CN105683359A - 将从间充质干细胞诱导的多能干细胞分化为肝细胞的方法 - Google Patents
将从间充质干细胞诱导的多能干细胞分化为肝细胞的方法 Download PDFInfo
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Abstract
本发明涉及包含腔昆布提取物(Ecklonia？cava)的诱导性多能干细胞脱分化用培养基组合物。并且，本发明涉及将利用上述培养基组合物制备的诱导性多能干细胞分化为肝细胞的方法。若利用本发明的培养基组合物，则可有效地将间充质干细胞制备成诱导性多能干细胞，并且由于所制备的多能干细胞可转化为肝细胞，因而可有效地用作细胞治疗剂。
Description
技术领域
本发明涉及利用间充质干细胞的多能干细胞诱导培养基组合物，制备诱导性多能干细胞，并将诱导性多能干细胞分化为肝细胞的方法。
背景技术
干细胞是能够在各组织中获取的分化之前步骤的未分化细胞的统称。干细胞具有在未分化状态下，规定期间内能够持续地制造出与自身相同的细胞的性质，以及在适当的条件下，能够分化为组成生物组织的多种细胞的性质。
根据分化能力和生成时期，干细胞大致可以分为胚胎干细胞(embryonicstemcell)和成体干细胞(adultstemcell)。并且，根据干细胞的分化能力，还可以分为多能性(pluripotency)、多潜能性(multipotency)及单能性(unipotency)干细胞。
成体干细胞可以分为多潜能性或单能性干细胞。作为典型的成体干细胞有间充质干细胞(mesenchymalstemcells；MSCs)和造血干细胞(hematopoieticstemcells；HSCs)。间充质干细胞分化为软骨细胞(chondrocyte)、成骨细胞(osteoblast)、脂肪细胞(adipocyte)、肌细胞(myocyte)、神经细胞(neuron)，造血干细胞主要分化为红细胞、白细胞、血小板等血液中的血球细胞。
相反，由于多能干细胞可以分化为构成生物体的3种全部胚叶(germlayer)，因而指称具有可以分化为人体的全部细胞或脏器组织的多功能性的干细胞，通常，胚胎干细胞属于多功能性干细胞。由于人体胚胎干细胞由可以成长为人类生命体的胚胎形成，因而存在诸多伦理性问题，但相对于成体干细胞，细胞增殖及分化能力优秀。虽然可以在骨髓、血液、大脑、皮肤等中获取成体干细胞而少存在伦理性问题，但相对于胚胎干细胞，具有有限的分化能力。
作为用于克服如上所述的问题的方案，尝试了用于使来源于成体的细胞脱分化，来制备与胚胎干细胞相似的定制型多能干细胞的各种方法。作为典型的方法有细胞融合法(fusionwithEScell)、体细胞核移植法(somaticcellnucleartransfer)、特定因子注入法(reprogrammingbygenefactor)等。由于诱导的细胞还具有2对基因，因而细胞融合法存在细胞的安全性方面的问题，而体细胞核移植法存在需要大量的卵子且效率非常低的问题。并且，特定因子注入法作为为了插入特异性基因来诱导脱分化而利用包含致癌基因的病毒的方法，由于引发癌症的危险性高、效率低、方法操作方面上的难度，因而在细胞治疗剂的开发可能性方面上存在问题。
为了成功地、大量地获得多能干细胞，在培养分离的脐带来源单核细胞的步骤中的培养组合物极其重要，需要进行通过更多量、高效率的诱导方法制备多能干细胞的研究。
作为如上所述的背景技术所说明的事项仅用于促进对本发明的背景的理解，不可以被认定为属于本领域所属的普通技术人员公知的以往的技术。
发明内容
技术问题
本发明人努力寻找高效率地诱导用于使安全性和生产效率高的细胞治疗剂的开发迈向实用化的多能干细胞的方法。结果，确认了在将作为安全的天然提取物的腔昆布提取物放入细胞培养基时，可以从间充质干细胞制备出诱导性多能干细胞，并能够以既安全又高的效率，使诱导性多能干细胞分化为肝细胞，从而完成了本发明。
