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Developmental Biology

在体外人骨髓间充质干细胞分化为心肌样细胞功能

Published: August 9, 2017 doi: 10.3791/55757
* These authors contributed equally

Summary

在这里,我们提出一种方法,有效地利用的心肌细胞分化潜力,年轻的人骨髓间充质干细胞的来源以生成功能、 订约、 心肌样细胞体外。

Abstract

心肌梗死和随后的缺血级联导致大量伤亡,心肌细胞,导致充血性心衰,全世界死亡的主要原因。骨髓间充质干细胞 (MSCs) 是一种有前景的细胞为基础的疗法,以取代当前的侵入性技术。骨髓间充质干细胞可以分化成间充质血统,其中包括心脏的细胞类型,但尚未实现完整功能的细胞分化。以前的分化的方法基于药物制剂或生长因子。然而,更多生理有关的战略也可以启用骨髓间充质干细胞向心肌细胞转化。在这里,我们提出一种利用心肌细胞饲养层上 MSC 聚合产生订约心肌样细胞的分化方法。

人脐带血管周围细胞 (HUCPVCs) 已被证明有更大的分化潜能比一般调查 MSC 类型,如骨髓间充质干细胞 (BMSCs)。作为一个 ontogenetically 年轻的来源,我们调查了早孕 (FTM) HUCPVCs 相比更旧的来源的心肌样潜力。FTM HUCPVCs 是新颖、 丰富的来源,保留其在子宫内理想属性时培养的间充质干细胞在体外。HUCPVCs 使用此分化协议、 FTM 和期限取得显著增加向心肌细胞分化与骨髓间充质干细胞,相比,增加表达的心肌细胞标记 (即,肌细胞增强因子 2 C、 心肌肌钙蛋白 T、 重链心肌肌球蛋白、 信号调节蛋白 α 和缝隙连接蛋白 43) 所示。他们还保持显著低免疫原性,证明了他们低 HLA A 表达和更高的 HLA — G 表达这一点。应用基于聚合的分化,FTM HUCPVCs 表明增加聚合形成潜在和生成的共培养对心脏饲养层 1 星期内收缩细胞集群,成为第一的 MSC 类型,这样做。

我们的结果表明,这种分化策略可以有效利用向心肌细胞的潜力,年轻的骨髓间充质干细胞,如 FTM HUCPVCs,表明那种体外预分化可能是一个潜在的战略,以增加其再生效果的体内

Introduction

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充血性心力衰竭 (CHF) 仍是全球发病率和死亡率的主要原因。CHF 经常发生后心肌细胞的巨大损失和心肌梗死 (MI)1的病理结果无细胞瘢痕组织的发展。虽然心脏是部分自我更新的器官,驻地干、 祖细胞池负责执行组织再生显著降低中丰度和老年患者,往往变得不足以最佳恢复的损伤后的功能。因此,是极大的兴趣发展涉及的健康供者细胞移植到受损心肌的实验性治疗。势在必行的是供者细胞不仅恢复结构的组织,但也达到心肌功能恢复的影响。

本机的心员工心组织居民和损伤后内源性的骨髓源性干细胞修复234。蓄热式导出细胞宿主和捐助者都必须有获得适当的表型和功能的重塑心肌,并有能力以高效、 安全地更换丢失的细胞微环境的能力。在体外分化方法曾经广泛实现高效率、 基于干细胞的心肌细胞生产56。表达谱的心脏沿袭标记用于定义向心脏沿袭7干细胞分化的过程。早期分化标记,如 NKX2.5,肌细胞增强因子 2 C (Mef2c) 和 GATA489,可以指示开始向心肌细胞进程。成熟的心肌细胞标记通常用于评估分化效果是信号调节蛋白 α (关于)10、 心肌肌钙蛋白 T (肌钙)11、 重链心肌肌球蛋白 (MYH6)81213和缝隙连接蛋白 43 (Cx43)141516。利用胚胎干细胞 (Esc) 和多能性干细胞 (Psc) 的方法彻底优化并讨论了有关的诱导因素,氧气和养分的渐变,细节和行动5671718的确切时间。尽管如此,基于 ESC 和 PSC 的技术仍然存在多个的伦理和安全问题,和次优的电生理及免疫学特征1920。主机经常用这些细胞移植免疫排斥的经验和需要永久免疫抑制。这主要是由于失主要组织相容性复合体 (MHC) 分子在东道国和捐助和由此产生的 T 细胞反应21。同时个别 MHC I 类匹配是一个可能的解决方案,更易于访问的临床实践需要普遍是理想来克服排斥反应的关切的细胞来源。

