CN105255826A - 人诱导多功能干细胞向睾丸间质细胞的诱导分化方法及其用途 - Google Patents
人诱导多功能干细胞向睾丸间质细胞的诱导分化方法及其用途 Download PDFInfo
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- CN105255826A CN105255826A CN201510845021.8A CN201510845021A CN105255826A CN 105255826 A CN105255826 A CN 105255826A CN 201510845021 A CN201510845021 A CN 201510845021A CN 105255826 A CN105255826 A CN 105255826A
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Abstract
本发明提供了一种人诱导多能干细胞(iPS)经神经嵴干细胞(NCSCs)分化为睾丸间质细胞(LCs)的体外定向诱导分化方法，并利用动物模型证实来源于iPS细胞的LCs具备再生衰老或损伤LCs的能力，为性腺功能低下的病人尤其是LOH病人提供新的补充睾酮的方案。
Description
技术领域
本发明涉及干细胞与组织工程技术领域，具体涉及人诱导多功能干细胞(iPS)细胞向睾丸间质细胞(LCs)的诱导分化方法及其用途。
背景技术
睾丸间质细胞(Leydigcells,LCs)分布于睾丸生精细胞管之间的疏松结缔组织，其分泌的睾酮是成年男性体内睾酮的主要来源。LCs移植是治疗LOH等睾酮缺乏相关疾病的最佳手段，它能够更好的模拟人体睾酮分泌的生理特点，使血清睾酮浓度和睾丸局部睾酮浓度都能得到有效的提高，可以获得更好的治疗效果的同时，尽量避免治疗过程中产生的不良后果。然而，由于LC发育起源不明，而且存在取材困难、数量稀少、扩增困难等缺陷明显制约了其临床应用前景。目前睾丸间质细胞常用的分离方法为密度梯度离心法(GeRS,DongQ,SottasCM,PapadopoulosV,ZirkinBR,HardyMP.InsearchofratstemLeydigcells:identification,isolation,andlineage-specificdevelopment.ProcNatlAcadSciUSA2006；103:2719-2724；StanleyE,LinCY,JinS,LiuJ,SottasCM,GeR,etal.Identification,proliferation,anddifferentiationofadultLeydigstemcells.Endocrinology2012；153:5002-5010.)。
诱导多能干细胞(inducedpluripotentstemcell，iPS细胞)具备了与胚胎干细胞相似的自我更新和多向分化潜能，同时因其可来源于自身成体细胞，克服了胚胎干细胞存在的伦理与免疫源性等诸多问题，成为研究人类疾病发病机制、开展组织细胞替代治疗的重要细胞来源。个体来源的成体细胞经过转录因子(KLF4,SOX2,OCT4和c-MYC)导入重编程为多能干细胞，从而使自体移植成为可能。以iPS细胞作为“种子”细胞，可以将其在体外大量扩增并诱导分化为特定的组织细胞。
LCs的可能来源包括肾上腺-性腺原基(adrenal-gonadalprimordium)、神经嵴(neuralcrest)、中肾(mesonephros)或者体腔上皮(coelomicepithelium)[Barsoum,I.B.andH.H.Yao,FetalLeydigcells:progenitorcellmaintenanceanddifferentiation.JAndrol,2010.31(1):p.11-5.]。Davidoff的研究发现，睾丸间质祖细胞表达神经干细胞标志Nestin和周细胞标志NG2，进一步证实了LC细胞有可能是神经嵴起源[Davidoff,M.S.,Middendorff,R.,Enikolopov,G.,Riethmacher,D.,Holstein,A.F.,Müller,D.,Progenitorcellsofthetestosterone-producingLeydigcellsrevealed.JCellBiol,2004.167(5):p.935-44.]。
发明内容
本发明人发现，诱导多能干细胞(inducedpluripotentstemcell，iPS)在体外经神经嵴干细胞(neuralcreststemcells，NCSCs)，可以分化为成熟睾丸间质细胞(Leydigcells,LCs)。本发明的目的是提供了一种人诱导多能干细胞(iPS)经神经嵴干细胞(NCSCs)分化为睾丸间质细胞(LCs)的体外定向诱导分化方法，并利用动物模型证实来源于iPS细胞的LCs具备再生衰老或损伤LCs的能力，为性腺功能低下的病人尤其是LOH病人提供新的补充睾酮的方案。
本发明的方案是将人iPS细胞系诱导分化为神经嵴干细胞(hNCSCs)，随后进一步诱导分化为睾丸间质细胞(LCs)。其具体步骤如下：
(1)人iPS细胞系(hiPSCs)向神经嵴干细胞(hNCSCs或称iPS-hNCSCs)的诱导分化：
①制备细胞悬液。将人iPS细胞系(hiPSCs)消化成小团块状后重悬。
其中可使用含有ROCK抑制剂的mTeSR培养液重悬细胞。
②诱导分化。将悬浮细胞接种在低粘附性的培养皿中，使用神经分化培养液进行培养形成胚体结构。
其中神经分化培养液可以，例如，含有80％KnockoutTMDMEM、18％KnockoutTMSR、1％双抗、1mML-谷氨酸、和0.1mM的β-巯基乙醇。
③贴壁培养。将形成的胚体接种于纤连蛋白包被的培养板中进行贴壁培养。
其中，接种时间可为胚体形成5天后。培养板可用多聚赖氨酸/明胶/纤连蛋白包被。培养板中可加入神经嵴干细胞培养液，所述神经嵴干细胞培养液可以是按1:1比例将DMEM/F12培养基与Neurobasal培养基混合，并添加1％(V/V)N2、2％(V/V)B27、1％(V/V)青链霉素混合液、1mML-谷氨酸和0.1mM的β-巯基乙醇，再加入10ng/mL的bFGF和10ng/mL的EGF。
④流式分选。使用流式细胞仪分选出P75+/HNK1+的细胞，即神经嵴干细胞(hNCSCs或称iPS-hNCSCs)。
所述神经嵴干细胞培养液可以是按1:1比例将DMEM/F12培养基与Neurobasal培养基混合，并添加1％(V/V)N2、2％(V/V)B27、1％(V/V)青链霉素混合液、1mML-谷氨酸和0.1mM的β-巯基乙醇，再加入10ng/mL的bFGF和10ng/mL的EGF。
(2)使步骤(1)获得的神经嵴干细胞(hNCSCs或称iPS-hNCSCs)向睾丸间质细胞(iPS-hNCSCs-LCs或称hNCSCs-LCs)诱导分化。
步骤(2)可以是扩增步骤(1)获得的神经嵴干细胞(hNCSCs)，并在睾丸间质细胞(LCs)分化培养液中诱导分化14天，获得睾丸间质细胞(hNCSCs-LCs)。
其中，可以在神经嵴干细胞培养液中扩增获得的神经嵴干细胞(hNCSCs)。进一步，可以在hNCSCs扩增达到60％的密度时换成LCs分化培养液。LCs分化培养液可以是在DMEM-F12培养基中加入2％FCS、10ngPDGF-BB、1ng/mlLH、1nM甲状腺激素(T3)和70ng/mlIGF-I。所获得的hNCSCs-LCs表达3β-HSD，P450C17，StAR，SF-1，并分泌睾酮。
将通过本发明方法获得的睾丸间质细胞(iPS-hNCSCs-LCs)移植到去除睾丸间质细胞的大鼠动物模型(应用大鼠睾丸间质细胞的特异性凋亡诱导剂二甲磺酸乙烷(EDS)建立的睾丸间质细胞的凋亡模型，即EDS模型)，评价该细胞在睾丸微环境中的作用。结果发现，该细胞移植后可以提高血清睾酮的浓度，因此本发明的方法得到的睾丸间质细胞(iPS-hNCSCs-LCs)可以用于制备治疗睾酮水平低下相关疾病的药物。
本发明将人iPS细胞经诱导分化及细胞分选获得神经嵴干细胞，并在体外诱导培养基中诱导分化神经嵴干细胞，获得睾丸间质细胞并分泌睾酮。将本发明的睾丸间质细胞(iPS-hNCSCs-LCs)移植到用EDS消除了睾丸间质细胞的大鼠模型中，可提高模型动物的血清睾酮水平。