因此，本发明的目的在于，提供用于使包含腔昆布提取物的间充质干细胞脱分化为诱导性多能干细胞的培养基组合物。
本发明的另一目的在于，提供在包含腔昆布提取物的培养基中，使间充质干细胞脱分化为诱导性多能干细胞，再次将上述诱导性多能干细胞分化为肝细胞的方法。
本发明的还有一目的在于，提供通过上述制备方法制备而成的肝细胞。
本发明的又一目的在于，提供包含上述肝细胞的细胞治疗用组合物。
通过以下的具体实施方式、发明要求保护范围及附图更加明确地说明本发明的目的及优点。
解决问题的手段
根据本发明的第一方面，本发明提供培养基组合物，包括腔昆布(Eckloniacava)提取物，用于使间充质干细胞(mesenchymalstemcell)脱分化为诱导性多能干细胞(inducedpluripotencystemcell)，其特征在于，上述诱导性多能干细胞分化为肝细胞。
根据本发明的另一方面，本发明提供使间充质干细胞分化为肝细胞(hepatocyte)的方法，包括：步骤(a)，将腔昆布提取物放入细胞培养基；步骤(b)，在上述培养基使间充质干细胞脱分化为诱导性多能干细胞；以及步骤(c)，使上述诱导性多能干细胞分化为肝细胞。
本发明人努力寻找在不存在迫害胚胎的伦理性问题的状态下，高效率地诱导用于使安全性和生产效率高的细胞治疗剂的开发迈向实用化的多能干细胞的方法。结果，在将作为安全的天然提取物的腔昆布提取物放入细胞培养基时，惊奇地发现能够以高效率制备出诱导性多能干细胞，再次将上述诱导性多能干细胞分化为肝细胞。
在本发明中使用的术语“胚胎干细胞”作为在受精后的发生初期的胚泡(blastocyst)的内细胞团(innercellmass)中分离来进行培养的细胞，是指称具有多能性(pluripotency)的细胞。在本发明中使用的术语“多能干细胞”指称具有分化为构成生物体的3种胚叶的多能性的干细胞，上述3种胚叶即为内胚叶(endoderm)、中胚叶(mesoderm)、外胚叶(ectoderm)。
在本发明中使用的术语“分化(differentiation)”是指在细胞通过分裂增殖生长的期间，互不相同的结构或功能被特异化的现象，即，为了执行生物的细胞、组织等应做的工作，而改变形态或功能。
在本发明中使用的术语“细胞治疗剂”作为通过从人体中分离、培养及特异的操作制备的细胞及组织，以治疗、诊断及预防的目的而所使用的医药品，指称如下医药品，即，为了恢复细胞或组织的功能而在体外增殖、筛选出同种细胞或异种细胞，或者通过其他方法改变细胞的生物学特性等的一系列行为，以治疗、诊断及预防的目的使用的医药品。根据细胞的分化程度，细胞治疗剂大致分为体细胞治疗剂、干细胞治疗剂，尤其，本发明涉及干细胞治疗剂。
本发明的间充质干细胞作为从哺乳动物来源胚胎干细胞或成体干细胞中分离出的细胞，优选地，上述间充质干细胞为脐带间充质干细胞，更优选地，上述间充质干细胞为人体脐带间充质干细胞。上述干细胞可以从用于连接人体的胎盘和胎儿的脐带中采集。从脐带中采集间充质干细胞的方法可以利用多种方法来执行，例如，采集人体脐带，由杜氏磷酸缓冲液(DPBS，Dulbecco’sPhosphateBufferedSaline)清洗，直至不流出血液，并借助手术用刀片进行铰碎，在37℃的温度下进行培养(incubation)，从而可以获取含有单核细胞的溶液。
以下，根据本发明的步骤详细地进行说明。
步骤(a)，将腔昆布提取物放入细胞培养基：
作为包含于本发明的培养基组合物的有效成分的腔昆布(甘苔，Eckloniacava)是主要栖息在韩国南海岸、济州岛一带及郁陵岛海岸一带的褐藻植物海带目海带科的多年生海藻类，主要成为鲍鱼和海螺等的食物，还用作制备海藻酸或碘·钾的主原料或可以食用。
本发明中所包含的腔昆布提取物可以利用水、(a)碳原子数为1-4的无水低级醇或含水低级醇(甲醇(methanol)、乙醇(ethanol)、丙醇(propanol)、丁醇(butanol)、正丙醇(normalpropanol)、异丙醇(isopropanol)及正丁醇(isobutanol)等)、(b)上述低级醇和水的混合溶剂、(c)丙酮(acetone)、(d)乙酸乙酯(ethylacetate)、(e)三氯甲烷(chloroform)、(f)1，3-丁二醇(1,3-butyleneglycol)、(g)己烷(hexane)、(h)二乙醚(diethylether)等的有机溶剂提取，优选地，可以利用甲醇和水或乙醇和水的混合溶剂提取。在利用混合溶剂提取的情况下，甲醇或乙醇的含量优选为50～80v/v％。
目前，虽然正增加用于将上述腔昆布提取物适用于化妆品等的皮肤组合物的事例(参照韩国公开专利第2013-0017159号、第2012-0040488号、第2010-0097293号等)，但没有被开发为多能干细胞诱导用培养基的事例。
在本发明中使用的术语“培养基”是指如下混合物，即，用于在包含糖、氨基酸、各种营养物质、血清、生长因子、无机质等对细胞的生长及增殖必要的因素的生物体外进行干细胞等的细胞的培养或分化的混合物。
在本领域的市场中销售多种培养基，还可以人为制造使用。作为市场上销售的培养基有达尔伯克改良伊格尔培养基(DMEM，Dulbecco’sModifiedEagle'sMedium)、最小必需培养基(MEM，MinimalEssentialMedium)、伊格尔基本培养基(BME，BasalMediumEagle)、洛斯维帕克培养基1640(RPMI1640)、F-10、F-12、达尔伯克改良伊格尔培养基F-12、α-最小必需培养基(α-MinimalEssentialMedium)、G-最小必需培养基(Glasgow'sMinimalEssentialMedium)、伊斯科夫氏改良杜尔贝科氏培养基(IMDM，Iscove’sModifiedDulbecco'sMedium)、羊水培养基(AmnioMax)、新型二代羊水培养基(AminoMaxⅡcompleteMedium)(Gibco公司，纽约，美国(Gibco，Newyork，USA))、张氏培养基(Chang'sMedium)、间充质干细胞无血清培养基(MesemCult-XFMedium)(细胞技术有限公司，温哥华，加拿大(STEMCELLTechnologies，Vancouver，Canada))等，与可以人为制造使用的培养基一同使用为本发明的培养基组合物中所包含的基本培养基。
在上述基本培养基中可以添加通常添加的血清成分(例如，胎牛血清(FBS，FetalBovineSerum))及抗生素(例如，青霉素、链霉素)等。在能够实现本发明的效果的范围内，可改变添加于上述基本培养基的血清成分或抗生素成分的浓度，优选地，可添加10％的胎牛血清，100unit/ml的青霉素，50μg/ml的链霉素等。
并且，本发明的培养基还可包含营养混合物(NutrientMixture)。上述营养混合物作为通常使用于细胞培养的包含各种氨基酸、维生素、无机盐等的混合物，可使用混合上述氨基酸、维生素、无机盐等来制备，或者商业制备的营养混合物。作为商业制备的营养混合物，可以举出M199、MCDB110、MCDB202、MCDB302等，但不限于此。
并且，为了多能干细胞的诱导和稳定化，本发明的培养基还可包含能量水。优选地，添加0.01至10v/v％的上述能量水，更优选地，添加0.05至0.5v/v％的上述能量水。
本发明的培养基组合物作为在多能干细胞诱导方面特异的培养基，可在上述基本培养基添加腔昆布提取物，从而实现本发明的培养基组合物，优先地，以全部培养基组合物为基准，能够以1至1000μg/ml的浓度包含腔昆布提取物，更优选地，能够以100至400μg/ml的浓度包含腔昆布提取物。
步骤(b)，使间充质干细胞脱分化为诱导性多能干细胞：
接着，利用上述培养基，使间充质干细胞脱分化为诱导性多能干细胞。
根据本发明的一实施例，可以确认如下内容：在利用本发明的包含腔昆布提取物的培养基组合物的情况下，与仅利用DMEMF-12培养基的情况不同地，在第8日～第10日，形成了多能干细胞群体(图2及图3)。
