臺灣博碩士論文加值系統

精原幹細胞為一群具有多功能潛力的幹原細胞，在適當調控下可以形成成熟的精子或是發展成為個胚層所需之細胞與組織。因此，為了研究早期精子生成作用相關的分子調控機制以及多功能性精原幹細胞發展的潛力，在體外建立不含血清的精原幹細胞培養系統使其可以長期培養及增生或是誘導其分化是所必需的，亦有助於進一步釐清精原幹細胞生長所需之生長因子及其作用機制。
我們成功地利用ICR品系雄性小鼠睪丸中的細胞在體外培養出具有多功能特性的精原幹細胞群落，我們的實驗結果得知在laminin (6.25ng/ml)的coating material及EGF (10ng/ml) +倍Insulin/ transferring–selenium的培養下，這些細胞群落具有專一性且高度表現鹼性磷酸脢的活性及Oct4蛋白，顯現出這些細胞群落具有精原幹細胞的潛力。我們利用反轉錄聚合酶連鎖反應以及細胞螢光免疫染色來鑑定精原幹細胞進一步專一性表現的基因及蛋白質，我們發現在mRNA層次上，細胞群落專一地表現精原幹細胞所特有的相關基因，包括Oct4, Mvh, Fragilis, Stella, Dazl, Tex14, Piwil2等但不表現c-kit及Gcnf (Oct4的抑制者)；在蛋白質層次上，則表現Oct4, SSEA1, CD29, CD49f等，但不表現CD117 (c-kit), CD30, CD34。
此外，我們利用EGFP-lentivirus感染這些具精原幹細胞潛力的細胞群落來追蹤，再使用細胞移植後以檢驗細胞的功能性，結果顯示我們成功地將精原幹細胞移植至原本未能正常進行精子生成作用的睪丸中，並且有明顯的螢光表現。我們的實驗結果證實，於本實驗室之未含血清的體外細胞培養模式，可以成功地培養處於早期精原幹細胞(c-kit negative)的細胞群落。

誌謝辭----------------------------------------------------------------------------- 4
縮寫表----------------------------------------------------------------------------- 5
中文摘要-------------------------------------------------------------------------- 7
英文摘要-------------------------------------------------------------------------- 8

第一章 緒論--------------------------------------------------------------------- 9
1.1 幹原細胞 (stem cells)---------------------------------------------------- 9
1.2 生命起源與幹細胞來源------------------------------------------------- 9
1.3 人類幹細胞研究的歷史------------------------------------------------- 10
1.4 幹原細胞功能上的分類------------------------------------------------- 11
1.4.1全能幹細胞 (totipoent stem cells)-------------------------------- 11
1.4.2多功能幹細胞 (pluripotent stem cells)-------------------------- 11
1.4.3多向性幹細胞 (multipotent stem cells)-------------------------- 12
1.4.4專能幹細胞 (unipotent stem cells)------------------------------- 12
1.5 幹原細胞的種類---------------------------------------------------------- 12
1.6 胚胎生殖細胞 (embryonic germ cells)-------------------------------- 13
1.7 精原幹細胞 (germ line stem cells, GSCs)---------------------------- 13
1.7.1原生殖細胞 (primordial germ cells, PGCs)--------------------- 13
1.7.2精原母細胞 (gonocytes)------------------------------------------- 14
1.7.3未分化A型-single精原細胞 ( undifferentiated type Asingle spermatogonial stem cells, SSCs)--------------------------------
14
1.8 精原幹細胞的標記物---------------------------------------------------- 15
1.8.1轉錄因子Oct4------------------------------------------------------- 15
1.8.2 Mvh-------------------------------------------------------------------- 16
1.8.3 Fragilis---------------------------------------------------------------- 17
1.8.4 Stella------------------------------------------------------------------- 17
1.8.5 Dazl-------------------------------------------------------------------- 18
1.8.6 Tex14 ------------------------------------------------------------------ 19
1.8.7 Piwi12----------------------------------------------------------------- 19
1.8.8 Gcnf-------------------------------------------------------------------- 20
1.8.9 CD117 (c-kit )-------------------------------------------------------- 20
1.8.10 CD29 (Integrin β1)、CD49f (Integrin α6)----------------------- 21
1.9 研究動機－體外未含血清之精原幹細胞培養系統的建立--- 22