附图说明
图1是hiPSCs向hNCSCs的诱导分化过程中形成胚体和悬浮培养后的白光照片和分化后的NCSCs标志物的免疫荧光染色图。其中A-C为白光照片，A.扩增培养的hiPSCs；B.悬浮培养的胚体；C.贴壁培养的胚体，其形成神经花环结构，并向外迁移；D是分化后细胞中NCSCs特异性标志物的表达(免疫荧光染色)。其中Scalebar＝100μm。
图2是对hNCSCs进行生物学特性分析的白光和荧光染色照片。A.用流式细胞仪检测NCSCs的标志物HNK1和P75在hiPSCs中的表达的图；B.流式细胞分选后贴壁培养的hNCSCs的白光照片；C.悬浮培养后的hNCSCs的白光照片；D.扩增培养的hNCSCs中NCSCs特异性标志物P75和Sox10的表达图。Scalebar＝100μm。
图3是hNCSCs向外周神经元和施旺细胞分化的白光和免疫荧光染色照片。A.白光光镜下观察hNCSCs向外周神经元分化前后的细胞形态。B-D为免疫荧光染色结果。B.hNCSCs分化形成peripherin+/Tuj+外周神经元；C.hNCSCs分化为TH+/Tuj+外周交感神经元；D.hNCSCs分化形成GFAP+/S100B+施旺细胞。Scalebar＝100μm。
图4是hNCSCs诱导分化为hNCSCs-MSC的白光和组织化学染色照片。A.白光光镜下观察hNCSCs诱导分化为hNCSCs-MSC前后的细胞形态；B.利用化学染色方法将hNCSCs-MSC在合适的诱导分化液中培养一段时间以后的AlizarinRedS染色图、TuluidineBlue染色图、和OilRedO染色图；C，将hNCSCs-MSC在合适的诱导分化液中培养一段时间以后的αSMA染色图。Scalebar＝100μm。
图5是hNCSCs-LCs中LC标志物表达的图。
图6是hNCSCs-LCs体外培养条件下分泌睾酮的图。
图7是说明hNCSCs-LCs移植对EDS模型大鼠的血清睾酮水平的影响的图。
具体实施方式
可以理解的是，在此描述的特定实施方式通过举例的方式来表示，其并不作为对本发明的限制。在不偏离于本发明范围的情况下，本发明的主要特征可以用于各种实施方式。本领域的技术人员将会意识到或能够确认，仅仅使用常规实验，许多等同物都能应用于本文所描述的特定步骤中。这些等同物被认为处在本发明的范围之内，并且被权利要求所覆盖。
本发明一方面提供了一种人诱导多能干细胞(iPS)向睾丸间质细胞(iPS-hNCSCs-LCs)的诱导分化方法，其特征在于：
(1)使人诱导多能干细胞(iPS)诱导分化为人神经嵴干细胞(iPS-hNCSCs)；
(2)使步骤(1)中获得的人神经嵴干细胞(iPS-hNCSCs)诱导分化为睾丸间质细胞(iPS-hNCSCs-LCs)。
在一个具体的实施方案中，本发明的人诱导多能干细胞(iPS)向睾丸间质细胞(iPS-hNCSCs-LCs)的诱导分化方法的步骤(1)包括将人诱导多能干细胞(iPS)消化后重悬，将悬浮细胞接种在低粘附性的培养皿，优选Petri培养皿中，使用神经分化培养液进行悬浮培养，形成胚体，随后将胚体接种到用纤连蛋白包被的培养板中用神经嵴干细胞培养液进行贴壁培养，将贴壁培养的细胞通过流式细胞仪分选出P75+/HNK1+双阳细胞，即为人神经嵴干细胞(iPS-hNCSCs)。
在一个具体的实施方案中，使用含有ROCK抑制剂的mTeSR培养液重悬人诱导多能干细胞(iPS)。
可以使用本领域已知的神经分化培养液。在一个优选的实施方案中，神经分化培养液含有80％KnockoutTMDMEM、18％KnockoutTMSR、1％(V/V)青链霉素混合液、1mML-谷氨酸、0.1mM的β-巯基乙醇。
可以使用本领域已知的神经嵴干细胞培养液。在一个优选的实施方案中，神经嵴干细胞培养液为按1:1比例将DMEM-F12培养基与Neurobasal培养基混合，并添加1％(V/V)N2、2％(V/V)B27、1％(V/V)青链霉素混合液、1mML-谷氨酸和0.1mM的β-巯基乙醇，再加入10ng/mL的bFGF和10ng/mL的EGF。
