成人小鼠心脏干细胞的分离、鉴定及分化
Summary
本文的总体目标是规范心脏干细胞 (csc) 与成年小鼠心脏分离、表征和分化的协议。在这里, 我们描述了一种密度梯度离心方法, 以分离小鼠 csc 和详细的方法, csc 培养, 增殖, 并分化为心肌细胞。
Abstract
心肌梗塞 (mi) 是全球发病率和死亡率的主要原因。再生医学的一个主要目标是补充心肌梗死后的死亡心肌。虽然有几种策略已被用于再生心肌, 干细胞治疗仍然是补充心肌梗死心脏的死亡心肌的主要方法。越来越多的证据表明, 成人心脏中存在常驻心脏干细胞 (csc), 其内分泌和 (或旁分泌) 对心脏再生有影响。然而, csc 分离及其对心肌细胞, 特别是心肌细胞的表征和分化, 仍然是一个技术挑战。在本研究中, 我们提供了一个简单的方法, 分离, 表征和分化 csc 从成年小鼠心脏。在这里, 我们描述了一种密度梯度方法隔离 csc, 其中心脏被消化0.2% 胶原酶 ii 溶液。为了表征分离的 csc, 我们评估了 cscs1、nkx2-5 和 gat4 和多能/干细胞的表达。我们还通过在培养培养培养皿和评估扩散标记 ki-67 的表达来确定孤立的 csc 的增殖潜力。为了评估 csc 的分化潜力, 我们选择了七到十天培养的 csc。我们把它们转移到一个有心肌细胞分化培养基的新盘子里。它们在细胞培养孵化器中孵育 12天, 而分化培养基每三天改变一次。分化的 csc 表达心肌细胞特异性标记: 放线素和肌钙蛋白 i。因此, 使用该协议分离的 csc 具有干细胞和心脏标记物, 它们有向心肌细胞谱系增殖和分化的潜力。
Introduction
缺血性心脏病, 包括心肌梗死 (mi), 是全世界死亡的主要原因1。干细胞治疗再生死亡心肌仍然是改善心肌梗死心脏 2,3,4, 5 的主要途径。不同类型的干细胞被用来补充死亡心肌和改善心肌梗死的心功能。它们大致可分为胚胎干细胞6和成人干细胞.在成人干细胞中, 使用了各种类型的干细胞, 如骨髓源的单个核细胞7,8, 骨髓间充质干细胞 9,10, 脂肪组织11、12和脐带13和 csc14、15。干细胞可以通过内分泌和/或辅助作用促进心脏再生16,17,18, 19,20.然而, 干细胞治疗的一个主要局限性是获得足够数量的干细胞, 这些干细胞能够增殖和/或分化到特定的心脏谱系21,22。干细胞自体同种异体移植是干细胞治疗中的一个重要挑战。csc 可能是心脏再生的更好方法, 因为它们来自心脏, 它们比非心脏干细胞更容易分化为心脏谱系。因此, 它降低了畸胎瘤的风险。此外, csc 的内分泌和副分泌效应, 如来自 csc 的外显子和 mirna, 可能比其他类型的干细胞更有效。因此, csc 仍然是心脏再生23,24 的更好选择。
尽管 csc 由于其心脏源, 是心肌心脏再生的较好候选, 但 csc 的一个主要限制是由于缺乏有效的隔离方法而导致的产量较低。另一个限制因素可能是 csc 向心肌细胞谱系 2、25、26、27 的分化受损。为了避免这些限制, 重要的是要为 csc 的分离、表征和向心脏谱系的分化制定一个有效的协议。csc 没有单一的可接受标记, 基于细胞表面标记的 csc 隔离可产生较少的 csc。在这里, 我们标准化了一种简单的梯度离心方法, 将 csc 从鼠标心脏中分离出来, 该方法具有成本效益, 并提高了 csc 的产量。这些孤立的 csc 可以选择特定的细胞表面标记通过荧光激活细胞短路。除了 csc 分离外, 我们还提供了 csc 培养、表征和向心肌细胞谱系分化的协议。因此, 我们提出了一种很好的方法来隔离、表征、培养和区分 csc 和成人鼠标心脏 (图 5)。
Protocol
小鼠的住房、麻醉和牺牲是按照内布拉斯加州大学医学中心批准的 iacuc 协议进行的。 1. 材料 使用在机构动物设施内部饲养的10至12周的 c57bl/6j 黑雄性小鼠, 对 csc 进行隔离, 还可以从非怀孕的雌性小鼠中分离。 消毒所有必要的手术器械, 包括手术剪刀, 精细的手术剪刀, 弯曲的小腿钳, 和手术刀片, 通过高压灭菌他们之前, 鼠标安乐死的小鼠。 在灭菌条件?…
Representative Results
在本研究中, 我们分离 csc 从10至12周的 c57bl/6j 雄性小鼠心脏。在这里, 我们提出了一个简单的方法, csc 隔离和表征使用标记的多能性。我们还提出了一个优雅的方法 csc 分化和描述的 csc 分化为心肌细胞谱系。我们在相对照显微镜下观察到2至3天培养的 csc 的纺锤形形态 (图 1 a 和1 b)。我们发现在维持培养基中培养7天的时候, csc 的形态发生?…
Discussion
此 csc 隔离协议的关键步骤如下所示。1) 从小鼠身上提取心脏时, 必须保持灭菌状态。心脏提取过程中的任何污染都可能损害 csc 的质量. 2) 在切碎心脏之前, 必须完全切除血液, 这是通过用 hbss 溶液对整个心脏和心脏碎片进行几次清洗来完成的。3) 心脏碎片必须完全裂解成单细胞悬浮液与胶原酶溶液。4) 用于分离细胞的多蔗糖和二甲酸钠梯度溶液必须预热。5) csc 培养的介质必须预热。6) 在培养皿中…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家卫生研究院向 paras kumar mishra 提供的 hl-113281 和 hl116205 赠款的部分支持。
Materials
| Mice | The Jackson laboratory, USA | Stock no. 000664 | |
| Antibodies: | |||
| OCT4- | Abcam | ab18976 (rabbit polyclonal) | OCT4-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution |
| SOX2 | Abcam | ab97959 (rabbit polyclonal) | SOX2-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution |
| Nanog | Abcam | ab80892 (rabbit polyclonal) | Nanog-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution |
| Ki67 | Abcam | ab16667 (rabbit polyclonal) | Ki67-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution |
| Sca I | Millipore | AB4336 (rabbit polyclonal) | Sca I Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution |
| NKX2-5 | Santa Cruz | sc-8697 (goat polyclonal) | NKX2-5-Primary antibody- 1:50 dilution, Secondar antibody- 1:200 dilution, in blocking solution |
| GATA4 | Abcam | ab84593 (rabbit polyclonal) | GATA4-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution |
| MEF2C | Santa Cruz | sc-13268 (goat polyclonal) | MEF2C-Primary antibody- 1:50 dilution, Secondar antibody- 1:200 dilution, in blocking solution |
