases.