Name: pan-myeloid differentiation of human cord blood derived cd34+ hematopoietic stem and progenitor cells
Uploaded: 2019-08-09T14:45:00
Description: Watch this Scientific Journal Video about Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells at JoVE.com

作者要感谢温迪·巴雷特、雷切尔·卡瓦列罗和马里科帕综合卫生系统的加布里埃拉·鲁伊斯为脱认和捐献脐带血单位,姆利纳利尼·卡拉为流动细胞学提供帮助,盖伊·克鲁克斯和克里斯托弗·西特关于前体骨髓分化的建议。这项工作得到了美国国家卫生研究院(R21CA170786和R01GM127464)和美国癌症协会(机构研究补助金74-001-34-IRG)的资助。内容完全由作者负责,不一定代表国家卫生研究院的官方观点。

用于实验的人类脐带血是由健康人捐赠后,知情同意马里科帕综合卫生系统(MIHS),凤凰城。被除名的单位是通过MIHS和亚利桑那大学之间的材料转移协议获得的。
1. 试剂和缓冲液
注:在无菌条件下在生物安全柜中制备所有试剂和缓冲液。
<ol><li>通过加入7.2 mL的无菌35%牛血清白蛋白(BSA;使用无菌组织培养等级35%BSA)和5 mL无菌0.5M乙烯二胺四乙酸(EDTA)?...

<ol>
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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=The+U2AF1S34F+mutation+induces+lineage-specific+splicing+alterations+in+myelodysplastic+syndromes.">The U2AF1S34F mutation induces lineage-specific splicing alterations in myelodysplastic syndromes.</a> Journal of Clinical Investigation. 127 (6), 2206-2221 (2017).</li><li>Yoo, E. S., 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=Myeloid+differentiation+of+human+cord+blood+CD34++cells+during+ex+vivo+expansion+using+thrombopoietin,+flt3-ligand+and/or+granulocyte-colony+stimulating+factor.">Myeloid differentiation of human cord blood CD34+ cells during ex vivo expansion using thrombopoietin, flt3-ligand and/or granulocyte-colony stimulating factor.</a> British Journal of Haematology. 105 (4), 1034-1040 (1999).</li><li>Hao, Q. L., Smogorzewska, E. M., Barsky, L. W., Crooks, G. 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Y., 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=Enforced+expression+of+an+Flt3+internal+tandem+duplication+in+human+CD34++cells+confers+properties+of+self-renewal+and+enhanced+erythropoiesis.">Enforced expression of an Flt3 internal tandem duplication in human CD34+ cells confers properties of self-renewal and enhanced erythropoiesis.</a> Blood. 105 (1), 77-84 (2005).</li><li>Ambrosini, P., 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=IL-1beta+inhibits+ILC3+while+favoring+NK-cell+maturation+of+umbilical+cord+blood+CD34(+)+precursors.">IL-1beta inhibits ILC3 while favoring NK-cell maturation of umbilical cord blood CD34(+) precursors.</a> European Journal of Immunology. 45 (7), 2061-2071 (2015).</li><li>Batard, P., 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=TGF-(beta)1+maintains+hematopoietic+immaturity+by+a+reversible+negative+control+of+cell+cycle+and+induces+CD34+antigen+up-modulation.">TGF-(beta)1 maintains hematopoietic immaturity by a reversible negative control of cell cycle and induces CD34 antigen up-modulation.</a> Journal of Cell Science. 113, 383-390 (2000).</li><li>Huang, N., Lou, M., Liu, H., Avila, C., Ma, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+potent+small+molecule+capable+of+regulating+polyploidization,+megakaryocyte+maturation,+and+platelet+production.">Identification of a potent small molecule capable of regulating polyploidization, megakaryocyte maturation, and platelet production.</a> Journal of Hematology &amp; Oncology. 9 (1), 136 (2016).</li></ol>

此处所述的协议适用于 UCB 衍生 CD34和HSPC 到四个骨髓谱系的体外分化。由SCF、TPO、Flt3L和IL3组成的细胞因子混合物的初始孵育刺激CD34+细胞。随后,通过SCF、IL3、Flt3L、EPO和TPO的混合物实现了差异化。在此组合中,SCF、IL3 和 Flt3L 对 CD34+ HSC 的生存和增殖非常重要。 EPO 和 TPO 分别促进对红细胞和巨核细胞的分化,IL3 促进早期粒细胞单细胞的分化前体和成熟细胞13,17,18。<sup class="xr...

