Name: generating self-assembling human heart organoids derived from pluripotent stem cells
Uploaded: 2021-09-15T15:00:00
Description: Watch this Scientific Journal Video about Generating Self-Assembling Human Heart Organoids Derived from Pluripotent Stem Cells at JoVE.com

这项工作得到了美国国立卫生研究院国家心脏，肺和血液研究所的支持，奖项编号为K01HL135464和R01HL151505，美国心脏协会的奖项编号为19IPLOI34660342。我们要感谢MSU高级显微镜核心和密歇根州立大学药理学和毒理学系的William Jackson博士获得共聚焦显微镜，IQ显微镜核心和密歇根州立大学基因组学核心的测序服务。我们还要感谢阿吉雷实验室的所有成员的宝贵意见和建议。

1. hPSC培养与维护
注意：人诱导的PSCs（hiPSCs）或人胚胎干细胞（hESCs）在解冻后需要培养至少连续传代2次，然后才能用于产生EB进行分化或进一步冷冻保存。hPSCs在PSC培养基（见 材料表）中基底膜 - 细胞外基质（BM-ECM）包被的6孔培养板上培养。当在6孔板中的hPSCs上进行培养基更换时，将培养基直接添加到孔的内侧，而不是直接在细胞顶部，以防止不必要的细胞脱离...

<ol>
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人类干细胞衍生的心肌细胞和其他心脏来源的细胞的最新进展已被用于模拟人类心脏发育22，24，25 和疾病26，27，28 ，并作为筛选治疗药物的工具29，30 和有毒物质31，32</sup...

