CN101044235A - 胚胎干细胞的培养基和培养 - Google Patents
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Abstract
以前培养人胚胎干细胞的方法需要成纤维细胞饲养细胞或曾经接触成纤维细胞饲养细胞的培养基以将干细胞维持在未分化状态。现在发现,如果在培养干细胞的培养基中加入高水平的成纤维细胞生长因子、γ氨基丁酸、2-哌啶酸、锂和转化生长因子β,即便没有饲养细胞或调理培养基,干细胞也将在多次传代中无限期地维持未分化。
Description
相关申请的交叉参考
本申请要求2004年9月8日提交的美国临时专利申请序列号60/608,040的优先权。
关于联邦政府资助研究或开发的声明
本发明是利用下述机构授予的美国政府资助下完成的:NIH RR17721。美国政府对本发明享有一定权利。
发明背景
干细胞被定义为能够分化成许多其它分化细胞类型的细胞。胚胎干细胞是来自胚胎的干细胞,能够分化成大多数,如果不是全部,成熟机体的分化细胞类型。干细胞被称为是多能的,这是指它能够分化成许多细胞类型。一类受到研究机构高度关注的多能干细胞是人胚胎干细胞,这里简称为人ES细胞,这是一种来自人胚胎来源的胚胎干细胞。人胚胎干细胞由于能够在培养基中无限增殖并由此而能够(至少原则上能够)提供细胞和组织以替代缺陷或受损的人组织而受到极大科学关注。人胚胎干细胞培养物的存在有可能提供无限量的人细胞和组织以用于各种有助于人体健康的治疗方案和研究计划。可以想像,在未来,将能够增殖人胚胎干细胞并使其定向分化成特定谱系以产生出于治疗目的可移植入人体的分化的细胞或组织。人胚胎干细胞和可由它们衍生的分化细胞也是用于研究人类细胞和发育系统的有力科学工具。
已经描述了产生和培养人胚胎干细胞的基础技术。现有技术的确有效,但一些目前用来培养人胚胎干细胞的方法有限制和缺点。一种限制意义特别重大。大多数现有人胚胎干细胞系在一定程度上或在其它程度上已经直接暴露于小鼠细胞或暴露于其中已经培养过小鼠细胞的培养基。发现来自现有细胞系的一些人ES细胞展示出人类细胞通常不产生的唾液酸残基Neu5Gc,这一事实倍受关注。最初的产生和培养人胚胎干细胞的技术要使用小鼠胚胎成纤维细胞(MEF)饲养细胞作为饲养层,在该饲养层上可培养人胚胎干细胞。成纤维细胞饲养层通过一些仍不十分清除的机制使得干细胞能够保持在未分化状态。随后发现,如果使干细胞接触“调理培养基(conditionedmedium)”也能获得相同的现象。调理培养基是已经培养过饲养细胞(如MEF)的干细胞培养基。无论饲养细胞是能赋予培养基某些因子还是能从培养基中除去某些因子,其结果都是调理培养基可用来培养干细胞使其不发生分化。任何一种培养条件,即人ES细胞直接在鼠饲养细胞上生长或使用调理培养基都涉及这样的问题,即一种或多种试剂如病毒能从小鼠细胞传递到人ES细胞。如果培养人胚胎干细胞的一个目的是产生最终可植入人体的组织,就高度需要干细胞从未暴露于其它物种的细胞或者暴露于曾用来培养其它物种的细胞的培养基。因此,在长期培养人胚胎干细胞技术的持续发展中最感兴趣的是找到能增殖和培养人胚胎干细胞但不含成纤维细胞饲养层的培养条件。
培养中的人胚胎干细胞的一个特有性状是,如果条件不够理想,这种细胞则有分化趋势。当在培养时过分要求维持人ES细胞处于未分化状态时则容易诱导人ES细胞分化。大多数培养条件将导致一定水平不希望的分化,尤其是在正在生长的ES细胞集落边缘周围。尽管培养ES细胞时可以有一定程度不希望的分化,但目的是定义能够尽可能使培养物维持未分化即得到尽可能少的分化细胞的培养条件。我们认为,我们已经采用特别严格的标准来定义将支持无限培养未分化的ES细胞培养物的条件。
