CN105899079A - 可扩展性骨骼肌谱系特化和培育的方法 - Google Patents
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Abstract
本公开涉及用于加强培养肉生产如家畜自主肉生产的方法。在某些方面,所述肉是源于任何多细胞动物的组织或细胞的食用产品,其预期用作人、伴侣动物、尸体预期用于食用用途的驯化或捕获动物、服务型动物、受保护动物物种、用于实验目的的动物或细胞培养物的食用食物或营养组分。
Description
对相关申请的交叉引用
本申请要求2013年10月30日提交的美国临时专利申请61/962,068的权益,所述专利申请以引用的方式整体并入本文中。
序列表的并入
与本申请一起提交的用电子方式提交的在ASCII文本档案(名称52553_136621_ST25.txt;大小:2,962字节;以及创建日期:2014年10月30日)中序列表的内容以引用的方式整体并入本文中。
关于联邦资助的研发的声明
本发明是在政府扶持下依据由美国国立卫生研究院(NationalInstitutes of Health)授予的许可编号R01HD069979/00032722所完成。政府在本发明中享有某些权利。
背景技术
本公开涉及用于加强培养肉生产如家畜自主肉生产的方法。“培养肉”(例如通过体外细胞培养、组织工程和食品技术方法进行动物自主肉生产)在概念上的期许包括相对于常规肉生产,生产效率提高、环境影响减小、烹饪应用效用扩大、营养价值增强、生产不野蛮并且食物安全性提高。然而到目前为止,技术还没有足够进步到支持可扩展的在经济上可持续的生产。目前对原型组织的实验室规模的培育已经利用一级动物组分如动物组织和血清,从而在很大程度上否定了动物自主肉生产的优势。因此,目前的方法不能充分解决培养肉生产对动物的依赖性来实现“培养肉”在概念上的期许并且提供在商业上有利的产品。因此,需要提供用于从自我更新性体外来源进行可扩展性肉培育以用于膳食营养和其它应用的新型且改善的方法。
发明内容
实例性实施方案提供一种用于骨骼肌培育的可扩展性平台,所述平台利用具有分化成骨骼肌的潜能的细胞系。在某些方面,细胞系来自家畜,如家养的牛、猪、绵羊、山羊、骆驼、水牛、兔等。在某些方面,细胞系来自家禽,如家养的鸡、火鸡、鸭、鹅、鸽子等。在某些方面,细胞系来自常见的游戏物种,如野鹿、鹑鸡类飞禽、水禽、野兔等。在某些方面,细胞系来自水生物种或半水生物种,所述物种从野生渔业或水产业操作商业收集或为好玩而收集,包括某些鱼、甲壳动物、软体动物、头足动物、鲸目动物、鳄目动物、海龟、蛙等。在某些方面,细胞系来自稀有的受保护或灭绝的动物物种。在某些方面,细胞系来自任何显示骨骼肌组织特化能力的多细胞动物物种。在某些方面,将细胞系用于研究目的或用于治疗目的,如人、灵长类动物、啮齿动物(包括大鼠和小鼠)和伴侣动物如狗、猫、马等。在某些特定方面,来自任何生物体的细胞系是自我更新性干细胞系。在某些方面,所选细胞系通过‘遗传转换’来修饰,以诱导细胞快速且有效地转化成骨骼肌以用于培养肉生产。在一个实例性实施方案中,上述方面或其它方面可以通过包括以下操作的方法来实现:通过肌生转录因子修饰所选自我更新细胞系,以产生以肌生转录因子修饰的细胞系,以及通过外源调控来诱导这种修饰的细胞系以引导交替的自我更新或分化过程。
在某些方面,自我更新细胞系选自具有肌生潜能的胚性干细胞、诱导型多能干细胞、体细胞系或胚外细胞系。在某些方面,细胞系源自预期用于膳食消耗的物种。肌生转录因子的说明性非限制性实例包括单独或组合的MYOD1、MYOG、MYF5、MYF6、PAX3、PAX7、其旁系同源物、直系同源物、遗传性变型,或如本文进一步描述的肌生转录因子的各别启动子识别DNA序列的转录活化激动剂。
在某些特定方面,诱导性MyoD转录因子可以用作分化谱系指示子。在某些特定方面,猪诱导型多能细胞系O2K可以用作自我更新细胞系。在一个特定方面,所述方法包括:用诱导性MyoD转录因子对O2K干细胞系修饰,以产生以肌生转录因子修饰的O2KM细胞系,以及通过外源调控来诱导这种O2KM细胞系以引导交替的自我更新或分化过程。上述修饰步骤可进一步包括用染色体整合的载体对细胞系进行修饰,所述染色体整合的载体组成性表达MYOD1转录因子与来自组成性活性启动子区域的ESR1配体结合的诱导性融合物。例如,所转译的融合转录物(例如MyoDER)的诱导活性可在ESR1激动剂(例如17-β***(E2))存在下有条件地活化。
在某些方面,诱导步骤可进一步包括自我更新子步骤和分化子步骤,由双开关机制调控。在自我更新子步骤中,保持修饰的细胞系处于未分化的基态,如在多西环素(DOX)存在下进行,从而通过多能性转基因POU5F1和KLF4的诱导表达使细胞系维持干细胞自我更新状态。在分化子步骤中,如用E2在无DOX存在下处理修饰的细胞系,而细胞系通过诱导性MyoD转录因子有效地指定为骨骼肌细胞,即肌生谱系,从而产生具有纺锤体样形态的特征性细长细胞。当在低***素培养基中进一步培养时,产生的肌细胞可融合于多核肌管,即骨骼肌纤维的前体中。在低***素培养基中长时间培养之后,多核肌管成熟成能够终末分化的骨骼肌纤维,所述终末分化如由增加的MYOG、DES、MYHC表达;融合;以及具有可收缩潜能的肌节肌原纤维的发育所证明。分化子步骤可进一步包括在培养基中添加某些试剂以活化经典WNT信号转导通路以防止细胞死亡并且有助于肌生分化,以及向培养基中添加后生调节剂以改变染色质结构以增强肌生性基因表达。
本公开的某些方面采用基因强化的细胞,以在无血清培养基中得到不受限制的更新能力以及与骨骼肌(构成非内脏肉制品的占优组织谱系)的有效会话。当与可扩展性组织工程方法关联时,这些方法可革命化肉的生产方式,并且通过实现以不受限制的量培育动物组织以进行动物自主培养肉生产来向消费者销售。其它所涵盖的应用包括体内异种移植用途,以及用于药物筛选、发育生理学和发育生物学的体外模型。
下文参考任何随附图式详述本公开的其它特征和优势,以及本公开的各个实施方案的结构和操作。
附图说明
图1.图1示出了MyoDER DNA序列。
图2.图2为一种方法的示意图,所述方法包括用于扩增未分化细胞系或骨骼肌谱系特化的双开关调控机制。
图3.图3为示出应用于由DOX或E2调控的肌生性经过修饰的O2KM细胞系的双开关机制的示意图。
图4A.图4A为可选MyoDER转基因表达盒的示意图。箭头和盒子分别表示启动子和基因序列。
图4B.图4B示出了抗MYOD1抗体在杀稻瘟菌素(blastidicin)所选O2K中的MyoDER转基因表达的西方墨点图像检测。将转基因表达盒修饰的O2K命名为O2KM。检测TUBA(α-微管蛋白)作为内负载对照物。
