Cardiac cell therapy using adult cells with stem- or progenitor-like properties has been under clinical investigation for more than 15 years. However, the cumulative results from dozens of trials suggest a slight, transient benefit at best.^1,2 The underlying rationale for these trials was derived from studies in small and large animals where a more consistent functional benefit has been observed,³ albeit by still unclear mechanisms. Most trials to date have used whole, unfractionated bone marrow mononuclear cells (MNCs) delivered systemically via the circulation, usually by intracoronary infusion.^1,2 This strategy was also based on previous rodent studies in which infused adult stem cells were reported to home to the injured heart and restore contractile function, in part through direct regeneration of myocardium. Our results here, and some recent data in the field, show that infused progenitor cells perish and do not penetrate the capillary network of the heart to generate new cardiac myocytes. Thus, cell infusion is now proposed to work through temporary secretion of rejuvenating factors from these cells, within the circulation or when lodged in other tissues.⁴ Here we critically re-evaluated the hypothesis that function of the injured rodent heart can be improved with systemic infusion of unfractionated bone marrow MNCs, versus MNCs delivered by direct intraparenchymal injection.

Eight-week-old male and female C57Bl/6J (wild-type) mice received acute ischemia/reperfusion (I/R) injury (120 minutes of ischemia) followed by MNC therapy or saline at 1 week post-I/R, to mimic clinical use of cell therapy in patients with pre-existing myocardial infarction (Figure, A).^1,2 All animal procedures were conducted in accordance with institutional guidelines and were approved by the Institutional Animal Care and Use Committee. For in vivo tracking, MNCs were isolated from strain-matched Rosa26-mTomato/mGFP reporter mice that express ubiquitous membrane-localized TdTomato (mTomato). MNCs were isolated from an equal number of 8-week-old male and female mice via Ficoll density centrifugation and delivered intravenously in sterile saline via tail vein at a dose of 1×10⁶ cells, in line with clinical studies of systemic infusion.^1,2 We first assessed MNC retention by detection of mTomato using flow cytometry. mTomato⁺ MNCs in circulation accounted for less than 0.2% of all blood cells, either 5 min after infusion or by 2 days post-infusion (Figure, B). MNCs were largely absent from all tissues examined, including heart and lung (Figure, C), accounting for less than 3 cells per milligram tissue weight, or roughly 450 MNCs in an entire mouse heart.

Figure. Bone marrow mononuclear cells delivered by vascular infusion do not persist or improve cardiac function in ischemia/reperfusion-injured mice.