第二章 材料與實驗方法------------------------------------------------------- 24
2.1 實驗動物與細胞株------------------------------------------------------- 24
2.2 免疫組織染色 (immunohistochemistry)------------------------------ 24
2.3 細胞培養------------------------------------------------------------------- 26
2.3.1睪丸組織精原幹細胞初級培養 (primary culture)------------- 26
2.3.2 TM4細胞株培養---------------------------------------------------- 28
2.4 鹼性磷酸酶 (alkaline phosphatase) 活性測定---------------------- 28
2.5 反轉錄聚合酶連鎖反應RT-PCR--------------------------------------- 29
2.5.1 RNA extraction from the SSC colonies--------------------------- 29
2.5.2 cDNA Synthesis Reaction------------------------------------------ 30
2.5.3聚合酶連鎖反應PCR---------------------------------------------- 31
2.5.4洋菜膠體電泳 (agarose gel)--------------------------------------- 33
2.6 細胞螢光免疫染色 (immunocytochemistry)------------------------- 34
2.7 使用busulfan 處理FVB mouse---------------------------------------- 35
2.8 以帶有綠色螢光蛋白(EGFP)基因的Lentivirus感染精原幹細胞 35
2.8.1VSVG (envelope), Δ8.9(package), GFP(transgenes: fluorescence)質體DNA的萃取------------------------------------
35
2.8.2 Lentivirus的製備---------------------------------------------------- 37
2.8.3以Lentivirus感染精原幹細胞------------------------------------ 38
2.9 以受綠色螢光蛋白基因EGFP-Lentivirus感染的精原幹細胞進
行移植作用-----------------------------------------------------------------
39

第三章 實驗結果---------------------------------------------------------------- 41
3.1 Oct4蛋白於不同天數ICR品系小鼠睪丸組織的表現-------------- 41
3.2精原幹細胞培養------------------------------------------------------------ 41
3.3鹼性磷酸酶------------------------------------------------------------------ 42
3.4以不同coating material所培養之細胞聚落形態變化及鹼性磷酸酶活性表現-----------------------------------------------------------------
42
3.5在不含血清培養液下處理不同生長因子之培養細胞群落型態
變化--------------------------------------------------------------------------
44
3.6利用RT-PCR方法檢測不同細胞與組織中Oct4基因表現情形--- 44
3.7利用RT-PCR方法檢測體外培養的細胞群落相關基因與蛋白質表現情形--------------------------------------------------------------------
44
3.8利用細胞螢光免疫染色檢測體外培養的細胞群落相關蛋白質表現情形--------------------------------------------------------------------
45
3.9以busulfan處理ICR老鼠睪丸組織切片 (蘇木素--伊紅染色)---- 46
3.10 EGFP-Lentivirus感染之精原幹細胞移植於睪丸內螢光表現情形 (精原幹細胞功能之分析)------------------------------------------
46

第四章 討論---------------------------------------------------------------------- 47
4.1 細胞的形態變化---------------------------------------------------------- 47
4.2 鹼性磷酸酶活性---------------------------------------------------------- 48
4.3 轉錄因子Oct4與其他標記物------------------------------------------ 48
4.4 移植作用------------------------------------------------------------------- 51
4.5 精原幹細胞發展的潛力與前景---------------------------------------- 51
4.5.1 精原幹細胞的多功能潛力-------------------------------------- 51
4.5.2 精原幹細胞的培養與細胞株的建立-------------------------- 52