图5.图5为示出O2KM细胞在如亲本O2K细胞系的自我更新条件下显示稳定的紧凑菌落形态的图像。
图6.图6为一组图像,所述图像示出了在以聚D-赖氨酸+层粘连蛋白+MATRIGEL涂布的皿上、在用于无酚自我更新培养基(SRM)的+/-0.25μΜ5-氮杂-胞嘧啶核苷(5AC)存在下、在5%O2下培养3天的O2KM,之后在补充有3μΜCHIR99021的无酚红肌生性诱导培养基(MIM)中在20%O2下用或不用17-β***(E2)诱导MyoDER融合蛋白2天。C.O2KM在E2诱导第2天的相差图。
图7.图7示出了在指定的2天E2诱导方案之后收集的分化O2KM细胞溶解产物中,MYOD1(MyoD)、MYF5(Myf5)和MYOG(成肌素)的西方墨点分析。MyoDER迁移:约75kD。预期的内源MYOD1迁移:45-50kD。
图8.图8示出了在2天的10μΜE2诱导时程之前和之后,在暴露于5AC的O2KM培养物中的肌细胞细胞表面标记NCAM(Alexa568)和核(DAPI)的免疫荧光检测图。
图9.图9示出了以下物质的一组相差图:i.在聚D赖氨酸+明胶+层粘连蛋白上在5%O2下在SRM中培养的未分化的基态O2K菌落,ii.-vi.从如图片i.中所示的基态、在补充有ii.0μΜ、iii.1μΜ、iv.3μΜ、v.6μΜ或vi.9μΜCHIR99021的分化培养基(DM)中在20%O2下分化2天的粘附菌落。当培养物中的拟胚体暴露于6μΜ(vii.)或9μΜ(viii.)CHIR99021时,非粘附菌落占优势。
图10.图10示出了说明在分化期间的粘附O2K细胞群体变化的条形图。百分比表示在指定浓度(示于图9的图片ii.-vi.中)的CHIR99021存在下2天之后培养物中所存在的粘附细胞相对于在从基态(示于图9的图片i.中)分化之前所存在的粘附细胞的比率。对于每一所列培养条件,n=3。
图11.图11示出了膜联蛋白V标记细胞的流式细胞术分析的条形图。在分析之前,在20%O2下在分化培养基中、在0μΜ、1μΜ、3μΜ或6μΜCHIR99021存在下培养未分化的O2K菌落1天。
图12.图12示出了在指定浓度的CHIR99021存在下从基态(如图9中所示)分化的培养物中GSK3β底物丝氨酸33、37和苏氨酸41处的相对CTNNB1(β-连环蛋白)水平和硫酰化(p-CTNNB1)的西方墨点分析。在收集之前,使培养物暴露于50nM花萼海绵诱癌素A(Calyculin A)和30μΜMG-132持续3小时以使用于比较分析的p-CTNNB1的可检测水平稳定。
图13A.图13A示出了说明如图12中所示的p-CTNNB1/CTNNB1带的密度比的图。
图13B.图13B示出了说明如图12中所示的CTNNB1/TUBA带的密度比的图。
图14.图14示出了说明分化标记时程西方墨点分析的图像。在CHIR99021存在下从基态分化的O2K培养物中多能性标记POU5F1和KLF4以及前肌生性上段标记PAX3的表达水平。
图15.图15示出了在无(左图)或有(右图)5-Aza-胞嘧啶核苷(5AC)存在下分化的O2KM肌细胞的终末分化的图像。注意左图具有平坦形态的肌细胞衍生物(-5AC),与左图中的细长多核肌管(+5AC)相反。
图16.图16示出了如在图9的图片i-ii中在培养物24小时的过渡之前和之后凋亡细胞的膜联蛋白V标记。
图17A.图17A示出了在分化环境过渡(12-48小时)之前(0小时)和之后(12-48小时),基态菌落中全长CPP32(约32kD酶原3a)和裂解大片段(约17kD裂解半胱天冬酶3a)的西方墨点检测。
图17B.图17B示出了在指定CHIR99021水平存在下向分化环境过渡42小时之后,菌落中的全长CPP32和裂解片段的西方墨点检测。
图18.图18示出了在含有6μΜCHIR99021分化环境中形成的拟胚体经2天以及在转移到以聚D赖氨酸+层粘连蛋白+MATRIGEL涂布的衬底再经1天(第3天,左图)或3天(第5天,右图)之后的过度生长形态。
图19A.图19A示出了来自在无3i、DOX、hLIF和E2存在下、在含有KOSR的分化环境中培养的未经过修饰的piPSC的溶解产物的西方墨点。
图19B.图19B示出了来自在DOX、LIF和3i存在下培养的MyoDER修饰piPSC的溶解产物的西方墨点。将MYODER修饰的piPSC在自我更新或扩增环境中培养3天。
图20A.图20A为O2KM扩增和诱导方案、接着为终末分化方案的示意图。
图20B.图20B示出了肌管形态和构象。诱导后(第2天),piPSC当在扩增和诱导方案期间暴露于5AC时发育成细长的各项异性的屈光肌管。
图20C.图20C示出了到第6天的肌球蛋白重链的均匀表达。
图20D.图20D示出了肌管多核化。左图:第4天终末分化培养物的放大图。带支架的箭头表示单一肌管内的多个核。右图:通过碘化丙啶标记和流式细胞术分析所显示的由肌管倍数表示的肌核分布,n=3,示出了标准偏差。
图20E.图20E的西方墨点法示出了历经8d的过程肌间线蛋白(DES)和成肌素(MYOG)的表达增加。
图20F.图20F示出了伴随终末分化的从细胞周期的退出。
图20G.图20G示出了第6天的肌管的透射电子显微镜术。使肌节结构单元在单行(左图)和交错的平行行(右图)中对齐。
图21A.图21A示出了在自收缩性第6天的肌管亚群中观测到不同步的单细胞瞬态周期(左图、中图和右图)。
图21B.图21B示出了通过在第6天的肌管中进行1.0Hz场刺激所实现的钙瞬态周期的FOV活化和同步。
图21C.图21C示出了通过10mM咖啡因使第6天的肌管发生FOV钙瞬态活化。
图21D.图21D对通过100nM乙酰胆碱在第7天的肌管中所发生的钙瞬态活化的单细胞分析。
具体实施方式
以提供描述性支持所必需的程度,随附权利要求书的主题和/或文本以引用方式整体并入本文中。本书面说明书的所有读者都将了解,在本文中所述且要求保护的示例性实施方案可以在无任何本文特别公开或没有特别公开的所引用特征、要素或步骤存在下适当地实践。
贯穿本公开,术语“一个(种)”实体是指所述实体中的一个(种)或多个(种);例如,将“一种聚核苷酸”理解为表示一种或多种聚核苷酸。因此,术语“一个(种)”、“一个(种)或多个(种)”和“至少一个(种)”可在本文中互换使用。
此外,“和/或”当用于本文中时被视为特定公开两种指定特征或组分(有或无其它)中的每一种。因此,如在本文中用于词语如“A和/或B”中的术语“和/或”意图包括“A和B”、“A或B”、“A”(单独)和“B”(单独)。同样地,如用于词语如“A、B和/或C”中的术语“和/或”意图涵盖以下方面中的每一个:A、B和C;A、B或C;A或C;A或B;B或C;A和C;A和B;B和C;A(单独);B(单独);和C(单独)。