造血干细胞(HSCs)的正常分化对于维持所有血细胞系的生理水平至关重要。在分化期间,在协调响应细胞外线索(包括生长因子和细胞因子)时,HSCs 首先产生具有淋巴-骨髓电位1、2、3 的多能祖细胞 (MPP) 细胞 ,4 (图 1)。MpP 产生常见的骨髓祖体 (CmPs) 和常见的淋巴原代后代 (CLP) 受血统限制。CLP分化成由B、T和自然杀伤细胞组成的淋巴状谱系。CmPs 通过两个更受限的祖体群体(巨核细胞红细胞祖(MEPs)和粒细胞单细胞原代 (GMPs) 生成骨髓系。MEP产生巨核细胞和红细胞,而GMP产生粒细胞和单核细胞。除了通过CmPs产生,兆核细胞也报告直接产生于HC或早期的MpPs通过非规范途径5,6。
造血干细胞和祖细胞(HSPCs)的特点是表面标记CD34和缺乏系系特异性标记(Lin-)。其他通常用于区分 HSC 和骨髓祖体群体的表面标记包括 CD38、CD45RA 和 CD123 2(图1)。HSC 和 Mp 分别为林-/CD34+/CD38-和林-/CD34+/CD38+。骨髓已提交祖细胞群体由CD45RA和CD123的存在或不存在区分。CmP 是林-/CD34+/CD38+/CD45RA-/CD123 lo,GMP 是林-/CD34+/CD38+/CD45RA+/CD123lo,MEP是林-/CD34|/CD38=/CD45RA-/CD123-.
CD34+干细胞和祖细胞的总数可以从人类脐带血 (UCB)、骨髓和外周血获得。CD34–细胞占人类UCB中单核细胞总数的0.02%至1.46%,而骨髓中细胞的百分比在0.5%至5.3%之间,在外周血7、8、9中,其比例要低得多,为±0.01%.UCB衍生CD34+细胞的增殖能力和分化潜力明显高于骨髓或外周血细胞1、10,为获得足够的材料用于分子分析,结合在分化期间对细胞进行免疫表征和形态表征。
脐带血衍生CD34+HSPCs的体外分化是研究正常造血和造血病机制的一种广泛应用模型。当用适当的细胞因子培养时,UCB CD34+ HSPC 可诱导沿骨髓或淋巴体谱11、12、13、14、15进行分化,16.在这里,我们描述了CD34和HSPCs从人类UCB分离和免疫表征表表征,以及它们分化为骨髓体代细胞的协议。这种培养系统基于在MS-5基质细胞存在的情况下,以细胞因子诱导的HSPCs分化,以模拟骨髓中的微环境。培养条件导致CD34+细胞的初始扩张,随后分化为表达四个骨髓系细胞标记的细胞,即粒细胞(CD66b)、单核细胞(CD14)、巨核细胞(CD41)和红细胞(CD235a)。CD34®细胞分化协议的应用包括控制造血的分子机制研究,以及研究骨髓疾病相关突变和小分子对自我更新和HSPC的差异化。

