Name: ex vivo expansion and genetic manipulation of mouse hematopoietic stem cells in polyvinyl alcohol-based cultures
Uploaded: 2023-02-10T13:45:00
Description: Watch this Scientific Journal Video about Ex Vivo Expansion and Genetic Manipulation of Mouse Hematopoietic Stem Cells in Polyvinyl Alcohol-Based Cultures at JoVE.com

我们感谢用于流式细胞术访问的WIMM流式细胞术核心，以及用于慢病毒载体生成的WIMM病毒筛选核心。这项工作由Kay Kendall白血病基金和英国医学研究委员会资助。

所有动物程序，包括繁殖和安乐死，都必须在机构和国家准则范围内进行。下面详述的实验已获得英国内政部的批准。有关本协议中使用的所有材料、试剂和设备的列表，请参阅 材料表 。
1. 准备储备溶液
<ol><li>聚苯乙烯原液解决方案<ol><li>取50 mL组织培养质量水放入小玻璃瓶中（适合高压灭菌）。用微波炉将水加热至接近沸腾。</li>
		<li>称出5克PVA?...

<ol>
	<li>Laurenti, E., G&#246;ttgens, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+haematopoietic+stem+cells+to+complex+differentiation+landscapes.">From haematopoietic stem cells to complex differentiation landscapes.</a> Nature. 553 (7689), 418-426 (2018).</li><li>Eaves, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem+cells:+concepts,+definitions,+and+the+new+reality.">Hematopoietic stem cells: concepts, definitions, and the new reality.</a> Blood. 125 (17), 2605-2613 (2015).</li><li>Wilkinson, A. C., Igarashi, K. J., Nakauchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haematopoietic+stem+cell+self-renewal+in+vivo+and+ex+vivo.">Haematopoietic stem cell self-renewal in vivo and ex vivo.</a> Nature Reviews Genetics. 21 (9), 541-554 (2020).</li><li>Crane, G. M., Jeffery, E., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+haematopoietic+stem+cell+niches.">Adult haematopoietic stem cell niches.</a> Nature Reviews Immunology. 17 (9), 573-590 (2017).</li><li>Osawa, M., Hanada, K., Hamada, H., Nakauchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+lymphohematopoietic+reconstitution+by+a+single+CD34-low/negative+hematopoietic+stem+cell.">Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell.</a> Science. 273 (5272), 242-245 (1996).</li><li>Granot, N., Storb, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=History+of+hematopoietic+cell+transplantation:+challenges+and+progress.">History of hematopoietic cell transplantation: challenges and progress.</a> Haematologica. 105 (12), 2716-2729 (2020).</li><li>Wilkinson, A. C., Nakauchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stabilizing+hematopoietic+stem+cells+in+vitro.">Stabilizing hematopoietic stem cells in vitro.</a> Current Opinion in Genetics &amp; Development. 64, 1-5 (2020).</li><li>Wilkinson, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+ex+vivo+haematopoietic-stem-cell+expansion+allows+nonconditioned+transplantation.">Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation.</a> Nature. 571 (7763), 117-121 (2019).</li><li>Nishimura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+polyvinyl+alcohol+for+chimeric+antigen+receptor+T-cell+expansion.">Use of polyvinyl alcohol for chimeric antigen receptor T-cell expansion.</a> Experimental Hematology. 80, 16-20 (2019).