合作伙伴:
  2006-2012
  教育部顧問室幹細胞與
  再生醫學教學資源中心
  2013
  教育部資料司幹細胞與
  再生醫學教學資源中心
 
 
 

 

   本核心實驗室提供高品質的胚幹細胞給國內相關研究學者。先前,我們的實驗室已經建立了三個胚幹細胞株,但我們期待能夠建立更多的胚幹細胞株,尤其是沒有異種汙染的細胞株,以提供持續且穩定的細胞數目及品質。

we establish a core laboratory to supply our high-quality human embryonic stem cells (hESC) for researchers within Taiwan. Three hESC lines (NTU1, NTU2, and NTU3) have been derived and well characterized in our laboratory (Chen et al., 2007). More cell lines (especially xeno-free cell lines) will be derived and characterized to make sure our resource of hESC won’t be running out in this project
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In our laboratory, we had established three hESC named NTU1, NTU2 and NTU3 (Chen et al., 2007). These three hESC lines grow at similar speed in both media (serum-containing or serum-free), but hESC culture in serum-containing medium yielded significantly higher percentages of morphologically good colonies. All three cell lines exhibit normal karyotype and express undifferentiated markers including Oct-4, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 (Figure 1, Chen et al., 2007).

Figure 1. Derivation and characterization of human embryonic stem cell (hESC) lines. Frozen-thawed human embryos were cultured to blastocyst stage, and hESCs were derived as described in Materials and methods. (A) A blastocyst after treatment with proteinase to remove the zona pellucida. (B) The trophoblast cells appeared destroyed after immunosurgery. (C) Seven days after plating the inner cell mass (ICM) on the feeder cells. (D) NTU1 hESC line at passage 4. (E) NTU2 line at passage 7 transferred by mechanical method. This is a morphologically good colony. (F) NTU1 line at passage 11 transferred by collagenase type IV. (G) A morphologically fair colony. (H) A morphologically poor colony. (I–N) These are representative marker expressions for NTU1 line. (I) Alkaline phosphatase. (J) Oct-4. (K) SSEA-3. (L) SSEA-4. (M) TRA-1-60. (N) TRA-1-81. (O) Karyotype of NTU1 line (46, XX). (P) Karyotype of NTU2 line (46, XX). Scale bars, 200μm.


Besides, we characterize hESCs with RT-PCT to identify the expression of many genes including Nanog, Oct-4, TERT, Dppa5, UTF1, Sox2, Rex1 and FoxD3, which are markers of undifferentiated stem cells (Figure 2A, Chen et al., 2007). We also present the evidence of gene expressions representing the three embryonic germ layers, including MAP2, SOX1 and GFAP (ectoderm), HAND1, cTnI, GATA6 and HBZ (mesoderm) and HNF-4A, AFP, GATA4 and insulin (endoderm), in NTU1 and NTU2 hESC lines (Figure 2B, Chen et al., 2007).

In the aspect of pluripotency, the hESC lines differentiate spontaneously in vitro into cells with or without embryoid body (EB) and those cells expressing markers belonging to all three embryonic germ layers and germ cell markers, including c-Kit, STELLA, VASA and growth differentiation factor 9 (GDF9), in directly adherent culture. In vivo, teratoma formation was noted since 4 weeks after injection of hESC into SCID mice (Figure 6A). The mouse with tumor formation was sacrificed, and the tumour was removed at 8–12 weeks when it reached a diameter about 2 cm. Examination of tumors from both cell lines (NTU1 and NTU2) revealed mature cystic teratomas, consisting of many cells from all three embryonic germ layers. These include neural tubules, tooth-like structure, optic disc (ectoderm), cartilage, skeletal muscle, smooth muscle (mesoderm), respiratory epithelium and gut epithelium (endoderm) (Figure 6B-F). No ovarian follicle-like structure was observed in these tumors. The in vivo differentiation potential of NTU3 needs to be verified in further experiments.

 

However, we still have to derive more new hESC lines to make sure our resource of hESC won’t be running out. To establish new hESC lines and maintain high quality of hESCs will be the first goal in this project. In order to train more students and technicians to practice the experiment of hESC lines, we will hold two workshops one year. We hope to retain the technique in hESC through workshops; meanwhile, we would like to consult about technique and new evidences for hESCs during the workshops. Also, providing accessible website for the scheduled reservation of the hESCs will be done as soon as possible.
On the other hand, we plan to set up a data bank providing the characteristics of hESC. To characterize the hESC lines including the current available and newly derived hESCs, we propose using the methods like immunocytometry, RT-PCR, microarray, proteomic analysis to reach this aim.

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1
提供胚幹細胞給計畫中研究學者做為相關實驗使用

2

建立長期且穩定鑑定胚幹細胞的技術以及資料庫

3

與台灣大學基因體醫學研究中心核心實驗室合作

4

提供胚幹細胞給國內相關研究學者作為相關實驗使用

5

促進幹細胞研究的國際合作

1
  • To supply hESC for projects within this project
2
To provide the techniques and routines for characterization of hESC.
3
To integrate the service provided by the core laboratories of NTU (National Taiwan University) Center of Genomic Medicine (CGM)
4
To supply hESC and technical supports for stem cell researchers nationwide.
5
To promote the international collaboration of stem cell research

 

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10055 台北市中正區徐州路2號6樓 臺大基因體中心-幹細胞核心實驗室 聯絡人:許莉苓 電話:02-23123456 #88637
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