在本发明制备的诱导性多能干细胞具有与胚胎干细胞相同的分化能力，在细胞的形状方面也几乎与胚胎干细胞相同。根据本发明的一实施例，调查胚胎干细胞中的特异性基因(Nanog，Oct4，Sox-2，Klf)及蛋白质(阶段特异性胚胎抗原4，stagespecificembryonicantigen4，SSEA4)是否表达的结果，确认了在通过本发明诱导的多能干细胞中，上述基因和蛋白质以与胚胎干细胞相同的方式表达(图4)。
步骤(c)，使诱导性多能干细胞分化为肝细胞：
接着，使制备的上述诱导性多能干细胞分化为肝细胞。
分化为上述肝细胞的步骤可利用本领域公知的多种分化培养基进行分化，优选地，利用包含地塞米松(dexamethason)、转铁蛋白(transferring)、亚硒酸钠(sodiumselenite)、胰岛素及生长因子(GrowthFactor)的分化培养基执行分化。上述成分的优选含量中包含1-100mM的地塞米松、1至10μg/ml的转铁蛋白、1至50ng/ml的亚硒酸钠、1至50ng/ml的胰岛素、10至500ng/ml的肝细胞生长因子(HGF，hepatocytegrowthfactor)和/或成纤维细胞生长因子(FGF，fibroblastgrowthfactor)等的生长因子。
作为上述分化培养基的基本培养基，可利用达尔伯克改良伊格尔培养基、最小必需培养基、伊格尔基本培养基、洛斯维帕克培养基1640、F-10、F-12、达尔伯克改良伊格尔培养基F-12、α-最小必需培养基、G-最小必需培养基、伊斯科夫氏改良杜尔贝科氏培养基、羊水培养基、新型二代羊水培养基(Gibco公司，纽约，美国(Gibco，Newyork，USA))、张氏培养基、间充质干细胞无血清培养基(细胞技术有限公司，温哥华，加拿大(STEMCELLTechnologies，Vancouver，Canada))等。
在本发明制备的诱导性多能干细胞具有与胚胎干细胞相同的多能分化能力(pluripotency)，根据本发明的一实施例，可以确认具有能够分化为外胚叶、中胚叶及内胚叶的多能性(图5)。
因此，本发明的诱导性多能干细胞可有效地分化为肝细胞。
根据本发明的一实施例，可以确认被预测为多能干细胞的细胞能够分化为肝细胞(图6)。
根据本发明的另一方面，本发明提供包含上述分化的肝细胞的细胞治疗用组合物。
本发明的组合物可借助任意的给药途径给药，具体地，可通过腹腔或胸腔给药、皮下给药、静脉或动脉血管内给药、肌肉内给药、基于注射的局部给药等的方法进行给药。
在本发明中，上述组合物可基于通常的方法，以注射剂、悬浮剂、乳化剂等的形态给药，根据需要，可以被悬浮于弗氏完全佐剂等的佐剂，或可以与卡介苗(BCG，BacillusCalmette-Guerin)等的具有佐剂活性的物质一同给药。上述组合物可含有进行灭菌处理的稳定剂、可湿性粉剂或乳化促进剂、用于调整渗透压的盐或缓冲剂等的佐剂及其他在治疗性方面有用的物质，可通过通常的混合、颗粒化或涂敷方法制备而成。基于本发明的细胞治疗用组合物可含有药剂学上允许的载体或添加剂，除了有效成分之外，可含有稀释剂(例如，葡萄糖、山梨醇、纤维素、甘氨酸、乳糖、蔗糖、甘露醇)、结合剂(例如，硅酸镁铝、淀粉糊、黄芪胶、羧甲基纤维素钠)、崩解剂(例如，淀粉、琼脂、海藻酸或其钠盐)或者沸腾混合物和/或吸收剂、甜味剂、香味剂及着色剂。
本发明的细胞治疗用组合物可适用于多种疾病，以后，根据对人的临床试验结果，还可适用于同种细胞治疗剂。
发明的效果
归纳本发明的特征及优点如下：
(i)本发明提供包含腔昆布提取物的诱导性多能干细胞脱分化用培养基组合物。
(ii)并且，本发明提供使利用上述培养基组合物制备的诱导性多能干细胞分化为肝细胞的方法。
(iii)若利用本发明的培养基组合物，则可有效地将间充质干细胞制备成诱导性多能干细胞，并且由于所制备的多能干细胞可转化为肝细胞，因而可有效地用作细胞治疗剂。