第五章 未來研究方向---------------------------------------------------------- 56

第六章 實驗結果圖次目錄---------------------------------------------------- 59
Fig. 1 不同天數ICR品系小鼠睪丸組織切片Oct-4免疫組織染色--- 60
Fig. 2 實驗用ICR品系新生小鼠(出生1天)及腹腔解剖圖(出自九十二學年度 精原幹細胞的培養與鑑定 論文 作者吳采蓉)-
62
Fig. 3 體外培養ICR新生小鼠(出生1天)睪丸組織內細胞形態變化 64
Fig. 4 細胞聚落(colonies)鹼性磷酸酶活性呈色測定------------------- 66
Fig. 5 不同coating material培養之細胞聚落形態變化及鹼性磷酸
. 酶活性表現-------------------------------------------------------------

68
Fig. 6 在不含血清培養液下處理不同生長因子之培養細胞群落型
. 態變化-------------------------------------------------------------------

70
Fig. 7 .利用RT-PCR檢測在不同細胞組織下Oct4表現情形-----------
72
Fig. 8 .利用RT-PCR檢測體外培養的細胞聚落相關基因表現情形--
74
Fig. 9 細胞螢光免疫染色檢測體外培養精原幹細胞群落相關蛋白
. 質表現------------------------------------------------------------------

76
Fig.10以busulfan處理ICR小鼠睪丸組織切片(蘇木素--伊紅染色)-- 78
Fig.11受EGFP-lentivirus感染的精原幹細胞表現情形----------------- 80
Fig.12受綠色螢光蛋白基因EGFP-Lentivirus感染的精原幹細胞進
. 行移植作用後睪丸內螢光表現情形-------------------------------

82

文獻出處---------------------------------------------------------------------------- 84

圖次目錄
圖一、 胚胎發育從受精卵至囊胚期發展過程---------------------- 10
圖二、 小鼠系統中具多功能性分化的幹原細胞------------------- 12
圖三、 小鼠生殖細胞循環過程---------------------------------------- 14
圖四、 Oct4在胚胎發育過程中的表現情形------------------------- 16
圖五、 Dazl在胚胎發育過程中的表現情形------------------------ 18
圖六、 Tex14在減數分裂過程中的表現情形----------------------- 19
圖七、 Gcnf在胚胎幹細胞中的調控機制---------------------------- 20
圖八、 精原幹細胞中各個基因的表現時---------------------------- 22
圖九、
以顯微注射(microinjection)將精原幹細胞注入睪丸輸精細管中----------------------------------------------------------
25
圖十、 我們實驗室以未含血清之培養系統所培養的精原幹細胞基因表現情形與其細胞群落所處時期(灰色長方框區域)(具有gonocytes至undifferentiated type Asingle SSCs之潛力)------------------------------------------------------------


50
圖十一、
第一株表現type As、Apr and early Aal SSCs特性的rat
精原幹細胞株：A303, A304------------------------------------
53
圖十二、
第一株表現Apr and early Aal SSCs特性的mouse精原
幹細胞株：S4-----------------------------------------------------
53
圖十三、
具有發展type As SSCs特性潛力的mouse精原幹細胞
株：C18-4----------------------------------------------------------
54

