CN114369577A - 牛诱导扩展多能性成体干细胞、建系方法及培养液 - Google Patents
牛诱导扩展多能性成体干细胞、建系方法及培养液 Download PDFInfo
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- CN114369577A CN114369577A CN202011105359.7A CN202011105359A CN114369577A CN 114369577 A CN114369577 A CN 114369577A CN 202011105359 A CN202011105359 A CN 202011105359A CN 114369577 A CN114369577 A CN 114369577A
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Abstract
本发明公开了一种牛诱导扩展多能性成体干细胞、建系方法及培养液，通过从牛成纤维体细胞中转基因、诱导培养得到，并能够在体外稳定传代培养。该干细胞具有全能性、稳定性和安全性，能在体内外向胚胎和胚胎外组织分化，可用于育种繁育、基因编辑、动物克隆、医学模型、药物开发载体和诱导生产牛生殖配子等多种生命科学以及医学领域的应用。
Description
技术领域
本发明属于细胞生物学和分子生物学技术领域，具体涉及一种牛诱导扩展多能性成体干细胞、建系方法及培养液。
背景技术
诱导多能干细胞(iPSCs)是将特定转录因子导入到体细胞，诱导其细胞核重编程而得到的干细胞，是通过在分化的细胞中表达特定的几个转录因子，诱导体细胞的重编程而获得的可不断自我更新且具有多向分化潜能的细胞。诱导多能干细胞(iPSCs)除了可以无限的自我更新并能分化为包括三个胚层在内的所有细胞类型外，最大的特点是不需使用胎儿或胚胎，而是将分化细胞通过基因表达的调整直接重编程到多能性阶段，在研究体细胞重编程和再生医学方面具有深远影响。
目前研究已经报道了小鼠(Establishment in culture of pluripotentialcells from mouse embryos[J].Nature，1981，292(5819):154-156.)、人(Embryonic StemCell Lines Derived from Human Blastocysts[J].Science，1998，282(5391):1145-1147.)、大鼠(Germline Competent Embryonic Stem Cells Derived from RatBlastocysts[J].Cell，2008，135(7):1299-1310.)、猪(Establishment of porcine andhuman expanded potential stem cells[J].Nature Cell Biology，2019，21(6):687-699.)、羊、马、牛的iPS细胞，但大动物iPS的多能性特别是嵌合体形成和生殖细胞传代还没有得到确认。李荣风等(Establishment of bovine trophoblast stem-Like cells fromIn vitro-produced blastocyst-stage embryos using two inhibitors[J].Stem Cellsand Development，2014，23(13):1501-1514.)研究报道从附植前的胚胎细胞中获得牛滋养层干细胞(BTSCs)，该细胞注射NOD-SCID小鼠能形成畸胎瘤，并在体外分化成胎盘样细胞。吴侠等(Establishment of bovine embryonic stem cells after knockdown of CDX2[J].Scientific Reports，2016，6(1):28343-28343.)从CDX2敲出的胚胎中获得牛扩展多能性胚胎干细胞(CDX2-KD bESCs)，具有体内外分化能力，但没有形成嵌合体。赵丽霞等(Characterization of the single-cell derived bovine induced pluripotent stemcells[J].Tissue&Cell，2017，49(5):521-527)转入牛源四因子Oct4、Sox2、Klf4、cMyc到牛体细胞中获得具有三胚层分化能力的牛诱导多功能干细胞。Yanina Soledad Bogliotti等(Efficient derivation of stable primed pluripotent embryonic stem cells frombovine blastocysts[J].Proceedings of the National Academy of Sciences of theUnited States of America，2018，115(9):2090-2095.)从牛胚胎中获得了激发态胚胎干细胞(primed bESCs)，该干细胞没有报道形成嵌合体，其全能性不如小鼠原始态胚胎干细胞(naive mESCs)。
发明内容
本研究首次从牛成纤维体细胞中转基因、诱导培养得到牛诱导扩展多能性成体干细胞(bEPSCiPS)，该干细胞能在体内外中向胚胎和胚胎外组织分化，具有全能性、稳定性和安全性。
上述技术问题，本发明通过以下技术方案实现：
一种牛诱导扩展多能性成体干细胞的建系方法，包括以下步骤:
(a)解冻牛成纤维细胞(BEF)，在牛成纤维细胞培养液上培养，待细胞汇合度达到80％以上，收集细胞计数1×106；
(b)转入外源基因Oct4、Sox2、Klf4、cMyc、LIN28、Nanog、LRH1和RARG，在提前解冻的饲养层细胞上培养，培养条件为M15+DOX培养液；
(c)待干细胞克隆长出后，挑入细胞因子培养液培养，隔天换液，1到5天构建成牛诱导扩展多能性成体干细胞系。
优选地，所述外源基因Oct4、Sox2、Klf4、cMyc、LIN28、Nanog、LRH1和RARG的核苷酸序列如SEQ.ID.NO：1-8所示。
优选地，所述饲养层细胞的制备方法为：解冻牛成纤维细胞，在牛成纤维细胞培养液上培养，待细胞汇合度达到80％以上，按照1：16进行细胞传代，待细胞的汇合度再次达到80％时，添加10μg/ml的丝裂霉素于培养箱中培养2.5-3h，胰酶消化后用BEF培养液中止消化，离心并去上清，经含10％DMSO、10％胎牛血清和80％牛成纤维细胞培养液的培养液重悬并冻存为饲养层细胞。
优选地，所述步骤(b)中的提前解冻的饲养层细胞为实验前一天，解冻冻存的饲养层细胞并将其接种于铺有0.1％明胶的培养皿中用牛成纤维细胞培养液培养。
优选地，所述BEF培养液是在DMEM培养基中，添加10％FBS，1×谷氨酰胺，1×非必需氨基酸，1×青链霉素。
优选地，所述M15+DOX培养液是在DMEM培养基中，添加15％FBS、1×谷氨酰胺、1×非必需氨基酸、1×青链霉素、强力霉素Dox。
一种牛诱导扩展多能性成体干细胞建系用细胞因子培养液，所述细胞因子培养液包括：(1)有效量的成纤维细胞生长因子或其等效物；(2)有效量的一种或多种Wnt信号通路抑制剂；(3)有效量的一种或多种GSK-3α/β信号通路抑制剂；(4)有效量的一种或多种Lck/Src信号通路抑制剂；(5)有效量的Activin A蛋白或其等效物。
优选地，所述Wnt信号通路抑制剂为IWR-1、XAV-939、ICG-001、Wnt-C59、LGK-974、LF3、CP21R7、NCB-0846、PNU-74654、SKL2001、KY02111、IWP-2、IWP-L6、FH535、WIKI4、PRI-724、IQ-l、KYA1797K、C59、ETC-159、G007-LK、G244-LM中的一种或多种。
优选地，所述GSK-3α/β信号通路抑制剂为CHIR99021、B216763、AT7519、CHIR-98014、TWS119、Tideglusib、SB415286、AZD2858、AZD1080、AR-A014418、TDZD-8、LY2090314、2-D08、6-溴靛玉红-3'-丙酮肟(BIO-acetoxime)、IM-12、1-氮杂坎帕罗酮(1-Azakenpaullone)、靛玉红(Indirubin)、Bikinin中的一种或多种。