附图说明
图1为示出在间充质干细胞中注入腔昆布提取物培养基来进行培养时，诱导了与胚胎干细胞几乎相同的多能干细胞的图。
图2为利用特异性蛋白质的表达将通过本发明的方法诱导的多能干细胞确定为多能干细胞的图。
图3为示出通过本发明的方法，根据腔昆布提取物的浓度诱导的多能干细胞的群体的形成的图。
图4为示出通过本发明的方法诱导的多能干细胞的基因表达的图。
图5为对通过本发明的方法诱导的多能干细胞生物体内的分化能力进行试验的结果。
图6为通过本发明的方法诱导的多能干细胞，使肝细胞分化用培养基分化为肝细胞的结果。
具体实施方式
以下，通过实施例更详细地说明本发明。这些实施例仅仅是用于更具体地说明本发明的，根据本发明的主旨，本发明的范围不局限于这些实施例对于本技术领域的普通技术人员来说是显而易见的。
实施例
实施例1：腔昆布提取物的制备
在韩国济州岛购买实验中要使用的生药试样，并经过专家的准确鉴定后使用于实验中。将100g的干燥的生药试样放入的70％甲醇，并进行回流提取16小时后，使用过滤纸进行过滤。在旋转减压蒸发器中浓缩滤液，并立即进行冷冻干燥。
实施例2：从人体脐带中分离间充质干细胞及培养间充质干细胞
实施例2-1：采集人体脐带
产后直接收集脐带组织。在将试样转移到实验室之前，首先清洗干净后，立即转移到装有添加转移用培养基(50IU/ml的青霉素、50μg/ml的链霉素(在美国英杰生命技术有限公司(Invitrogen)购买))的F-12培养基的500ml的灭菌玻璃瓶。在实验室，在灭菌状态、100等级的流罩下提取干细胞。试样先转移至灭菌不锈钢容器。用磷酸缓冲盐溶液(PBS)清洗几次后，将脐带组织试样切为2cm长度，并转移至10cm直径的细胞培养皿，在这里进行额外的清洗，并用70％乙醇进行抗感染处理，并用添加有抗生素混合物(50IU/ml的青霉素、50μg/ml的链霉素(在美国英杰生命技术有限公司购买))的磷酸缓冲盐溶液进行多次清洗，直至上述溶液变干净为止。
实施例2-2：从人体脐带中分离干细胞及培养干细胞
为了从脐带的血管及其他内部因素中分离华通氏胶(脐带的基质)，先切开脐带组织。为了提取细胞，将去除血管后分离的华通氏胶切成小块(0.5cm×0.5cm)大小。在具有适合提取上皮干细胞或间充质干细胞的细胞培养条件的互不相同的组织培养皿放入脐带华通氏胶的切块，由此执行外植(explant)过程。
为了分离/培养间充质细胞，将上述外植的组织放入添加有10％的胎牛血清(FBS，海克隆公司(Hyclone))的5ml的达尔伯克改良伊格尔培养基F-12(玉博生物科技公司(Gibco))、10％的胎牛血清、100unit/ml的青霉素、50μg/ml的链霉素，并在二氧化碳细胞培养基维持了37℃。隔3天或4天更换一次培养基。使用光学显微镜对细胞的生长(outgrowth)进行了监视。为了进一步的扩张及冷冻保存(利用达尔伯克改良伊格尔培养基/10％的胎牛血清)伸长的细胞，对其进行了胰蛋白酶处理(0.125％的胰蛋白酶/0.05％的乙二胺四乙酸二钠(EDTA))。
隔3天或4天更换一次上述培养基。通过光学显微镜，对外植组织的细胞的生长进行了监视。
为了提取间充质干细胞，细胞的团块在达尔伯克改良伊格尔培养基F-12(Gibco公司)、10％的胎牛血清、100unit/ml的青霉素、50μg/ml链霉素中再悬浮并被计数，在10cm组织培养皿中以1×106细胞/皿的密度接种。隔3天或4天更换一次上述培养基。通过光学显微镜，对细胞的生长及克隆形成进行了监视。在约90％的细胞数(confluence)中，以如上所述的方式对细胞进行传代培养(sub-culture)。
实验例1：将间充质干细胞诱导为多能干细胞
实验例1-1：基于腔昆布提取物浓度的人类间充质干细胞的多能干细胞的制备