应了解,本文中无论在哪里用措辞“包含(包括)”描述方面,还提供以术语“由...组成”和/或“基本上由...组成”描述的另外的类似方面。
除非另外定义,否则本文使用的所有技术术语和科学术语都具有与本公开所涉及领域的普通技术人员通常所理解相同的含义。例如,《生物医学和分子生物学简明词典(Concise Dictionary of Biomedicine and MolecularBiology)》,Juo,Pei-Show,第2版,2002,CRC出版社;《细胞分子生物学词典(Dictionary of Cell and Molecular Biology)》,第3版,1999,学术出版社;和《牛津生物化学与分子生物学词典(Oxford Dictionary Of Biochemistry AndMolecular Biology)》,修订,2000,牛津大学出版社向本领域技术人员提供本公开中所用的许多术语的通用词典。
以Système International de Unites(SI)接受的形式表示单位、前缀和符号。数值范围包括界定范围的数字。除非另有规定,否则从5'到3'书写核酸序列并且从左到右以氨基到羧基的取向书写氨基酸序列。在本文中提供的标题不是本公开的各个方面的限制,可通过总体参考说明书来取标题。
除非在本文中另有规定,否则所有本文所述的方法都可以任何合适的顺序进行。
不应将本说明书中的措辞或术语视为指示任何未要求保护的要素为必要的或重要的。
除非在本文中另有规定,否则在本文中叙述值的范围仅旨在充当个别地提及每一属于所述范围的独立值的速记方法,并且将每一独立值并入说明书中,就如同其在本文中被个别地叙述一般。当提供具体的值范围时,应了解每一居中值都意图包括于其中,并且还包括所有较小的子范围。
本文提供用于从体外细胞来源产生功能性骨骼肌的新型且改善的方法,所述功能性骨骼肌用于培育肉,如工程化组织或其它食用产品。涵盖这些方法,例如用于家畜自主肉生产的方法,其中肉是预期用作人、伴侣动物、尸体预期用于食用用途的驯化或捕获动物、服务型动物、受保护动物物种、用于实验目的的动物或细胞培养物的食用食物或营养组分的源于任何多细胞动物组织或细胞的食用产品。
还提供体外产生的动物组织,如通过本文所公开的各个方面中的任一个制备的肌肉组织。在某些方面,细胞来源为干细胞来源,例如可自我更新的干细胞系。所述方法的某些方面采用源自预期物种的肌生性、诱导性转基因修饰的可自我更新的细胞系。在某些方面,预期物种可为任何可食用物种,包括家畜和家禽物种。在某些方面,预期物种为家畜物种,如家养的牛、猪、绵羊、山羊、骆驼、水牛、兔等。在某些方面,预期物种为家禽物种,如家养的鸡、火鸡、鸭、鹅、鸽子等。在某些方面,预期物种为常见的游戏物种,如野鹿、鹑鸡类飞禽、水禽、野兔等。在某些方面,预期物种为水生物种或半水生物种,所述物种从野生渔业或水产业操作商业收集或为好玩而收集,包括某些鱼、甲壳动物、软体动物、头足动物、鲸目动物、鳄目动物、海龟、蛙等。在某些方面,预期物种为稀有的受保护或灭绝的动物物种。在某些方面,预期物种为任何显示骨骼肌组织特化能力的多细胞动物物种。在某些方面,预期物种为用于研究目的或治疗目的,如人、灵长类动物、啮齿动物(包括大鼠和小鼠)和伴侣动物如狗、猫、马等。
在某些方面,由双开关机制调控细胞系以维持细胞系处于自我更新过程或者直接肌生分化中。
本文所公开的方面的亲本/宿主细胞系具有以下性质:永生(自我更新性)并且具有分化、改编、指定或以其它方式转化成骨骼肌谱系的潜能,如以下方案包括引导肌生性转化的一种或多种组分。三类干细胞可以用作可扩展性培育的细胞来源:(1)谱系限制性一级成年祖细胞干细胞分离物、(2)谱系限制性永生化细胞系,以及(3)多能干细胞系。已经确定这些方法中的每一个在充当培养肉生产用细胞来源方面具有优势和缺陷。
谱系限制性一级成年祖细胞干细胞分离物。其包括定型为构成肉制品的谱系如骨骼肌的成年祖细胞。骨骼肌祖细胞包括但不限于卫星细胞、成肌细胞和肌细胞。其优势包括:i)一级成年祖细胞局限于特定谱系并且需要极少或不需要对所需谱系的体外特化;以及ii)一级成年祖细胞不需要遗传修饰来谱系特化。其缺陷包括:i)其必须从新鲜屠宰的动物尸体收集或者从有创生检获得。任一方法都传达着对家畜的依赖,并且使家畜自主生产的益处折衷,到家畜用于所述过程中的程度;ii)一级细胞分离是极其低效的过程。所需细胞包含来源组织的一部分。所需细胞的一部分存活过分离过程。所需细胞谱系必须从存活细胞的混合群体分离,从而需要另外的纯化和扩增步骤。iii)一级成年祖细胞服从‘海佛烈克极限(Hayflick Limit)’,其中细胞在丧失其增殖能力之前仅可***有限次数。此外,一级成年祖细胞丧失其以与长久传代相符的方式终末分化的能力。因此,必须从一级细胞分离物获得其它细胞,从而限制从单一分离物的培育规模可扩大性;以及iv)适用于培养肉生产的组织如骨骼肌的谱系的一级细胞培养物具有锚定依赖性,从而限制了培养物的体积规模可扩大方法。在混悬液培养物中,这些细胞可能易于因失巢凋亡而发生细胞死亡。
谱系限制性永生化细胞系。其为谱系定型一级细胞,所述一级细胞经过基因改变以无限地自我更新,同时保留其终末分化的能力或者受到谱系限制。其优势包括:i)“永恒地自我更新”(即不服从‘海佛烈克极限’)并且可无限地扩增以进行可扩展性家畜自主培育;ii)局限于特定谱系并且需要极少或不需要进一步的体外特化。其缺陷包括:i)来自某些能按照适用于培养肉生产的谱系(例如骨骼肌)分化的物种的永生化谱系限制性细胞系可能需要发育;ii)谱系定型细胞系的培养物具有锚定依赖性,从而限制了规模可扩大性。在混悬液培养物中,谱系定型细胞系可能易于因失巢凋亡而发生细胞死亡;以及iii)可实现‘永生化’的细胞转型使基因修饰成为必需。还没有充分表征永生化适用的一级细胞群体而不干扰其终末分化能力所必需的基因修饰。
多能干细胞系:多能干细胞系包括维持以未分化状态自我更新或替代地分化成任何组织谱系的能力的胚胎干细胞或诱导型多能干细胞(iPSC)。其优势包括:i)一般来说,多能干细胞系以大于一级或永生化谱系限制性细胞系的速率增殖,从而减少在生产过程中生物质扩增所需的时间;ii)多能干细胞可以在混悬液培养物中培育成拟胚体,从而增强每单位培养物体积的培养物规模可扩大性。此外,可以将拟胚体培养成与微模具和生物印刷组织装配方法相容的‘生物油墨’;以及iii)如永生化谱系限制性细胞系那样,多能干细胞不服从‘海佛烈克极限’并且可无限地扩增以进行可扩展性家畜自主培育。其缺陷包括:i)源自某些物种的可靠的胚胎干细胞系可能需要发育;ii)iPSC改编和自我更新的方法可能具有转基因依赖性。因此,iPSC多能性可能需要基因修饰来进行未分化状态的诱导和自我更新。有效的iPSC分化需要使改编和维持与分化状态互斥的未分化状态所用的转基因沉默的机制,以避免抵触对所需谱系特化不利的转录网络活化;以及iii)相对于谱系限制性一级成年祖细胞干细胞和永生化细胞系,多能干细胞通常需要其它谱系特化步骤来发展和丰富所需谱系特化。