A, Experimental scheme and timeline for cell retention studies using genetically labeled bone marrow mononuclear cells (MNCs) isolated from Rosa26-mTomato mice on the C57Bl/6J background. 1×10⁶ mTomato⁺ MNCs or sterile saline was infused intravenously (i.v.) by tail vein into C57Bl/6J (wild-type) mice, 1 week after ischemia/reperfusion (I/R) injury via 120 minutes of left coronary artery ischemia. Male and female mice were used in all experiments and for MNC isolations. B, Quantitative flow cytometry analysis of mTomato fluorescence from peripheral blood of post-I/R C57Bl/6J mice infused with MNCs or saline. mTomato⁺ MNCs accounted for <0.2% of all circulating nucleated blood cells at 5 minutes (min) or 2 days (d) after cell infusion. C, Quantitative flow cytometry analysis of single-cell suspensions prepared from dissociated whole hearts, lungs, or livers of post-I/R C57Bl/6J mice infused with MNCs or saline, 2 d postinfusion. mTomato⁺ MNCs accounted for less than 3 cells per milligram of tissue across each organ. D and E, Representative confocal micrographs showing histological sections from hearts (D) or lungs, livers, kidneys, and spleens (E) from n=3 C57Bl/6J mice infused at 1 week post-I/R with mTomato⁺ MNCs and harvested for analysis 5 min after infusion. Immunohistochemistry was performed against laminin (green) and CD31 (white) along with endogenous mTomato fluorescence (red). Nuclei were visualized with DAPI (blue). Scale bars in D, 100 µm. Yellow dashed lines in D denote a rare mTomato⁺ MNC (mT) within the cardiac vasculature, as shown in D´ with high magnification insets. Scale bars in D´, 10 µm. Images in E show additional organs from the mice in D. Scale bars, 100 µm. Yellow dashed lines denote rare mTomato⁺ MNCs, shown in enlarged insets above. Inset scale bars, 50 µm. F, Experimental scheme and timeline for post-I/R functional studies in C57Bl/6J mice after MNC delivery. At 1 week post-I/R, animals received either intravenous infusion (i.v.) of 1x10⁶ mTomato⁺ MNCs, direct intramyocardial injection (i.car) of 1×10⁵ mTomato⁺ MNCs (1/10^th the infusion dose, over 2 injection sites flanking the infarct border zone), or sterile saline. Animals that received infusion vs intramyocardial injection of saline showed no difference in cardiac function or structure post-I/R, so these groups were combined (denoted as Sal.). G through I, Experimental groups as described in F were assessed by 2-dimensional M-mode echocardiography, 2 weeks (w) after cell therapy (3 weeks post-I/R). Left ventricular fractional shortening (%FS; G) was significantly decreased in all post-I/R groups vs sham-operated controls at 2 weeks. Direct intramyocardial injection of MNCs but not MNC infusion attenuated this cardiac dysfunction. *P<0.05 vs Sham; #P<0.05 vs Sal.; %P<0.05 vs i.v. MNC, all by 1-way ANOVA with Tukey multiple comparisons test. Heart rates (H) were equivalent in all groups. Cardiac hypertrophy (I) as assessed via gravimetric analysis of heart weight/body weight (HW/BW) ratio was attenuated at 2 weeks posttherapy by MNC direct injection, but not MNC infusion. *P<0.05 vs Sal. by Kruskal-Wallis with Dunn multiple comparisons test.

Interestingly, confocal microscopy showed the few rare mTomato⁺ MNCs observed in the heart were always within CD31⁺ capillaries and not free within the parenchyma (Figure, D). Similarly, minimal retention was observed in other organs, including lung, liver, kidney, and spleen (Figure, E). Thus, MNCs infused into mice with acute I/R injury are rapidly cleared within minutes and are not retained at physiologically meaningful levels in any tissue, injured or otherwise.

We also assessed whether the delivery of MNCs could improve function of the injured heart, as suggested in various human clinical trials.^1,2 At 1 week post-I/R, mice were either infused with 1×10⁶ MNCs, or we delivered MNCs directly into either side of the infarct border zone via intramyocardial injection at a dose of 1×10⁵ total MNCs, 1/10^th the infusion amount (Figure, F). Systemic infusion of MNCs did not alter cardiac dysfunction or adverse remodeling compared with saline controls when assessed 2 weeks later (Figure, G through I). However, direct intramyocardial injection of 1/10^th the dosage of the same MNCs significantly improved systolic function (Figure, G and H) and attenuated cardiac hypertrophy (Figure, I) post-I/R. Taken together, these data indicate that the previously reported beneficial effects of MNC therapy in the rodent heart are not achieved with systemic infusion. In contrast, direct intramuscular delivery of MNCs flanking the infarcted region of the heart mildly, albeit significantly, improved cardiac function over 2 weeks.

Our data do not support a mechanism whereby systemically infused stem- or progenitor-like cells seed distal organs and produce paracrine-mediated cardiac rejuvenation (Figure, G). The minuscule amount of MNCs retained after infusion were trapped within the vasculature (Figure, B through E) and seemingly below a quantitative level that could have a global paracrine secretory effect on the heart, although local signaling via secreted factors could be occurring in regions where the few MNCs were present. In contrast, direct injection of MNCs on both sides of the infarct region was beneficial post-I/R in mice. For patient safety reasons, many clinical trials conducted to date have used vascular infusion, with little understanding of the efficacy of such a route, especially because most animal model data used direct intraparenchymal injection. We have recently shown that direct injection of nearly any cell type into the parenchyma of the heart surrounding the injured region produces a localized innate immune response that further enhances healing by affecting the extracellular matrix and microvasculature.⁵ Future uses of cardiac cell–based therapy in humans warrants careful re-examination as to why systemic vascular infusion would be selected, especially because we now understand that these cells do not make it through the capillaries, nor persist from a distal region as a global source of secreted paracrine factors.

