CN108601356A - 生成功能性造血干细胞的方法 - Google Patents
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Abstract
提供使用机械拉伸或瞬时受体电位阳离子通道‑香草酸亚家族成员4(Trpv4)激动剂制备如自体和/或异基因HSC等造血干细胞(HSC)群体的方法，和HSC在移植中的使用的方法。具体而言，所述方法包括提供包含生血内皮(HE)细胞的群体细胞，使HE细胞与一定量的Trpv4的激动剂接触；和/或使细胞经历周期性2维拉伸，持续一段时间并且在足以刺激内皮‑向‑HSC的转变的条件下进行。还公开了治疗恶性或非恶性血液、代谢或免疫失调的方法，其包括向受试者给予治疗有效量的通过所述方法得到的HSC。
Description
优先权声明
本申请要求2015年12月3日提交的美国临时申请系列号62/262,464和2016年5月6日提交的美国临时申请系列号62/332,853的权益。将前述的全部内容引入于此以作参考。
联邦政府资助的研究或开发
本发明是在国立卫生研究院授予的基金号5K01DK085217、2R03DK100672和5R01HL131645下由政府支持完成的。政府拥有本发明的某些权利。
技术领域
本文记载了使用机械拉伸或Trpv4激动剂来制备如自体和/或异基因HSC等造血干细胞(HSC)群体的方法，和HSC在移植中的使用的方法。
背景技术
HSC移植(HSCT)广泛用于治疗具有血液、骨髓、代谢和免疫疾病的患者(1-5)。尽管脐带和单倍同一性干细胞移植改进，HSC移植的治疗用途经常由于难以及时发现合适的人白细胞抗原(HLA)匹配的供体而受到限制，特别是在少数民族和缺乏国家无关的供体登记的国家(6-9)。尽管混血人群占美国人口的1.6％(970万)，但混血志愿者仅占登记册中7百万人的3％(21,000)，还有6,000名患者没有骨髓匹配。即使有人发现了合适的匹配，免疫并发症如移植物抗宿主病(GVHD)、供体排斥、和高的治疗相关死亡率也可能影响患者的生存。然而，这些并发症被自体移植所消除。尽管自体HSC不会完全替代异基因HSC，特别是在恶性血液病的背景下，但是他们将克服HSCT中的主要障碍，包括对于具有广泛跨度的恶性和非恶性血液、免疫和代谢失调的患者而言缺乏供体可用性和GVHD等障碍(3-5,15)。
发明内容
本文记述了开发人生血内皮细胞源的、临床级别的HSC用于治疗恶性和非恶性血液、代谢和免疫失调的方法。
因此，本文记述了用于制备造血干细胞(HSC)群体的方法。所述方法包括提供包括生血内皮(HE)细胞的群体，和(i)使HE细胞与一定量的瞬时受体电位阳离子通道-香草酸亚家族成员4(Trpv4)的激动剂接触；和/或(ii)使所述细胞经历周期性2维拉伸，持续一段时间并在足以刺激内皮-向-HSC的转变的条件下进行。
在一些实施方案中，所述受试者不具有恶性血液病。
本文还提供治疗具有血液、骨髓、代谢和免疫疾病的受试者的方法；所述方法包括向所述受试者给予治疗有效量的造血干细胞(HSC)，所述造血干细胞通过本文记述的方法、例如包括以下的方法获得：提供包括生血内皮(HE)细胞的群体，和(i)使HE细胞与一定量的瞬时受体电位阳离子通道-香草酸亚家族成员4(transient receptor potential cationchannel-subfamily vanilloid member 4,Trpv4)的激动剂接触；和/或(ii)使所述细胞经历周期性2维拉伸，持续一段时间并在足以刺激内皮-向-HSC的转变的条件下进行。
在一些实施方案中，用于本文记述的方法的HE细胞从诱导多能干细胞(iPSC)获得，例如使用既定方法首先将皮肤或CD34+细胞转变成诱导多能干细胞(优选人iPSC)。然后使用本领域公知的和/或本文记述的方法将此类人iPSC分化成人HE细胞。例如，人iPSC向EB的分化可以在药物、生长因子、细胞因子混合剂(cocktails)的存在下发生从而诱导生血内皮命运，接着在7-9天之间分选细胞并进一步用已确定的混合剂温育来富集HE细胞。
在一些实施方案中，Trpv4的激动剂选自由以下组成的组：花生四烯酸、5,6-EET、8,9-EET，双穿心莲内酯A(BAA)，佛波酯(如4α-PDD和4α-PDH)，RN-1747，取代的1,4-二氨基丁烷或1,3-二氨基丙烷类似物；和GSK10116790A。
在一些实施方案中，HE细胞或IPSC从具有血液、骨髓、代谢或免疫疾病的受试者获得。
在一些实施方案中，Trpv4的激动剂选自由以下组成的组：花生四烯酸、5,6-EET、8,9-EET，双穿心莲内酯A(BAA)，佛波酯(如4α-PDD和4α-PDH)，RN-1747，取代的1,4-二氨基丁烷或1,3-二氨基丙烷类似物；和GSK10116790A。
在一些实施方案中，受试者为哺乳动物，如人。
在一些实施方案中，受试者具有多发性骨髓瘤；非何杰金淋巴瘤；何杰金病；急性髓性白血病；神经母细胞瘤；生殖细胞瘤；自身免疫性失调(系统性红斑狼疮(SLE)或系统性硬化病)；或淀粉样变性。
除非另外定义，否则本文使用的所有技术和科学术语具有与本发明所属领域的普通技术人员通常理解的含义相同的含义。本文描述了用于本发明的方法和材料；也可以使用本领域公知的其他合适的方法和材料。材料、方法和实施例仅是说明性的且不意欲限制。本文提及的所有出版物、专利申请、专利、序列、数据库条目和其他参考文献都以其全部内容引入以作参考。在冲突的情况下，本说明书包括定义将作出控制。
从以下详细的说明和附图、以及从权利要求中，本发明的其他特征和优点将显而易见。
附图说明
图1A-G.尽管剪切应力通路的抑制和早期循环停滞但造血干细胞出现。(A-B)30-42hpf之间对照和cdh5沉默的胚胎中从flk1:mCherry+内皮细胞出现的cd41:eGFP+HSC的延时共焦成像，表明cdh5的缺失和因而受损的循环，并没有影响cd41:eGFP+HSC的内皮出现(实施例用箭头标记)。(C)zf-runx1和zf-c-Myb表达水平的qRT-PCR分析，证实了尽管cdh5-吗啡啉突变体(morphant)胚胎中cdh5缺失和NOS通路抑制，但HSC替代标志物的表达水平正常。(D-E)野生型(对照)flk1:mCherry和malbec::flk1:mCherry的共焦成像证实动脉和静脉在缺乏cdh5下形成。(F-G)21-48hpf之间的lcr:eGFP+红细胞以及flk1:mCherry+血管结构形成的延时共焦成像。在21hpf时，来自原始造血作用的红细胞存在于AGM区。23hpf时心跳一开始，红细胞就开始在对照胚胎中循环，且在25-48hpf之间内皮标志物的表达明显，这表明血管形成。与之相反，尽管心跳开始且血管形成，但lcr:eGFP+红细胞在cdh5-吗啡啉突变体的AGM区中累积，表明早期循环停滞。
图2A-E.cdh5-吗啡啉突变体胚胎具有正常的心率、受损的心输出量和异常的心脏形态学。(A)对照和cdh5-吗啡啉突变体胚胎(0.2-0.4mM)中斑马鱼心脏的心电描记法，表明cdh5-吗啡啉突变体具有正常的心率。(B)对照和cdh5-吗啡啉突变体胚胎(0.2-0.4mM)中的心输出量，表明cdh5-吗啡啉突变体不具有心输出量。(C)cdh5-吗啡啉突变体的微血管造影，表明在心房中捕获cdh5-吗啡啉突变体心脏中注射的荧光染料，如心包水肿所见。(D)从对照和cdh5-吗啡啉突变体分离的心脏使用抗心肌细胞肌球蛋白重链(MF20)和内皮细胞(Flk1)的抗体的免疫组织化学，表明心房、心室和流出道扭曲。*P<0.05