除上文提供的三种干细胞类别以外,本文还涵盖其它亲本/宿主细胞系。例如,肌生潜能之前已由畸胎瘤测定确立的诱导型滋养层细胞系(表示外胚型非多能、非体永生化细胞)也可适于肌生转化。例如,针对多能性局部改编的体细胞系可以具有肌生潜能,但不能形成呈现三个胚胎胚层的畸胎瘤。例如,虽然其存在有争议,但是STAP细胞系(多能性的刺激触发性获得)可具有肌强效性和自我更新性。
O2K细胞系。O2K细胞系为从植入前猪胚胎的内细胞群建立的诱导型多能干细胞系。已经研究了O2K细胞系,并且发现O2K的自我更新状态可通过使用‘Tet-On’诱导体系由多西环素(即DOX)转录性活化POU5F1和KLF4转基因来维持。
MYOD1转录因子。MYOD1(即MyoD)为骨骼肌谱系定型的首要调控剂。先前已经描述了MyoDER构筑体,其由鼠MYOD1基因与编码人***受体α的配体结合域的序列的基因融合物组成,示于图1中(SEQ IDNO:1)。在图1中,MyoDER由在鼠MYODI基因在Nar I限制酶消化位点与ESR1(即人***受体α)核苷酸844-1781的配体结合域编码序列之间的基因融合物组成。还存在未指定的连接子序列和Cla 1连接子序列。通过添加***受体α配体17β-***(即E2)来转译后诱导MyoDER融合构筑体的肌生特化活性。在无17β-***存在下,MyoDER保持非活性状态。MyoDER构筑体在本文中称为“诱导性MyoD”。
现在参考图2,一个方面为细胞-备料-扩增,即在维持细胞备料以继续可扩展性培育所必需的自我更新条件下扩增细胞系。另一方面为谱系-特化/分化,即诱导肌生性谱系分化以进行进一步组织培育过程。因此,某些方面可概括成包括两个主要步骤:i)用肌生转录因子修饰所选自我更新细胞系,以产生以肌生转录因子修饰的细胞系,以及ii)通过外源调控诱导这些修饰的细胞系以维持于自我更新过程或进入分化过程。如本文中使用,用肌生转录因子“修饰”细胞系是指将可操作地编码肌生转录因子(如通过转染、转导、转型等来实现)的核酸载体或构筑体***细胞系中,其中经过修饰的细胞系表达所述肌生转录因子。在某些方面,使所***的肌生转录因子诱导式表达以产生诱导性-肌生转录因子细胞修饰的细胞系。如本文中使用,“诱导式”、“诱导性”等是指可用于外源调控基因产物如肌生转录因子的活性的任何基因工程化方法。诱导方法包括但不限于通过配体诱导性转录因子技术(例如tet-on、tet-off、RheoSwitch)、定点再组合技术(例如Cre-LoxP、flp-FRT)、转座子技术(例如Sleeping Beauty,PiggyBac)、配体结合受体融合技术(例如***、孕酮、雄激素、甲状腺激素、糖皮质激素、他莫昔芬配体激动剂)和对带有肌生转录因子基因的染色体外表达载体进行瞬态转染来调控肌生转录因子活性。在某些方面,将核酸构筑体或载体以染色体方式整合到经过修饰的细胞系中。自我更新细胞系的代表性实例包括选自胚胎干细胞、诱导型多能干细胞和永生谱系限制性细胞系的那些。在某些方面,这些自我更新细胞系源自预期用于膳食消费或用于研究目的或治疗目的的物种。肌生转录因子的代表性实例包括单独或组合使用的MYOD1、MYOG、MYF5、MYF6、PAX3、PAX7、其旁系同源物、直系同源物、遗传性变型,或本文所公开的肌生转录因子的各别启动子识别DNA序列的转录活化激动剂。
现在参考图3,图3包括由DOX或E2调控的肌生性修饰的O2KM细胞系的双开关机制的示例性示意图。如图3中所示,在O2KM干细胞自我更新过程期间,在DOX存在下在自我更新培养基(SRM)中诱导多能性转基因POU5F1和KLF4表达,而当不存在DOX时这种表达受到抑制。类似地,在O2KM肌生分化过程期间,在E2存在下在肌生诱导培养基(MIM)中通过诱导性MyoD转基因活化肌生分化,而当不存在E2时这种定向分化无活性。
某些方面提供分化/特化方法,所述方法在MIM中包括除E2以外的其它试剂以防止细胞死亡并且调节染色质的后生状态。例如,可将糖原合成酶激酶-3β(Glyogen Synthase Kinase-3β;GSK3β)抑制剂添加到SRM和MIM中以活化经典WNT信号转导通路,这又会在DOX退出之时增强肌生分化并且减少细胞死亡。不受理论限制,在某些方面,后生模块化改变肌生转录因子对染色质的活化和/或增强肌生转录因子如MYF5的表达。
应了解GSK3β抑制包括用小分子进行靶向。基因编辑也是一种通过WNT信号转导活化来增强骨骼肌特化的有希望的方法。例如,在宿主/亲本细胞系中可以通过使GSK3β等位基因突变来抑制GSK3β,使GSK3β等位基因突变通过序列特异性***或缺失技术(即锌指核酸酶(Zinc-FingerNuclease)、TALEN、CRISPR)来实现。内源启动子区域的突变可用于抑制GSK3β表达。或者,GSK3β开放阅读框可以使用相同方法缺失或突变以消除GSK3β活性。同样地,GSK3β的下游硫酰化目标:β-连环蛋白(CTNNB1)可以在编码由GSK3β磷酸化的残基的密码子处突变,从而防止GSK3β将CTNNB1硫酰化,从而产生组成性活性的稳定CTNNB1。这种“基因编辑”法将降低通过小分子靶向进行GSK3β抑制的成本,并且可能改善肉制品的安全概況,因为在所述过程期间不需要其它化学物质来抑制GSK3β。涵盖进行Wnt信号转导抑制的第三种方法包括使用GSK3β的反义核酸抑制剂或拮抗WNT通路的其它因子。其可以包括使用序列靶向性shRNA或miRNA的RNA干涉法。
某些特定方面提供GSK3β抑制剂促进例如在DOX退出之时的细胞存活的用途。GSK3β抑制剂的一个说明性实例为CHIR99021。其它代表性GSK3β抑制剂可以包括不限于:氯化锂、BIO、SB216763、CHIR-98014、TWS119、太古昔布(Tideglusib)、IM-12、1-Azakenpullone、AR-A014418和SB415286。在不受理论束缚的情况下,据信伴随有DOX,通过涉及GSK3β抑制的WNT信号转导通路来维持未分化细胞的两种自我更新。
