"Derivation of Pluripotent Stem Cells from Cultured Human Primordial Germ Cells" (1998), by John Gearhart et al.
"Derivation of Pluripotent Stem Cells from Cultured Human Primordial Germ Cells" (1998), by John Gearhart et al.
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2012-05
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Ke Wu
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作者：
Ke Wu
1998年11月，发表了两份关于首次分离人类多能干细胞[5]的独立报告，其中一份是《从培养的人类原始生殖细胞中衍生多能干细胞》。这篇由John D. Gearhart及其研究小组（Michael J. Shamblott [6], Joyce Axelman [7], Shunping Wang [8], Elizabeth M. Bugg [9], John W. Littlefield [10], Peter J. Donovan [11], Paul D. Blumenthal [12], George R. huggins）撰写的论文，在James A. Thomson及其研究小组在《科学》杂志上发表了“来自人类囊胚的胚胎干细胞系”之后不久，发表在《美国国家科学院院刊》上。Gearhart的论文表明，多能性人类干细胞[5]能够发育成构成人体的所有细胞类型，它可能来源于从胚胎中分离出来的原始生殖细胞[14]，而原始生殖细胞[14]是完全分化的生殖细胞[14]的前体。当时，吉尔哈特是约翰霍普金斯大学医学院的妇产科教授。他有遗传学背景，他的大部分研究都是关于基因如何调节组织和胚胎形成。然而，小鼠胚胎干细胞[17]的成功分离鼓励Gearhart继续在人类[18]中分离类似细胞。人类胚胎干细胞（由Thomson的研究小组获得）和人类胚胎生殖细胞（由Gearhart的研究小组获得）的主要区别在于，人类胚胎生殖细胞[19]来源于早期生殖细胞[14]。尽管如此，它们被认为与人类胚胎干细胞b[17]具有相似的特性。Gearhart和脊[21]，性腺的发育前体，以及肠系膜[21]来自流产的5至9周大的人类胚胎/胎儿。胚胎是在捐赠者给予知情同意后使用的。机械分解后，施加力将样本组织分离成单个细胞，将细胞培养并放置在有丝分裂失活的小鼠[23]成纤维细胞饲养层上。小鼠成纤维细胞来自山德士近交小鼠[24]，是迄今为止唯一能够产生人类EG细胞的细胞类型。分解后的细胞在含人重组白血病抑制因子（hrLIF）、人重组碱性成纤维细胞生长因子（hrbGFG）和福斯克林的环境中生长。这些因素能使细胞分裂，但不能使细胞分化。检测细胞的碱性磷酸酶活性以及五种细胞表面标记物的存在：碱性磷酸酶、阶段特异性抗原(SSEA)-1、SSEA-3、SSEA-4、TRA-1-60和TRA-1-81。对一批胚状体（分化的细胞聚集体）进行免疫组织化学分析，最终在不含hrLIF、hrbFGF和forskolin的情况下培养14天，然后包埋石蜡。经过60 - 70天的培养（传代8-10），来自5种不同培养的细胞进行核型[26]分析，以检测人类染色体。这篇论文由John D. Gearhart和他的研究小组——Michael J Shamblott， Joyce Axelman, Shunping Wang, elizabeth M. Bugg, John W. Littlefield, Peter J. Donovan, Paul D. Blumenthal和George R. Huggins——在James A. Thomson和他的研究小组在《科学》杂志上发表了“来自人类囊胚的胚胎干细胞系”之后不久发表在《美国国家科学院院刊》上。吉尔哈特的论文提出，多能性人类干细胞可以从从胚胎中分离出来的完全分化的生殖细胞的前体原始生殖细胞中提取出来，这种干细胞有能力发育成构成人体的所有细胞类型。当时，吉尔哈特是约翰霍普金斯大学医学院的妇产科教授。他有遗传学背景，他的大部分研究都是关于基因如何调节组织和胚胎形成。然而，小鼠胚胎干细胞的成功分离鼓励吉尔哈特继续在人类中分离类似的细胞。人类胚胎干细胞（由汤姆森的研究小组获得）和人类胚胎生殖细胞（由吉尔哈特的研究小组获得）的主要区别在于，人类胚胎生殖细胞是从早期生殖细胞中获得的。尽管如此，它们被认为与人类胚胎干细胞具有相似的特性。
In November 1998, two independent reports were published concerning the first isolation of pluripotent human stem cells [5] , one of which was “Derivation of Pluripotent Stem Cells from Cultured Human Primordial Germ Cells.” This paper, authored by John D. Gearhart and his research team— Michael J. Shamblott [6] , Joyce Axelman [7] , Shunping Wang [8] , Elizabeth M. Bugg [9] , John W. Littlefield [10] , Peter J. Donovan [11] , Paul D. Blumenthal [12] , and George R. Huggins—was published in Proceedings of the National Academy of Science soon after James A. Thomson [13] and his research team published “Embryonic Stem Cell Lines Derived from Human Blastocysts” in Science . Gearhart’s paper suggested that pluripotent human stem cells [5] , which have the ability to develop into all cell types that make up the body, could be derived from primordial germ cells [14] , which are precursors of fully differentiated germ cells [14] , isolated from embryos. At the time, Gearhart was a professor of obstetrics and gynecology at Johns Hopkins University School of Medicine [15] . With a background in genetics, he had devoted the majority of his research to how genes [16] regulate tissue and embryo formation. However, the