优选地，所述Lck/Src信号通路抑制剂为WH-4-023、达沙替尼(Dasatinib)、塞卡替尼(Saracatinib)、普纳替尼(Ponatinib)、SKI-606、KX2-391(Tirbanibulin)、NVP-BHG712、PP2、PP121中的一种或多种；
优选地，所述细胞因子培养液组成为：
1×谷氨酰胺
1×非必需氨基酸
1×青链霉素
0.1mM 2-巯基乙醇
1μM CHIR99021
0.3μM WH-4-023
5μM XAV939 or IWR-1
50μg/ml vitamin C
10ng/ml LIF
20.0ng/ml Activin A
0.3％FBS
mTeSRTM1培养基
其中，IWR-1结构式为：
CHIR99021结构式为：
WH-4-023结构式为：
XAV939结构式为：
一种牛诱导扩展多能性成体干细胞的建系方法得到的牛诱导扩展多能性成体干细胞。
本发明的有益效果是：
(1)首次从牛成纤维体细胞中转基因、诱导培养得到牛诱导扩展多能性成体干细胞(bEPSCiPS)；
(2)构建的牛诱导扩展多能性成体干细胞，具有全能性、稳定性和安全性；
(3)利用该方法的牛诱导扩展多能性成体干细胞建系效率快并能够在体外稳定传代；
(4)该干细胞能在体内外中向胚胎和胚胎外组织分化；
(5)该方法制备的牛诱导扩展多能性成体干细胞能够用于育种繁育、基因编辑、动物克隆、医学模型、药物开发载体和诱导生产牛生殖配子等多种生命科学以及医学领域的应用。
附图说明
图1是本发明所述的牛诱导扩展多能性成体干细胞(克隆形态良好界限清晰，符合干细胞典型形态结构)；
图2是本发明所述的牛诱导扩展多能性成体干细胞AP染色图片(干细胞特有的特异性蛋白阳性表达)；
图3是本发明所述的牛诱导扩展多能性成体干细胞多能基因检测图片(关键多能基因阳性表达)；
图4是本发明所述的牛诱导扩展多能性成体干细胞畸胎瘤图片(具有异种三胚层分化能力)；
具体实施方式
实施例1
饲养层细胞的制备及培养
1.1饲养层细胞的制备及培养：
解冻牛成纤维细胞(BEF)，BEF培养液为含有10％FBS(BI)、1×谷氨酰胺、1×非必需氨基酸、1×青链霉素的Knockout DMEM培养基。待细胞汇合度达到80％以上，按照1：16进行细胞传代。待细胞的汇合度再次达到80％时，添加10μg/ml的丝裂霉素于培养箱中培养2.5-3h。胰酶消化后用BEF培养液中止消化，离心并去上清，经含10％DMSO、10％胎牛血清和80％BEF培养液的培养液重悬并冻存为饲养层细胞。
1.2饲养层细胞的培养：
实验前一天，解冻饲养层细胞并将其接种于铺有0.1％明胶的培养皿中用BEF培养液培养。
实施例2
牛诱导扩展多能性成体干细胞的建系、传代及冻存
2.1牛诱导扩展多能性成体干细胞的建系：
解冻牛成纤维细胞(BEF)，BEF培养液为含有10％FBS(BI)、1×谷氨酰胺、1×非必需氨基酸、1×青链霉素的Knockout DMEM培养基。待细胞汇合度达到80％以上，收集细胞计数1×106。转入外源基因Oct4、Sox2、Klf4、cMyc、LIN28、Nanog、LRH1和RARG。转入的外源基因序列见SEQUENCE LISTING。在提前解冻好的饲养层细胞上培养，培养条件为M15+DOX培养液，M15+DOX培养液15％FBS(BI)、1×谷氨酰胺、1×非必需氨基酸、1×青链霉素、强力霉素Dox的Knockout DMEM培养基。待干细胞克隆长出后挑入细胞因子培养液，培养条件为培养液含有1×谷氨酰胺、1×非必需氨基酸、1×青链霉素、0.1mM 2-巯基乙醇、1μM CHIR99021(Tocris，cat.no.4423)、0.3μM WH-4-023(Tocris，cat.no.5413)、5μM XAV939(Sigma，cat.no.X3004)or 5μM IWR-1(Tocris，cat.no.3532)、50μg ml-1vitamin C(Sigma，cat.no.49752-100G)、10ng ml-1LIF(Millipore)，20.0ng ml-1Activin A(R&D)、0.3％FBS(Gibco，cat.no.10270)和mTeSRTM1(STEMCELL)培养基。