</li><li>Becker, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+cell+cloning+platform+for+gene+edited+functional+murine+hematopoietic+stem+cells.">A single cell cloning platform for gene edited functional murine hematopoietic stem cells.</a> bioRxiv. , (2022).</li><li>Kobayashi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+optimization+enables+maintenance+of+quiescent+hematopoietic+stem+cells+ex+vivo.">Environmental optimization enables maintenance of quiescent hematopoietic stem cells ex vivo.</a> Cell Reports. 28 (1), 145-158 (2019).</li><li>Kobayashi, H., Takubo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+culture+method+to+maintain+quiescent+human+hematopoietic+stem+cells.">A culture method to maintain quiescent human hematopoietic stem cells.</a> Journal of Visualized Experiments. (171), e61938 (2021).</li><li>Aal Wilson, ., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem+cells+reversibly+switch+from+dormancy+to+self-renewal+during+homeostasis+and+repair.">Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.</a> Cell. 135 (6), 1118-1129 (2008).</li><li>Ochi, K., Morita, M., Wilkinson, A. C., Iwama, A., Yamazaki, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-conditioned+bone+marrow+chimeric+mouse+generation+using+culture-based+enrichment+of+hematopoietic+stem+and+progenitor+cells.">Non-conditioned bone marrow chimeric mouse generation using culture-based enrichment of hematopoietic stem and progenitor cells.</a> Nature Communications. 12 (1), 3568 (2021).</li><li>Wilkinson, A. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+in+vitro+expanded+hematopoietic+stem+cells.">Identification and characterization of in vitro expanded hematopoietic stem cells.</a> EMBO Reports. 23 (10), e55502 (2022).</li><li>Zhang, Q., Konturek-Ciesla, A., Yuan, O., Bryder, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+expansion+potential+of+murine+hematopoietic+stem+cells:+a+rare+property+only+partially+predicted+by+phenotype.">Ex vivo expansion potential of murine hematopoietic stem cells: a rare property only partially predicted by phenotype.</a> bioRxiv. , (2022).</li><li>Schindele, P., Wolter, F., Puchta, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR+guide+RNA+design+guidelines+for+efficient+genome+editing.">CRISPR guide RNA design guidelines for efficient genome editing.</a> Methods in Molecular Biology. 2166, 331-342 (2020).</li><li>Hanna, R. 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C., Ishida, R., Nakauchi, H., Yamazaki, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+ex+vivo+expansion+of+mouse+hematopoietic+stem+cells.">Long-term ex vivo expansion of mouse hematopoietic stem cells.</a> Nature Protocols. 15 (2), 628-648 (2020).</li><li>Ieyasu, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+all-recombinant+protein-based+culture+system+specifically+identifies+hematopoietic+stem+cell+maintenance+factors.">An all-recombinant protein-based culture system specifically identifies hematopoietic stem cell maintenance factors.</a> Stem Cell Reports. 8 (3), 500-508 (2017).</li></ol>