图3A-C.血管中的脉动是由心跳造成的。(A)3D多普勒分析来测量野生型和cdh5-吗啡啉突变体胚胎中的脉动速率，表明cdh5-吗啡啉突变体在缺乏血液流动下具有正常的脉动速率。(B-C)对照胚胎中lcr:eGFP::flk1:mCherry的延时共焦成像，接着脉动血管的运动-频率相关值的基于机器学习(人工智能)的测量，表明血管中的脉动与心跳和血液流动同步。
图4.使用标准化定制生物反应器对E11.5 AGM源分选的内皮、生血内皮、血管和间充质基质细胞重现2D周向拉伸条件的示意图。我们使用安装至柔性底培养板的尼龙膜的计算机控制的真空泵系统(FlexCell拉伸系统)，接着使用CFU、基因表达、FACS、长期植入(包括连续移植)和有限稀释测定来分析出现HSC的功能性潜能。
图5A-C.2D周向拉伸刺激HSC形成。(A)E11.5 AGM细胞的2D周期性应变(6％,24h)和瞬时受体电位阳离子通道-香草酸亚家族成员4(Trpv4)激动剂(GSK10116790A，本文称作GSK101)处理后的集落形成单位测定来测量多能GEMM集落(HSPC的标志物)。(B)8周后在移植有静态E11.5 AGM、2D周期性应变处理的E11.5 AGM、和Trpv4激动剂(GSK 101)处理的AGM的受体中CD45.2+外周血嵌合体的百分比。各受体植入有2e.e.AGM。(C)8周后来自移植有静态E11.5 AGM、2D周期性应变处理的E11.5 AGM、和Trpv4激动剂(GSK 101)处理的E11.5 AGM的小鼠的外周血的定量谱系分析。在该定制的器官芯片(organ-on-a-chip)模型中GEMM、％CD45.2外周血嵌合体、和％多谱系重构的增加表明2D周期性应变、和Trpv4激活刺激了HSC形成。n＝6；*P<0.05vs静态对照。
图6解释对E11.5 AGM源的分选细胞的2D周向拉伸施加后的潜在结果的决策树。
图7.拉伸激活离子通道的抑制减少HSC形成。GdCl3-、钌红-、GSK205(Trpv4拮抗剂)-、和GSK101(Trpv4激动剂)-处理的斑马鱼胚胎中zf-runx1和zf-c-Myb表达的qRT-PCR分析。*P<0.05vs对照；各组中n＝120。
图8A-D.Trpv4的激活增强正常胚胎中HSPC形成并挽救隐性心脏病(sih)胚胎中HSC形成。HSPC标志物(c-myb)表达的整胚原位杂交(Whole-mount in situhybridization)表明，用Trpv4激动剂(GSK101)温育对照胚胎增强HSPC形成(图8A vs 8B)，而用Trpv4激动剂(GSK101)温育隐性心脏病(sih)-MO胚胎挽救HSPC形成(图8A vs 8C vs8D)。各组中n＝120；c-myb+HSPC集群的实例由箭头表示。
图9A-9B.Trpv4的激活刺激HSPC形成。(A)用拉伸敏感的离子通道调节剂的外植体培养中的小鼠E11.5 AGM源生血内皮细胞的图解，接着分析它们的造血功能。(B)我们使用外植体培养系统将整个E11.5 AGM与拉伸激活离子通道(SAC)的非特异性抑制剂(GdCl3)、Trpv的泛抑制剂(钌红；R.R.)、Trpv4激动剂(GSK101和4α-PDD)、Trpv4拮抗剂(GSK205)、以及Trpv4激动剂(GSK101)和CREB磷酸化抑制剂(KT5720)温育24小时，接着进行集落形成分析。我们发现Trpv4的激活刺激多能祖细胞(GEMM)形成，而SAI和Trpv4的抑制减弱GEMM形成。我们还确定了在HSPC形成中CREB磷酸化(KT5720)的抑制减弱Trpv4激动剂(GSK101)的刺激的影响。n＝8；*P<0.05vs静态对照；**P<0.05vs GSK101处理。
图10A-D.用拉伸激活离子通道调节剂的离体小鼠胚胎培养的示意图，接着进行它们造血功能的分析。我们已使用我们定制的小鼠胚胎温育单元优化了培养E9.5至E10.5(A)、E10.5至E11.5(B)和E10.5至E12.5(C)的小鼠胚胎的条件。我们通过计数各发育阶段的体节数来验证胚胎生长并分析造血功能(D)。
图11：所提出的Trpv4激活后HSC形成的机理。
具体实施方式
在胎儿发育期间，主动脉-性腺-中肾(AGM)中的一部分内皮细胞，称为生血内皮细胞，改变其命运而成为最终定植于胎肝和骨髓的HSC(10,11)。然而，刺激生血内皮细胞的因子的身份仍然是难以捉摸的，限制了生血内皮细胞作为功能性HSC的潜在来源的效用。血液流动介导的对内皮衬里的剪切应力是唯一已知的刺激HSC的内皮出现的生物机械力(12,13)。使用Cdh5-无效(null)的斑马鱼和小鼠模型，最近确定了尽管早期循环停滞但功能性HSC出现(14)。这些cdh5沉默模型用作研究触发功能性HSC出现的不依赖于剪切应力的生物机械力的关键，从而研究脉动压(pulse-pressure)介导的周向拉伸控制HSC出现的其他机理。
实验室中由生血内皮细胞生成HSC的尝试大多都未成功，部分是由于缺乏有关于从生血内皮细胞刺激HSC出现的因子的知识。
如本文所述，微血管造影、超声心动描记术、3D数字多普勒超声和延时共焦成像确定了由于来自跳动心脏的脉动所引起的周向血管拉伸触发功能性HSC从生血内皮细胞出现，其可最终植入并分化成明确的谱系。另外在没有心跳和血液流动的情况下，拉伸敏感的瞬时受体电位阳离子通道-香草酸亚家族成员4(Trpv4)通道的激活挽救了在隐性心脏病(tnnt2；sih)-沉默的胚胎中的HSC形成。
本发现确立了新的离体方法(采用2D周向拉伸)和新的药物靶点(Trpv4和/或立早反应基因(immediate early response genes)(IEG，一类转录因子；CREB、c-Fos、Elk1、NFAT)的调节子)，从而刺激功能性HSC形成。本方法允许确立生血内皮细胞作为在治疗血液、骨髓衰竭、代谢组学和免疫失调中的功能性HSC的骨髓非依赖性来源。
本文记述的不带偏见的斑马鱼遗传筛选研究了造血细胞的起源和发育，并且导致可以刺激斑马鱼、小鼠和人中内皮转化为HSC的新的细胞外在和细胞内在因子的鉴定。血液流动和剪切应力介导的NOS的活化已经表明作为胎儿发育期间从生血内皮的HSC出现的触发剂(12,13)。在该模型中，可预测血液流动的缺乏会损害造血细胞形成。然而，使用斑马鱼和小鼠模型确定了功能性HSC在没有血液流动的情况下出现(14)。为了分析刺激内皮-向-HSC的转变的新的生物机械力，本实验说明脉动压介导的周向拉伸经由Trpv4信号传导的活化刺激功能性HSC形成，并确立周向拉伸作为新的生物机械力以及Trpv4作为一种新的刺激HSC的内皮出现的分子机理。因此，本方法提供了一种平台，其使用生血内皮细胞作为治疗人血液、免疫、代谢和骨髓衰竭疾病时的功能性HSC的新的和安全的来源。