作为替代细胞来源,用于在临床应用中,骨髓间充质干细胞和骨髓间充质干细胞,特别被进行了自其最初被描述在 1995年22组织再生使用。骨髓间充质干细胞据说是驻地的再生细胞在几乎任何带血管蒂的组织23中可以发现。骨髓间充质干细胞后从所需的源的隔离,很容易可以扩大在文化,有广泛的旁分泌能力,经常拥有理想或免疫调节属性2425。其安全性和有效性已经进行了几个临床前研究,特别是心脏再生326

许多 MSC 分化策略利用药理剂 5-氮杂胞苷22和二甲基亚砜27,和增长或人骨的因素,像 BMPs572829或血管紧张素 II30,与变量效率。这些策略,不过,不基于天真再生细胞有望后归巢或传递到损伤的部位,遇到的障碍体内。更多生理有关的战略,同时更难定义和操纵,都基于的前提下,可以通过从组织微环境本身的信号诱导 MSC 分化。先前的研究显示,暴露于心肌细胞裂解物31或左室心肌3233,或直接与原发性心肌细胞体外1534,接触可以增加心肌标志物检测在骨髓间充质干细胞中表达。别人有后,表现出自发再生治疗心肌损伤与骨髓间充质干细胞35363738,尽管部分,骨髓间充质干细胞和心肌细胞3940融合生成新生心肌。我们所知,从人骨髓间充质干细胞 (hMSCs) 的任何组织源的功能、 自发订约心肌细胞尚未报告。

目前的共识是所有骨髓间充质干细胞产生的血管周围细胞23。年轻骨髓间充质干细胞与周细胞属性可以从人脐带组织414243的血管周围地区隔离。与骨髓间充质干细胞,HUCPVCs 拥有更多的分化潜能和几个其他再生的优势,两个体外4144体内454647。值得注意的是,被母胎界面的来源,HUCPVCs 有显著较低免疫原性相比成人骨髓间充质干细胞的来源。我们的研究重点的表征和 FTM HUCPVCs,年轻源间充质干细胞研究,临床前应用,我们以前已经增加了增殖能力和较高的 multilineage 分化的能力,包括在向心肌细胞谱系41

在这里,我们提出一种协议,结合聚合形成和原发性心脏细胞饲养层,归纳部队达到完成向心肌细胞分化的骨料骨髓间充质干细胞。 提供 3D 的环境,更好地模型条件体内相比 2D 遗民文化。利用心脏饲养层提供一种环境,是对骨髓间充质干细胞的最终移植网站的代表。我们证明年轻来源的间充质干细胞分离产前或产后的脐带有较高的能力,与窗体集料,达到相比成人骨髓间充质干细胞,同时仍然保持其免疫特权心肌表型。除了陡海拔及心脏沿袭标记基因诱导的表达的细胞内 (即, cTnT 与 MYH6) 和细胞表面蛋白 (即,关于和 Cx43) 具体为心肌细胞,我们表明可以用这种方法利用 FTM HUCPVCs 分化潜力,它们可以导致自发性收缩心肌样细胞。

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Discussion

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2 年来,发展与几个不同的策略被用来从 MSC 来源生成心肌样细胞受到了心脏干细胞分化。许多这些策略,然而,是低效的而且使用的条件往往不代表环境移植细胞遇到的体内