我们希望该协议为更普遍地研究HSC生物学，造血和血液学提供有用的方法。自从最初开发用于FACS纯化的HSC8的基于PVA的培养方法以来，该方法已经扩展。例如，该方法已被证明适用于富含骨髓的c-Kit和负表面带电板14。它与转导和电穿孔的相容性也已得到证实14，15。这些HSC和c-Kit+ HSPC培养物的体内验证可...

造血系统通过专门的血液和免疫细胞类型支持哺乳动物的一系列基本过程，从氧气供应到对抗病原体。需要持续的血液生成（造血）来支持血液系统稳态，而血液系统稳态由造血干细胞和祖细胞（HSPC）维持1。最原始的造血细胞是造血干细胞（HSC），它具有独特的自我更新和多谱系分化能力2，3。这是一种罕见的细胞群，主要存在于成人骨髓4中，它们以大约每30，000个细胞中一个的频率发生。HSC被认为支持终生造血，并有助于在血液应激后重建造血。这些能力还允许HSC在移植到受辐照的受体后稳定地重建整个造血系统5。这代表了HSC的功能定义，也构成了HSC移植疗法的科学基础，HSC移植疗法是一系列血液和免疫疾病的治愈性治疗方法6。由于这些原因，HSC是实验血液学的主要焦点。
尽管研究重点很大，但稳定地将HSCs从体外扩增7仍然具有挑战性。我们最近开发了第一个用于小鼠HSC8的长期离体扩增培养系统。该方法可以在 4 周培养中将可移植 HSC 扩增 234-899 倍。与其他方法相比，该方案的主要变化是去除血清白蛋白并用合成聚合物代替。聚乙烯醇（PVA）被确定为小鼠HSC培养物8的最佳聚合物，现在也已用于培养其他造血细胞类型9。然而，最近还发现了另一种名为Soluplus（聚乙烯基己内酰胺-醋酸酯-聚乙二醇接枝共聚物）的聚合物，它似乎可以改善克隆HSC扩增10。在使用聚合物之前，使用胎牛血清、牛血清白蛋白级分V或重组血清白蛋白形式的血清白蛋白，但这些对HSC扩增的支持有限，仅支持短期（~1周）离体培养7。然而，应该注意的是，将HSC保持在静止状态的HSC培养方案可以支持更长的离体培养时间11，12。
与其他培养方法相比，基于PVA的培养物的一个主要优点是可以产生的细胞数量以及该方案可用于体外跟踪HSC的时间长度。这克服了实验血液学领域的几个障碍，例如每只小鼠可分离的HSC数量少（只有几千个）以及难以在体内随时间追踪HSC。然而，重要的是要记住，这些培养物刺激HSC增殖，而体内HSC池主要处于稳定状态13。此外，尽管培养物对HSC具有选择性，但随着时间的推移，其他细胞类型确实会随着培养物而积累，并且可移植HSC在1个月后仅代表约34个细胞中的一个。髓系造血祖细胞似乎是这些HSC培养物中的主要污染细胞类型8。然而，我们可以使用这些培养物来富集来自异质细胞群的HSC（例如，c-Kit+骨髓HSPCs14）。它还支持用于遗传操作的HSC的转导或电穿孔14，15，16。为了帮助从异质培养的HSPC群体中鉴定HSC，CD201（EPCR）最近被确定为有用的离体HSC标志物10，17，18，可移植HSC仅限于CD201 + CD150 + c-Kit+ Sca1 + Lineage-部分。
该协议描述了启动，维持和评估基于PVA的小鼠HSC扩增培养的方法，以及使用电穿孔或慢病毒载体转导在这些培养物中进行遗传操作的方案。这些方法有望对一系列实验血液学家有用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection kit</td>
			<td>Fisher Scientific</td>
			<td>12764416</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Appleton Woods Ltd</td>
			<td>HC002</td>
			<td></td>
		</tr>
		<tr>
			<td>P3 Primary Cell 4D-Nucleofector X Kit</td>
			<td>Lonza&#160;</td>
			<td>V4XP-3024</td>
			<td></td>
		</tr>
		<tr>
			<td>Pestle and mortar</td>
			<td>Scientific Laboratory Supplies Limited</td>
			<td>X18000</td>
			<td></td>
		</tr>
		<tr>
			<td>QuadroMACS separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-976</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL syringe</td>
			<td>VWR International Ltd</td>
			<td>720-2519</td>
			<td></td>
		</tr>
		<tr>
			<td>19 G needle</td>
			<td>VWR International Ltd</td>
			<td>613-5394</td>
			<td></td>
		</tr>
		<tr>
			<td>50 &#956;m cell strainer</td>
			<td>Sysmex</td>
			<td>04-004-2317</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m cell strainer</td>
			<td>Corning</td>
			<td>431751</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimtech wipes</td>
			<td>VWR International Ltd</td>
			<td>115-2075</td>
			<td></td>
		</tr>
		<tr>
			<td>LS MACS column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alt-R S.p. Cas9 Nuclease V3, 100 &#956;g</td>
			<td>IDT&#160;</td>
			<td>1081058</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal free recombinant mouse stem cell factor&#160;</td>
			<td>Peprotech</td>
			<td>AF-250-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal free recombinant mouse thrombopoietin</td>
			<td>Peprotech</td>
			<td>AF-315-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD117 APC (clone: 2B8)</td>
			<td>ThermoFisher</td>
			<td>17-1171-83</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD117 BV421 (clone: 2B8)</td>
			<td>Biolegend</td>
			<td>105828</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD127&#160;APC/Cy7 (clone: A7R34)</td>
			<td>Biolegend</td>
			<td>135040</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD127 biotin (clone: A7R34)</td>