本公开记述了在胎儿发育期间生血内皮是如何转变为HSC的，并至少部分地提供了再现该过程的方法。如本文所述，HSC出现、迁移、自我更新、植入和分化，而不论血液循环受损和/或NOS抑制与否。因此，必然存在调节内皮出现和HSC发育的血液流动和剪切应力非依赖的、细胞外在机理。微血管造影、共焦成像、3D多普勒超声、基因表达分析和超声心动描记术的数据表明，脉动压介导的周向拉伸刺激内皮向HSC的转变。器官芯片用于对小鼠E11.5 AGM和/或AGM源的内皮细胞、生血内皮细胞、造血细胞、血管细胞和间充质基质细胞原位再现周向拉伸条件。周向拉伸活化了Trpv4离子通道，其在内皮和造血组织上表达。另外，Trpv4活化增加了HSC形成并挽救了隐性心脏病-沉默的胚胎中的造血作用。周向拉伸作为刺激HSC出现的新的生物机械、细胞外在因子的鉴定将使生血内皮作为功能性HSC的潜在来源的模型。
生血内皮细胞
本发明包括使用生血内皮细胞来生成HSC。生血内皮细胞(如，Flk1+CD45+细胞、Flk1+CD41+细胞或CD31+CD43+细胞)可以以任何方式获得，包括来自异基因供体或来自要用HSC治疗的受试者(即，自体生血内皮细胞，如从使用来自受体的细胞建立的iPSC生成)。生血内皮细胞的分离方法是本领域已知的，且包括从人多能干细胞生成。参见，例如本文的实施例3和Ditadi等人，Nature Cell Biol.17(5)580-591(2015)；Nakajima-Takagi等人，Blood.2013；121(3):447-458；Zambidis等人，Blood.2008Nov 1；112(9):3601-14和Park等人，Cytometry A.2013 Jan；83(1):114–126(基于人类胚胎体(hEB)用于有效的hiPSC分化的血-内皮分化方法)；Choi等人，Cell Rep.2012Sep 27；2(3):553–567.(与OP9共培养中的hPSC分化)；Sandler等人，2014 July 17；511(17509):312-318(内皮细胞向造血细胞)；还参见Sluvkin,Blood 2013 122:4035-4046。
从生血内皮(HE)细胞生成HSC
本方法可以用于在体外从HE细胞生成HSC，并且可以包括在Trpv4激动剂的存在下温育细胞和/或使所述细胞经历拉伸如周期性拉伸。
Trpv4激动剂
Trpv4为瞬时受体电位(TRP)离子通道的TRPV亚家族的成员。该通道可以由物理刺激(例如，细胞溶胀或拉伸和无害加温至例如约27-35℃)和包括下列物质的化学配体激活：花生四烯酸、5,6-EET和8,9-EET(Watanabe等人，Nature 424:434-438(2003),Vincent和Duncton,Current topics in medicinal chemistry 11(17):2216-26(2011))；双穿心莲内酯A(BAA,Smith等人，J Biol Chem 281:29897-29904(2006))；佛波酯如4α-PDD和4α-PDH(Watanabe等人，J Biol Chem 277:13569-13577(2002)；Vincent和Duncton,Currenttopics in medicinal chemistry 11(17):2216-26(2011))；RN-1747；取代的1,4-二氨基丁烷或1,3-二氨基丙烷类似物(Vincent和Duncton,Current topics in medicinalchemistry 11(17):2216-26(2011))；和GSK10116790A(Thorneloe等人，J Pharmacol ExpTher 326:432-442(2008))。参见，例如下列文献中公开的化合物：Vriens等人，MolPharmacol 75(6)1262-1279(2009)；Vriens等人，Curr Neuropharmacol.6(1):79–96(2008)；Vincent和Duncton,Current topics in medicinal chemistry 11(17):2216-26(2011)；WO2007098393；WO2007030761；WO2006105475；WO2007070865；WO2007082262；WO2007082262；和Jeong等人，在美国Washington,DC举行的第238届ACS国家会议中，2009年8月16-20日；Washington,DC，美国，2009；MEDI-392。
拉伸
可选地或另外，机械手段可用于将拉伸力施加至细胞。例如，安装于柔性底培养板的尼龙膜的计算机控制的真空泵系统(如，FlexCellTM拉伸系统)可用于在限定的和受控的周期应变条件、例如如本文所述的条件下将2D周向拉伸离体施加至HE源细胞。
使用方法
本文所述的方法可用于生成HSC群体供移植方案使用，从而例如治疗血液(恶性或非恶性)、骨髓、代谢和免疫疾病。在一些实施方案中，HSC源自自体细胞，例如由使用来自受体受试者的细胞建立的iPSC生成。在一些实施方案中，例如，其中使用自体源细胞，受体受试者具有选自如下的病症：多发性骨髓瘤；非何杰金淋巴瘤；何杰金病；急性髓性白血病；神经母细胞瘤；生殖细胞瘤；自身免疫性失调(系统性红斑狼疮(SLE)、系统性硬化病)；淀粉样变性；或其他可使用自体HSC移植物治疗的病症。在一些实施方案中，其中使用自体源细胞(如，其中HSC由来自受体受试者的细胞生成)，受体受试者不具有恶性血液病。在一些实施方案中，受体受试者具有急性髓性白血病；急性成淋巴细胞性白血病；慢性髓细胞样白血病；慢性淋巴细胞性白血病；骨髓增生病；骨髓异常增生综合征；多发性骨髓瘤；非何杰金淋巴瘤；何杰金病；再生障碍性贫血；单纯红细胞再生障碍；阵发性夜间血红蛋白尿；范康尼贫血；重型地中海贫血；镰状细胞贫血；重度联合免疫缺陷(SCID)；威-奥综合征；噬血细胞性淋巴组织细胞增多症(Hemophagocytic lymphohistiocytosis)；先天性代谢异常(Inbornerrors of metabolism)；大疱性表皮松解；重型先天性中性粒细胞减少症；舒-戴综合征；戴-布贫血；或白细胞粘附缺陷；在这些实施方案中，优选使用异基因源细胞(如，从来自除了受体受试者以外的受试者、例如基于血型和人白细胞抗原(HLA)分型与受体受试者配型的受试者的细胞生成的HSC)。
所述方案可包括将使用本文所述的方法生成的HSC例如通过静脉输注或骨髓内移植向受试者给予。所述方法可在清髓、非清髓或基于免疫毒素的(例如抗-c-Kit等)调理方案之后进行。
实施例
进一步在下列实施例中描述本发明，但这些实施例并不限制权利要求中所述的本发明的范围。
实施例1：周向拉伸对HSC的内皮出现的作用的细胞生理学分析。
引言
在胚胎发育期间，血液流动对主动脉内皮衬里的腹壁产生剪切应力并刺激向HSC的内皮转变(12)。剪切应力进一步激活内皮NOS通路，这触发HSC的内皮出现(13)。近来证实了在不存在主动的血液循环下功能性HSC出现、发育、植入并重构多谱系成人血液(14)，因此，本实施例研究了刺激HSC的内皮出现的新的细胞外在机理。使用3D多普勒超声、微血管造影、超声心动描记术、和共焦成像，我们阐明了脉动压对血管产生周期性拉伸(图1-3)。我们开发了AGM芯片(AGM-on-a-chip)从而通过向FlexCell孔的柔性尼龙膜上覆盖的E11.5AGM-生血内皮细胞单细胞悬浮液施加周期性应变来离体重现周向拉伸条件(图4)。我们发现，对E11.5 AGM细胞的2D周向拉伸刺激了HSC形成(图5).