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	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">0.4% Trypan blue solution</td>
			<td style="width:112px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15250-061</td>
			<td style="width:339px;">Dilute working stock to 0.2% in sterile 1x PBS</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">0.5 M UltraPure Ethylene diamine tetra acetic acid, pH 8.0</td>
			<td style="width:112px;">Gibco&#160;</td>
			<td style="width:119px;">15575-038</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">10x Hanks Balanced Salt Solution (HBSS)</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:119px;">14185052</td>
			<td style="width:339px;">Dilute to 1x with sterile distilled water &#38; pH to 7.2</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">2.5% Trypsin, no phenol red</td>
			<td style="width:112px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15090046</td>
			<td style="width:339px;">Dilute working stock to 1x with sterile 1x PBS</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">30 &#181;m Pre-separation filters</td>
			<td style="width:112px;">Miltenyi biotech</td>
			<td style="width:119px;">130-041-407</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">35% sterile Bovine serum albumin</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7979</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">7-AAD</td>
			<td style="width:112px;">Biolegend</td>
			<td style="width:119px;">420404</td>
			<td style="width:339px;">Used as a live/dead stain to eliminate dead cells from FACS analysis</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-human CD10-FITC antibody (Clone HI10a)</td>
			<td style="width:112px;">Biolegend</td>
			<td style="width:119px;">312207</td>
			<td style="width:339px;">Use 1:20 dilution</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Anti-human CD11b-FITC (activated) antibody (Clone CBRM1/5)</td>
			<td style="width:112px;">Biolegend</td>
			<td style="width:119px;">301403</td>
			<td style="width:339px;">Use 1:5 dilution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-human CD123-APC antibody (Clone 6H6)</td>
			<td style="width:112px;">Biolegend</td>
			<td style="width:119px;">306012</td>
			<td style="width:339px;">Use 1:20 dilution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-human CD14-PE antibody (Clone M5E2)</td>
			<td style="width:112px;">Biolegend</td>
			<td style="width:119px;">301806</td>
			<td style="width:339px;">Use 1:20 dilution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-human CD19-FITC antibody (Clone 4G7)</td>
			<td style="width:112px;">BD Biosciences</td>
			<td style="width:119px;">347543</td>
			<td style="width:339px;">Use 1:5 dilution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-human CD235a-APC antibody (Clone GA-R2 (HIR2))</td>
			<td style="width:112px;">BD Biosciences</td>
			<td style="width:119px;">551336</td>
			<td style="width:339px;">Use 1:20 dilution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-human CD235a-FITC antibody (Clone HIR2)</td>
			<td style="width:112px;">Biolegend</td>
			<td style="width:119px;">306609</td>
			<td style="width:339px;">Use 1:50 dilution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-human CD34-APC-Cy7 antibody (Clone 581)</td>
			<td style="width:112px;">Biolegend</td>
			<td style="width:119px;">343514</td>
			<td style="width:339px;">Use 1:20 dilution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-human CD38-PE antibody (Clone HIT2)</td>
			<td style="width:112px;">Biolegend</td>
			<td style="width:119px;">303506</td>
			<td style="width:339px;">Use 1:20 dilution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-human CD3-FITC antibody (Clone UCHT1)</td>
			<td style="width:112px;">Biolegend</td>
			<td style="width:119px;">300405</td>
			<td style="width:339px;">Use 1:20 dilution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-human CD41a-PerCP-Cy5.5 antibody (Clone HIP8)</td>
			<td style="width:112px;">Biolegend</td>
			<td style="width:119px;">303720</td>
			<td style="width:339px;">Use 1:20 dilution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-human CD45Ra-PE-Cy7 antibody (Clone HI100)</td>
			<td style="width:112px;">Biolegend</td>
			<td style="width:119px;">304126</td>
			<td style="width:339px;">Use 1:20 dilution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-human CD66b-PE-Cy7 antibody (Clone G10F5)</td>
			<td style="width:112px;">Biolegend</td>
			<td style="width:119px;">305116</td>
			<td style="width:339px;">Use 1:20 dilution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-human CD7-FITC antibody (Clone CD7-6B7)</td>
			<td style="width:112px;">Biolegend</td>
			<td style="width:119px;">343103</td>
			<td style="width:339px;">Use 1:20 dilution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:112px;">Fisher Scientific</td>
			<td style="width:119px;">BP231-100</td>
			<td style="width:339px;">Filter sterilize before use</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Dulbecco&#8217;s Modified Eagle Medium (DMEM) powder with L-Glutamine&#160;</td>
			<td style="width:112px;">Gibco</td>
			<td style="width:119px;">12100046</td>
			<td style="width:339px;">Reconstitute 1 packet to make 1 L of DMEM media&#160; with sodium bicarbonate, 10% FBS &#38; 1% penicillin &#38; streptomycin&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fetal bovine serum, Australian source, heat inactivated</td>
			<td style="width:112px;">Omega Scientific</td>