在不受理论束缚或不受任何特定代表性实例限制的情况下,观测到DOX与GSK3β抑制剂CHIR99021从补充有N-2和B-27血清替代物的培养基同时退出会加速分化中O2K亲本系在48小时内的大规模细胞死亡。在CHIR99021存在下退出DOX可实现分化中O2K的存活。通过形态学、细胞粘附测定、裂解半胱天冬酶-3积累和膜联蛋白V标记来证实在无GSK3β抑制剂存在下分化中O2K细胞的死亡以及在所述GSK3β抑制剂存在下的相应存活率。在亲本O2K细胞系中,GSK3β的抑制和谐地引起其硫酰化底物CTNNB1(经典WNT信号转导通路的下游阳性效应物)得到稳定化和活化。WNT信号转导在从未分化基态多能性的两种中胚层特化,即从未成形中胚层的前肌生性丰富以及体内与体外定型肌细胞的终末分化中起到重要作用。一致地,在无DOX存在下以及在GSK3β抑制剂存在下O2K系的长久培养支持向着前肌生性上段分化,如由PAX3表达和伴随的POU5F1和KLF4表达损失所证明。此外,与5AC组合,GSK3β抑制剂增强肌生转录因子MYF5在分化中O2K中的表达。此外,GSK3β抑制剂如CHIR99021增强肌生性鼠C2C12细胞系终末分化成多核肌管,如表1中所示。
表1列出细胞外基质效应物(即明胶、聚D-赖氨酸、层粘连蛋白、MATRIGEL)和可溶性因子(E2、CHIR99021)对鼠C2C12成肌细胞的终末分化的影响,其通过评估行进5天分化时程的肌管形成的尺寸和程度来得到。(根据指示稳健肌管形成的[****]到指示不能检测到肌管形成的[-]对培养物进行评分。)
表1.细胞外基质效应物和可溶性因子对终末分化的影响。
在某些方面,由于其多项功能:(1)抑制DOX退出之后的细胞死亡、(2)支持向着前肌生状态分化、(3)增强肌生性特化以及(4)增强终末分化,因此GSK3β抑制剂当在分化期间(在无DOX存在下)保留于培养基中时被认为与衍生肌细胞的诱导性肌生转录因子定向谱系特化和后续终末分化条件相容。例如,在后续的E2诱导型谱系特化和终末分化过程期间,在DOX退出之后CHIR99021保留于O2KM培养物中。
然而,虽然上述补充有GSK3β抑制剂的培养方案支持细胞存活,以及O2KM使肌生性谱系特化为具有独特的肌细胞样纺锤体形态的分化细胞,但是衍生细胞不能终末分化成细长的屈光肌管。因此,使用后生调节剂以增强否则不允许发生肌生成的细胞中的肌生性基因的表达,并且增强谱系定型肌细胞终末分化为成熟肌纤维。为了使O2KM能够终末分化,在DOX退出之前在5-氮杂-胞嘧啶核苷(5AC)存在下培养细胞72小时,持续E2诱导期间的48小时,接着是另外最多6天的终末分化阶段。在5AC存在下培养的O2KM源性肌细胞中,MYF5表达增强并且细胞显示屈光肌原纤维形态,而在无5AC存在下衍生的肌细胞表达减少的MYF5并且显示对于成熟肌原纤维非典型的平坦形态。这种差别可以由以下来解释:在DOX退出用了48小时之前和之后仅在暴露于5AC的O2KM中观测到肌生转录因子MYF5的表达增强。
结合已知为活性反式激活子的较低分子量的未磷酸化的成肌素同种型的增浓,在这些培养物中形态学差别可以由5AC暴露会增强MYF5表达来解释。应了解,后生调节要求通过改变核小体相关组蛋白的DNA甲基化模式和转译后修饰来改变影响转录因子结合和目标转录性活化的染色质结构。应了解,后生调节可能需要靶向后生通路或包含后生机构的表达蛋白质的小分子激动剂或拮抗剂。小分子后生调节剂的一个说明性实例为5-氮杂-胞嘧啶核苷(5AC)。小分子后生调节剂的其它代表性实例包括5-氮杂-2'-脱氧胞苷、RG108、Scriptaid、丁酸钠、曲古抑菌素A、辛二酰苯胺异羟肟酸、MS-275、CI-994、BML-210、M344、MGCD0103、PXD101、LBH-589、Tubastatin A、NSC3825、NCH-51、NSC-3852、HNHA、BML-281、CBHA、Salermide、庚二酸二苯酰胺、ITF-2357、PCI-24781、APHA化合物8、Droxinostat和SB-939。后生调节中所涉及的蛋白质的代表性实例包括组蛋白脱乙酰酶旁系同源物、组蛋白乙酰转移酶旁系同源物、tet-甲基胞嘧啶加双氧酶旁系同源物、组蛋白脱甲基酶旁系同源物、组蛋白甲基转移酶旁系同源物和DNA甲基转移酶旁系同源物、组蛋白,以及染色质重构复合物的亚单元,包括Mi-2/NuRD(和其组分如甲基-CpG-结合域蛋白3(MBD2))和SWI/SNF(和其组分如BAF60和BAF60C)。进一步了解到,蛋白质后生调节剂的各别活性可能受代表性形式的影响,如由小分子因子靶向、各别外源转录物的过度表达、反义RNA靶向的各别转录物降解、RNAi和在基因位点的目标突变。
以下所公开的实施方案仅为代表。因此,在以下实施例中所公开的特定结构细节、功能细节和程序细节不会被视为具有限制性。
实施例
方法
O2KM细胞系。通过杀稻瘟菌素(含有MyoDER开放阅读框序列(ORF)的可选转基因盒)的慢病毒***从亲本O2K细胞系获得O2KM细胞系。如在本实例性部分中所提及,O2K细胞/细胞系与piPSC细胞可互换使用。如下制备图4A中所说明的慢病毒载体:从含有PCR扩增的MyoDER ORF的pENTR/D-TOPO入门载体(Life Technologies#K2435-20)克隆,克隆在attR1与attR2再组合位点之间的pLentiCMVBlast目的载体(Addgene#17451)的CMV启动子的下游的MyoDER ORF(Addgene#13494)。为了制备慢病毒上清液,使用PolyJet转染试剂(Signagen#SL100688)将293FT细胞与所制备的pLentiCMVBlast[MyoDER]质粒、pMD2.G包膜质粒(Addgene#12259)和psPAX2包装质粒(Addgene#12260)共转染。用从293FT上清液浓缩的假病毒转导O2K。在无酚红培养基中培养转导的O2K,并且用10μg/mL灭瘟素选择4天,接着用15μg/mL灭瘟素再选择2天。将所选细胞命名为O2KM。通过西方墨点法验证(图4B)验证O2KM备料中MyoDER的表达。
在DOX存在下的O2KM干细胞备料扩增。关于亲本O2K系实施O2KM干细胞更新环境,例外如下:用DMEM/F-12的无酚红制剂和神经基础培养基代替含有酚红的制剂以防止对MyoDER的多效性激动作用(即活化)。O2KM细胞备料自我更新培养基(SRM)由以下组分组成:无酚红神经基础培养基(Life Technologies#12348-017)、无酚红DMEM-F12(LifeTechnologies#11039-021)、1X非必需氨基酸(Sigma-Aldrich#M7145)、0.5XGlutamax(Life Technologies#35050061)、0.000007%β-巯基乙醇、0.5X N2补充物(Life Technologies#17502048)、0.5X B27补充物减去维生素A(SupplementMinus Vitamin A)(Life Technologies#12587010)、0.1mg/mL牛血清白蛋白、2μg/mL海克酸强力霉素(即DOX)、10ng/mL人白血病抑制因子(hLIF,Millipore#LIF1050)、3μΜCHIR99021、0.8μΜPD032591和0.1μΜPD 173074。在下文中,三种抑制剂CHIR99021、PD032591和PD173074一起被视作'3i'。或者,使用15%KnockOut Serum Replecement(KOSR;Life Technologies#A15870)用N-2和B-27血清替代物进行替代。通过菌落的酶促解离以及细胞在5%O2下每3天传代到用含聚D赖氨酸和鼠层粘连蛋白的无苯酚SRM涂布的培养皿上来维持O2KM。在这些自我更新条件下,O2KM维持如亲本O2K系那样的紧凑的干细胞样形态,如图5中所示。
