Introduction

Acute myocardial infarction (AMI) and resulting cardiac damage and heart failure are leading causes of morbidity and mortality worldwide. Multiple studies have examined the utility of CD34⁺ cells for the treatment of acute and ischemic heart disease. However, the optimal strategy to enrich CD34 cells from clinical sources is not known. We examined the efficacy of fluorescence activated cell sorting (FACS) and magnetic beads cell sorting (MACS) methods for CD34 cell isolation from mobilized human mononuclear peripheral blood cells (mhPBMNCs).

Methods

mhPBCs were processed following acquisition using FACS or MACS according to clinically established protocols. Cell viability, CD34 cell purity and characterization of surface marker expression were assessed using a flow cytometer. For in vivo characterization of cardiac repair, we conducted LAD ligation surgery on 8–10 weeks female NOD/SCID mice followed by intramyocardial transplantation of unselected mhPBMNCs, FACS or MACS enriched CD34⁺ cells.

Results

Both MACS and FACS isolation methods achieved high purity rates, viability, and enrichment of CD34⁺ cells. In vivo studies following myocardial infarction demonstrated retention of CD34⁺ in the peri-infarct region for up to 30 days after transplantation. Retained CD34⁺ cells were associated with enhanced angiogenesis and reduced inflammation compared to unselected mhPBMNCs or PBS treatment arms. Cardiac scar and fibrosis as assessed by immunohistochemistry were reduced in FACS and MACS CD34⁺ treatment groups. Finally, reduced scar and augmented angiogenesis resulted in improved cardiac functional recovery, both on the global and regional function and remodeling assessments by echocardiography.

Conclusion

Cell based therapy using enriched CD34⁺ cells sorted by FACS or MACS result in better cardiac recovery after ischemic injury compared to unselected mhPBMNCs. Both enrichment techniques offer excellent recovery and purity and can be equally used for clinical applications.

中文翻译：

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使用荧光和磁激活细胞分选法分离人CD34亚集的方法：一项体内比较研究。

介绍

急性心肌梗塞（AMI）以及由此引起的心脏损害和心力衰竭是全球发病率和死亡率的主要原因。多项研究检查了CD34 ⁺细胞在治疗急性和缺血性心脏病中的实用性。但是，从临床来源富集CD34细胞的最佳策略尚不清楚。我们检查了荧光活化细胞分选（FACS）和磁珠细胞分选（MACS）方法从动员的人单核外周血细胞（mhPBMNCs）中分离CD34细胞的功效。

方法

根据临床确定的方案，使用FACS或MACS在采集后处理mhPBC。使用流式细胞仪评估细胞活力，CD34细胞纯度和表面标志物表达的特征。对于体内心脏修复的特征，我们对8-10周的雌性NOD / SCID小鼠进行了LAD结扎手术，然后进行了未选择的mhPBMNCs，FACS或MACS富集的CD34 ⁺细胞的心肌内移植。

结果

MACS和FACS分离方法均实现了高纯度，高活力和CD34 ⁺细胞富集。心肌梗死后的体内研究表明，移植后长达30天，CD34 ⁺在梗死周围区域保留。与未选择的mhPBMNCs或PBS治疗组相比，保留的CD34 ⁺细胞与增强的血管生成和减少的炎症相关。通过免疫组织化学评估，心脏疤痕和纤维化在FACS和MACS CD34 ⁺治疗组中减少。最后，疤痕的减少和血管生成的增强导致心脏功能恢复的改善，无论是整体功能还是区域功能以及超声心动图对重塑的评估。

结论

与未选择的mhPBMNC相比，使用通过FACS或MACS分选的富集CD34 ⁺细胞进行的基于细胞的治疗可改善缺血性损伤后的心脏恢复。两种富集技术均提供出色的回收率和纯度，并且可以等同地用于临床应用。