一些培养基配方能够在一段时间内使人ES细胞维持未分化状态,但长期培养时将不能维持这种状态。具体地说,我们定义来自最初的种子培养物的人ES细胞在培养容器上生长至细胞铺满该相同的培养容器为一个“传代”。我们发现一些培养基配方允许培养人ES细胞一或两个传代不发生严重分化,但在随后的传代中细胞迅速分化。我们已经开始相信,为得到确实能够支持人ES细胞无限增殖而不分化且不含饲养细胞或调理培养基的培养基,培养基必需支持培养人ES细胞处于基本均匀且未分化状态至少5代。培养物在培养期间维持相对同质和未分化并保留人ES细胞所有重要特征也是重要的。
可通过形态学特征非常容易地评价干细胞培养物的分化状态。未分化的干细胞具有一个特征形态,即具有清晰细胞边界的小且致密的细胞,通过在显微镜下检测干细胞培养物可容易观察到这种形态。相反,已经分化的细胞看上去较大且更加散开,并具有不明显的边界。尽管一些分化的细胞可能会在未分化细胞集落边缘出现(通常是这样),但最理想的干细胞培养物是这样一种细胞培养物,它在培养容器中增殖,在似乎分化的培养物周围仅具有最少量的细胞。根据经验可以在视觉上准确判断人ES细胞培养物的分化状况和健康状况。用来追踪ES细胞未分化状态的生化标记是转录因子Oct4,它被认为是ES细胞未分化状态最可靠的标记,并且是当未分化的细胞开始分化时最先失去的标记之一。
发明概述
本发明可概述为一种不需要饲养细胞或调理培养基的培养人胚胎干细胞的方法,该方法包括在含有盐、维生素、氨基酸、葡萄糖、成纤维细胞生长因子、γ氨基丁酸、2-哌啶酸(pipecholic acid)、锂和转化生长因子β的培养基中培养人胚胎干细胞的步骤,所述物质的含量都足以在多次传代培养中维持干细胞处于未分化状态。
本发明还涉及一种人胚胎干细胞的体外细胞培养物,该人胚胎干细胞在含有高水平成纤维细胞生长因子、γ氨基丁酸、2-哌啶酸、锂和转化生长因子β的培养基中培养,因此干细胞在不需要成纤维细胞饲养细胞或调理培养基的情况下便可以未分化状态无限期地培养。
本发明的一个目的是定义人胚胎干细胞的长期培养条件,该培养条件避免使用或暴露于动物细胞和动物蛋白,不论是饲养细胞还是用于调理要培养干细胞的培养基的其它动物细胞和动物蛋白。
本发明的另一个目的是定义用于人胚胎干细胞的培养条件,该培养条件尽可能地确定,同时尽可能维持培养中最大比例的细胞处于未分化状态。
通过以下描述将了解本发明的其它目的、特征和优点。
附图简述
图1展示了由以下说明书描述的试验工作获得的数据,显示了培养基组分减少了生长于其中的人ES细胞培养物中分化细胞的比例。
图2是显示了含有人基质蛋白的培养基使培养的人ES细胞不展示非人源唾液酸残基的数据的图示。
图3是显示了人干细胞培养物中高水平未分化细胞的数据的图示。
图4是显示了这里所述的培养基使干细胞培养物健壮生长的数据的图示。
发明详述
我们已经鉴定了许多能够使人胚胎干细胞以未分化状态无限期地培养和强壮增殖且同时完全不需要饲养细胞和调理培养基的培养条件和培养基。开发这些培养基和培养条件能够无需直接或间接接触任何种类的动物细胞而在确定的和受控的条件下衍生和维持人ES细胞系。这些培养基已被证实能支持未分化的ES细胞增殖多代,至少5代,这是这些培养基将支持这种无限期地培养的有力证据。
用来培养和增殖人ES细胞的确定的(defined)人源化培养基通常包含盐、维生素、葡萄糖源、矿物质和氨基酸。为补充培养基和提供支持细胞生长的条件,最初的干细胞培养基含有来自一种来源或另一种来源的血清。之前还报道加入成纤维细胞生长因子和替代血清添加物可在不需要血清的情况下培养人ES细胞。可通过商业获得用于此目的的替代血清,或者替代血清可以是用蛋白质如血清白蛋白、维生素、矿物质、运铁蛋白或运铁蛋白替代品、以及胰岛素或胰岛素替代品配制的混合物。也可在这种替代血清组分中添加硒。这里优选用确定的替代血清来代替培养人ES细胞的任何来源的血清,以便避免血清组成变化的问题以及使用尽可能确定的培养基。我们已经定义了充分的培养基,这里提到的培养基的所有组分列于下面的表1,该表列出了被称为TeSR1的我们的培养基的所有组分以及各成分的浓度。TeSR1培养基由添加了人血清白蛋白、维生素、抗氧化剂、痕量矿物质、特定脂类和克隆生长因子的DMEM/DF12基础培养基构成。