与现有的方法,相比议定书 》 在这里提出了一种利用原发性心脏饲养层和 MSC 聚合形成的组合。原发性心脏饲养层提供类似于间充质干细胞移植后暴露的分化环境。聚合生成环境与氧气和养分的梯度,可以有诱导效应对启动系的承诺。基于 PSC 和 esc 键的使用低氧预适应的心肌细胞分化战略和聚合形成建立了这对干细胞55253的诱导作用。此外,3D 结构更好地类似于细胞间连接提供的体内。我们使用这种方法,成功地生产 hMSC 类型是能够做到这一点,我们所知的同步订约 hMSC 集料第一次。重要的是,只有最年轻成人骨髓间充质干细胞来源的应用,FTM HUCPVCs 表现出这种能力。

在使用的天真或预分化骨髓间充质干细胞胚胎干细胞或物业服务公司为心脏再生的显著优势在于其免疫调节或理想性质。然而,即使是在骨髓间充质干细胞,免疫调节特性随其年龄和源54。骨髓间充质干细胞已经改变其免疫原性的属性后分化,往往会导致他们拒绝后植入55。如上文所述,从脐带间充质干细胞的来源有特殊免疫赦免56由于他们在子宫内的起源。使用协议上文所述,甚至后分化为心肌样细胞,词和 FTM HUCPVCs 表现出高水平的 HLA-G (即积极衰减先天免疫反应) 和维持低水平的 HLA A (即确定 T 细胞介导的细胞毒作用)。

这个分化协议的优点是方便地允许为多个层次的分析。首先,实时成像功能,承包骨料是较为可行的不是区分单个细胞为饲养层集成。如议定书 》 利用混合物种共同文化,人特异性引物的主要适用于 qPCR 分析,而外地资产管制系统和国际刑事法院提供的快速鉴定表型 — — 具体而言,心脏标记物的表达 (图 2)。使用议定书 》 所述,检测到的关于、 肌钙、 MYH6、 aSarc、 Mef2c,Cx43 水平升高。在与 HuNu 一起,有可能查明 Mef2c 和分化间充质干细胞中的 aSarc 的表达,鼠源性心肌细胞饲养层或融合的细胞相区别。虽然订约集料在饲养层上的识别是相对比较容易,收集或收获他们可以构成限制。配备了微操作机器人的显微镜可以用来应对这一挑战。或者,从使用人类抗体抗-TRA-1-85 的共同文化,可以有效地排序人类细胞。虽然共同文化也存在潜在的细胞融合的挑战,这也是可以克服核和细胞表面的人类标记示踪剂 (补充图 1) 结合使用。

可能会修改我们的鉴别方法是调整集料的形成。我们的研究结果表明,骨料粒径可以最优分化的关键参数。假定聚合是选择性的多的粘接的细胞,它可以作为 CD49f 阳性间充质干细胞的一个预选步骤。增强的 MSC 领域形成有链接,增加 CD49f 表达49,和 CD49f 也是与更高的细胞相关联的因为它直接连接到 SOX2 和 OCT449。我们观察到在更旧的来源的间充质干细胞相比,FTM HUCPVCs,这就可以解释观察到的减少集料粒径 CD49f 阳性细胞较低的数字。我们理论化,选择此选项可以通过增加每集料以实现更高的集料粒径,间充质干细胞的数量赔偿因此可能提高效率的聚合诱导分化。

未来的应用程序可能需要修改议定书。就必须预先分化间充质干细胞的临床应用发生在异种自由的条件下。订约的轻骨料产品使用此协议也可以用于创造的人造的心脏肌肉组织。临床适用的产品,cgmp 工作流是必要的。虽然异种免费培养基是供人间充质干细胞,提供无动物饲养层可能一个挑战。然而,应用预分化的饲养层的其他干细胞来源,如诱导多能干细胞,是一个可能的解决方案。