			<td>Biolegend</td>
			<td>135006</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD150 PE/Cy7 (clone: TC15-12F12.2)</td>
			<td>Biolegend</td>
			<td>115914</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD201&#160;APC (clone: eBio1560)</td>
			<td>ThermoFisher</td>
			<td>17-2012-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD34 FITC (clone: RAM34)</td>
			<td>ThermoFisher</td>
			<td>11-0341-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD4&#160;APC/Cy7 (clone: RM4-5)</td>
			<td>Biolegend</td>
			<td>100526</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD4 biotin (clone: RM4-5)</td>
			<td>Biolegend</td>
			<td>100508</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD45R&#160;APC/Cy7 (clone: RA3-6B2)</td>
			<td>Biolegend</td>
			<td>103224</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD45R biotin (clone: RA3-6B2)</td>
			<td>Biolegend</td>
			<td>103204</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD48 BV421 (clone: HM48-1)</td>
			<td>Biolegend</td>
			<td>103428</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD8 biotin (clone: 53-6.7)</td>
			<td>Biolegend</td>
			<td>100704</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD8a&#160;APC/Cy7 (clone: 53-6.7)</td>
			<td>Biolegend</td>
			<td>100714</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse Ly6G/Ly6C&#160;APC/Cy7 (clone: RB6-8C5)</td>
			<td>Biolegend</td>
			<td>108424</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse Ly6G/Ly6C biotin (clone: RB6-8C5)</td>
			<td>Biolegend</td>
			<td>108404</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse Sca1 PE (clone: D7)</td>
			<td>Biolegend</td>
			<td>108108</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse Ter119&#160;APC/Cy7 (clone: TER-119)</td>
			<td>Biolegend</td>
			<td>116223</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse Ter119 biotin (clone: TER-119)</td>
			<td>Biolegend</td>
			<td>116204</td>
			<td></td>
		</tr>
		<tr>
			<td>CellBIND plates, 24-well</td>
			<td>Corning</td>
			<td>3337</td>
			<td>negative surface charged</td>
		</tr>
		<tr>
			<td>CellBIND plates, 96-well&#160;</td>
			<td>Corning</td>
			<td>3330</td>
			<td>negative surface charged</td>
		</tr>
		<tr>
			<td>Custom synthetic sgRNA&#160;</td>
			<td>Synthego, Sigma Aldrich, IDT</td>
			<td>Custom order</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Merck Life Science UK Limited</td>
			<td>F7524-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin Coated plates, 96-well</td>
			<td>BD Biosciences</td>
			<td>354409</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#39;s F-12 Nutrient Mix</td>
			<td>Gibco</td>
			<td>11765054</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium-X (100x)</td>
			<td>Gibco</td>
			<td>51500.056</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Alfa Aesar</td>
			<td>J61196.AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinyl alcohol</td>
			<td>Sigma Aldrich</td>
			<td>P8136</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Enzo Life Sciences (UK) Ltd</td>
			<td>EXB-0018</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin APC/Cy7</td>
			<td>Biolegend</td>
			<td>405208</td>
			<td></td>
		</tr>
		<tr>
			<td>T&#252;rks&#8217; solution</td>
			<td>Sigma Aldrich</td>
			<td>109277</td>
			<td></td>
		</tr>
		<tr>
			<td>Virkon</td>
			<td>Mettler-Toledo Ltd</td>
			<td>95015662</td>
			<td></td>
		</tr>
	</tbody>
</table>