研究设计
在小鼠中，HSC开始从E10.5和E12.5之间的AGM-生血内皮细胞中出现。这些HSC定殖在E12.5和E15.5之间的胎肝，且最终植入骨髓中并重构多谱系血液(10,21-26)。我们从定时交配的怀孕小鼠中收获了E10.5或11.5胚胎，解剖AGM，并制备单细胞悬浮液来分选内皮、生血内皮、造血、血管和/或间充质干细胞。
为了使用AGM源细胞重现原位周向拉伸条件，我们使用了基于FlexCell FX-5000拉伸系统的器官盘(organ-in-a-dish)(亦称器官芯片(organ-on-a-chip)或生物反应器)法。我们的计算机调控的、基于真空压力的生物反应器通过对在柔性底培养板的尼龙膜上生长的细胞施加限定的和受控的周期性应变条件而对AGM源细胞产生离体2D周向拉伸(图4)。
我们首先使用纤连蛋白/基质胶(Matrigel)涂布了6孔FlexCell板的各个孔并使其沉降在各孔的尼龙膜上。然后我们在各FlexCell孔中接种了两个AGM的胚胎相当(e.e.)的单细胞悬浮液(由内皮、生血内皮、造血、血管平滑肌(VSMC)和间充质干细胞组成)源细胞，其中纤连蛋白/基质胶的涂层使AGM源(分选或未分选的)细胞粘附至壁底。我们用含有Myelocult和生长因子的外植体培养基填充各孔(27,28)。我们将这些6孔板放入FlexCell机中并使用定制的销钩在各个孔的底部施加周期性应变(4-12％)6-48小时。我们还将定制的橡胶垫圈插入多个孔中来刺激静态控制条件。接下来，我们在经历外部周期性应变的几个孔中添加了调节拉伸激活离子通道信号传导的药物。在6-48小时的周向拉伸的外部刺激后，我们从FlexCell板收集了AGM源细胞并分析了它们制造造血干细胞的能力：(I)我们使用Methocult培养基进行了CFU分析，从而计数在接种AGM源分选细胞七天后多潜能粒细胞、红细胞、巨噬细胞、巨核细胞祖细胞(GEMM)、粒细胞-巨噬细胞祖细胞(CFU-GM,CFU-M,CFU-G)、和红细胞祖细胞(BFU-E)的集落数。(II)我们分析了Runx1、c-Myb、Lmo2、Gata2、CD144和Gata1的表达水平。(III)我们还用荧光标记的缀合抗体染色AGM源细胞，从而测量Flk1、CD144、CD41、c-Kit和CD45.2的表达水平。(IV)我们将两种e.e.AGM(供体；CD45.2)源细胞注入亚致死照射的CD45.1(SJL；受体)小鼠。我们分析了供体细胞的长期植入和多谱系重构潜能达16周，接着连续移植以区分HSC和生血内皮细胞。(V)我们还进行了有限稀释测定来分析2D周向拉伸后每AGM源细胞生成的HSC数。
结果
无偏见的斑马鱼化学诱变筛选产生malbec，一种血管内皮钙粘蛋白(ve-cdh,cdh5)的突变体。Cdh5是一种细胞粘附分子，其有助于保护内皮渗透性和内皮衬里的完整性(16-18)。cdh5的缺失导致无心输出量及主动的血液循环受损。malbec,cdh5-吗啡啉突变体胚胎，Cdh5-/-Gfp+:Cdh5+/+Gfp-嵌合体，和Cdh5fl/fl:Scl-Cre-ERT小鼠胚胎，尽管早期循环停滞，但具有正常的原始的和确定的造血功能(14)。
在斑马鱼中，在约23hpf心跳开始，在大约24-26phf血液循环开始，在26-48hpf之间确定的HSC从AGM区中的生血内皮细胞出现(11)。血液流动对主动脉内皮的腹壁产生剪切应力并通过激活NOS通路来刺激内皮-向-HSC的转变(12,13)。当我们在30-48hpf之间进行了对照和cdh5沉默的cd41:eGFP::flk1:mCherry胚胎二者的延时共焦成像时，我们发现尽管早期循环停滞，但cdh5沉默的cd41:eGFP低HSC从flk1:mCherry+内皮细胞中出现(图1A-B)。另外，我们在19-48hpf之间在L-NAME、一种NOS的药理学抑制剂的存在下温育了cdh5沉默的胚胎(12)。我们发现NOS抑制并未消除cdh5沉默的胚胎中的HSC形成(图1C)。因此，HSC将从生血内皮细胞出现，而不论循环受损和NOS抑制与否。这些引人注目的数据使我们研究了负责内皮向HSC转变的不依赖于剪切应力的生物力学和分子机理。
为了记录cdh5沉默的胚胎中循环是否受损，我们分析了chd5沉默的胚胎中血管结构和血液循环。我们首先进行了对照和cdh5沉默的flk1:mCherry胚胎中血管的共焦成像。我们发现cdh5沉默的胚胎中动脉和静脉均是完整的(图1D-E)。接下来，我们使用对照和cdh5沉默的lcr:eGFP::flk1:mCherry胚胎的延时共焦成像分析了心跳开始前后血管中红血细胞的循环。我们发现即使在心跳开始之后lcr:eGFP+红细胞也在cdh5沉默的胚胎的血管中累积(图1F-G)。这些数据表明尽管血管形成，但cdh5-吗啡啉突变体不具有主动的循环。
为了开始分析cdh5沉默的斑马鱼胚胎中双室心脏的形态和功能，我们进行了cdh5-吗啡啉突变体心脏的微血管造影、免疫组织化学、超声心动描记术和电生理学评估。我们使用了电生理学和超声心动描记术证实了cdh5-吗啡啉突变体中的心率与对照相当(图2A)，但cdh5-吗啡啉突变体中的心搏量接近无效。因此，我们还发现cdh5-吗啡啉突变体中心输出量(＝心搏量×心率)受损(图2B)。我们将荧光珠注入cdh5-吗啡啉突变体的心脏中并追踪其通路。与对照胚胎不同，我们发现荧光珠并未进入cdh5-吗啡啉突变体胚胎中的主要的主动脉循环，这是因为并未在心脏中捕获荧光珠(图2C-D)。为了检查心脏的结构完整性，我们从对照和cdh5沉默的flk1:mCherry::cmlc2:eGFP胚胎分离了心脏并进行内皮衬里(GFP)和心肌细胞(MF20)的免疫组织化学。我们发现cdh5-吗啡啉突变体中形成了心房、房室(AV)瓣、心室和流出道，正在收缩但伸长且扭曲(图2E)。另外，我们在cdh5-吗啡啉突变体心腔中观察到显著的心包水肿，这可能是由于血液从心脏的回流(图2C)。心包空间中的液体累积导致减少的心室充盈和随后的血流动力学紊乱(hemodynamic compromise)(19,20)。为了检查心包填塞是否是心包腔中液体累积的因素，我们刺穿了心腔(如心包穿刺术中的)并抽吸心包液，从而减少对心脏的液体压力累积。然而，我们无法挽救cdh5-吗啡啉突变体心脏的心输出量缺陷(视频数据未示出)。这些数据表明cdh5-吗啡啉突变体中心跳是正常的，但由于心脏的结构缺陷，它们的心输出量受损，导致血液在心包腔中的累积。