细胞死亡的CHIR99021抑制。亲本O2K系在无支持自我更新的培养基组分hLIF、DOX、CHIR99021、PD032591和PD173074SRM以及KOSR存在下的分化引起大规模细胞死亡,如通过以下来确定:(1)如图9的图片i.-ii.所示的相差显微技术、(2)如图17A中所示的CPP32裂解,以及(3)图16中所示的膜联蛋白V标记。然而,包括CHIR99021的培养基制剂当保留于不存在hLIF、DOX、PD032591和PD173074的SRM基础培养基中时支持分化期间两种细胞存活,如由以下来确定:(1)如图9的图片iii.-viii.所示的相差显微技术、(2)如图10中所示的细胞粘附测定、(3)如图17B中所示的CPP32裂解抑制,以及(4)如图11中所示的膜联蛋白V标记。此外,在原始分化期间暴露于CHIR99021稳定化并调节GSK3β底物CTNNB1(如图12、13A和13B中所示,已知在胚胎谱系特化期间引导中胚层分化的经典WNT信号转导通路的磷酸调控的下游效应物)的硫酰化状态、中胚层祖细胞的肌生性增浓和骨骼肌细胞的终末分化。与这些发现相符地,补充有CHIR99021的基础培养基支持分化中O2K的前肌生性上段谱系特化,如图14中所示,并且当包括于肌生性鼠C2C12细胞系的低***素分化培养物(2%马血清/DMEM)中时,支持向骨骼肌管的终末分化增强,如表1中所列。因为CHIR99021抑制细胞死亡、分化中O2K细胞系被支持的向着上段的分化,以及C2C12细胞系增强的终末分化,所以确立了先例以将化合物保留于所有培养期。因此,除非另有说明,否则在扩增、诱导和终末分化步骤(图20A)期间将3μΜCHIR99021保留于培养基中。
O2KM肌生性诱导。将O2KM细胞以密度4.1×103个细胞/cm2接种于涂有聚D赖氨酸、鼠层粘连蛋白和MATRIGEL的培养皿上,并且在5%O2下在自我更新培养基中培养3天。为了有助于分化,将培养物转移到20%O2基础分化环境,所述环境通过退出PD032591、PD173074、DOX、hLIF和β-巯基乙醇来指定。为了条件诱导所表达的MyoDER蛋白,将10μΜE2添加到培养基中。如下证实在2天的诱导培养之后E2定向肌生性谱系特化:(1)采用经过处理的培养物中的骨骼肌细胞的纺锤体样形态特征,如图6中所示,(2)内源MYOG骨骼肌转录因子的表达,如图7中所示,以及(3)骨骼肌细胞细胞表面标记NCAM的均匀表达,如图8中所示。
5AC作用。在'MyoDER'的E2介导性诱导之前、期间和之后暴露于5AC使O2KM能够从肌细胞终末分化成屈光的丝状肌管。将鼠C2C12细胞系用作阳性对照物以筛选对于O2KM的终末分化最优的条件。这些条件包括:MATRIGEL细胞外基质和CHIR99021补充,不存在E2,如表1中所列。然而,在无E2(确定对C2C12终末分化最优的条件)的分化培养基中传代到涂有MATRIGEL的培养皿上的O2KM源性肌细胞不能建立屈光肌管,如图15和图20B中所示。因此,O2KM源性肌细胞中的基因表达程序不足以实现根据所确立的条件进行终末分化。先前测定了5AC,即小分子后生调节剂,以使不允许发生肌生成的细胞系中能够骨骼肌转录。此外,进一步确定5AC暴露会增强C2C12细胞系的终末分化。因此,在如图20A中所示的增殖性O2KM扩增和诱导方案期间包括250nM 5AC,即未分化O2KM所耐受的最高剂量。
终末分化。在E2诱导2天之后,培养物进行原位终末分化,或者在终末分化培养基(TDM)中以1.56×105个细胞/cm2传代到涂有MATRIGEL的培养皿中以进行终末分化。从与MIM相同的组分配制TDM,不同之处在于以下修改:退出E2、添加4μΜA 83-01和100nM IGF-1。N-2和B-27补充物仅用作血清替代物。在诱导方案之后,培养物在20%O2下分化最多6天(图20A)。在扩增和诱导方案期间暴露于5AC的培养物在终末分化方案期间形成各项异性的屈光肌管,如图20B中所示。图20C示出了到第6天肌球蛋白重链的均匀表达,并且图20E示出了历经8天的过程肌间线蛋白(DES)和成肌素(MYOG)递增的表达。在终末分化期间观测到肌管多倍性,如图20D中左图所示。图20D中右图示出根据倍性的第8天肌管中肌核的相对分布。如图20F中所示,更新环境(第3天菌落)、扩增环境(第0天培养物,图20A)和终末分化环境(第8天培养物,图20A)中的S期核的相对普及表示在终末分化之后退出细胞周期。通过以下验证了终末分化中的骨骼肌肌管的收缩潜能:(1)组织良好的肌节的结构发育,如图20G中所示;(2)不同步的自发性收缩,如图21A中所示;(3)通过场刺激实现的可收缩刺激和同步,如图21B中所示;(4)咖啡因刺激的收缩,如图21C中所示;以及(5)乙酰胆碱所刺激的收缩,如图21D中所示。
结果
通过CHIR99021调节调亡和分化轴。图9示出了基态piPSC菌落在5%O2下在自我更新环境(i.)中培养以及在20%O2下在不存在DOX、LIF和3i下(ii.)48小时之后的相差图像。图16示出了在培养物24小时的过渡之前和之后凋亡细胞的膜联蛋白V标记。图17A示出了在分化环境过渡(12-48小时)之前(0小时)和之后(12-48小时),基态菌落中全长CPP32(约32kD酶原3a)和裂解大片段(约17kD裂解半胱天冬酶3a)的西方墨点检测。图9示出了在如所示补充有CHIR99021的分化环境中培养48小时之后粘附培养物(iii、iv、vi和vi)和非粘附拟胚体培养物(vii和viii)相差图像。图10示出了相对于基态(即0小时)培养物(针对100%校正),如所示补充有CHIR99021的分化环境培养物持续48小时的粘附细胞百分比。*作为拟胚体存活的非粘附细胞,对于每一培养条件n=3。图11示出了在培养物持续24小时过渡到如所示补充有CHIR99021的分化环境之后凋亡细胞的膜联蛋白V标记。图17B示出了在指定CHIR99021水平存在下向分化环境过渡42小时之后,菌落中的全长CPP32和裂解片段的西方墨点检测。TUBA被检测为内部蛋白质负载对照物。