在一个具体的实施方案中，胚体形成5天后接种到用纤连蛋白包被的培养板中。在一个优选的实施方案中，所述用纤连蛋白包被的培养板是用多聚赖氨酸/明胶/纤连蛋白包被的。
在一个具体的实施方案中，贴壁培养的细胞在贴壁5-7天后用流式细胞仪进行分选。
在一个具体的实施方案中，本发明的人诱导多能干细胞(iPS)向睾丸间质细胞(iPS-hNCSCs-LCs)的诱导分化方法的步骤(2)包括扩增步骤(1)获得的人神经嵴干细胞(iPS-hNCSCs)，随后更换为睾丸间质细胞(LCs)分化培养液诱导分化，获得睾丸间质细胞(iPS-hNCSCs-LCs)。
在一个具体的实施方案中，在上述步骤(2)中，扩增人神经嵴干细胞(iPS-hNCSCs)使之达到60％的密度时更换为LC分化培养液。
在优选的实施方案中，LCs分化培养液为在DMEM-F12培养基中加入2％FCS、10ngPDGF-BB、1ng/mlLH、1nM甲状腺激素(T3)和70ng/mlIGF-I。
在一个具体的实施方案中，上述步骤(2)中的诱导时间为14天。
本发明的又一方面提供本发明的睾丸间质细胞(iPS-hNCSCs-LCs)在制备提高睾酮水平的药物中的应用，以及在制备治疗睾酮水平低下导致的相关疾病的药物中的应用。
在一个具体的实施方案中，本发明的睾丸间质细胞(iPS-hNCSCs-LCs)可以提高血清睾酮水平。
本发明中所使用的“hNCSCs”或“iPS-hNCSCs”是指由人诱导多能干细胞(iPS)诱导分化获得的人神经嵴干细胞(hNCSCs)。
本发明中所使用的“iPS-hNCSCs-LCs”或“hNCSCs-LCs”是指由人诱导多能干细胞(iPS)诱导分化的人神经嵴干细胞(hNCSCs)诱导分化获得的睾丸间质细胞(LCs)。
为使本发明的目的、技术方案和优点更加清楚明了，下面结合具体实施方式并参照附图，对本发明进一步详细说明。应该理解，这些描述只是示例性的，而并非要限制本发明的范围。此外，在以下说明中，省略了对公知结构和技术的描述，以避免不必要地混淆本发明的概念。
实施例1从iPS细胞系向hNCSCs分化
1)制备细胞悬液：使用已建立的人iPS细胞系(hiPSCs，HumMolGenet.2013,22(11):2221-33)，hiPSCs细胞在Matrigel上扩增培养时呈扁平的克隆样生长，细胞排列紧密，如图1A所示。将hiPSCs细胞用0.5mmol/L的EDTA消化成小块状，用含有ROCK抑制剂的mTeSR培养液重悬细胞。其中所使用的ROCK抑制剂为Y27632(Calbiochem,SanDiego,CA)。
2)诱导分化：收集悬浮细胞，接种在Petri培养皿中，使用神经分化培养液(80％KnockoutTMDMEM、18％KnockoutTMSR、1％(V/V)青链霉素混合液、1mML-谷氨酸、0.1mM的β-巯基乙醇)悬浮培养，形成透亮球状的胚体，如图1B所示。其中KnockoutTMDMEM和KnockoutTMSR均购自Invitrogen,Carlsbad，CA。
3)贴壁培养：悬浮培养形成胚体5天后，将球状胚体接种到用多聚赖氨酸/明胶/纤连蛋白包被的培养板中进行贴壁培养，使用神经嵴干细胞培养液(按1:1比例将DMEM-F12培养基与Neurobasal培养基混合，并添加1％(V/V)N2、2％(V/V)B27、1％(V/V)青链霉素混合液、1mML-谷氨酸和0.1mM的β-巯基乙醇，再加入10ng/mL的bFGF和10ng/mL的EGF)，隔天换液。贴壁2天后即可见细胞团中央部位出现明显的神经花环结构，并见细胞向外迁移，如图1C所示。
对贴壁培养的细胞进行免疫荧光染色检测发现，如图1D所示，Pax6、Sox2等标志物主要表达在神经花环中央部位的细胞，神经嵴干细胞特异性标志物AP2α、Sox10、P75、HNK1等主要表达在向外迁移的细胞中，表明经过胚体培养阶段再贴壁培养后，可以将hiPSCs诱导分化成hNCSCs。