先天性心脏缺陷（CHDs）是人类最常见的先天性缺陷类型，影响约1%的活产1，2，3。在大多数情况下，冠心病的原因仍然未知。在实验室中创建与发育中的人类心脏非常相似的人类心脏模型的能力是直接研究人类而不是代孕动物模型中CHDs的根本原因的重要一步。
实验室培养的组织模型的缩影是类器官，类似于细胞组成和生理功能中感兴趣的器官的3D细胞结构。类器官通常来自干细胞或祖细胞，并已成功用于模拟许多器官，如脑4，5，肾6，7，肠8，9，肺10，11，肝脏12，13和胰腺14，15，仅举几例。最近的研究表明，创造自组装心脏类器官在体外研究心脏发育是可行的。这些模型包括使用小鼠胚胎干细胞（mESCs）对早期心脏发育进行建模16，17直至房室规格18，并使用人类多能干细胞（hPSCs）来生成具有高度复杂细胞组成的多生殖层心内胚类器官19和腔室心形体20。
本文提出了一种新颖的3步WNT调制方案，以高效且具有成本效益的方式生成高度复杂的hHO。类器官在96孔板中生成，从而形成一个可扩展的高通量系统，可以轻松自动化。该方法依赖于创建hPSC聚集体并触发心脏发生的发育步骤，包括中胚层和心脏中胚层形成，第一和第二心场规范，心前器官形成和房室规范。分化15天后，hHO包含心脏中发现的所有主要细胞谱系，明确定义的内腔，心房和心室腔以及整个类器官的血管网络。这种高度复杂且可重复的心脏类器官系统适合在心脏发育，疾病和药理学筛查研究中研究结构，功能，分子和转录组学分析。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Donkey anti- mouse</td>
			<td>Invitrogen</td>
			<td>A-21202</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Donkey anti- rabbit</td>
			<td>Invitrogen</td>
			<td>A-21206</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 Donkey anti- mouse</td>
			<td>Invitrogen</td>
			<td>A-21203</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 Donkey anti- rabbit</td>
			<td>Invitrogen</td>
			<td>A-21207</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 Donkey anti- goat</td>
			<td>Invitrogen</td>
			<td>A32849</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>HAND1</td>
			<td>Abcam</td>
			<td>ab196622</td>
			<td>Rabbit; 1:200</td>
		</tr>
		<tr>
			<td>HAND2</td>
			<td>Abcam</td>
			<td>ab200040</td>
			<td>Rabbit; 1:200</td>
		</tr>
		<tr>
			<td>NFAT2</td>
			<td>Abcam</td>
			<td>ab25916</td>
			<td>Rabbit; 1:100</td>
		</tr>
		<tr>
			<td>PECAM1</td>
			<td>DSHB</td>
			<td>P2B1</td>
			<td>Rabbit; 1:50</td>
		</tr>
		<tr>
			<td>TNNT2</td>
			<td>Abcam</td>
			<td>ab8295</td>
			<td>Mouse; 1:200</td>
		</tr>
		<tr>
			<td>THY1</td>
			<td>Abcam</td>
			<td>ab133350</td>
			<td>Rabbit; 1:200</td>
		</tr>
		<tr>
			<td>TJP1</td>
			<td>Invitrogen</td>
			<td>PA5-19090</td>
			<td>Goat; 1:250</td>
		</tr>
		<tr>
			<td>VIM</td>
			<td>Abcam</td>
			<td>ab11256</td>
			<td>Goat; 1:250</td>
		</tr>
		<tr>
			<td>WT1</td>
			<td>Abcam</td>
			<td>ab89901</td>
			<td>Rabbit; 1:200</td>
		</tr>
		<tr>
			<td>Media and Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Innovative Cell Technologies</td>
			<td>NC9464543</td>
			<td>cell dissociation reagent</td>
		</tr>
		<tr>
			<td>Activin A</td>
			<td>R&#38;D Systems</td>
			<td>338AC010</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement (Minus Insulin)</td>
			<td>Gibco</td>
			<td>A1895601</td>
			<td>insulin-free cell culture supplement</td>
		</tr>
		<tr>
			<td>B-27 Supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>cell culture supplement</td>
		</tr>
		<tr>
			<td>BMP-4</td>
			<td>Gibco</td>
			<td>PHC9534</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Bioworld</td>
			<td>50253966</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR-99021</td>
			<td>Selleck</td>
			<td>442310</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(-)-Fructose</td>
			<td>Millipore Sigma</td>
			<td>F0127</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Scientific</td>
			<td>62248</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Millipore Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>10566016</td>
			<td></td>
		</tr>
		<tr>
			<td>Essential 8 Flex Medium Kit</td>
			<td>Gibco</td>
			<td>A2858501</td>
			<td>pluripotent stem cell (PSC) medium containing 1% penicillin-streptomycin</td>
		</tr>
		<tr>
			<td>Fluo4-AM</td>
			<td>Invitrogen</td>
			<td>F14201</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Millipore Sigma</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Millipore Sigma</td>
			<td>410225</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel GFR</td>
			<td>Corning</td>
			<td>CB40230</td>
			<td>Basement membrane extracellular matrix (BM-ECM)</td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>Millipore Sigma</td>
			<td>S30-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>MP Biomedicals</td>
			<td>IC15014601</td>
			<td>Powder dissolved in PBS Buffer &#8211; use at 4%</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Solution</td>
			<td>Gibco</td>
			<td>10010049</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Solution (10x)</td>
			<td>Gibco</td>
			<td>70011044</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybead Microspheres</td>
			<td>Polysciences, Inc.</td>
			<td>73155</td>
			<td>90 &#181;m</td>
		</tr>
		<tr>
			<td>ReLeSR</td>
			<td>Stem Cell Technologies</td>
			<td>NC0729236</td>
			<td>dissociation reagent for hPSCs</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Gibco</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiazovivin</td>
			<td>Millipore Sigma</td>
			<td>SML1045</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Millipore Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution</td>
			<td>Gibco</td>
			<td>1525006</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASHIELD Vibrance Antifade Mounting Medium</td>
			<td>Vector Laboratories</td>
			<td>H170010</td>
			<td></td>
		</tr>
		<tr>
			<td>WNT-C59</td>
			<td>Selleck</td>
			<td>NC0710557</td>
			<td></td>
		</tr>
		<tr>
			<td>Other</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Microcentrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>02682002</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Falcon Tubes</td>
			<td>Fisher Scientific</td>
			<td>1495970C</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL Cryogenic Vials</td>
			<td>Corning</td>
			<td>13-700-500</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Reagent Reservoirs</td>
			<td>Fisherbrand</td>
			<td>13681502</td>
			<td></td>
		</tr>
		<tr>
			<td>6-Well Flat Bottom Cell Culture Plates</td>
			<td>Corning</td>
			<td>0720083</td>
			<td></td>
		</tr>
		<tr>
			<td>8 Well chambered cover Glass with #1.5 high performance cover glass</td>
			<td>Cellvis</td>
			<td>C8-1.5H-N</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well Clear Ultra Low Attachment Microplates</td>
			<td>Costar</td>
			<td>07201680</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td>Image processing software</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark Professional</td>
			<td>06-666</td>
			<td>laboratory wipes</td>
		</tr>
		<tr>
			<td>Micro Cover Glass</td>
			<td>VWR</td>
			<td>48393-241</td>
			<td>24 x 50 mm No. 1.5</td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>Fisherbrand</td>
			<td>1255015</td>
			<td></td>
		</tr>
		<tr>
			<td>Moxi Cell Counter</td>
			<td>Orflo Technologies</td>
			<td>&#160;MXZ001</td>
			<td></td>
		</tr>
		<tr>
			<td>Moxi Z Cell Count Cassette &#8211; Type M</td>
			<td>Orflo&#160;Technologies</td>
			<td>MXC001</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel Pipettes</td>
			<td>Fisherbrand</td>
			<td>FBE1200300</td>
			<td>30-300 &#181;L</td>
		</tr>
		<tr>
			<td>Olympus cellVivo</td>
			<td>Olympus</td>
			<td></td>
			<td>For Caclium Imaging, analysis with Imagej</td>
		</tr>
		<tr>
			<td>Sorvall Legend X1 Centrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>75004261</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermoshaker</td>
			<td>ThermoFisher Scientific</td>
			<td>13-687-711PM</td>
			<td></td>
		</tr>
		<tr>
			<td>Top Coat Nail Varish</td>
			<td>Seche Vite</td>
			<td></td>
			<td>Can purchase from any supermarket</td>
		</tr>
	</tbody>
</table>