最后,描述的鉴别方法可以用于生产功能性年轻来源的间充质干细胞的心肌样细胞在方便、 重现性好的方式。

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Disclosures

克利福德 · L.Librach 博士是联合专利持有人:隔离和使用来自第一孕期胎儿脐带组织细胞的方法,授予在加拿大和澳大利亚。

Acknowledgments

作者感谢以下人员和研究人员所作的贡献: 马修 Librach、 莱拉 Maghen、 坦尼娅 A.Baretto,Shlomit Kenigsberg 和安德鲁 Gauthier 费舍尔。这项工作被支持由安大略省研究基金-研究卓越 (ORF RE,圆 #7) 和创建程序公司。

Materials

Name Company Catalog Number Comments
0.25% Trypsin/EDTA Gibco 25200056 For cell dissociation
Alpha-MEM Gibco 12571071 For HUCPVC and BMSC culture media.
PE-conjugated anti-human/mouse CD49f antibody Biolegend 313612 Integrin marker for FC
APC-conjugated human Cx43/GJA1 antibody R&D Systems FAB7737A Connexin 43 marker for FC
FITC-conjugated HLA-A2 antibody Genway Biotech Inc. GWB-66FBD2 Immunogenicity marker for FC
FITC-conjugated anti-HLA-G [MEM-G/9] antibody Abcam ab7904 Immunogenicity marker for FC
FITC-conjugated mouse anti-human SIRPA/CD172a antibody AbD Serotec/Bio-Rad MCA2518F Cardiac marker for FC
APC-conjugated human TRA-1-85/CD147 antibody R&D Systems FAB3195A Human cell marker for FC and FACS
FITC-conjugated human TRA-1-85/CD147 antibody R&D Systems FAB3195F Human cell marker for FC and FACS
Anti-connexin 43/GJA1 antibody Abcam ab11370 Cx43. For ICC
Goat anti-rabbit IgG (H+L) cross-absorbed secondary antibody, Alexa Fluor 555 Life Technologies A-21428 For ICC
Anti-sarcomeric alpha actinin [EA-53] antibody Abcam ab9465 aSARC. For ICC
Goat anti-mouse IgM heavy chain cross-absorbed secondary antibody, Alexa Fluor 555 Life Technologies A-21426 For ICC
Mef2C (D80C1) XP rabbit antibody New England BioLabs Ltd. 5030S For ICC
Donkey anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor 488 Life Technologies A-21206 For ICC
Anti-nuclei (HuNu) (clone 235-1) antibody EMD Millipore MAB1281 For ICC
MZ9.5 Stereomicroscope Leica For imaging aggregates.
1.5 ml centrifuge microtubes Axygen MCT-150-C For staining MSCs with fluorescent dye.
ImageJ Open source image processing software.
Aria II  BD UHN SickKids FC Facility. For cell sorting.
Bone marrow mesechymal stromal cells Lonza PT-2501 BMSCs
Bovine serum albumin Sigma-Aldrich A7030-100G BSA. To prepare solutions for ICC
BrdU EMD Millipore MAB3424 Caution: BrdU is a strong teratogen and suspected mutagen. Please ensure proper training and refer to the SDS before use.
Canto II BD UHN SickKids FC Facility. For flow cytometry.
cDNA EcoDry Premix Clontech/Takara 639570 For preparation of cDNA for qPCR
CellTracker Green CMFDA Dye Life Technologies C7025 Fluorescent imaging of cell cytoplasm
Countess automated cell counter Invitrogen Inc. C10227 For cell counting
DMEM-F12 Sigma-Aldrich D6421 For rat primary cardiomyocyte culture medium.
Dulbecco's Phosphate Buffered Saline Gibco 10010023 D-PBS, without Ca2+, Mg2+
EVOS Life Technologies In-house fluorescent microscope
FACSCalibur BD In-house. For flow cytometry.
Fetal bovine serum (Hyclone) GE Healthcare SH3039603 FBS. Component of cell culture medium.
IDT Prime Time qPCR probes Integrated Data Technologies FAM fluorophore http://www.idtdna.com/pages/products/gene-expression/primetime-qpcr-assays-and-primers
Lab Vision PermaFluor Aqueous Mounting Medium ThermoScientific TA-030-FM For storage of cells to undergo ICC