为避免对之前认为是维持人ES细胞处于未分化状态所必需的成纤维细胞饲养层的需要,这里报道将较高浓度的FGF(10-1000ng/ml)和GABA(γ氨基丁酸)、2-哌啶酸(PA)、锂(LiCl)和转化生长因子β(TGFβ)组合使用将能够使培养基支持未分化的干细胞生长。已经发现这些添加物的组合足以维持人ES细胞培养物无限期地处于未分化状态而不需接触饲养细胞或调理培养基。这些添加物被证实是足够的。然而并不是每一种培养基配方都需要它们中的全部。通过选择性删除这些添加剂,可删除这些组分中的一种或多种,产生仍将生长但培养物中未分化状态的纯度有损失的人ES细胞培养物。这种培养物可在许多代中保持或不保持稳定。然而,显然这种组合足以使各种培养基能支持在不含饲养细胞或调理培养基的情况下在无限次数的传代中长期培养和增殖未分化的人ES细胞。
我们最初对这些生长因子进行主观筛选,根据人ES细胞表达的受体进行选择,鉴定了一些对未分化细胞的增殖有正面效果的因子。这些因子中有bFGF、LiCl、γ-氨基丁酸(GABA)、2-哌啶酸和TGFβ最终含在TeSR1中。对所测试的四种细胞系中的每一种而言,TeSR1中持续表达人ES细胞特征标记的细胞的增殖率和百分比高于培养在成纤维细胞调理的培养基内的对照细胞,并且,除去这5种因子中的任何一种减低了培养的效果。这些数据中的一些示于图1,该图显示,生长在省略了这些成分中任何一种的培养基中的培养物显示出的未分化细胞的比例低于含有所有这4种培养基成分的培养物。注意,Oct4、SSEA-1、SSEA-4、Tral-60和Tral-80都是用来追踪干细胞分化状态的细胞表面标记或转录因子(Oct4)。图4显示了类似的试验,该试验证实,当培养基中含有所有这些组分时,在多次传代中培养物的生长速率最高。
对人ES细胞的培养条件而言,在培养容器中含有生物基质也是有益的。之前使用的一种此类材料是MatrigelTM,这是来自小鼠细胞的人造基底膜,它以不含小鼠细胞的市售产品提供。现在已知的用于类似目的的另一种人源材料是纤连蛋白,它是一种人糖蛋白,以不溶形式使用以形成也用作ES细胞培养的基底膜的纤维基质。就我们掌握的情况,仅纤连蛋白基质是不够的。然而,现在还发现,人基质材料可由人基质蛋白胶原IV、纤连蛋白、层粘连蛋白和玻连蛋白的组合制成,这种基质足以支持人ES细胞在TeSR1培养基中永远处于未分化状态。
在系统地测试了超过80种生长因子之后得到了上面列出的培养基添加物。虽然这些添加物中的一些在至少几次传代中支持培养中的人ES细胞生长,但许多在随后的传代中无法维持ES细胞处于未分化状态。我们未鉴定产生了下面的实施例中描述的培养基添加物的结果的这些因子的其它组合。这并不是说不能对这些成分进行改变。例如,由于LiCl能刺激wnt途径而将其用于该培养基。Wnt自身或该途径的其它刺激物如活化素可作为LiCl的等效物,用于代替LiCl,虽然LiCl可能是用于此目的的最经济的试剂。类似地,GABA被认为与GABA受体反应,科学文献中鉴定了一些作为该相同受体的激动剂的分子,这些分子也可作为等效物,用于代替培养基中的GABA。还认为PA也与GABA受体反应。虽然发现PA和GABA在这里所用浓度下都对培养基有帮助,但也可以想像,可以显著升高这些成分中的一种成分或其它成分的浓度而避免对其它成分的需要。