			<td style="width:119px;">FB-22 Lot #609716</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Human CD34 microbead kit&#160;</td>
			<td style="width:112px;">Miltenyi biotech</td>
			<td style="width:119px;">130-046-702</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Human Thrombopoietin (TPO), research grade</td>
			<td style="width:112px;">Miltenyi biotech</td>
			<td style="width:119px;">130-094-011</td>
			<td style="width:339px;">Make a stock of 100 &#181;g/mL in 1x PBS + 0.1% BSA. Use 50 ng/mL for both myeloid differentiation &#38; stimulation medium</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">L-Glutamine</td>
			<td style="width:112px;">Omega Scientific</td>
			<td style="width:119px;">GS-60</td>
			<td style="width:339px;">2 mM concentration in stimulation medium</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">LS Columns</td>
			<td style="width:112px;">Miltenyi biotech</td>
			<td style="width:119px;">130-042-401</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">MACS Multi stand</td>
			<td style="width:112px;">Miltenyi biotech</td>
			<td style="width:119px;">130-042-303</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">MidiMACS magnetic separator</td>
			<td style="width:112px;">Miltenyi biotech</td>
			<td style="width:119px;">130-042-302</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">MNC fractionation media (Ficol-Paque PLUS)</td>
			<td style="width:112px;">GE Healthcare Biosciences</td>
			<td style="width:119px;">17-1440-03</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">MS-5 cells</td>
			<td style="width:112px;"></td>
			<td style="width:119px;"></td>
			<td style="width:339px;">Gift from the laboratory of Gay Crooks, UCLA</td>
		</tr>
		<tr height="148">
			<td height="148" style="height:148px;width:216px;">Paraformaldehyde</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:119px;">P6148</td>
			<td style="width:339px;">Heat 800 mL of 1x PBS in a glass beaker on a stir plate in a chemical hood to ~65 &#176;C. Add 10 g of paraformaldehyde powder. To completely dissolve the paraformaldehyde, raise the pH by adding 1 N NaOH. Cool and filter the solution and make up the volume to 1 L with 1x PBS. Adjust the pH to 7.2.&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Penicillin &#38; Streptomycin</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:119px;">P4458-100ml</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Poly-L lysine</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:119px;">P2636</td>
			<td style="width:339px;">Make a 10 mg/mL stock in 1x PBS</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Recombinant human erythropoietin-alpha (rHu EPO-&#945;)</td>
			<td style="width:112px;">BioBasic</td>
			<td style="width:119px;">RC213-15</td>
			<td style="width:339px;">Make a stock of 2000 units/mL in 1x PBS + 0.1% BSA. Use 4 units/mL for myeloid differentiation</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Recombinant human fibronectin fragment (RetroNectin)</td>
			<td style="width:112px;">Takara&#160;</td>
			<td style="width:119px;">T100B</td>
			<td style="width:339px;">Use 20 &#181;g/mL diluted in sterile 1x PBS to coat wells prior to stimulation of CD34+ HSCs.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Recombinant human Flt-3 ligand (rHu Flt-3L)</td>
			<td style="width:112px;">BioBasic</td>
			<td style="width:119px;">RC214-16</td>
			<td style="width:339px;">Make a stock of 100 &#181;g/mL in 1x PBS + 0.1% BSA. Use 5 ng/mL for myeloid differentiation &#38; 50 ng/mL in stimulation medium</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Recombinant human interleukin-3 (rHu IL-3)</td>
			<td style="width:112px;">BioBasic</td>
			<td style="width:119px;">RC212-14</td>
			<td style="width:339px;">Make a stock of 100 &#181;g/mL in 1x PBS + 0.1% BSA. Use 5 ng/mL for myeloid differentiation &#38; 20 ng/mL in stimulation medium</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Recombinant human stem cell factor (rHu SCF)</td>
			<td style="width:112px;">BioBasic</td>
			<td style="width:119px;">RC213-12</td>
			<td style="width:339px;">Make a stock of 100 &#181;g/mL in 1x PBS + 0.1% BSA. Use 5 ng/mL for myeloid differentiation &#38; 50 ng/mL in stimulation medium</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Serum free medium (X-Vivo-15)</td>
			<td style="width:112px;">Lonza&#160;</td>
			<td style="width:119px;">04-418Q</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Sodium bicarbonate</td>
			<td style="width:112px;">Fisher Scientific</td>
			<td style="width:119px;">BP328-500</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Wright-Giemsa stain, modified</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:119px;">WG16-500</td>
			<td style="width:339px;">Use according to manufacturer&#39;s instructions</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Equipment&#160;</td>
			<td style="width:112px;"></td>
			<td style="width:119px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">BD LSR II flow cytometer</td>
			<td style="width:112px;">BD Biosciences</td>
			<td style="width:119px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Centrifuge</td>
			<td style="width:112px;">Sorvall Legend RT</td>
			<td style="width:119px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Light microscope</td>
			<td style="width:112px;">Olympus</td>
			<td style="width:119px;"></td>
			<td style="width:339px;"></td>
		</tr>
	</tbody>
</table>