LSR II  BD UHN SickKids FC Facility. For flow cytometry.
MoFlo Astrios Beckman Coulter UHN SickKids FC Facility. For cell sorting.
Normal goat serum Cell Signaling Technology 5425S NGS. Used in blocking solution for ICC
Nunc Lab-Tek II Chamber Coverglass, 8-wells Thermo Scientific Nunc 155409 To prepare samples for ICC
OmniPur Triton X-100 Surfactant EMD Millipore 9410-OP As a component of permeabilizing solution when preparing cells for ICC
Paraformaldehyde, 16% Solution, EM Grade Electron Microscopy Sciences 15710 For fixing cells for ICC.
Penicillin/streptomycin Gibco 15140122 Component of cell culture medium.
Primers Sigma Custom Standard DNA Oligos, Desalted, 0.2 μmol CTnT_F: GGC AGC GGA AGA GGA TGC TGA A; CTnT_R: GAG GCA CCA AGT TGG GCA TGA ACG A; MYH6 F: GCA AAG TAC TGG ATG ACA CGC T; MYH6 R: GTC ATT GCT GAA ACC GAG AAT G
Quorum Spinning Disk Confocal Zeiss SickKids Imaging Facility
ReproCardio hiPS cell derived cardiomyocytes ReproCell RCD001N Positive control for qPCR
RNeasy mini kit Qiagen 74106 To isolate RNA for qPCR
Rotor-Gene SYBR Green PCR Kit Qiagen 204074 For qPCR with master mix
RPMI 1640 Gibco A1049101 For MSC, monocyte coculture medium.
TaqMan qPCR primer assays Thermo Fisher Scientific 4444556 For qPCR
Trypan Blue Life Technologies T10282 Staining of cells for viability and counting
Trypsin Gibco 272500108 For cell dissociation
Volocity Perkin-Elmer Volocity 6.3 Imaging software
0.2 μm pore filter Thermo Fisher Scientific 566-0020 For sterilizing tissue culture media
HERAcell 150i CO2 Incubator Thermo Fisher Scientific 51026410 For incubating cells
Dulbecco's phosphate buffered saline Sigma-Aldrich D8537 PBS. 1X, Without calcium chloride and magnesium chloride
Forceps Almedic 7727-A10-704 For handing rat heart. Can use any similar forceps.
Scissors Fine Science Tools 14059-11 For mincing rat heart. Curved scissors recommended.
50 mL tube BD Falcon 352070 For collection during cardiomyocyte collection and general tissue culture procedures
15 mL tube BD Falcon 352096 For general tissue culture procedures
6-well plates Thermo Scientific Nunc CA73520-906 For tissue culture
10 cm tissue culture dishes Corning 25382-428 For aggregate formation
Axiovert 40C Microscope Zeiss For bright-field imaging through out tissue culture and the rest of the protocol
70 μm cell strainer Fisherbrand 22363548 To ensure a single cell suspension before flow cytometry or sorting
Triton X-100 EMD Millipore 9410-1L Used in permeabilization solution for ICC
Hoechst 33342 Thermo Fisher Scientific H1399 Stain used during visualization of Cx43 localization

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<em>在体外</em>人骨髓间充质干细胞分化为心肌样细胞功能
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Szaraz, P., Gratch, Y. S., Iqbal, F., Librach, C. L. In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells. J. Vis. Exp. (126), e55757, doi:10.3791/55757 (2017).More

Szaraz, P., Gratch, Y. S., Iqbal, F., Librach, C. L. In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells. J. Vis. Exp. (126), e55757, doi:10.3791/55757 (2017).

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