The authors thank Jeff Bailey and Victoria Summey in the Cincinnati Children’s Hospital Medical Center Comprehensive Mouse and Cancer Core for assistance with intravenous infusions. All flow cytometric data were acquired using equipment maintained by the Research Flow Cytometry Core in the Division of Rheumatology at Cincinnati Children’s Hospital Medical Center.

This study was supported by grants from the National Institutes of Health (NIH) and by the Howard Hughes Medical Institute and American Heart Association MERIT award (to J.D.M.). R.J.V. was supported by a National Research Service Award from the NIH (F32 HL128083) and a Career Development Award from the American Heart Association (19CDA34670044).

None.

Requests of materials, datasets, and protocols used in this study should be directed to the corresponding author and will be made available to investigators upon reasonable request.

https://www.ahajournals.org/journal/circ

使用具有干细胞或祖细胞样特性的成年细胞进行的心脏细胞治疗已经进行了超过15年的临床研究。但是，数十项试验的累积结果表明，充其量只是暂时的获益。^1,2这些试验的基本原理源自对小型和大型动物的研究，这些动物观察到了更一致的功能益处，尽管机制尚不清楚，³。迄今为止，大多数试验使用的是通常通过冠状动脉内输注通过循环系统递送的完整，完整的骨髓单个核细胞（MNC）。^1,2该策略也基于先前的啮齿动物研究，在该啮齿动物研究中，注入的成年干细胞据报道归巢于受伤的心脏并恢复收缩功能，部分是通过心肌的直接再生。我们在这里的结果以及该领域的一些最新数据表明，注入的祖细胞会消失并且不会穿透心脏的毛细血管网络以生成新的心肌细胞。因此，现在提出细胞输注可以通过在循环内或当其滞留在其他组织中时从这些细胞暂时分泌再生因子来起作用。⁴在这里，我们严格地重新评估了假说，即通过全身输注未分级的骨髓MNC相对于通过实质实质内注射递送的MNC可以改善啮齿动物心脏的功能。

八周龄的雄性和雌性C57Bl / 6J（野生型）小鼠遭受急性缺血/再灌注（I / R）损伤（缺血120分钟），然后在I / R后1周接受MNC治疗或盐水治疗，模仿已存在心肌梗塞的患者进行细胞疗法的临床使用（图A）。^1,2所有动物程序均按照机构指南进行，并得到机构动物护理和使用委员会的批准。为了进行体内跟踪，从菌株匹配的Rosa26-mTomato / mGFP中分离了MNC表达无处不在的膜定位TdTomato（mTomato）的记者小鼠。根据全身输注的临床研究，通过Ficoll密度离心从相等数量的8周龄雄性和雌性小鼠中分离出MNC，并以1×10 ^6个细胞的剂量通过尾静脉在无菌盐水中静脉内递送MNC ，符合全身输注的临床研究。^1,2我们首先通过流式细胞术检测mTomato来评估MNC保留。输注后5分钟或输注后2天，循环中的mTomato ⁺ MNC占所有血细胞的比例不到0.2％（图B）。所检查的所有组织（包括心脏和肺）都基本上不存在MNC（每毫克组织重量少于3个细胞），或整个小鼠心脏中约450个MNC。