较高浓度(40-100ng/ml)的成纤维细胞生长因子似乎可以排除对饲养细胞的需要。优选的FGF是碱性FGF,也称为bFGF和FGF2,但至少包括FGF4、FGF9、FGF17和FGF18的其它FGF也足以满足该目的。其它FGF即便是在更高浓度可能也是有效的。
之前只有在存在成纤维细胞饲养细胞时或在调理培养基中培养人胚胎干(ES)细胞培养物时才能使其维持在未分化状态的这一观察结果导致人们推测成纤维细胞将某种能抑制ES细胞分化的因子释放到培养基中。然而,无论由成纤维细胞饲养细胞介导的对培养基的效应如何,现在将清楚下面描述的培养基将代替这种效应。下面描述的三种培养基是确定的,不含动物细胞,并能长期培养未分化的人ES细胞处于未分化状态。提供了一个培养基例子,其中培养基中的蛋白质都是人蛋白质,具有“人源化”培养基和培养条件,从而避免任何可能的涉及动物来源的亚细胞产品的问题。
实施例
除非另有说明,用于这里描述的所有培养的TeSR1培养基的组成列于下面的表1。我们预备试验提示,未分化的人ES细胞在pH 7.2、同渗容摩(osmolality)350mOsMol和10%CO2/5%O2大气下最适宜增殖。这些条件被用于这里所述的所有随后的培养。
人ES细胞系H1、H7、H9和H14的细胞在TeSR1中分别强壮增殖11、7、25和17代(2-6个月)。证实细胞系H14在7代之后、H9在8代和21代之后核型正常。证实H1和H9在11和20代后形成畸胎瘤。
已知之前的ES细胞培养物由于存在不是由人制造的唾液酸Neu5Gc而不是最佳的。由于人基质组分胶原、纤连蛋白、层粘连蛋白和玻连蛋白将最终的动物产品排除出入ES细胞的TeSR1培养条件,我们检测了当在这种培养基中培养时现有人ES细胞系是否消除了Neu5Gc。我们证实了培养在成纤维细胞调理培养基中的人ES细胞上存在Neu5Gc,培养在Matrigel上的TeSR1的细胞上检测到减少的但可检测的Neu5Gc,培养在使用四种人基质组分的TeSR1中的ES细胞上检测不到任何Neu5Gc。这些数据示于图2。因此,培养在TeSR1和人蛋白质基质上的人ES细胞不展示培养在鼠科饲养细胞上的细胞中发现的非人唾液酸残基。
为检测ES细胞集落的条件和长期维持人ES细胞培养物的培养适用性,将TeSR1培养基与之前最好的培养条件(就我们掌握的情况是使用调理培养基)进行比较。发现TeSR1培养基能够维持人ES细胞处于这样一种未分化状态,即超过90%的细胞即使在长期培养后仍继续呈Oct4阳性。该检测结果示于图3。这首次说明,不含任何饲养细胞且不含调理培养基的培养基能维持人ES细胞的未分化生长水平至培养中90%以上的细胞在所有阶段维持未分化的程度。
在TeSR1培养基和TeSR培养基中测试了培养了3代的H1细胞的生长曲线和FACS分析,TeSR培养基省略了以下各组分:TGFβ、PA、LiCl、GABA和bFGF。开始培养时,在第1代的第0天将3×105各细胞系的细胞铺板。计算三个复孔的细胞数以评价贴壁情况(第2-3天)和传代时的最终细胞数(第6-7天)。重复最初的铺板密度和取样时间,可能的话进行5代。在第3代的第6天通过FACS分析细胞的细胞表面标记SSEA1、SSEA4、Tra1-60和Tra1-81和转录因子Oct4。该数据示于图1。这些数据显示,人ES细胞可培养在缺乏这些组分中的每一种的培养基中,但代价是培养时细胞会出现一些不希望的分化,且只有使用所有这些组分才能获得未分化水平最高的培养物。用其它细胞系也获得了类似结果。
测试了维持在TeSR1培养基中的人ES细胞系的多能性。将分别培养在Matrigel基质上TeSR1培养基中11代和20代的新出现的细胞系WA01和WA09的细胞分别注射入SCID-米色小鼠。接种6-8后在小鼠中出现了展示复杂分化的畸胎瘤。
表1TeSR1培养基的完整配方