根据济州岛腔昆布提取物的浓度，将人类脐带干细胞诱导为多能干细胞的实验中，作为间充质干细胞的专用培养基，对照组使用了达尔伯克改良伊格尔培养基F-12(Gibco公司)、10％的胎牛血清、100unit/ml的青霉素、50μg/ml的链霉素作为基本培养基，实验组使用经过三次继代培养的人类脐带间充质干细胞，并在培养基添加了1μg/ml、10μg/ml、100μg/ml、200μg/ml、400μg/ml、800μg/ml、1000μg/ml浓度的济州岛腔昆布提取物和0.1v/v％的能量水(含有SiO2、Al2O3、TiO3、Fe2O3、CaO、Na2O、K2O、LiO的纯化去离子水，STCnara公司)(图1)。在6-孔板(dish)接种1×104个细胞，并维持37℃和5％的CO2来培养了对人类脐带间充质干细胞进行分离并清洗的单核细胞。
针对通过本发明的方法诱导的多能干细胞，胚胎干细胞的特异蛋白质的八聚体结合转录因子4(OCT4，octamer-bindingtranscriptionfactor-4)、性别决定区Y框蛋白基因2(SOX2，sexdeterminingregionY-box2)、阶段特异性胚胎抗原4(stage-specificembryonicantigen4)是否表达通过使用对此的抗体，并使用免疫化学染色法，从而分析了蛋白质是否表达。染色过程首先利用4％的多聚甲醛(Paraformaldehyde)固定细胞之后，用磷酸缓冲盐溶液清洗，并用1％的牛血清白蛋白(BSA，BovineSerumAlbumin)溶液阻断(blocking)。对OCT4、SOX3、SSEA4处理第一抗体，并在4℃的温度下反应18小时之后，用磷酸缓冲盐溶液清洗，并处理具有第一抗体的荧光(FITC)的第二抗体，并在常温条件下反应1小时。用磷酸缓冲盐溶液清洗之后，使用共聚焦显微镜(confocalmicroscope)分析了表达与否，并将结果表示在图2。BF是指明视野(brightfield)，第二个图表示对各蛋白质表达的染色结果，第三个图表示上述两个图片的结合(图2)。
结果，实验组中，观察到济州岛腔昆布提取物的浓度仅为100～400μg/ml时，10天之后才形成了群体(图3)，作为多能干细胞特异标记的OCT4、SOX2、SSEA4仅在群体染色，从而确认了多能干细胞。
实验例1-2：多能干细胞基因的分析比较
借助显微镜，观察在上述实施例2-1制备的多能干细胞，并使用200μl吸液管摘除群体之后，使用TRIzol试剂(美国英杰生命技术有限公司制造)分离了全部RNA。利用逆转录聚合酶链式反应(RT-PCR)合成cDNA之后，利用OCT4、性别决定区Y框蛋白基因2(Sox-2，SRY-relatedHMG-box2)、胚胎干细胞转录因子(Nanog)、原癌基因(c-Myc)及作为对照基因的3-磷酸甘油醛脱氢酶(GAPDH，glyceraldehyde3-phosphatedehydrogenase)基因特异性引物进行了聚合酶链式反应。Nanog、OCT4、Sox-2为胚胎干细胞中可视的特异性基因，c-Myc基因均可在胚胎干细胞及成体细胞中视为阳性，是非特异性基因。通过琼脂糖凝胶电泳法对聚合酶链式反应产物进行分析，并将确认上述基因的表达的结果表示在图4。根据图4，未经诱导过程的间充质干细胞中，作为多能干细胞的特异性基因的OCT4的表达率低，相反，通过本发明的方法诱导的多能干细胞(STC2013-F002)中，这些特异性基因的表达率显著。作为干细胞基因的SOX2和Nanog的表现程度相似，作为非特异性基因的c-Myc，经过诱导过程的细胞(STC2013-F002)的表达率比未经诱导过程的细胞低。
实验例2基于畸胎瘤试验的多能干细胞的确认
为了对通过本发明的方法诱导的多能干细胞的生物体内的分化能力进行分析，将在支持细胞上培养的未分化多能干细胞群体的第五天，对胰蛋白酶-乙二胺四乙酸二钠进行处理，摘除群体之后，放入胶原酶(collagenase)，并放置于培养器30分钟。回收未分化多能干细胞，并对患有严重联合免疫缺陷病(severecombinedimmunedeficiency、SCID)的小鼠皮下注射(subcutaneousinjection)了1x106细胞。四周后，获取所形成的畸胎瘤，用4％的多聚甲醛(paraformaldehyde)固定之后，实施了常规石蜡包埋(embedding)。以10μm的厚度切开组织，并对其进行了苏木精(Hematoxylin)及伊红(Eosin)染色。