pan-myeloid differentiation of human cord blood derived cd34+ hematopoietic stem and progenitor cells

人类造血干细胞的体外分化是研究造血干细胞的一个广泛应用的模型。此处描述的方案用于细胞因子诱导的CD34分化 -造血干细胞和祖细胞到四个骨髓体体细胞。CD34–细胞从人类脐带血中分离出来,在细胞因子存在的情况下与MS-5基质细胞共同培养。描述了干细胞和祖细胞的免疫表征特征,以及分化的骨髓体系细胞。使用该协议,CD34+细胞可以用小分子孵育,或用慢病毒转导,以表达骨髓疾病突变,以研究其对骨髓分化的影响。

应用上述协议可产生 5.6 (± 0.5) x 108 MDC 和 1 (± 0.3) x 106 CD34+来自脐带血单位 ±100 mL 的细胞。CD34+细胞总数的百分比在 80-90% 之间 (图 2A,B)。Manz等人描述方案的免疫表位分析表明,CD34+细胞通常由+20%的HSC和+72%的MpPs组成,即林-/CD34+/CD38-和林-/CD34|/CD38=分别 (<strong...

Watch this Scientific Journal Video about Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells at JoVE.com

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

在这里,我们提出了免疫表征表表征和细胞因子诱导分化脐带血衍生CD34-造血干细胞和祖细胞到四个骨髓系的方案。该协议的应用包括研究骨髓疾病突变或小分子对CD34+细胞骨髓分化的影响。

人脐带血衍生CD34的泛骨髓分化–造血干细胞和祖细胞

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine ...