4)流式分选：贴壁5天后，将胚体消化成单个细胞，用抗P75和HNK1的流式抗体标记细胞后，进行P75+/HNK1+双阳细胞的流式分选。分选时吸管吸去培养液，用PBS洗2遍，加入Accutase消化已分化的hiPSCs，37℃作用3-5分钟后观察细胞变圆变透亮，加入培养基终止消化并用枪头把细胞吹散后，利用尼龙筛，过筛细胞并1500rmp离心5min、弃去上清，加入1mLPBS后混匀，吸取20μL细胞悬液进行细胞计数。余下细胞分为四组进行抗体标记：IgG阴性对照组、P75抗体单标组、HNK1抗体单标组和P75+/HNK1+抗体样本组，每106细胞量加入20μL抗体标记。使用流式细胞仪(BDinfluxcellsorter)先对IgG阴性对照组细胞悬液进行流式上样，选出阴性荧光信号区域作为阴性对照，收集荧光强度高于阴性对照10倍以上的细胞。流式检测分析可见诱导分化后约80-90％的hiPSCs细胞表达NCSC特异性标志物HNK1和P75(图2A)，说明经过诱导后多数细胞已分化为hNCSCs。
通过流式分选即可获得纯化的hNCSCs，按5×104-1×105细胞/cm2进行贴壁培养，如图2B所示，贴壁培养的细胞形态较为均一。将hNCSCs消化传代后接种在低粘附性培养板中，如图2C所示，细胞形成大小较为均一的神经球体。对分选获得的hNCSCs细胞进行免疫荧光检测，如图2D所示，可见细胞维持表达NCSCs特异性的标志物P75、Sox10等。这表明在现有的培养扩增条件下，细胞能够维持NCSCs的特性。
对hNCSCs进行生物学特性鉴定。将hNCSCs接种至用多聚赖氨酸/明胶/纤连蛋白包被的多孔板中后，换成相应细胞的诱导培养基进行诱导分化。如图3A所示，外周神经元诱导分化2周后可见细胞形态发生明显改变，细胞胞体变圆并有细长的丝状突起出现。3-4周后对细胞进行染色鉴定，如图3B和3C所示，可见有peripherin+/Tuj+外周神经元、TH+/Tuj+的交感神经元出现。对hNCSCs用施旺细胞诱导液进行诱导分化，4周后进行染色，如图3D所示，可以见有GFAP+/S100b+的施旺细胞出现。
将hNCSCs在MSC细胞培养液(低糖DMEM，10％FBS)中诱导分化7天后，分化为MSC(mesenchymalstemcells,间充质干细胞)细胞，如图4A所示，可见细胞形态变为梭形，并呈漩涡状生长。对hNCSCs诱导而来的MSC(hNCSCs-MSC)进一步进行多向分化能力鉴定。在合适的诱导分化液中培养一定时间后，如图4B所示，AlizarinRedS染色钙结节形成证实hNCSCs-MSC分化为成骨细胞，TuluidineBlue染色证实hNCSCs-MSC已向软骨细胞分化，OilRedO染色脂滴形成表明hNCSCs-MSC已向脂肪细胞分化。在合适的诱导分化液中培养一定时间后，如图4C所示，αSMA染色显示hNCSCs-MSC已分化为平滑肌细胞。说明培养的hNCSCs具有多向分化能力。
实施例2从hNCSCs诱导分化为睾丸间质细胞(iPS-hNCSCs-LCs或称hNCSCs-LCs)
扩增获得的hNCSCs细胞，达到60％的密度时换成睾丸间质细胞(LCs)分化培养液(DMEM-F12培养基中加入2％FCS、10ngPDGF-BB、1ng/mlLH,1nM甲状腺激素(T3)、70ng/mlIGF-I)，诱导14天，进行细胞分化，收集细胞上清，固定细胞。通过免疫荧光检测成熟LCs相关标志物的表达(LCs标志物3β-HSD、P450c17、steroidogenicacuteregulatoryprotein(StAR)、以及steroidogenicfactor1(SF-1))，如图5所示，免疫染色分析表明hNCSCs诱导分化的细胞(hNCSCs-LCs)表达3β-HSD，P450C17，StAR，SF-1。通过睾酮ELISA检测来测定培养液中睾酮水平，如图6所示,发现在体外分化后的hNCSCs-LCs逐渐增加睾酮分泌，表明hNCSCs可以分化为成熟的睾丸间质细胞。
实施例3iPS-hNCSCs-LCs(或称hNCSCs-LCs)在体内的作用