Reference List

[1]K.D.van der, S.Weiss, Why stem cells? Science 287, (2000) 1441.
[2]M.J.Evans, M.H.Kaufman, Establishment in culture of pluripotential cells from mouse embryos. Nature 292, (1981) 154-156.
[3]G.R.Martin, Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc.Natl.Acad.Sci.U.S.A 78, (1981) 7634-7638.
[4]J.L.Resnick, L.S.Bixler, L.Cheng, P.J.Donovan, Long-term proliferation of mouse primordial germ cells in culture. Nature 359, (1992) 550-551.
[5]Y.Matsui, D.Toksoz, S.Nishikawa, S.Nishikawa, D.Williams, K.Zsebo, B.L.Hogan, Effect of steel factor and leukaemia inhibitory factor on murine primordial germ cells in culture. Nature 353, (1991) 750-752.
[6]J.A.Thomson, J.r. Itskovitz-Eldo, S.S. Shapiro, M.A. Waknitz., J.J.Swiergiel, V.S.Marshall, J.M.Jones, Embryonic stem cell lines derived from human blastocysts. Science 282, (1998) 1145-1147.
[8]M.J.Shamblott, J.Axelman, Wang Huggins S., Bugg E.M., Littlefield J.W., Donovan P.J., Blumenthal P.D., G.R., Gearhart J.D., Derivation of pluripotent stem cells from cultured human primordial germ cells. Proc.Natl.Acad.Sci.U.S.A 95, (1998) 13726-13731.
[9]E. Marshall, Embryo greeted with skepticism. Science 282, (1998) 1390-1391.
[10]M.C.Hofmann, L. Braydich-Stolle, L. Dettin, E.M.Dym, Immortalization of mouse germ line stem cells. Stem Cells 23, (2005) 200-210.
[11]F.A.Brook, R.L.Gardner, Germline transmission of donor haplotype following spermatogonial transplantation. Proc.Natl.Acad.Sci.U.S.A 94, (1997) 5709-5712.
[12]A.G.Smith, Embryo-derived stem cells: of mice and men. Annu.Rev.Cell Dev.Biol. 17, (2001) 435-462.
[13]L.Labosky, Embryonic germ cell lines and their derivation from mouse primordial germ cells. Ciba Found.Symp 182, (1994) 157-168.
[14]B.L.Hogan, Morphogenesis. Cell 96, (1999) 225-233.
[15]T.A.Pelton, M.D.Bettess, J.Lake, J.Rathjen, P.D.Rathjen, Developmental complexity of early mammalian pluripotent cell populations in vivo and in vitro. Reprod.Fertil.Dev. 10, (1998) 535-549.
[16]K.A.Lawson, R.A.Pedersen, Clonal analysis of cell fate during gastrulation and early neurulation in the mouse. Ciba Found.Symp. 165, (1992) 3-21.
[17]Y.Matsui, Developmental fates of the mouse germ cell line. Int.J.Dev.Biol. 42, (1998) 1037-1042.
[18]P.P.Tam, M.H.Snow, Proliferation and migration of primordial germ cells during compensatory growth in mouse embryos. J.Embryol.Exp.Morphol. 64, (1981) 133-147.
[19]S.P.MacGregor, Tissue non-specific alkaline phosphatase is expressed in both embryonic and extraembryonic lineages during mouse embryogenesis but is not required for migration of primordial germ cells. Development 121, (1995) 1487-1496.
[20]P.J.Donovan, D.Stott, L.A.Cairns, J.Heasman, C.C.Wylie, Migratory and postmigratory mouse primordial germ cells behave differently in culture. Cell 44, (1986) 831-838.
[21]C.Ffrench-Constant, A.Hollingsworth, J.Heasman, C.C.Wylie, Response to fibronectin of mouse primordial germ cells before, during and after migration. Development 113, (1991) 1365-1373.
[22]M.Buehr, S.Gu, A.McLaren, Mesonephric contribution to testis differentiation in the fetal mouse. Development 117, (1993) 273-281.
[23]M.Buehr, A.McLaren, Isolation and culture of primordial germ cells. Methods Enzymol. 225, (1993) 58-77.
[24]N.N.S.Chuma, Transition into meiosis of mouse fetal germ cells in vitro and its inhibition by gp130-mediated signaling. Dev.Biol. 229, (2001) 468-479.