CHIR99021使CTNNB1稳定并且支持从基态分化。图12示出了如所示在CHIR99021存在下向分化环境过渡24小时之后,CTNNB1和p-CTNNB1(分别为总的和磷酸S33,37,T41β-连环蛋白)的西方墨点检测。TUBA被检测为内部蛋白质负载对照物。图13A表示p-CTNNB1/CTNNB1带的比率。图13B表示CTNNB1/TUBA带的比率。图13A和图13B呈现来自如图12中所示的西方墨点的密度计定量;n=3。图18示出了在含有6μΜCHIR99021分化环境中形成的拟胚体经2天以及在转移到以聚D赖氨酸+层粘连蛋白+MATRIGEL涂布的衬底再经1天(第3天,左图)或3天(第5天,右图)之后的过度生长形态。图14示出了根据在本文别处所述的一个方案方面在基态环境(第0天)和分化环境培养物(第1天至第5天)中的PAX3、POU5F1和KLF4表达的西方墨点分析。将TUBA检测示为内部蛋白质负载对照。
内源MRF的5-氮杂-胞嘧啶核苷和MyoDER活化。用整合的MyoDER表达盒修饰piPSC。图4A示出了灭瘟素(BLAST)可选MyoDER表达盒。箭头和盒子分别表示启动子和基因序列。图4B示出了未经过修饰的(O2K)和MyoDER表达盒-经过修饰的(O2KM)piPSC系中的MyoDER的西方墨点检测。用针对MyoD肽产生的抗体检测MyoDER。图7示出了在piPSC诱导2天之后MyoDER(约75kD)、MYF5和MYOG的西方墨点检测。没有检测到内源MYOD1(预期45kD)。将O2KM接种到以聚D赖氨酸+Martigel+鼠层粘连蛋白涂布的培养皿上,并且在5AC存在(即扩增,图20A)或不存在下与hLIF、3i和DOX一起培养3天,接着各别地过渡到补充有E2(即17β***)和3μΜCHIR99021的-/+5AC分化环境持续2天。双箭头:部分解析的MYOG同种型。图19A和19B。MYF5活化的决定子:西方墨点分析。图19A,在聚D-赖氨酸+层粘连蛋白+MATRIGEL上培养3天之后,在不存在3i、DOX、hLIF和E2下在如所示补充有5AC(如在图7中)和CHIR99021的分化环境中培养未经过修饰的piPSC 2天。在不存在CHIR99021下,如与在补充有N-2和B-27的分化环境(图17A、图19A)中的观测结果相反,未在补充有KOSR的分化环境中观测到17kD裂解半胱天冬酶同种型。内源MYF5表达的5AC增强的活化取决于同时的CHIR99021暴露,如图19A中所示。图19B示出了在DOX、LIF和3i存在下的MYF5活化。在自我更新环境或扩增环境中培养MYODER修饰的piPSC 3天。在DOX和CHIR99021存在下,在扩增培养3天之后,5AC暴露支持可检测的MYF5表达水平,如图19B中所示。相比之下,在更新培养(-5AC)3天之后没有检测到MYF5,如图19B中所示。TUBA被检测为内部蛋白质负载对照物。图6示出了在本文别处所述的谱系特化诱导培养方案(图20A)之后所选piPSC培养物的形态。图8示出了在5AC存在下暴露于10μΜE2之前(第0天,左图)和之后(第2天,右图),piPSC-MyoDER培养物中的核(DAPI)和NCAM的免疫细胞荧光检测。
谱系指定的piPSC的终末肌生成。在终末分化之前,在5AC存在下扩增培养物3天、在E2和5AC存在下诱导2天,并且在不存在5AC和E2下终末分化,如图20A中所示。图20B示出了肌管形态和构象。诱导后(第2天),piPSC发育成细长的各项异性的屈光肌管。当扩增和诱导步骤中不包括5AC时,细胞显示平坦的非屈光形态。图20C和图20E示出终末肌生成蛋白表达。用泛MyHC单克隆抗体克隆MF20,针对肌球蛋白重链(MyHC)同种型对第0天和第6天培养物染色。图20D示出了肌管多核化。左图:第4天终末分化培养物的放大图。带支架的箭头表示单一肌管内的多个核。右图:通过碘化丙啶标记和流式细胞术分析所显示的由肌管倍数表示的肌核分布,n=3,示出了标准偏差。图20E示出了西方墨点分析。第0天至第8天MyoD(MYOD1)、成肌素(MYOG)和肌间线蛋白(DES)表达。TUBA被检测为内部蛋白质负载对照物。图20F示出了伴随终末分化的从细胞周期的退出。用EdU标记第3天的经过修饰的piPSC更新、扩增培养物和第8天的终末分化培养物,并且通过流式细胞术测定S期分数。n=3,示出了标准偏差。图20G:透射电子显微镜术,第6天的肌管。使肌节结构单元在单行(左图)和交错的平行行(右图)中对齐。
piPSC源性肌管中的自发性和刺激诱导型钙瞬态活性。为了定量收缩周期,用Fluo-4AM钙染料对肌管培养物染色,洗涤并且通过共焦显微术捕捉图像序列。追踪整个视场(FOV)或单细胞的动态信号转导事件并且在成像过程对其进行绘图,[s]=秒。图21A示出了在自收缩性第6天的肌管亚群中观测到代表性非同步的单细胞瞬态周期(左图、中图和右图)。图21B示出了通过在第6天的肌管中进行1.0Hz场刺激所实现的钙瞬态周期的FOV活化和同步。图21C示出了通过10mM咖啡因使第6天的肌管发生FOV钙瞬态活化。图21D:对通过100nM乙酰胆碱在第7天的肌管中所发生的代表性钙瞬态活化的单细胞分析。
讨论
综上所述,已经发现本公开中所述的示例性实施方案的某些方面证明了对于培养肉应用的以下意外优势中的一个或多个:
(i)快速的细胞增殖速率;
(ii)快速的分化:17β-***诱导型MyoDER活化和多西环素退出用时少至48小时便使O2KM细胞系体外分化成肌生性谱系。不需要漫长的分化程序;
(iii)有效的分化:CHIR99021/5AC/MyoDER定向谱系特化确保以高保真度广泛转化成功能性骨骼肌细胞。不需要细胞分选;
(iv)无限的自我更新:在自我更新环境中严格地强制性支持未分化的O2KM进行自我更新;
(v)无血清培养基中的自我更新和终末分化:在无血清培养基中验证了O2KM系的自我更新与终末肌生分化;
(vi)与作为拟胚体的混悬液培养体系相容:在多能状态下,亲本O2K和经过修饰的O2KM对失巢凋亡具有抗性并且可以从混悬液培养物中的单细胞培育成拟胚体。O2KM源性拟胚体可以与用于培养肉生产的多细胞球体组装技术如生物印刷和微模具相容;以及
(vii)自体收缩:终末分化成骨骼肌的O2KM显示肌节成熟和自体收缩。因此,可能没有必要进行外部刺激(例如机械拉伸、乙酰胆碱受体活化、电刺激)来促进肌纤维成熟。