| Troponin I | Millipore | MAB1691 (mouse monoclonal) | Troponin I-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution |
| Actinin | Millipore | MAB1682 (mouse monoclonal) | Actinin-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution |
| ANP | Millipore | AB5490 (mouse polyclonal) | ANP-Primary antibody- 1:100 dilution, Secondar antibody- 1:200 dilution, in blocking solution |
| Alex Fluor-488 checken anti-rabbit | Life technology | Ref no. A21441 | |
| Alex Fluor-594 goat anti-rabbit | Life technology | Ref no. A11012 | |
| Alex Fluor-594 rabbit anti-goat | Life technology | Ref no. A11078 | |
| Alex Fluor-488 checken anti-mouse | Life technology | Ref no. A21200 | |
| Alex Fluor-594 checken anti-goat | Life technology | Ref no. A21468 | |
| Name | Company | Catalog Number | Comments |
| Culture medium: | |||
| CSC maintenance medium | Millipore | SCM101 | Note: For CSC culture, PBS or incomplete DMEM medium was used for washing the cells |
| cardiomyocytes differentiation medium | Millipore | SCM102 | |
| DMEM | Sigma-Aldrich | D5546 | |
| Name | Company | Catalog Number | Comments |
| Cell Isolation buffer: | |||
| polysucrose and sodium diatrizoate solution (Histopaque1077) | Sigma | 10771 | |
| HBSS | Gibco | 2018-03 | |
| Collagenase I | Sigma | C0130 | |
| Dispase solution | STEMCELL Technologies | 7913 | |
| PBS | LONZA | S1226 | |
| StemPro Accutase Cell Dissociation Reagent | Thermoscientific | A1110501 | |
| Other reagents: | |||
| BSA | Sigma | A7030 | |
| Normal checken serum | Vector laboratory | S3000 | |
| DAPI solution | Applichem | A100,0010 | Dapi, working concentration-1 µg/mL |
| Trypan blue | Biorad | 145-0013 | |
| Trypsin | Sigma | T4049 | |
| StemPro Accutase Cell Dissociation Reagent | Thermo Fisher Scientific | A1110501 | |
| Formaldehyde | Sigma | 158127 | |
| Triton X-100 | ACROS | Cas No. 900-293-1 | |
| Tween 20 | Fisher Sceintific | Lot No. 160170 | |
| Ethanol | Thermo Scientific | ||
| Name | Company | Catalog Number | Comments |
| Tissue culture materials: | |||
| 100 mm petri dish | Thermo Scientific | ||
| 6-well plate | Thermo Scientific | ||
| 24-well plate | Thermo Scientific | ||
| T-25 flask | Thermo Scientific | ||
| T-75 flask | Thermo Scientific | ||
| 15 ml conical tube | Thermo Scientific | ||
| 50 mL conical tube | Thermo Scientific | ||
| 40 µm cell stainer | Fisher Scientific | 22363547 | |
| 100 µm cell stainer | Fisher Scientific | 22363549 | |
| 0.22 µm filter | Fisher Scientific | 09-719C | |
| 10 mL syring | BD | Ref no. 309604 | |
| 10 µL, 200 µL, 1000 µL pipette tips | Fisher Scientific | ||
| 5 mL, 10mL, 25 mL disposible plastic pipette | Thermo Scientific | ||
| Name | Company | Catalog Number | Comments |
| Instruments | |||
| Centrufuge machine | Thermo Scientific | LEGEND X1R centrifuge | |
| EVOS microscope | Life technology | ||
| Automated cell counter | Biorad | ||
| Cell counting slide | Biorad | 145-0011 | |
| Pippte aid | Thermo Scientific | S1 pipet filler | |
| Name | Company | Catalog Number | Comments |
| Surgical Instruments: | |||
| Surgical scissors | Fine Scientific Tool | ||
| Fine surgical scissors | Fine Scientific Tool | ||
| Curve shank forceps | Fine Scientific Tool | ||
| Surgical blade | Fine Scientific Tool |
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Cite This Article
Yadav, S. K., Mishra, P. K. Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart. J. Vis. Exp. (143), e58448, doi:10.3791/58448 (2019).