generating self-assembling human heart organoids derived from pluripotent stem cells

研究人类心脏在健康和疾病中发育的能力受到 在体外模拟人类心脏复杂性的能力的高度限制。开发更有效的器官样平台，可以模拟复杂的 体内 表型，如类器官和器官芯片，将增强研究人类心脏发育和疾病的能力。本文描述了一种方案，通过使用人类多能干细胞的自组织和使用小分子抑制剂逐步激活发育途径来产生高度复杂的人心脏类器官（hHO）。胚胎体（EB）在具有圆底，超低附着孔的96孔板中产生，促进个体化结构的悬浮培养。
EB通过三步Wnt信号调节策略分化为hHO，该策略涉及初始Wnt通路激活以诱导心脏中胚层命运，第二步Wnt抑制以创建确定的心脏谱系，以及第三个Wnt激活步骤以诱导心前器官组织。这些步骤以96孔的形式进行，效率高，可重复，每次运行产生大量类器官。通过免疫荧光成像从第3天到第11天的分化分析揭示了第15天hHO内的第一和第二心场规格和高度复杂的组织，包括具有心房和心室心肌细胞区域的心肌组织，以及衬有心内膜组织的内腔。类器官还在整个结构和心外膜组织的外衬里中表现出复杂的血管网络。从功能的角度来看，hHO具有强大的水平，并表现出由Flu-4活体成像确定的正常钙活性。总体而言，该协议构成了人体器官样心脏组织 体外 研究的坚实平台。

为了在体外实现自组织hHO，我们使用Wnt通路调节剂修改和组合了先前描述的心肌细胞21和心外膜细胞22的2D单层分化方案，以及使用生长因子BMP4和Activin A的3D心前类器官16。，优化了Wnt途径激活剂CHIR99021的浓度和暴露持续时间，以产生来自人类PSC的高度可重复性和复杂的hHO.hPSCs或hESCs在PSC培养基中培养至60-80%的融合在...