尽管心脏跳动且血管已形成，心输出量几近为零且血细胞并没有在血管中主动循环。因此我们专注于刺激血管中HSC出现的不依赖于剪切应力的生物力学线索。出人意料的是，我们发现尽管缺乏血液流动，但血管是脉动的。这些引人注目的数据使我们研究血管中的脉动是否是由于它们的固有特性，就如淋巴管所见，或者是否是由于由心跳产生的脉动压。因此，我们使用了3D数字多普勒来测量对照胚胎和cdh5-吗啡啉突变体中的脉动频率。我们发现正常的脉动频率，但与对照相比cdh5-吗啡啉突变体中的振幅较低(图3A)。由于脉动速率的振幅受控于心跳和血液流动，预期cdh5-吗啡啉突变体中较低的振幅是由于血液流动的缺乏。然而，据信对照和cdh5-吗啡啉突变体二者都具有最佳的脉动频率，从而在血管上产生周向拉伸。因此，cdh5-吗啡啉突变体胚胎成为我们研究脉动压介导的周向拉伸对HSC的内皮出现的作用的关键。
为了证明不依赖于循环和剪切应力的生物力学机理在野生型胚胎中也是活跃的，我们在flk1:mCherry::lcr:eGFP胚胎中进行了脉动血管的延时共焦成像来研究脉动的来源并检查血管中的脉动是否与心跳同步(图3B)。我们随后使用了机器学习算法(人工智能)来测量描绘脉动血管的来自flk1:mCherry通道的个体运动频率相关值、和代表血液循环中的红细胞的lcr:eGFP通道(图3C)。当我们将GFP和mCherry通道的运动频率相关值叠加时，我们发现它们的频率重叠。这些数据消除了脉动为血管固有特性的可能性。因此，心跳介导的脉动压引起血管振荡，随后产生周向拉伸。由于HSC在没有主动血液循环下出现，我们研究了HSC形成时周向拉伸对生血内皮细胞的直接作用。
总之，这些分析表明心跳和脉动介导的周向拉伸刺激功能性HSC的内皮出现。
使用基于器官芯片的定制生物反应器(图4)，我们向两种e.e.E11.5 AGM细胞的单细胞悬浮液施加2D周向拉伸(6％周期性应变，24小时)。同时，我们还用Trpv4激动剂(GSK101(29))温育了两种e.e.E11.5 AGM静态细胞。我们发现2D周向拉伸和Trpv4激动剂处理二者不仅增强了GEMM形成，而且还增加了植入和多谱系重构的百分比。这些数据表明对于E11.5 AGM源细胞的2D周向拉伸和Trpv4通道的激活可以增强功能性HSC形成。
小鼠AGM为异基因组织。为了进一步剖析细胞水平上的周向拉伸作用，我们使用荧光激活细胞分选(FACS)或磁性激活细胞分选(MACS)从E11.5 AGM中分选了内皮细胞、生血内皮细胞、血管平滑肌细胞、间充质基质细胞、和造血细胞。我们将各细胞型的单细胞悬浮液铺在FlexCell孔上，施加2D周向拉伸，并分析CFU形成能力、基因表达变化(FACS和qRT-PCR)、有限稀释、以及长期植入和多谱系重构能力，接着进行连续移植测定(图4,6)。
我们还分析了周向拉伸介导的内皮向HSC的转变是否是由于AGM的内皮或生血内皮细胞与VSMC或间充质细胞之间的信号交叉。因此，我们将2D周期性应变施加到预混有VSMC或间充质细胞的AGM源内皮细胞或生血内皮细胞上，接着进行HSC功能性测定(图4,6)。
为了排除来自生血内皮细胞植入的潜在噪声并区分生血内皮细胞和HSC，我们使用既定方法(14,30-32)进行有限稀释和自我更新(连续长期移植)测定(图4,6)。
内皮细胞的生血特化(specification)和HSC的内皮出现的过程是动态且互补的(13,14,24-26)。如本文所述，由于不依赖于剪切应力的机理，在不存在主动血液循环下HSC出现、发育、植入和分化。我们已经成功地优化了实验条件用于重现对FlexCell拉伸系统中正在生长的细胞的受控的2D周向拉伸条件(图4,5)。由于心跳引起血管中的脉动因而产生周向拉伸(图1-3)，周向拉伸的离体重构可以以刺激内皮向HSC转变的独立的、相加的或协同的细胞外在的生物机械力而出现。
由于对E11.5 AGM细胞的单细胞悬浮液的2D周向拉伸刺激了HSC形成(图5)，预期施加至分选的生血内皮细胞的2D周向拉伸还增加HSC形成。
人内皮细胞重编程为造血细胞需要血管诱导(33)。另外，血管微环境(vascularniche)促进造血多能祖细胞形成(34)。因此，预期对预混有分选的VSMC的内皮或生血细胞的2D周向拉伸会比对单独的内皮或生血内皮细胞的2D周向拉伸具有实质上高的生血影响。
我们首先将人诱导多能干细胞(hiPSC)分化成类胚胎体(EB)然后用药物和生长因子处理它们来诱导中胚层以及随后的造血和内皮特化(参见实施例3)。在EB分化的7-8天之间，我们分选了CD34+CD43-细胞并用药物和生长因子处理它们七天来富集生血内皮细胞。然后我们将人生血内皮细胞(35)接种在柔性尼龙膜上并使用CFU测定、基因表达分析和移植到NOD-SCID免疫紊乱(immunocompromised)小鼠中来试验2D离体周向拉伸是否刺激它们的造血细胞形成潜能。
实施例2：检查HSC形成期间Trpv4信号传导的分子机理。
引言
周向拉伸的影响通过K1选择性家族、Trp家族、ENaC/DEG家族和/或Piezo家族的一个或多个成员来传达(36-39)。我们的基因表达分析表明Trpv4在内皮和造血细胞中都有表达。因此，我们研究了Trpv4在内皮向HSC转变中的作用。我们证实了拉伸激活离子通道(40,41)和Trpv家族通道(41,42)的非特异性抑制，以及Trpv4的靶向抑制(42)降低斑马鱼和小鼠中HSPC标志物的表达及HSPC的数量(图7,9)。我们还通过证实trpv4通道的激活(29)增加了斑马鱼和小鼠中的HSC形成来建立trpv4信号传导和HSC生成之间的功能连接(图7,8A-B,9)，同时在没有心跳和血液流动的情况下挽救sih(tnnt2)沉默的胚胎中的造血缺陷(图8A,C-D)。
我们通过进行斑马鱼胚胎-胚胎移植以及E10.5小鼠胚胎的离体(ex vivo)培养、接着长期植入和多谱系重构测定来分析了Trpv4激活后产生的额外的HSC的功能潜力。
我们的预期是拉伸激活的Trpv4离子通道的刺激将是朝向在血液和骨髓的恶性和非恶性失调的治疗中开发患者源内皮细胞作为临床级HSC的来源的巨大飞跃。