[25]D.G.de Rooij, Proliferation and differentiation of spermatogonial stem cells. Reproduction. Reproduction. 121, (2001) 347-354.
[26]M.H.Rosner, M.A.Vigano, K.Ozato, P.M.Timmons, F.Poirier, P.W.Rigby, L.M.Staudt, A POU-domain transcription factor in early stem cells and germ cells of the mammalian embryo. Nature 345, (1990) 686-692.
[27]K.Okamoto, H.Okazawa, A.Okuda, M.Sakai, M.Muramatsu, H.Hamada, A novel octamer binding transcription factor is differentially expressed in mouse embryonic cells. Cell 60, (1990) 461-472.
[28]H.R.Scholer, S.Ruppert, N.Suzuki, K.Chowdhury, P.Gruss, New type of POU domain in germ line-specific protein Oct-4. Nature 344, (1990) 435-439.
[29]H.R.Scholer, G.R.Dressler, R.Balling, H.Rohdewohld, P.Gruss, Oct-4: a germline-specific transcription factor mapping to the mouse t-complex. EMBO J. 9, (1990) 2185-2195.
[30]Y.I.Yeom, G.Fuhrmann, C.E.Ovitt, A.Brehm, K.Ohbo, M.Gross, K.Hubner, H.R.Scholer, Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development 122, (1996) 881-894.
[31]M.Pesce, X.Wang, D.J.Wolgemuth, H.Scholer, Differential expression of the Oct-4 transcription factor during mouse germ cell differentiation. Mech.Dev. 71, (1998) 89-98.
[32]L.R.Gavis, A conserved 90 nucleotide element mediateds translational repression of nanos RNA. Development 122, (1996) 2791-2800.
[33]M.A Ginsburg., Primordial germ cells in the mouse embry during gastrulation. Development 110, (1990) 520-528.
[34]Y.Fujiwara, T.Komiya, H.Kawabata, M.Sato, H.Fujimoto, M.Furusawa, T.Noce, Isolation of a DEAD-family protein gene that encodes a murine homolog of Drosophila vasa and its specific expression in germ cell lineage. Proc.Natl.Acad.Sci.U.S.A 91, (1994) 12258-12262.
[35]S.S.Tanaka, Y.Toyooka, R.Akasu, Y.Katoh-Fukui, Y.Nakahara, R.Suzuki, M.Yokoyama, T.Noce, The mouse homolog of Drosophila Vasa is required for the development of male germ cells. Genes Dev. 14, (2000) 841-853.
[36]Tashiaki Noce, Vasa homolog genes in mammalian germ cell. Development cell structure and function 26, (2001) 131-136.
[37]M.Azim Surani, A molecular programme for the specification of germ cell fate in mice. Nature 418, (2002) 293-300.
[38]M.Saitou, S.C.Barton, M.A.Surani, A molecular programme for the specification of germ cell fate in mice. Nature 418, (2002) 293-300.
[39]M.Sato, T.Kimura, K.Kurokawa, Y. Fujita, K.Abe, M. Masuhara, T.Yasunaga, Ryo A., M. Yamamoto, T. Nakano, Identification of PGC7, a new gene expressed specifically in preimplantation embryos and germ cells Mechanisms. Development 113, (2002) 91-94.
[40]Bortvin Alex, Dppa3 / Pgc7 / stella is a maternal factor and is not required for germ cell specification in mice BMC Developmental Biology 4, (2004) 1-5.
[41]H.Yen Pauline, Putative biological functions of the DAZ family. International Journal of Andrology 27, (2004) 125-129.
[42]A.D.Johnson, R.F.Bachvarova, M.Drum, T.Masi, Expression of axolotl DAZL RNA, a marker of germ plasm: widespread maternal RNA and onset of expression in germ cells approaching the gonad. Dev.Biol. 234, (2001) 402-415.
[43]M.Ruggiu, R.Speed, M.Taggart, S.J.McKay, F.Kilanowski, P.Saunders, J.Dorin, H.J.Cooke, The mouse Dazla gene encodes a cytoplasmic protein essential for gametogenesis. Nature 389, (1997) 73-77.