Watch this Scientific Journal Video about Generating Self-Assembling Human Heart Organoids Derived from Pluripotent Stem Cells at JoVE.com

Generating Self-Assembling Human Heart Organoids Derived from Pluripotent Stem Cells

在这里，我们描述了一种方案，通过自组织有效地使用人类多能干细胞来创建与发育相关的人类心脏类器官（hHO）。该协议依赖于发育线索的顺序激活，并产生高度复杂，功能相关的人体心脏组织。

产生来自多能干细胞的自组装人心脏类器官

The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in ...

该协议使我们能够以一种简单且具有成本效益的方式创建一个复杂的3D结构，该结构与发育中的人类心脏非常相似，并且可以跨细胞系重复。它由具有三个分化步骤的单一方案组成，从而产生了心脏的所有细胞类型，包括腔室和血管网络。这项技术使我们能够模拟各种人类心血管疾病，并筛选有效的药物治疗。由于其设计的高吞吐量特性。这种方法可用于研究发育中的人类心脏以及通常与之相关的疾病。其中之一是先天性心脏缺陷。该协议需要基本的细胞培养技能。我们建议细心和细致的移液以及轻柔地处理类器官。首先在分化前两天创建胚胎体或EB。将亚汇合的 HPSC 与 DBPS 洗涤至少 10 秒钟。然后吸出DPBS。加入一毫升室温ACUTA，轻轻敲击板，每分钟轻轻敲击五次，持续三到五分钟以诱导脱离，同时在显微镜下检查。然后加入一毫升PSC培养基和Thias Avin或Thias以停止反应。在孔中上下移液培养基，两到三次以产生单个细胞悬浮液并收集细胞。然后将单细胞悬浮液转移到离心管中，以300倍G.吸出上清液旋转五分钟，并将细胞重新消耗在一毫升PSC培养基和Thias中。稀释DPBS中的细胞。使用细胞计数器或血囊肿计计数细胞，并将PSC培养基中的细胞与Thias稀释至每毫升100， 000个细胞的浓度。向圆底超低连接孔板96的每个孔中加入100微升稀释的细胞悬浮液。将板以100倍G离心3分钟，并在37摄氏度和5%二氧化碳下孵育24小时。24小时后，小心地从每个孔中取出50微升培养基，并加入200个核心凋落的新鲜PSC培养基，加热至37摄氏度，每孔体积为250微升。将细胞在37摄氏度和5%二氧化碳下孵育24小时。从每个孔的一侧除去166微升或三分之二的培养基，并加入166微升含有无胰岛素B27补充剂的RPMI 1640，6微摩尔欢呼990211，0.875纳克每毫升骨形态发生蛋白4和1.5纳克每毫升Activin A.在37摄氏度和5%二氧化碳下孵育24小时。24小时后，从每个孔的一侧取出166微升培养基，并加入166微升新鲜RPMI 1640和无胰岛素B27补充剂。在37摄氏度和5%二氧化碳下孵育24小时。为了在第二天诱导心脏中胚层规格，从每个孔中取出166微升培养基，加入166微升RPMI 1640，含有无胰岛素B27补充剂和三微摩尔Wnt-C59，并继续孵育48小时。在第四天，从每个孔中取出166微升培养基，加入166微升新鲜RPMI 1640和无胰岛素B27补充剂，然后再孵育48小时。在第六天，从每个孔中取出166微升培养基，并加入166微升RPMI 1640和B27补充剂。对于这种和所有后续的培养基变化，使用B27补充胰岛素，再孵育24小时。在第七天，细胞培养物已准备好进行前心外膜分化。从每个孔中取出166微升培养基，加入166微升含有B27补充剂和三微摩尔cheer 99021的新鲜RPMI 1640。然后在孵育一小时后，从培养箱中取出板，从每个孔的一侧取出166微升培养基，并加入166微升含有B27补充剂的新鲜RPMI 1640继续孵育48小时。每48小时重复一次此过程，直到收集。类器官已准备好在第15天进行分析和实验 要转移整个类器官，请将P200移液器的尖端从开口处切开5至10毫米，直径约为2至3毫米。按下移液器柱塞，将吸头垂直插入带有类器官的圆底孔中。吸收大约100至200微升的培养基，足以收集类器官并将其转移到目标目的地。在分化EBS表现为黑色球形团块之前，分化过程从第0天开始，Wnt途径激活和生长因子添加正好24小时。此外，在第二天对Wnt C59的编辑导致类器官从大约200微米扩大到最大一毫米。人类心脏类器官早在第六天就开始跳动，到第10天，100%的类器官显示出可见的跳动。第七天类器官的高放大倍率图像显示，大多数表达一的手细胞是起源于心肌细胞的，而第二手是来自第二个心场的非肌分裂细胞，即祖细胞。第一手和第二手的RNA转录本从第三天开始在RNA测序数据中表达。FHF标记在第3天和第11天表达高度，而SF标记在第13天后表达得更高。免疫荧光染色揭示了构成人类心脏的各种人类心脏细胞类型谱系的存在。例如心肌和心外膜组织、心内膜细胞、内皮细胞和心脏成纤维细胞。在RNA SAC基因表达谱中也观察到表达上述细胞谱系的标记。使用整个类器官中单个细胞的活钙成像来测量类器官的电生理功能。Fluo-4荧光强度随时间而变化，因为钙从细胞中进入，显示出规律的动作电位。热图显示类器官高放大率区域的钙强度，显示由于钙瞬变和单个细胞而增加的强度。在尝试此方案时，请记住，EB和随后的类器官不会附着在板的底部，除了需要仔细更换培养基的培养基外，我们还将随着移除而移动。类器官可以培养更长的时间引入成熟技术，甚至暴露于外部刺激，如机械或电调节，甚至压力源，如毒素。该技术允许进入人类胎儿心脏发育的阶段，否则这些阶段在研究中是无法进入的，为心脏发育和疾病病因学的新见解铺平了道路。