虽然已经与其实例性实施方案关联描述了本发明,但是应了解本发明的方法能够进一步修改。本专利申请意图涵盖本发明的任何变化、用途或改适,所述变化、用途或改适通常遵循本发明的原理并且包括如下对本公开的偏离:所述偏离处于本发明所属领域的已知或惯用实践范围内,并且可以适用于前文和在上文随附权利要求书的范围中所述的基本特征。
Claims (28)
1.一种生产培养肌肉组织的方法,所述方法包括:
用肌生转录因子修饰动物物种的自我更新细胞系以产生以肌生转录因子修饰的细胞系,以及
通过外源调控诱导所述经过修饰的细胞系以维持所述细胞系处于自我更新过程或使所述细胞系进入肌生分化过程。
2.如权利要求1所述的方法,其中所述肌肉组织为家畜或家禽物种的肌肉组织。
3.如权利要求2所述的方法,其中所述肌肉组织为家畜物种的肌肉组织。
4.如权利要求3所述的方法,其中所述家畜物种为猪或牛。
5.如权利要求1至4中任一项所述的方法,其中所述产生的肌肉组织适于人和非人膳食消费。
6.如权利要求1至5中任一项所述的方法,其中所述自我更新细胞系选自胚胎干细胞、诱导型多能干细胞、胚外细胞系和体细胞系。
7.如权利要求6所述的方法,其中所述自我更新性干细胞系来自家畜或家禽物种。
8.如权利要求7所述的方法,其中所述自我更新性干细胞系来自家畜物种。
9.如权利要求8所述的方法,其中所述家畜物种为猪或牛。
10.如权利要求6至9中任一项所述的方法,其中所述自我更新性干细胞系来自预期用于人或非人膳食消费的任何动物物种、任何伴侣动物,和/或来自用于治疗剂研发的任何动物物种。
11.如权利要求1至10中任一项所述的方法,其中所述肌生转录因子选自MYOD1、MYOG、MYF5、MYF6、PAX3、PAX7、其旁系同源物、直系同源物和遗传性变型。
12.如权利要求1至11中任一项所述的方法,其中所述肌生转录因子为MYOD1。
13.如权利要求1至12中任一项所述的方法,其进一步包括:将所述经过修饰的细胞系维持在第一培养基中以使未分化的细胞备料扩增。
14.如权利要求13所述的方法,其进一步包括:将所述经过修饰的细胞系维持在包含多西环素的第一培养基中以使细胞备料扩增。
15.如权利要求1至14中任一项所述的方法,其进一步包括将所述经过修饰的细胞系转移到第二培养基中以进行谱系特异性分化。
16.如权利要求15的方法,其进一步包括:在包含E2的第二培养基中处理所述经过修饰的细胞系以进行谱系特异性分化。
17.如权利要求15或16所述的方法,其中所述第二培养基进一步包含用于活化经典WNT信号转导通路的试剂、用于后生调节的试剂或其组合。
18.如权利要求17所述的方法,其中所述第二培养基进一步包含用于活化所述经典WNT信号转导通路的试剂。
19.如权利要求18所述的方法,其中用于活化所述经典WNT信号转导通路的所述试剂包括GSK3β抑制剂。
20.如权利要求18所述的方法,其中所述GSK3β抑制剂选自以下项中的一个或多个成员:CHIR99021、氯化锂、BIO、CHIR-99021、SB216763、CHIR-98014、TWS119、太古昔布、IM-12、1-Azakenpullone、AR-A014418和SB415286。
21.如权利要求20所述的方法,其中所述GSK3β抑制剂为CHIR99021。
22.如权利要求17所述的方法,其中所述第二培养基进一步包含用于后生调节的试剂。
23.如权利要求22所述的方法,其中所述后生调节剂选自以下项中的一个或多个成员:5AC、RG108、Scriptaid、丁酸钠、曲古抑菌素A、辛二酰苯胺异羟肟酸、MS-275、CI-994、BML-210、M344、MGCD0103、PXD101、LBH-589、Tubastatin A、NSC3825、NCH-51、NSC-3852、HNHA、BML-281、CBHA、Salermide、庚二酸二苯酰胺、ITF-2357、PCI-24781、APHA化合物8、Droxinostat和SB-939、组蛋白脱乙酰酶旁系同源物、组蛋白乙酰转移酶旁系同源物、tet-甲基胞嘧啶加双氧酶旁系同源物、组蛋白脱甲基酶旁系同源物、组蛋白甲基转移酶旁系同源物和DNA甲基转移酶旁系同源物、组蛋白以及染色质重构复合物的亚单元,包括Mi-2/NuRD和SWI/SNF。
24.如权利要求22或23所述的方法,其中所述后生调节剂为DNA甲基化的抑制剂。
25.如权利要求1至24中任一项所述的方法,其中所述方法包括形成多核肌管。
26.如权利要求1至25中任一项所述的方法,其中所述肌肉组织包括骨骼肌纤维。
27.如权利要求1至26中任一项所述的方法,其中所述经典WNT信号转导通路通过对GSK3β或其底物CTNNB1的基因编辑抑制来活化。
28.一种体外产生的肌肉组织,其通过如权利要求1至27中任一项所述的方法来制备。
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BR112016009803A2 (pt) | 2017-12-05 |
JP2020089381A (ja) | 2020-06-11 |
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CN111154726A (zh) | 2020-05-15 |
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IL296889A (en) | 2022-12-01 |
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JP2016535601A (ja) | 2016-11-17 |
US20160227830A1 (en) | 2016-08-11 |
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AU2014342180B2 (en) | 2019-01-03 |
WO2015066377A1 (en) | 2015-05-07 |
BR112016009803A8 (pt) | 2018-01-30 |
AU2019202244A1 (en) | 2019-04-18 |
EP3071040A1 (en) | 2016-09-28 |
AU2019202244B2 (en) | 2021-09-23 |
JP6728049B2 (ja) | 2020-07-22 |
BR112016009803B1 (pt) | 2022-02-08 |
CA2928726A1 (en) | 2015-05-07 |
ES2782380T3 (es) | 2020-09-14 |
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