[44]E.Y.Xu, F.L.Moore, R.A.Pera, A gene family required for human germ cell development evolved from an ancient meiotic gene conserved in metazoans. Proc.Natl.Acad.Sci.U.S.A 98, (2001) 7414-7419.
[45]N.Geijsen, M.Horoschak, K.Kim, J.Gribnau, K.Eggan, G.Q.Daley, Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 427, (2004) 148-154.
[46]R.L.T.Y.S.P.A.R.B.L.G.R.M.e.al.Reijo, Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA-binding protein gene. Nature Genetics 10, (1995) 383-393.
[47]M.&.C.H.J.Ruggiu, In vivo and in vitro analysis of homodimerization activity of the mouse Dazl1 protein. Gene 252, (2000) 119-126.
[48]Schrans-Stassend.R.D.G., Nature of the spermatogenic arrest in Dazl -/-mice Biol Reprod 65, (2001) 771-776.
[49]J.G.M.ROCCHIETTI-MARCH, Dazl protein expression in adult rat testis is up-regulated at meiosis and not hormonally regulated International Journal of Andrology 23, (2000) 51-56.
[50]M.H.Wu, A.Rajkovic, K.H.Burns, W.Yan, Y.N.Lin, M.M.Matzuk, Sequence and expression of testis-expressed gene 14 (Tex14): a gene encoding a protein kinase preferentially expressed during spermatogenesis. Gene Expression Patterns 3, (2003) 231-236.
[51]W.Deng, H.Lin, Miwi, a murine homolog of piwi, encodes a cytoplasmic protein essential for spermatogenesis. Dev.Cell 2, (2002) 819-830.
[52]T.K.Satomi, Kuramochi-Miyagawa1, Mili, a mammalian member of piwi family gene, is essential for spermatogenesis. Development(2004) 839-849.
[53]H.a.S.A.C.Lin, A novel group of pumilio mutations affects the asymmetric division of germline stem cells in the Drosophila ovary. Development 124, (1997) 2463-2476.
[54]W.a.L.H.Deng, miwi, a murine homolog of piwi, encodes a cytoplasmic protein essential for spermatogenesis. Dev.Cell 2, (2002) 819-830.
[55]S.Kuramochi-Miyagaw, T. Kimura, T.W. Ijiri, T.Isobe, N.Asada, Y. Fujita, M. Ikawa, N. Iwai, M. Okabe, W. Deng, H. Lin, Y. Matsuda, T. Nakano, Mili, a mammalian member of piwi family gene, is essential for spermatogenesis Development 131, (2004) 839-849.
[56]Peili Gu, Damien LeMenuet, Arthur C.-K.Chung, Michael Mancini, David A.Wheeler, Austin J.Cooney, Orphan nuclear receptor GCNF is required for the repression of pluripotency genes during retinoic acid-induced embryonic stem cell differentiation Mol Cell Biol. 25, (2006) 8507-8519.
[57]D.Katz, C.Niederberger, G.R.Slaughter, A.J.Cooney, Characterization of germ cell-specific expression of the orphan nuclear receptor, germ cell nuclear factor. Endocrinology 138, (1997) 4364-4372.
[58]G.Fuhrmann, A.C.Chung, K.J. Jackson, G. Hummelke, A. Baniahmad, J.Sutter, J.Sutter, Mouse germline restriction of Oct4 expression by germ cell nuclear factor. Dev.Cell 1, (2001) 377-387.
[59]Y.L.Zhang, K.M.Akmal, J.K.Tsuruta, Q.Shang, T.Hirose, A.M.Jetten, K.H.Kim, D.A.O'Brien, Expression of germ cell nuclear factor (GCNF/RTR) during spermatogenesis. Mol.Reprod.Dev. 50, (1998) 93-102.
[60]F.Chen, A.J.Cooney, Y.Wang, S.W.Law, B.W.O'Malley, Cloning of a novel orphan receptor (GCNF) expressed during germ cell development. Mol.Endocrinol. 8, (1994) 1434-1444.
[61]D.Katz, C.Niederberger, G.R.Slaughter, A.J.Cooney, Characterization of germ cell-specific expression of the orphan nuclear receptor, germ cell nuclear factor. Endocrinology 138, (1997) 4364-4372.
[62]G.Fuhrmann, A.C.Chung, K.J.Jackson, G.Hummelke, A.Baniahmad, J.Sutter, I.Sylvester, H.R.Scholer, A.J.Cooney, Mouse germline restriction of Oct4 expression by germ cell nuclear factor. Dev.Cell 1, (2001) 377-387.