Research

JoVE Journal

Biochemistry

Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

从人体二尖瓣提取蛋白质的优化方案

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit ...

科研

生物化学

Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

人死后脑中洗涤剂不溶性蛋白的富集

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human ...

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

一种用于小数目造血干细胞的西方印迹协议

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western ...

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

结合 x 射线晶体学与小角度 x 射线散射到模型的非结构化区域的 Nsa1 从s 酿酒酵母

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the ...

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

在利什曼原虫、人体细胞和小鼠睾丸中的 Polysome 分析

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

Rapid Isolation of the Mitoribosome from HEK Cells

HEK 细胞 Mitoribosome 的快速分离

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative ...

Dynamic Proteomic and miRNA Analysis of Polysomes from Isolated Mouse Heart After Langendorff Perfusion

Langendorff 灌注后离体小鼠心脏 Polysomes 的动态蛋白质组学及 miRNA 分析

Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ...

Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity

通过对Rho激酶活性的暂时抑制,增强人类诱导多能干细胞衍生心肌细胞的诱导

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. ...

Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

The ongoing worldwide epidemic of diabetes increases the demand for the identification of environmental, nutritional, endocrine, genetic, and epigenetic ...

Human Mesenchymal Stem Cell Processing for Clinical Applications Using a Closed Semi-Automated Workflow

使用封闭式半自动化工作流程进行临床应用的人间充质干细胞处理

Human mesenchymal stem cells (hMSCs) are currently being explored as a promising cell-based therapeutic modality for various diseases, with more market ...