该协议可用于研究人类CD34阳性造血干细胞和祖细胞的正常骨髓分化过程中发生的分子和细胞变化。该协议的主要优点是，它允许CD34正细胞沿着所有四个骨髓血统，包括巨核细胞，红细胞，粒细胞和单核细胞分化。演示程序将是阿迪提巴帕特，一个后医生从我的实验室。要从人体脐带血样本中分离出单核细胞，在将袋内脂肪转移到生物安全柜内的无菌 250 毫升塑料瓶中之前，用 70% 乙醇对一袋脐带血的外表面进行消毒。用同等体积的室温稀释血液汉克缓冲盐水溶液，并在五个无菌的50毫升锥形管中加入15毫升的单核细胞分馏介质。轻轻混合后，小心地将30毫升稀释的脐带血倒入每个管的单核分馏介质上，以一定角度握住50毫升管，以避免混合层。通过密度梯度离心分离细胞，并去除单核细胞层上方的15至20毫升等离子体。使用移液器小心地将暴露的单核细胞层转移到新的50毫升管中，将单核细胞从两个管汇集到三个管，并将每个管中的最终体积与冷PBE缓冲液一起带到50毫升。通过离心对单核细胞样品进行浸渍，并吸进超盐。轻轻敲击每个管以松开细胞颗粒，并在五毫升冰冷的PBE缓冲液中稀释颗粒。将三到四管的细胞组合成一个50毫升的圆锥管，用五毫升的新鲜PBE缓冲液清洗所有管，收集任何剩余的细胞。数数后，用新鲜的冰冷PBE缓冲液将管子中的最终体积调至50毫升，通过离心收集细胞。要分离CD34正单核细胞，在收集细胞进行另一离心之前，将颗粒重新悬浮在50毫升新鲜PBE缓冲液中。丢弃上清液后，轻轻敲击以松开颗粒，并在每300微升冰冷PBE缓冲液浓度的1倍10至8个单核细胞下重新悬浮。用100微升Fc受体阻断试剂进行阻断，轻轻混合，然后从磁性活性细胞分拣人类CD34微珠试剂盒中加入100微升CD34微珠，每10次加入8个细胞，轻轻混合，在4摄氏度下孵育细胞30分钟。当细胞孵育时，将LS柱和预分离滤波器放在MACS分离器磁场中。用两毫升的冰冷 PBE 缓冲液和一毫升缓冲液平衡柱和预分离过滤器。让柱空入 15 毫升锥形管中，然后丢弃流经。在孵育结束时，将单核细胞样本的最终体积与新鲜冰冷PBE缓冲液一起带到15毫升，通过离心沉淀细胞。将颗粒重新悬浮在一毫升冰冷的 PBE 缓冲液中，将电池加载到柱上的预分离过滤器上。用三毫升新鲜冰冷 PBE 缓冲液冲洗管子，并将缓冲液添加到预分离过滤器中。每次洗涤时，无需过滤器，则使用额外的三毫升冰冷 PBE 缓冲液清洗柱四次，使柱在每次洗涤之间完全排空。最后洗涤后，将柱移入新的 15 毫升管中，将 5 毫升新鲜冰冷 PBE 通过柱插入管中。要确定新鲜分离的CD34阳性造血干细胞和祖细胞中干细胞和祖细胞群的分数，通过离心收集细胞，并在每50微升流动细胞体缓冲液浓度的10至6个细胞下重新悬浮细胞。接下来，根据实验流细胞仪染色方案，每5毫升荧光激活细胞分拣管中转移50微升等分的细胞。向细胞中加入适当的抗体鸡尾酒和活/死染色，将体积调整为100微升。在黑暗中20分钟后，每管用一毫升新鲜流动细胞学缓冲液清洗细胞，每管用500微升新鲜流动细胞测量缓冲液重新悬浮细胞。然后根据标准流量细胞测量方案分析流细胞仪上的细胞。为了将微珠分离的CD34正茎和祖细胞分化成骨髓血统细胞，首先在PBS中每孔重组人类纤维素素溶液中，用200微升覆盖无菌、未涂覆的48孔板的孔，在室温下两小时。在孵化结束时，丢弃每口井中的纤维素溶液，用200微升2%牛血清白蛋白阻断油井。在室温下30分钟后，每次洗涤用500微升无菌PBS洗两次。重新悬浮新鲜分离的CD34阳性细胞，每毫升温暖刺激中等浓度的5倍10。接下来，将200微升细胞种子放入纤维素片段涂层板的每个井中，将该细胞放在细胞培养培养箱中48小时。在孵化结束时，用温和的移液收集细胞，用500微升加热Dulbecco的改良鹰介质清洗孔，用细胞悬浮液将洗涤液集中起来。计数后，将细胞以每毫升骨髓分化介质浓度的2.5倍10重新悬浮到4个细胞。每井CD34阳性细胞层200微升，MS-5频闪细胞种子48井组织培养板。然后将盘子放在细胞培养培养箱中，每井用100微升新鲜2X骨髓分化介质轻轻更换100微升。第21天，通过流式细胞学收集细胞，用于分析骨髓谱谱标记表达，正如刚刚证明的。通过微珠分离从脐带血分离出的CD34阳性细胞的典型百分比，如80至90%的免疫细胞分析显示，这些CD34阳性细胞通常由大约20%的血统阴性CD34阳性CD38阴性造血干细胞组和72%的血统阴性CD34阳性的多能祖细胞群体组成。在多能祖细胞群体中，约25%的细胞表达常见的骨髓祖细胞标记，约15%表达粒细胞单细胞祖细胞祖先标记，约56%表达巨核细胞红细胞祖细胞祖细胞标记。免疫细胞分位还表明，CD34阳性细胞的百分比从隔离时约90%降低到第21天的25%以下。对CD34负种群的分析表明，表达成熟骨髓血统标记的细胞的百分比也同时上升。此外，Wright-Giemsa染色细胞在第1天和21天表现出明显的形态特征，未分化的培养物表现出大、圆核和极小的细胞质，分化培养物表现出血统细胞的形态特征，包括单细胞、粒细胞和红细胞。请记住，在不混合层的情况下将稀释的脐带血倒入单核细胞分馏介质上，在改变介质时不要干扰MS-5和CD34阳性细胞。按照此分化协议，CD34正细胞培养物可以分析形态变化、凋亡效应、细胞周期模式变化和分化程度。

Research

JoVE Journal

Developmental Biology

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

人类神经干细胞从外周血造血祖细胞直接诱导

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

科研

发育生物学

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

从脑室周围地区的成年大鼠和人类脊髓神经分离干/祖细胞

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

隔离和脂肪干细胞分化的猪从皮下脂肪组织

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

脂肪干细胞的软骨细胞分化诱导离心重力

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

分化软骨细胞与外周血源性人诱导多能干细胞

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

脐带血诱导的多能干细胞的软骨形成颗粒形成

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...