无机盐 | mM | 氨基酸 | mM |
氯化钙(无水)HEPES氯化镁(无水)硫酸镁(MgSO4)氯化钾(KCl)碳酸氢钠(NaHCO3)氯化钠(NaCl)磷酸氢二钠(无水)磷酸二氢钠(NaH2PO4-H2O)痕量矿物质硝酸铁(Fe(NO3)3-9H2O)硫酸铁(FeSO4-7H2O)硫酸铜(CuSO4-5H2O)硫酸锌(ZnSO4-7H2O)偏钒酸铵NH4VO3硫酸锰MnSO4H2O | 0.823211.760.23520.3190883.2614411.211294.558240.3920.3551520.000094080.0011764.0768E-060.0011760.0000561.00592E-05 | L-丙氨酸盐酸L-精氨酸L-天冬酰胺-H2OL-天冬氨酸L-半胱氨酸-HCl-H2OL-胱氨酸2HClL-谷氨酸L-谷氨酰胺甘氨酸L-组氨酸-HCl-H2OL-异亮氨酸L-亮氨酸盐酸L-赖氨酸L-甲硫氨酸L-苯丙氨酸L-脯氨酸L-丝氨酸 | 0.13920.54880.13920.13920.07840.07840.13922.960.2960.11760.3261440.3535840.3912160.0909440.168560.21760.296 |
钼酸铵NiSO4 6H2O偏硅酸钠Na2SiO3 9H2OSnCl2CdCl2CrCl3AgNO3AlCl3 6H2OBa(C2H3O2)2CoCl2 6H2OGeO2KBrKINaFRbClZrOCl2 8H2O生长因子GABA2-哌啶酸bFGFLiClTGFβ1脂类亚油酸硫辛酸花生四烯酸胆固醇 | 1.00404E-054.94861E-060.0049261085.32544E-066.21931E-059.41176E-065.00293E-062.4855E-054.99217E-055.0021E-052.5337E-055.04202E-065.12048E-060.0005001195.00414E-059.03834E-050.9790.0009845.80E-060.9792.35E-080.00709760.000399840.0013120.0113798 | L-苏氨酸L-色氨酸L-酪氨酸2Na 2H2OL-缬氨酸维生素抗坏血酸生物素胆碱盐酸盐D-泛酸钙叶酸i-肌醇烟酰胺盐酸吡哆素核黄素盐酸硫胺素维生素B12能量物质D-葡萄糖丙酮酸钠蛋白质人胰岛素人全运铁蛋白(Holo-Transferrin)人血清白蛋白其它组分谷胱甘肽(还原型) | 0.3520160.03465280.1677760.3543680.3751.12112E-050.05025440.00360640.0047040.054880.0129360.00760480.00047040.024602170.00039213.727840.3920.00344380.14199.70.00592996 |
DL-α生育酚-醋酸盐亚麻酸肉豆蔻酸油酸棕榈油酸硬脂酸 | 0.029620.0071840.0087580.007080.0078620.00703 | 次黄嘌呤Na酚红腐胺-2HCl胸苷2-巯基乙醇硒Pluronic F-68Tween 80 | 0.011760.01599360.0003943520.0011760.10.0001773040.2380.3358 |
Claims (16)
1.一种不需要饲养细胞或调理培养基的在基质上培养未分化状态的人干细胞的方法,所述方法包括以下步骤:
在含有含量足以将人干细胞历经多次连续培养传代而维持未分化状态的盐、维生素、氨基酸、葡萄糖、成纤维细胞生长因子、γ氨基丁酸、2-哌啶酸、锂和转化生长因子β的培养基中培养人干细胞。