其中，IWR-1结构式为：
CHIR99021结构式为：
WH-4-023结构式为：
XAV939结构式为：
隔天换液，第1到5天牛诱导扩展多能性成体干细胞建系成功。细胞建系形态结果见图1，本发明所述的牛诱导扩展多能性成体干细胞克隆形态良好界限清晰，符合干细胞典型形态结构。
2.2牛诱导扩展多能性成体干细胞的传代培养：
待牛诱导扩展多能性成体干细胞的汇合度达到80％时，胰酶消化后用K10培养液中止消化。K10培养液含有10％KSR(GIBCO)、1×谷氨酰胺、1×非必需氨基酸、1×青链霉素的F12 DMEM培养基。按1：2或1:4隔日传代。
2.3牛诱导扩展多能性成体干细胞的冻存：
待牛诱导扩展多能性成体干细胞的汇合度达到80％时，胰酶消化后用K10培养液中止消化。
离心并去上清，经含10％DMSO、90％胎牛血清和培养液重悬并冻存。
实施例3
牛诱导扩展多能性成体干细胞的检测
3.1牛诱导扩展多能性成体干细胞AP染色：
牛诱导扩展多能性成体干细胞培养汇合度达到80％时，将样品细胞经柠檬酸盐(citrate)-丙酮(acetone)–甲醛(formaldehyde)固定后，按照碱性磷酸酶染色试剂盒[theAlkaline Phosphatase Kit(Sigma-Aldrich)]的操作说明严格进行。室温避光反应30-40分钟，镜检。检测结果见图2，AP染色结果显示碱性磷酸酶活性很强，该干细胞处于未分化状态。
3.2牛诱导扩展多能性成体干细胞多能基因检测：
参照Qiagen公司的RNeasy Mini Kit提取方法获得牛诱导扩展多能性成体干细胞的总RNA，并用RNase-Free水溶解。取其中1.8μl用于测量RNA的浓度和纯度后，吸取1μg RNA利用Qiagen公司的QuantiTect Reverse Transcription Kit试剂盒反转录合成20μl(50ng/μl)cDNA并用于定量PCR检测牛诱导扩展多能性成体干细胞多能基因OCT4，SOX2和Nanog的表达。
实时定量PCR(Q-PCR)的反应条件如下：94℃反应30s，60℃反应30s，and 68℃反应30s，循环反应30次。在所有的Q-PCR实验中，用到的Taqman Probe(Assay ID:Mm01232884_m1)和荧光定量PCR仪(9700HT Fast Real-Time PCR System)均购于Applied Biosciences公司。基因表达量的计算我们应用的是ΔCt算法，内参为GAPDH。检测结果见图3，如图所示牛诱导扩展多能性成体干细胞多能性基因的检测表达高于牛囊胚。
3.3牛诱导扩展多能性成体干细胞畸胎瘤实验
收集牛诱导扩展多能性成体干细胞1×106个到15ml离心管，1300rpm离心3min，弃上清，加500μlPBS吹悬，将细胞悬液吸入到1ml注射器。将免疫缺陷鼠从鼠房取出，请专业人员将小鼠用手固定抓牢，露出大腿，在大腿外侧先擦碘酒，再擦酒精，然后大腿外侧皮下注射细胞悬液。放回IVC系统照看小鼠，一个月后观察小鼠大腿是否长出肿瘤，当肿瘤有豌豆大时，将小鼠解剖，肿瘤取出，用固定液固定好后做组织切片苏木精-伊红染色(Hematoxylin-eosin staining，HE)，判断细胞是否具有向三胚层组织细胞分化的潜能。如图4所示该干细胞具有异种三胚层分化能力。
本发明提供了一种牛诱导扩展多能性成体干细胞、建系方法及培养液，结合实施例加以具体说明，实施例中所述的原料均为市售原料，相关领域的人员完全可以根据本发明提供的方法进行适当改动或变更与组合，来实现该技术。需要特别说明的是，所有这些通过对本发明提供的系统进行相类似的改动或变更与重新组合，都被视为在本发明的保护范围和内容中。