先前的研究表明用大鼠睾丸间质细胞的特异性凋亡诱导剂二甲磺酸乙烷(EDS)处理4天能够耗尽睾丸间质细胞，因此向大鼠腹腔注射EDS建立EDS模型,。选择三组成年大鼠，分别为正常对照组、EDS-对照组、细胞移植组。正常对照组在第0天和第4天，分别腹腔注射相同体积的生理盐水。EDS-对照组大鼠在第0天向腹腔注射EDS(75mg/kg体重)，在第4天向睾丸内注射20μl生理盐水(10μl/单侧睾丸)。细胞组的大鼠在第0天向腹腔内注射EDS(75mg/kg体重)，在第4天,将LC培养液中培养了5-7天的hNCSCs-LCs(1.5x106重悬于10μlPBS/单侧睾丸)移植到大鼠睾丸内。移植后第10天时测量血清睾酮浓度。结果如图7所示，移植hNCSCs-LCs能够提高血清睾酮的水平。
应当理解的是，本发明的上述具体实施方式仅仅用于示例性说明或解释本发明的原理，而不构成对本发明的限制。因此，在不偏离本发明的精神和范围的情况下所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。此外，本发明所附权利要求旨在涵盖落入所附权利要求范围和边界、或者这种范围和边界的等同形式内的全部变化和修改例。
Claims (9)
1.一种人诱导多能干细胞(iPS)向睾丸间质细胞(iPS-hNCSCs-LCs)的诱导分化方法，其特征在于：
(1)使人诱导多能干细胞(iPS)诱导分化为人神经嵴干细胞(iPS-hNCSCs)；
(2)使步骤(1)中获得的人神经嵴干细胞(iPS-hNCSCs)诱导分化为睾丸间质细胞(iPS-hNCSCs-LCs)。
2.根据权利要求1的方法，其中步骤(1)包括将人诱导多能干细胞(iPS)消化后重悬，将悬浮细胞接种在低粘附性的培养皿，优选Petri培养皿中，使用神经分化培养液进行悬浮培养，形成胚体，随后将胚体接种到用纤连蛋白包被的培养板中，用神经嵴干细胞培养液进行贴壁培养，将贴壁培养的细胞通过流式细胞仪分选出P75+/HNK1+双阳细胞，即为人神经嵴干细胞(iPS-hNCSCs)。
3.根据权利要求2的方法，其中所述神经分化培养液含有80％KnockoutTMDMEM、18％KnockoutTMSR、1％(V/V)青链霉素混合液、1mML-谷氨酸、0.1mM的β-巯基乙醇。
4.根据权利要求2的方法，其中所述神经嵴干细胞培养液为按1:1比例将DMEM-F12培养基与Neurobasal培养基混合，并添加1％(V/V)N2、2％(V/V)B27、1％(V/V)青链霉素混合液、1mML-谷氨酸，0.1mM的β-巯基乙醇，再加入10ng/mL的bFGF和10ng/mL的EGF。
5.根据权利要求1的方法，其中步骤(2)包括扩增步骤(1)获得的iPS-hNCSCs，随后更换为睾丸间质细胞(LCs)分化培养液诱导分化，获得睾丸间质细胞(iPS-hNCSCs-LCs)。
6.根据权利要求5的方法，其中LCs分化培养液为在DMEM-F12培养基中加入2％FCS、10ngPDGF-BB、1ng/mlLH、1nM甲状腺激素(T3)和70ng/mlIGF-I。
7.根据权利要求1-6任一项的方法获得的睾丸间质细胞(iPS-hNCSCs-LCs)在制备提高睾酮水平的药物中的应用。
8.根据权利要求1-6任一项的方法获得的睾丸间质细胞(iPS-hNCSCs-LCs)在制备治疗睾酮水平低下导致的相关疾病的药物中的应用。
9.根据权利要求7或8的方法，其中睾酮水平为血清睾酮水平。
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| WO2018088240A1 (ja) * | 2016-11-10 | 2018-05-17 | 国立大学法人神戸大学 | ヒト多能性幹細胞由来ライディッヒ様細胞の作製方法及びヒト多能性幹細胞由来ライディッヒ様細胞 |
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| CN117431209A (zh) * | 2023-12-22 | 2024-01-23 | 上海元戊医学技术有限公司 | 通过神经嵴细胞系制备间充质干细胞的方法及作为骨关节炎药物的应用 |
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