结果
为了证实拉伸激活离子通道的激活是否影响HSC出现，我们在19-42hpf之间用拉伸激活离子通道(SAC)的非特异性抑制剂(氯化钆,GdCl3；(40))温育了斑马鱼胚胎。使用斑马鱼WISH和定量RT-PCR，我们发现在GdCl3处理的斑马鱼胚胎中HSPC替代标志物runx1和c-myb的表达减少(图7)。这些数据表明由拉伸激活离子通道触发的细胞信号传导调节HSC的内皮出现。
周向拉伸的影响由K1选择性超家族、Trp超家族、ENaC/DEG超家族和/或Piezo家族的一个或多个成员来传达(36-39)。生物信息学(in silico)基因表达分析证实Trpv4在内皮和造血细胞中都有表达。然而，Trpv4在HSC出现中的直接作用尚未确定。因此，我们在19-42hpf之间使用Trpv通道的泛抑制剂(钌红；(41))、Trpv4拮抗剂(GSK 205；(42))和Trpv4激动剂(GSK101；(29))处理斑马鱼胚胎，接着进行HSPC替代标志物(c-myb和runx1)的qRT-PCR和WISH。在Trpv家族通道的泛抑制(使用钌红(41))或Trpv4的特异性抑制(使用Trpv4拮抗剂；GSK205；(42))下，我们发现runx1和c-myb表达的减少(图7)。我们进一步证实了对照胚胎用trpv4激动剂(GSK101；(29))温育增加HSPC标志物c-myb(图7,8A-B)和runx1的表达。这些数据暗示了Trpv4离子通道的激活是HSC出现中的关键信号传导因素。总之，这些发现表明周向拉伸刺激Trpv4信号传导并因此触发HSC的内皮出现。
隐性心脏病心脏肌钙蛋白(sih,tnnt2)突变体胚胎缺乏心跳和血液循环(43)。我们和其他人(12)已证实了sih胚胎缺乏造血干/祖细胞(HSPC)标志物如c-myb(图8A vs.8C)和runx1(数据未示出)的表达。由于血管上心跳介导的脉动压刺激HSC出现，我们研究了在没有心跳和血液循环的情况下Trpv4离子通道的激活是否触发HSC形成。因此，我们在19-42hpf之间用trpv4激动剂(GSK101)温育了sih沉默的胚胎(casper或cd41:eGFP::flk1:mCherry)。我们使用WISH(图8A,8C,vs 8D)和qRT-PCR技术基于runx1和c-myb的mRNA表达水平证实了trpv4激动剂(GSK101)挽救了sih胚胎中的造血干/祖细胞(HSPC)形成。我们还确定了trpv4激活挽救了trpv4激动剂处理的sih沉默的cd41-eGFP:flk1-mCherry胚胎中cd41:eGFP+HSC的形成。
小鼠中，第一次心跳发生在E8.25，在E10.5-E12.5之间HSC开始从AGM源造血内皮出现(10,11)。为了研究周向拉伸激活的离子通道是否也参与哺乳动物HSC出现，我们将E11.5 AGM与非特异性SAC通道抑制剂(GdCl3)和泛-Trpv通道抑制剂(钌红)在外植体培养基中一起温育并分析它们的造血CFU能力、内皮和造血基因(CD41、CD45.2、c-Kit、Flk1、CD144)的表达。我们发现GdCl3和钌红减少造血基因的表达(数据未示出)和GEMM集落形成能力(图9B)。这些数据表明HSC出现和发育中的周向拉伸信号传导通路在哺乳动物系统中是保守的。
为了进一步研究Trpv4对HSC从生血内皮细胞形成的影响，我们将E11.5AGM(28)与Trpv4激动剂(GSK101、4α-PDD(44))和Trpv4拮抗剂(GSK 205)一起温育24-48小时(图9)。我们发现Trpv4的药理学激活刺激了多能GEMM形成，而Trpv4的抑制减弱了多能GEMM形成(图9B)。这些数据表明Trpv4通道的调节调控内皮细胞向HSC的转变。
总之，数据突出指示了Trpvr4、即周向拉伸信号传导分子的激活，刺激了内皮向HSC的转变。因此，这些发现确立了Trpv4的药理学激活作为刺激HSC形成的新途径。
实施例2A：研究Trpv4激活后生成的HSC的功能性效用。
基本原理、策略和分析计划：周向拉伸激活内皮细胞上的trpv4(45)。这里我们证实了周向拉伸介导的trpv4激活刺激内皮-向-HSC的转变。为了示出Trpv4激活后产生的HSC是功能性的，我们将斑马鱼胚胎与Trpv4激动剂一起温育，接着进行胚胎-胚胎HSC移植以及E10.5小鼠胚胎与Trpv4激动剂的离体温育，随后进行AGM移植。
斑马鱼功能分析和胚胎-胚胎HSC移植：
我们利用斑马鱼转基因系cd41:eGFP用于HSC，flk1:mCherry用于内皮细胞表达分析(14)。我们在发育的19和42hpf之间在斑马鱼胚胎中注射了trpv4吗啉环(morpholino)和/或温育cd41:eGFP::flk1:mCherry转基因胚胎与trpv4激动剂(GSK101、4αPDD)。在42hpf时，我们将进行FACS分析来测量cd41:eGFP+HSC、cd41:eGFP+flk1:mCherry+生血内皮细胞、和flk1:mCherry+内皮细胞的相对水平。另外，我们将药物处理的或吗啉环注射的cd41:eGFP::flk1:mCherry胚胎置于低熔点蔗糖中，并进行延时共焦成像来定量从flk1:mCherry+内皮细胞中出现的cd41:eGFP+HSC。这些分析检查了trpv4离子通道通路的激活或沉默可以如何影响生血内皮细胞的HSC生成能力。这些为既定技术(图1A-B)。
斑马鱼HSC移植：cd41:eGFP低HSC植入受辐射的斑马鱼胚胎中，之前已成功地促进受体肾髓中的血液祖细胞，表明cd41:eGFP低HSC的功能性潜能(46,47)。为了检查trpv4激活后生成的HSC的功能性潜能，我们从trpv4激动剂处理的cd41:eGFP+胚胎中分选了cd41:eGFP+HSC并制备400个细胞/微升的悬浮液，其中以0.5％若丹明-葡聚糖作为注射用标记物。我们用细胞悬浮液回填显微注射针并校准来确保各胚胎可以注射有1、2或4滴。这提供0.4、0.8或1.6细胞/胚胎的估计细胞剂量。我们将液滴注入48hpf野生型胚胎受体的静脉窦(即，居维叶管)，置于蔗糖注射斜坡(ramps)。基于我们之前的经验，我们每剂量注射大约30个胚胎，并期望每组12-26个胚胎存活至成年期(3-5个月)。我们针对一定百分比的植入的cd41+细胞使用FACS机分析受体胚胎和/或全肾髓中的cd41:eGFP+血小板。我们以高于背景的GFP+阳性细胞(>0.001％的WKM)作为植入，对任何受体进行评分。该方法已在之前记述过(14,48)。