ex vivo expansion and genetic manipulation of mouse hematopoietic stem cells in polyvinyl alcohol-based cultures

自我更新的多能造血干细胞（HSC）是一种重要的细胞类型，因为它们能够在一生中支持造血并在移植后重建整个血液系统。HSC在临床上用于干细胞移植疗法，代表了一系列血液疾病的治愈性治疗。人们对了解调节HSC活性和造血的机制以及开发新的基于HSC的疗法都非常感兴趣。然而，HSCs离体的稳定培养和扩增一直是在易于处理的离体系统中研究这些干细胞的主要障碍。我们最近开发了一种基于聚乙烯醇的培养系统，该系统可以支持可移植小鼠HSC的长期和大规模扩增以及对其进行基因编辑的方法。该协议描述了通过电穿孔和慢病毒转导培养和遗传操作小鼠HSC的方法。该协议有望对对HSC生物学和造血感兴趣的广泛实验血液学家有用。

对于HSC的FACS纯化，我们预计在富含c-Kit的骨髓中，~0.2%的细胞是年轻（8-12周龄）C57BL / 6小鼠的CD150 + CD34-c-Kit+Sca1 + Lineage-群体（图1）。然而，转基因小鼠或不同年龄的小鼠可能表现出不同的HSC频率。培养4周后，我们预计CD201 + CD150 + c-Kit+ Sca1 + 谱系-分数为~10%（图2）。这?...

Watch this Scientific Journal Video about Ex Vivo Expansion and Genetic Manipulation of Mouse Hematopoietic Stem Cells in Polyvinyl Alcohol-Based Cultures at JoVE.com

Ex Vivo Expansion and Genetic Manipulation of Mouse Hematopoietic Stem Cells in Polyvinyl Alcohol-Based Cultures

这里介绍的是使用 离体 聚乙烯醇基扩增启动、维持和分析小鼠造血干细胞培养物的方案，以及通过慢病毒转导和电穿孔进行遗传操作的方法。

离体 聚乙烯醇基培养物中小鼠造血干细胞的扩增和遗传操作

Self-renewing&#160;multipotent&#160;hematopoietic stem cells (HSCs) are an important cell type due to their abilities to support hematopoiesis throughout ...