2.如权利要求1所述的方法,其特征在于,所述培养基含有浓度至少为40ng/ml的成纤维细胞生长因子。
3.如权利要求1所述的方法,其特征在于,所述培养基还含有运铁蛋白和胰岛素。
4.如权利要求1所述的方法,其特征在于,所述人干细胞是胚胎干细胞。
5.一种在基质上和含有盐、维生素、氨基酸和成纤维细胞生长因子的培养基中培养人胚胎干细胞的方法,该方法的改进之处包括,在培养基中加入含量足以将所述细胞历经多次连续培养传代而维持未分化状态的γ氨基丁酸、2-哌啶酸、锂和转化生长因子β。
6.一种体外细胞培养物,包含处于培养容器中的:
人干细胞;
干细胞能够生长于其上的基质;和
培养基,所述培养基含有盐、维生素、氨基酸、葡萄糖、成纤维细胞生长因子、γ氨基丁酸、2-哌啶酸、锂和转化生长因子β,它们的含量足以将所述人干细胞历经多次培养传代而维持未分化状态,培养基不含饲养细胞且从未接触饲养细胞。
7.如权利要求6所述的细胞培养物,其特征在于,所述培养基含有浓度至少为40ng/ml的成纤维细胞生长因子。
8.如权利要求6所述的细胞培养物,其特征在于,所述培养基含有浓度至少为100ng/ml的成纤维细胞生长因子。
9.如权利要求6所述的细胞培养物,其特征在于,所述培养基还含有选自白蛋白、胰岛素和运铁蛋白的蛋白质。
10.如权利要求6所述的细胞培养物,其特征在于,所述蛋白是人蛋白。
11.如权利要求10所述的细胞培养物,其特征在于,所述胰岛素和运铁蛋白是重组蛋白。
12.如权利要求6所述的细胞培养物,其特征在于,所述干细胞是人胚胎干细胞。
13.一种含有未分化的人干细胞、基质和培养基的人胚胎干细胞培养物,所述培养物不含饲养细胞或接触过饲养细胞的培养基,所述培养基也不含非人动物产品,所述人胚胎干细胞在未分化状态下增殖,并且,所述人胚胎干细胞经长期培养后有至少90%为转录因子Oct4阳性。
14.如权利要求13所述的人胚胎干细胞培养物,其特征在于,所述细胞不展示唾液酸残基Neu5Gc。
15.一种培养干细胞的培养基,该培养基含有盐、维生素、氨基酸、葡萄糖、成纤维细胞生长因子、γ氨基丁酸、2-哌啶酸、锂和转化生长因子β,它们的含量足以将所述干细胞历经多次培养传代而维持未分化状态。
16.如权利要求15所述的培养基,其特征在于,所述培养基还含有选自白蛋白、胰岛素和运铁蛋白的蛋白质。
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CN102559587A (zh) * | 2010-12-16 | 2012-07-11 | 中国科学院上海药物研究所 | 诱导性多能干细胞的制备方法以及用于制备诱导性多能干细胞的培养基 |
CN102399742A (zh) * | 2011-09-30 | 2012-04-04 | 东莞中山大学研究院 | 用于哺乳动物唾液腺上皮细胞和唾液腺干细胞培养的无血清培养液 |
CN102399742B (zh) * | 2011-09-30 | 2014-07-30 | 东莞中山大学研究院 | 用于哺乳动物唾液腺上皮细胞和唾液腺干细胞培养的无血清培养液 |
CN105452446A (zh) * | 2013-07-15 | 2016-03-30 | 鹿特丹伊拉斯姆斯大学医疗中心 | 用于体外培养干细胞的方法和培养基 |
CN104830772A (zh) * | 2015-05-28 | 2015-08-12 | 深圳富利鑫健康产业发展有限公司 | 一种造血干细胞培养基及其应用和干细胞培养方法 |
CN112029708A (zh) * | 2020-09-09 | 2020-12-04 | 江苏育瑞康生物科技有限公司 | 一种干细胞培养系统 |
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