Claims (10)
1.一种牛诱导扩展多能性成体干细胞的建系方法，其特征在于，包括以下步骤:
(a)解冻牛成纤维细胞，在牛成纤维细胞培养液上培养，待细胞汇合度达到80％以上，收集细胞计数1×106；
(b)转入外源基因Oct4、Sox2、Klf4、cMyc、LIN28、Nanog、LRH1和RARG，在提前解冻的饲养层细胞上培养，培养条件为M15+DOX培养液；
(c)待干细胞克隆长出后，挑入细胞因子培养液培养，隔天换液，1到5天构建成牛诱导扩展多能性成体干细胞系。
2.根据权利要求1所述的牛诱导扩展多能性成体干细胞的建系方法，其特征在于，所述外源基因Oct4、Sox2、Klf4、cMyc、LIN28、Nanog、LRH1和RARG的核苷酸序列如SEQ.ID.NO：1-8所示。
3.根据权利要求1所述的牛诱导扩展多能性成体干细胞的建系方法，其特征在于：所述牛成纤维细胞培养液是在DMEM培养基中，添加10％FBS，1×谷氨酰胺，1×非必需氨基酸，1×青链霉素。
4.根据权利要求1所述的牛诱导扩展多能性成体干细胞的建系方法，其特征在于，所述M15+DOX培养液是在DMEM培养基中，添加15％FBS、1×谷氨酰胺、1×非必需氨基酸、1×青链霉素及强力霉素Dox。
5.根据权利要求1所述的牛诱导扩展多能性成体干细胞的建系方法，其特征在于，所述细胞因子培养液包括：(1)有效量的成纤维细胞生长因子或其等效物；(2)有效量的一种或多种Wnt信号通路抑制剂；(3)有效量的一种或多种GSK-3α/β信号通路抑制剂；(4)有效量的一种或多种Lck/Src信号通路抑制剂；(5)有效量的Activin A蛋白或其等效物。
6.根据权利要求5所述的牛诱导扩展多能性成体干细胞的建系方法，其特征在于，所述Wnt信号通路抑制剂为IWR-1、XAV-939、ICG-001、Wnt-C59、LGK-974、LF3、CP21R7、NCB-0846、PNU-74654、SKL2001、KY02111、IWP-2、IWP-L6、FH535、WIKI4、PRI-724、IQ-l、KYA1797K、C59、ETC-159、G007-LK、G244-LM中的一种或多种。
7.根据权利要求5所述的牛诱导扩展多能性成体干细胞的建系方法，其特征在于，所述GSK-3α/β信号通路抑制剂为CHIR99021、B216763、AT7519、CHIR-98014、TWS119、Tideglusib、SB415286、AZD2858、AZD1080、AR-A014418、TDZD-8、LY2090314、2-D08、6-溴靛玉红-3'-丙酮肟(BIO-acetoxime)、IM-12、1-氮杂坎帕罗酮(1-Azakenpaullone)、靛玉红(Indirubin)、Bikinin中的一种或多种。
8.根据权利要求5所述的牛诱导扩展多能性成体干细胞的建系方法，其特征在于，所述Lck/Src信号通路抑制剂为WH-4-023、达沙替尼(Dasatinib)、塞卡替尼(Saracatinib)、普纳替尼(Ponatinib)、SKI-606、KX2-391(Tirbanibulin)、NVP-BHG712、PP2、PP121中的一种或多种。
9.一种权利要求1-4任一所述牛诱导扩展多能性成体干细胞建系方法用细胞因子培养液，其特征在于，所述培养液组成为：
1×谷氨酰胺
1×非必需氨基酸
1×青链霉素
0.1mM 2-巯基乙醇
1μM CHIR99021
0.3μM WH-4-023
5μM XAV939 or IWR-150μg/ml vitamin C
10ng/ml LIF
20.0ng/ml Activin A
0.3％FBS
mTeSRTM1培养基
其中，IWR-1结构式为：
CHIR99021结构式为：
WH-4-023结构式为：
XAV939结构式为：
10.权利要求1-4任一所述牛诱导扩展多能性成体干细胞的建系方法得到的牛诱导扩展多能性成体干细胞。
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