根据图5，可以用肉眼看出在注入通过本发明的方法制备的诱导多能干细胞的位置形成了畸胎瘤(teratoma)，更详细地，示出了形成组织学上可分化为来源于外胚叶的神经组织(图5a)、来源于中胚叶的肌肉组织(图5b)以及来源于内胚叶的胃肠组织(柱状细胞图5c)等的畸胎瘤。通过上述实验，可确认通过本发明的方法诱导的细胞实际上具有与生物体内的胚胎干细胞相同的分化能力，即具有可分化为外胚叶、中胚叶及内胚叶的多能性。
实验例3分化为肝细胞
为了诱导分化为肝细胞，使用混合腔昆布提取物和能量水的培养基，在湿度为95％、37℃、5％，CO2条件的培养基进行培养，由此从间充质干细胞诱导多能干细胞之后，在达尔伯克改良伊格尔培养基F-12、20nM的地塞米松、5.5μg/ml的转铁蛋白、7ng/ml的亚硒酸钠、100ng/ml的肝细胞生长因子、50ng/ml的成纤维细胞生长因子、10μg/ml的胰岛素中培养了肝细胞分化溶液3周。为了验证分化为肝细胞，通过甲胎蛋白(α-fetoprotein)免疫组织化学染色进行了确认的结果，如图6所示，在对分化培养基进行处理之前为阴性反应(图6A及图6B)，处理之后被染色为绿荧光色，呈现出阳性反应(图6C及图6D)，从而确认了被预测为多能干细胞的细胞可分化为肝细胞。免疫化学组织染色方法与上述免疫组织化学法相同。
以上，详细说明了本发明的特定部分，对于本领域所属的普通技术人员来说，这些具体地说明仅仅为优选事例，本发明的范围并不局限于此是显而易见的。因此，本发明的实质范围应以所附的发明要求保护范围和其等同物定义。
Claims (9)
1.一种使间充质干细胞分化为肝细胞的方法，其特征在于，包括：
步骤(a)，将腔昆布提取物放入细胞培养基；
步骤(b)，在上述培养基使间充质干细胞脱分化为诱导性多能干细胞；以及
步骤(c)，使上述诱导性多能干细胞分化为肝细胞。
2.根据权利要求1所述的方法，其特征在于，上述腔昆布提取物包含于选自由达尔伯克改良伊格尔培养基、最小必需培养基、伊格尔基本培养基、洛斯维帕克培养基1640、F-10、F-12、达尔伯克改良伊格尔培养基-F12、α-最小必需培养基、G-最小必需培养基、伊斯科夫氏改良杜尔贝科氏培养基、麦科伊5A培养基、羊水培养基、新型二代羊水培养基及张氏培养基、间充质干细胞无血清培养基组成的组中的培养基。
3.根据权利要求1所述的方法，其特征在于，以培养基组合物为基准，包含100～400μg/ml的上述腔昆布提取物。
4.根据权利要求1所述的方法，上述培养基组合物还包含0.01至10v/v％的能量水。
5.根据权利要求1所述的方法，上述步骤(c)利用包含地塞米松、转铁蛋白、亚硒酸钠、胰岛素及生长因子的培养基来执行。
6.根据权利要求5所述的方法，其特征在于，上述培养基为在选自由达尔伯克改良伊格尔培养基、最小必需培养基、伊格尔基本培养基、洛斯维帕克培养基1640、F-10、F-12、达尔伯克改良伊格尔培养基-F12、α-最小必需培养基、G-最小必需培养基、伊斯科夫氏改良杜尔贝科氏平培养基、麦科伊5A培养基、羊水培养基、新型二代羊水培养基及张氏培养基、间充质干细胞无血清培养基组成的组中的培养基中包含3-异丁基-1-甲基黄嘌呤、氢化可的松及吲哚美辛的培养基。
7.一种肝细胞，其特征在于，通过权利要求1所述的方法制备而成。
8.一种细胞治疗用组合物，其特征在于，包含权利要求7所述的肝细胞。
9.一种培养基组合物，包含腔昆布提取物，用于使间充质干细胞脱分化为诱导性多能干细胞，其特征在于，上述诱导性多能干细胞分化为肝细胞。
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