小鼠离体胚胎温育系统：
为了研究刺激HSC的内皮出现的因素，我们已经开发了离体小鼠胚胎培养系统，从而通过优化我们最先进的离体温育室的培养基和温度条件、湿气混合物和湿度水平从E9.5至E12.5生长小鼠胚胎(图10A-C)。因此，我们在E9.5和E12.5之间用靶向Trpv4的药理学试剂处理小鼠胚胎，从而试验它们对HSC的内皮出现的影响。这些分析验证了Trpv4离子通道调节剂的功能性HSC产生效率(图10D)。
为了研究离体条件下Trpv4是如何影响哺乳动物HSC形成的，我们用Trpv4调节剂温育了E10.5或E11.5小鼠胚胎24-48小时(图10A-C)。随后，我们解剖了来自E11.5或E12.5小鼠胚胎的AGM组织来进行CFU分析，并使用FACS和qRT-PCR分析测量CD41、c-Kit、CD144和CD45.2表达(图10D)。我们还将AGM源的确定性HSC植入亚致死辐射的和/或免疫缺陷的小鼠，从而通过评价受体中血液-谱系分布长达16周来验证它们的长期植入以及多谱系重构潜能。为了剖析Trpv4在细胞水平下的作用，我们使用已知的CFC测定、FACS和qRT-PCR分析以及长期植入分析在移植前从E11.5 AGM分选了内皮、生血内皮和造血细胞(14,30-32,49,50)。
实施例2B：HSC发育期间Trpv4激活的分子机理。
基本原理和策略：Trpv4的激活刺激细胞内钙水平(51-56)。细胞内钙水平([Ca+2]i)的瞬时增加进一步刺激立早基因类转录因子(57,58)，例如CREB(38,59-61)、Fos/Jun(62-64)、SRF/Elk1(57,65)、NFAT(66,67)、TCF(68,69)、TGF-β1/MRTF-A(63)。由于Trpv4激活刺激内皮向HSC形成(图5,7-9)，我们研究了Trpv4信号传导是如何调节向HSC的内皮细胞命运特化中细胞离子水平和转录变化的。
我们用Trpv4激动剂(GSK101)温育人脐静脉内皮细胞(HUVEC)，导致[Ca+2]i水平增加。为了分析内皮向HSC转变期间Trpv4和CREB是否信号交叉，我们使用E11.5 AGM外植体培养(图9A)并发现CREB磷酸化的药理学抑制(使用KT5720；(70))减弱多能GEMM形成中Trpv4激动剂介导的增加(图9B)。
为了建立HSC形成过程中Trpv4激活、[Ca+2]I、CREB磷酸化和/或其它转录因子之间的功能相关性，我们用Trpv4激动剂和特异性转录因子抑制剂温育斑马鱼和/或小鼠胚胎(图9B,10)。我们在斑马鱼胚胎中进行qRT-PCR、整胚原位杂交、和延时共焦成像，以及针对源自药物处理的E11.5小鼠胚胎的AGM细胞进行CFU、FACS和长期植入分析。
这些结果为功能性HSC的内皮出现中Trpv4的直接作用提供了第一证据。由于我们证实了用Trpv4激动剂的E11.5 AGM处理刺激功能性HSC形成，有理由预期在Trpv4激活后生成的cd41:eGFP+HSC将植入并因此将起作用。类似地，我们的用Trpv4激动剂离体温育E10.5小鼠胚胎接着进行移植，还可以证实较高水平的功能性HSC形成。由于斑马鱼胚胎用trp4激动剂温育增加了HSPC基因表达，小鼠胚胎和/或整个AGM的温育将产生增加的功能性HSC形成，导致增加的GEMM形成、造血基因表达、以及较高的长期植入和多谱系重构。由于Trpv4激活增强HUVEC中的[Ca+2]i水平，我们将看到Trpv4激动剂处理的生血内皮细胞中[Ca+2]i水平的增加。IEG由细胞内钙水平的瞬时增加而激活(57,58)。因此，我们预期看到Trpv4激活的生血内皮细胞中增加的CREB的磷酸化(图9B)和/或其它IEG的较高的表达/活性。因此，基于斑马鱼和小鼠的互补方法可以建立Trpv4在功能性HSC出现和发育中的保守作用。我们将遗传学和药理学工具与外植体和离体小鼠胚胎培养方法相结合，从而证实了Trpv4和/或IEG调节剂在人类血液疾病的治疗中建立内皮细胞作为功能性HSC的来源中的有希望的效用。
实施例3：从人iPSC生成HE细胞的示例性方案。
以下方案基于公开内容Ditadi等人，Nat Cell Biol.2015；17(5):580-91。
聚集培养基D0-D2
375ml IMDM(Invitrogen 10639-011)
125ml Ham's F-12
5ml P/S(10ng/ml)
5ml N2(LifeTech 17502-048)
10ml B27(LifeTech 17504-044)
3.3ml BSA(0.05％；原液7.5％，储存在+4℃下)
(5ml L-谷氨酰胺(2mM)；通常在IMDM中增补)
880uL抗坏血酸(1mM；H2O中的100mg/mL原液(0.57M)，储存在-20℃下)
750uL全铁转铁蛋白(Holo-Transferrin)(150ug/ml；Sigma T0665-1G；IMDM中的100mg/ml原液，储存在-20℃下)
1mL MTG 500x(0.4mM；制备500x原液：35uL/2ml PBS，过滤器灭菌，接受时储存在+4℃下)
来自D3的hEB培养基
500ml含增补剂的StemPro-34(Invitrogen 10639-011)
5ml L-谷氨酰胺(2mM)
880uL抗坏血酸(1mM；H2O中的100mg/mL原液(0.57M)，储存在-20℃下)
750uL全铁转铁蛋白(150ug/ml；Sigma T0665-1G；IMDM中的100mg/ml原液，储存在-20℃下)
1mL MTG 500x(0.4mM；制备500x原液：35uL/2ml PBS，过滤器灭菌，接受时储存在+4℃下)
5ml P/S(10ng/ml)
D0.EB的生成
1)通常，在分割hiPSC后一周，准备好细胞来制备EB。集落应看起来厚、密、白且相对无分化。
2)从每个15cm的盘中吸出培养基。用在0.22uM过滤的DMEM/F12中稀释的8mL 1X胶原酶IV代替。
胶原酶IV(Gibco Cat#17104-019)Receiving fridge.