该协议允许功能性小鼠造血干细胞的扩增和遗传操作，从而更深入地了解造血干细胞生物学和造血。该技术的优点是它可以支持造血干细胞进行长期离体培养，并允许遗传操作来询问造血的遗传学。该培养系统为造血干细胞生物学和造血的各种研究提供了平台技术，包括遗传、分子和生化研究。疑难解答表列出了使用此技术遇到的常见难题的原因和解决方案。展示如何正确提取骨髓细胞并进行完整的培养基更换比阅读它要容易得多，也更有效。要开始造血干细胞或HSC提取，请使用不起毛的精致工作湿巾清洁从适当安乐死的小鼠中新鲜分离的骨骼。从骨骼中去除肌肉和脊髓，然后将它们添加到含有约三毫升PBS的研钵中。使用杵将骨头压碎而不研磨，以尽量减少剪切力。然后，使用连接到5毫升注射器的19号针头，分解释放到PBS中的大骨髓碎片，并通过70微米过滤器将悬浮液转移到50毫升锥形管中。转移悬浮液后，用新鲜的PBS重复该过程，直到骨骼漂白。目标是每只小鼠的最终体积约为30毫升，两只小鼠的最终体积为50毫升，以漂白骨骼，直到看不到更多的骨髓。通过将磁性过滤柱放入磁性柱分离器的磁铁中来准备色谱柱富集，顶部有一个 50 微米的过滤器，下面有一个 15 毫升的锥形管。接下来，用别藻蓝蛋白抗c-Kit抗体和别藻蓝蛋白磁性微珠处理细胞后，通过50微米过滤器和过滤柱运行三毫升无菌PBS。一旦PBS通过，将细胞悬液通过色谱柱，然后每次洗涤三次三毫升冷PBS。每次洗涤后，等待色谱柱停止滴落，然后再添加PBS进行下一次洗涤。接下来，从磁铁上取下色谱柱并将其放在新鲜的15毫升管顶部，并在色谱柱中加入5毫升冷PBS，然后将色谱柱塞安装到其上并通过推动柱塞洗脱细胞。按照文中描述的方案制备足够的HSC培养基，用于所需数量的细胞或孔，以每毫升接种0.5至100万个细胞。然后旋转富含c-Kit的造血干细胞和祖细胞或HSPC，并以所需的细胞密度将沉淀重悬于新鲜制备的HSC培养基中。将细胞转移到纤连蛋白包被或负表面带电的板上，保持适当的体积。将细胞置于37摄氏度和5%二氧化碳的组织培养箱中。在不干扰细胞培养物的情况下从组织培养箱中轻轻取出板后，使用移液器或真空泵从每个孔的液体弯月面中缓慢吸出约90%至95%的培养基。避免从孔底部抽取培养基。然后将一毫升预热至37摄氏度的新鲜培养基加入孔中。将板放回组织培养箱，每两到三天更换一次培养基，直到实验需要。将细胞重悬于核转染缓冲液中后，立即将细胞悬液转移到含有络合核糖核蛋白的PCR管中，并通过缓慢上下移液轻轻混合。现在非常缓慢地将混合物转移到适当体积的电穿孔比色皿中，保持连续的流体运动，以避免在比色皿内形成气泡。然后，在电穿孔机上，选择被电穿孔孔的位置。使用触摸屏，选择细胞类型程序为 CD34 阳性人或输入脉冲代码 EO100，然后按 OK 按钮。将比色皿转移到电穿孔器上，然后按触摸屏上的“开始”按钮启动电穿孔。电穿孔后，立即向比色皿中加入培养基反应体积的五倍。现在轻轻地将细胞转移到准备好的板上，然后将板放回组织培养箱中。如前所述进行培养基更换后，收集10微升细胞并使用Turk溶液用血细胞计数器以1：2的稀释度计数。将所需剂量的细胞重新铺在纤连蛋白包被的平板中进行转导，并分别铺板未转导的阴性对照细胞。然后将慢病毒载体添加到每个含有细胞的孔中，保持每个细胞20个转导单位的剂量，以达到约30%的转导效率。但是，根据实验要求根据经验确定慢病毒载体剂量。最后，将细胞放回组织培养箱中6小时。如前所述执行介质更改。富含c-Kit的HSC的荧光激活细胞分选产生了约0.2%的CD150阳性，CD34阴性，cKit阳性，Sca1阳性和谱系阴性的细胞。HSPC培养四周后，发现CD201阳性，CD150阳性，c-Kit阳性，Sca1阳性和谱系阴性部分约为10%用表达GFP的慢病毒载体转导后，GFP阳性细胞约为30%当汇合时，培养物的预期密度约为每毫升200万个细胞。在进行第一次培养基更换的前24至48小时内观察到大约50%的细胞死亡。然而，一周后，细胞数量恢复到接种细胞数的80%至100%。使用新鲜和预热培养基进行准确的培养基更换对于这种HSC培养方法的长期稳定性至关重要。自2019年原始方法发表以来，该领域已开始以不同的方式使用它来研究HSC生物学。

Research

JoVE Journal

Biology

Ex Vivo Expansion and Genetic Manipulation of Mouse Hematopoietic Stem Cells in Polyvinyl Alcohol-Based Cultures

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

分离和造血干细胞移植（造血干细胞）

科研

生物学

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

从小鼠胚胎干细胞的造血干细胞的推导

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Method for Culture of Early Chick Embryos ex vivo (New Culture)

Method for Culture of Early Chick Embryos ex vivo New Culture

文化早期鸡胚体外（新文化）的方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

慢病毒介导的击倒在体外</em人造血干细胞>红细胞

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

表型分析和分离小鼠造血干细胞和传承的承诺祖

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

另类文化的人多能干细胞的生产，维护和遗传分析

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

代谢谱的实时分析<em&gt;离体</em&gt;鼠标肠隐窝类器官培养

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

测量的刚度<em&gt;离体</em&gt;鼠标主动脉用原子力显微镜

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

胚胎干细胞的跨内细胞质量注射导致嵌合体率增高

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

离体 聚乙烯醇基培养物中小鼠造血干细胞的扩增和遗传操作

离体聚乙烯醇基培养物中小鼠造血干细胞的扩增和遗传操作