[63]K.Yoshinaga, S.Nishikawa, M.Ogawa, S.Hayashi, T.Kunisada, T.Fujimoto, S.Nishikawa, Role of c-kit in mouse spermatogenesis: identification of spermatogonia as a specific site of c-kit expression and function. Development 113, (1991) 689-699.
[64]P.Rossi, C.Sette, S.Dolci, R.Geremia, Role of c-kit in mammalian spermatogenesis. J.Endocrinol.Invest. 23, (2006) 609-915.
[65]C.Sette, S.Dolci, R.Geremia, P.Rossi, The role of stem cell factor and of alternative c-kit gene products in the establishment, maintenance and function of germ cells. Int.J.Dev.Biol. 44, (2000) 599-608.
[66]R.A.O.R.Y.J.Bennett, Immunohistochemical localization of transforming growth factor-, epidermal growth factor (EGF), and EGF receptor in the human fetal ovary. J.Clin.Endocrinol.Metab. 81, (1996) 3073-3076.
[67]V.C.Broudy, Stem cell factor and hematopoiesis. blood 90, (1997) 1345-1364.
[68]P.H.Jones, F.M.Watt, Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression. Cell 73, (1993) 713-724.
[69]D.E.Williams, S.D.Lyman, Characterization of the gene-product of the Steel locus. Prog.Growth Factor Res. 3, (1991) 235-242.
[70]T.Shinohara, M.R.Avarbock, R.L.Brinster, beta1- and alpha6-integrin are surface markers on mouse spermatogonial stem cells. Proc.Natl.Acad.Sci.U.S.A 96, (1999) 5504-5509.
[71]T.Shinohara, M.R.Avarbock, R.L.Brinster, beta 1- and alpha 6-integrin are surface markers on mouse spermatogonial stem cells. Proc.Natl.Acad.Sci.U.S.A 96, (1999) 5504-5509.
[72]A.R.Bellve, Introduction: the male germ cell; origin, migration, proliferation and differentiation. Semin.Cell Dev. 9, (1998) 379-391.
[73]A.Spradling, D.Drummond-Barbosa, T.Kai, Stem cells find their niche. Nature 414, (2001) 98-104.
[74]A.R.Bellve, J.C.Cavicchia, C.F.Millette, D.A.O'Brien, Y.M.Bhatnagar, M.Dym, Isolation and morphological characterization. J.Cell Biol. 74, (1977) 68-85.
[75]R.L.Brinster, M.R.Avarbock, Germline transmission of donor haplotype following spermatogonial transplantation. Proc.Natl.Acad.Sci.U.S.A 91, (1994) 11303-11307.
[76]G.G.Parreira, T.Ogawa, M.R.Avarbock, L.R.Franca, C.L.Hausler, R.L.Brinster, L.D.Russell, Development of germ cell transplants: morphometric and ultrastructural studies. Tissue Cell 31, (1999) 242-254.
[77]P.Vecino, J.A.Uranga, J.Arechaga, Suppression of spermatogenesis for cell transplantation in adult mice. Protoplasma 217, (2001) 191-198.
[78]R.L.Brinster, Spermatogenesis following male germ-cell transplantation Proc.Natl.Acad.Sci.U.S.A 91, (1994) 11298-11302.
[79]M.Kanatsu-Shinohara, K. Inoue, J. Lee, M. Yoshimoto, N. Ogonuki, H. Miki, S. Baba, T. Kato, Y. Kazuki, S. Toyokuni, M. Toyoshima,O. Niwa,M. Oshimura,T. Heike,T. Nakahata,A.Ogura,F. Ishino, T. Shinohara, Generation of pluripotent stem cells from neonatal mouse testis. Cell 119, (2004) 1001-1012.
[80]Anonymous, Pluripotency of spermatogonial stem cells from adult mouse testis. Nature latters(2006).
[81]A.M.M.van Pelt, H.L.Roepers-Gajadien, I.S.Gademan, L.B.Creemers, D.G.de Rooij, F.M.F.van Dissel-Emiliani, Establishment of Cell Lines with Rat Spermatogonial Stem Cell Characteristics Endocrinology 143, (2002) 1845-1850.
[82]L.X.Feng, Y.Chen, L.Dettin, R.A.R.Pera, J.C.Herr, E.Goldberg, M.Dym, Generation and in Vitro Differentiation of a Spermatogonial Cell Line Science 297, (2002) 392-395.