量出0.5g胶原酶IV并用50mL DMEM/F12稀释。用0.22uM steriflip(500mg/50mL＝10X)过滤。使用前，用DMEM/F12将10X胶原酶稀释至1X(1mg/mL)。
3)在室温下在防护罩(hood)中孵育5分钟。
4)吸出胶原酶IV并用8mL过滤的DMEM/F12代替。
5)使用细胞刮刀，只刮取一次集落，注意保留整个菌落。
6)使用5mL玻璃移液器轻轻缓慢地将细胞转移至Falcon-15管。如果有残留的集落，用另外5mL洗涤板并加入相同的Falcon-15。
7)使细胞通过重力在Falcon-15中沉降(～3-5分钟)。吸出上清液从而除去大部分保留在悬浮液中的MEF。轻轻加入～10mL DMEM/F12来洗涤细胞，并以1000rpm离心1min。
8)在细胞旋转的同时，将11mL聚集培养基加入低粘附的10cm培养皿。
9)吸出培养基，用1mL聚集培养基轻轻重悬细胞。
10)轻轻地将1mL细胞转移到每个含有聚集培养基的低粘附10cm培养皿。使用相同的移液器，从板的一个区域吸取1mL而不带任何细胞，并清洗Falcon-15。将1mL加回到Falcon-15中；现在含有2个hiPSC起始板的每个板的最终体积为12mL。
11)转移至37℃的低氧培养箱。4-5个板可以堆叠在一起，其中一个板在堆的底部填充有PBS从而防止蒸发。这是hEB培养的第0天。
D0.添加10ng/ml BMP4。低氧下培养(5％CO2/5％O2/90％N2)0-8天。
D1.10ng/ml BMP4+5ng/ml bFGF。直接加入培养基。
D2.10ng/ml BMP4+5ng/ml bFGF+6μM SB-431542+3μM CHIR99021。直接加入培养基，除非颜色变化或有许多细胞碎片。
D3.5ng/ml bFGF+15ng/ml VEGF.收集培养基，1,000rpm旋转1min，用hEB培养基替代培养基。
D4-5.5ng/ml bFGF+15ng/ml VEGF.直接加入培养基。
D6-7.5ng/ml bFGF+15ng/ml VEGF+10g/ml IL-6+5ng/ml IL-11+25ng/ml IGF-1+50ng/ml SCF+2U/ml终EPO。收集培养基，1,000rpm旋转1min，用hEB培养基和造血细胞因子替代培养基。
D8.5ng/ml bFGF+15ng/ml VEGF+10ng/ml IL-6+5ng/ml IL-11+25ng/ml IGF-1+50ng/ml SCF+2U/ml终EPO+30ng/ml+TPO+10ng/ml Flt-3L+30ng/ml IL-3
D9.5ng/ml bFGF+15ng/ml VEGF+10ng/ml IL-6+5ng/ml IL-11+25ng/ml IGF-1+50ng/ml SCF+2U/ml终EPO+30ng/ml+TPO+10ng/ml Flt-3L+30ng/ml IL-3。将细胞移至正常O2培养箱。
表1.细胞处理方案
| 第0天 | 第1天 | 第2天 | 第3天 | 第4天 | 第5天 | 第6天 | 第7天 | 第8天 | 第9天 |
| BMP4 | BMP4 | BMP4 | |||||||
| bFGF | bFGF | bFGF | bFGF | bFGF | bFGF | bFGF | bFGF | bFGF | |
| SB | VEGF | VEGF | VEGF | VEGF | VEGF | VEGF | VEGF | ||
| CHIR | |||||||||
| IL-6 | IL-6 | IL-6 | IL-6 | ||||||
| IL-11 | IL-11 | IL-11 | IL-11 | ||||||
| IGF-1 | IGF-1 | IGF-1 | IGF-1 | ||||||
| SCF | SCF | SCF | SCF | ||||||
| EPO | EPO | EPO | EPO | ||||||
| TPO | TPO | ||||||||
| Flt-3 | Flt-3 | ||||||||
| IL-3 | IL-3 |
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其他实施方案
应理解的是，虽然已经结合本发明的详细描述记述了本发明，但是前文的描述旨在说明而不是限制本发明的范围，本发明的范围由所附权利要求的范围限定。其他方面、优点和修改在所附权利要求的范围内。
Claims (10)
1.一种制备造血干细胞(HSC)群体的方法，所述方法包括：
提供包括生血内皮(HE)细胞的群体，和
(i)使所述HE细胞与一定量的瞬时受体电位阳离子通道-香草酸亚家族成员4(Trpv4)的激动剂接触；和/或
(ii)使所述细胞经历周期性2维拉伸，
持续一段时间并在足以刺激内皮-向-HSC的转变的条件下进行。
2.根据权利要求1所述的方法，其中所述HE细胞从iPSC获得。
3.根据权利要求1所述的方法，其中所述Trpv4的激动剂选自由以下组成的组：花生四烯酸、5,6-EET、8,9-EET，双穿心莲内酯A(BAA)，佛波酯(如4α-PDD和4α-PDH)，RN-1747，取代的1,4-二氨基丁烷或1,3-二氨基丙烷类似物；和GSK10116790A。
4.根据权利要求1所述的方法，其中所述细胞从具有血液、骨髓、代谢或免疫疾病的受试者获得。
5.根据权利要求1所述的方法，其中受试者不具有恶性血液病。
6.一种治疗具有血液、骨髓、代谢和免疫疾病的受试者的方法，所述方法包括向所述受试者给予治疗有效量的造血干细胞(HSC)，所述造血干细胞通过包括以下的方法获得：
提供包括生血内皮(HE)细胞的群体，和
(i)使所述HE细胞与一定量的瞬时受体电位阳离子通道-香草酸亚家族成员4(Trpv4)的激动剂接触；和/或
(ii)使所述细胞经历周期性2维拉伸，
持续一段时间并在足以刺激内皮-向-HSC的转变的条件下进行。
7.根据权利要求6所述的方法，其中所述HE细胞从iPSC获得。
8.根据权利要求6所述的方法，其中所述Trpv4的激动剂选自由以下组成的组：花生四烯酸、5,6-EET、8,9-EET，双穿心莲内酯A(BAA)，佛波酯(如4α-PDD和4α-PDH)，RN-1747，取代的1,4-二氨基丁烷或1,3-二氨基丙烷类似物；和GSK10116790A。
9.根据权利要求6所述的方法，其中所述受试者为人。
10.根据权利要求6所述的方法，其中所述受试者具有多发性骨髓瘤；非何杰金淋巴瘤；何杰金病；急性髓性白血病；神经母细胞瘤；生殖细胞瘤；自身免疫性失调(系统性红斑狼疮(SLE)或系统性硬化病)；或淀粉样变性。
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