successful isolation of mice embryonic stem cells [17] encouraged Gearhart to pursue the isolation of similar cells in humans [18] . The principal difference between human embryonic stem (ES) cells, which Thomson’s team derived, and human embryonic germ (EG) cells, which Gearhart’s team derived, is that human embryonic germ cells [19] are derived from early germ cells [14] . Nonetheless, they are thought to share similar properties to human embryonic stem cells [17] . Gearhart and ridges [20] , the developmental precursors to gonads, and mesenteries [21] from aborted 5-to-9-week-old human embryos/fetuses. The embryos were used after the donors gave informed consent [22] . After mechanical disaggregation, where force is applied to separate a sample tissue into individual cells, the cells were cultured and placed on a mitotically inactivated mouse [23] fibroblast feeder layer. Mouse fibroblasts come from the Sandoz inbred mouse [24] and, to date, are the only cell type capable of generating human EG cells. The disaggregated cells were grown in an environment that included human recombinant leukemia inhibitory factor (hrLIF), human recombinant basic fibroblast growth factor (hrbGFG), and forskolin. These factors enable cell division, but not cell differentiation [25] . The cells were tested for alkaline phosphatase activity as well as the presence of five cell surface markers: alkaline phosphatase, stage specific antigen (SSEA)-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Immunohistochemical analysis was performed on a batch of embryoid bodies (differentiated cell aggregates) that were eventually cultured for fourteen days in absence of hrLIF, hrbFGF, and forskolin before being embedded in paraffin. After sixty to seventy days in culture (passage 8-10), cells from five different cultures underwent karyotype [26] analyses to detect human chromosomes. This paper, authored by John D. Gearhart and his research team - Michael J Shamblott, Joyce Axelman, Shunping Wang, Elizabeith M. Bugg, John W. Littlefield, Peter J. Donovan, Paul D. Blumenthal, and George R. Huggins - was published in Proceedings of the National Academy of Science soon after James A. Thomson and his research team published "Embryonic Stem Cell Lines Derived from Human Blastocysts" in Science. Gearhart 's paper suggested that pluripotent human stem cells, which have the ability to develop into all cell types that make up the body, could be derived from primordial germ cells, which are precursors of fully differentiated germ cells, isolated from embryos. At the time, Gearhart was a professor of obstetrics and gynecology at Johns Hopkins University School of Medicine. With a background in genetics, he had devoted the majority of his research to how genes regulate tissue and embryo formation. However, the successful isolation of mice embryonic stem cells encouraged Gearhart to pursue the isolation of similar cells in humans. The principal difference between human embryonic stem (ES) cells, which Thomson 's team derived, and human embryonic germ (EG) cells, which Gearhart 's team derived, is that human embryonic germ cells are derived from early germ cells. Nonetheless, they are thought to share similar properties to human embryonic stem cells.