数字。 在缺血/再灌注损伤的小鼠中，通过血管输注递送的骨髓单个核细胞不能持续存在或不能改善心脏功能。

A，使用从C57Bl / 6J背景上的Rosa26 -mTomato小鼠分离的基因标记的骨髓单核细胞（MNC）进行细胞保留研究的实验方案和时间表。在缺血/再灌注（I / R）损伤后120周左冠状动脉缺血1周后，通过尾静脉将1×10 ⁶ mTomato ⁺ MNC或无菌盐水经尾静脉静脉内（iv）注入C57Bl / 6J（野生型）小鼠中。在所有实验中均使用雄性和雌性小鼠进行MNC分离。B，定量注入MNC或盐水的I / R C57Bl / 6J小鼠的外周血mTomato荧光的流式细胞术分析。mTomato ⁺在输注细胞后5分钟（分钟）或2天（d），MNC占所有循环有核血细胞的<0.2％。C，从输注了MNC或盐水的I / R C57Bl / 6J小鼠的离体全心，肺或肝制备的单细胞悬液的定量流式细胞术分析，在输注后2天。mTomato ⁺ MNC占每个器官每毫克组织少于3个细胞。D和E，具有代表性的共聚焦显微照片，显示了在I / R后1周注入mTomato的n = 3只C57Bl / 6J小鼠的心脏（D）或肺，肝，肾和脾（E）的组织学切片⁺ MNCs，并在输注后5分钟收获进行分析。针对层粘连蛋白（绿色）和CD31（白色）以及内源性mTomato荧光（红色）进行了免疫组织化学。用DAPI（蓝色）可视化细胞核。标尺，D，100 µm。D中的黄色虚线表示心脏脉管系统内罕见的mTomato ⁺ MNC（mT），如D´所示，具有高放大倍数。标尺为D´，10 µm。E中的图像显示了D中来自小鼠的其他器官。比例尺，100 µm。黄色虚线表示稀有的mTomato ⁺ MNC，如上方放大图所示。插入比例尺，50 µm。F，MNC递送后在C57Bl / 6J小鼠中进行I / R后功能研究的实验方案和时间表。1周后的I / R，动物接受静脉内输注（IV）的1×10 ⁶ mTomato ⁺跨国公司，1×10直接心肌内注射（i.car）⁵ mTomato ⁺跨国公司（1/10^个输注剂量，经在梗塞边界区两侧的2个注射部位）或无菌生理盐水。输注与心肌内注射盐水的动物在I / R后心脏功能或结构无差异，因此将这些组合并（称为Sal。）。G至I，如F中所述的实验组在细胞治疗后2周（w）（I / R后3周），通过二维M型超声心动图对患者进行评估。与假手术组相比，所有I / R后组的左室分数缩短（％FS; G）在第2周时均显着降低。心肌内直接注射MNC而不是MNC输注可减轻这种心脏功能障碍。* P <0.05 vs深水; ＃P <0.05，与萨尔.; 与iv MNC相比，％P <0.05，均通过1向ANOVA与Tukey多重比较测试进行比较。所有组的心率（H）均相等。心脏肥大（我通过重量分析法评估的心率/体重（HW / BW）比值在治疗后2周通过MNC直接注射（而非MNC输注）减弱。* P <0.05 vs Sal。由Kruskal-Wallis与Dunn进行多重比较测试。

有趣的是，共聚焦显微镜检查发现，在心脏中观察到的少数罕见的mTomato ⁺ MNC总是在CD31 ⁺毛细血管内，而在薄壁组织内并非游离（图D）。同样，在其他器官（包括肺，肝，肾和脾脏）中也观察到了最小的保留（图E）。因此，注入急性I / R损伤小鼠的MNC在数分钟内迅速清除，并且在受伤或其他任何组织中均未保持生理上有意义的水平。

正如各种人类临床试验中所建议的，我们还评估了MNC的递送是否可以改善受伤心脏的功能。^1,2在I / R后1周，给小鼠灌输1×10 ^6个MNC，或者我们通过心肌内注射以1×10 ^5个总MNC的剂量将MNC直接递送至梗死边界区的任一侧， 1/10^个输液量（图中的F）。2周后评估，与盐水对照组相比，MNC的全身输注没有改变心脏功能障碍或不良重塑（图G至I）。然而，1/10的直接心肌内注射^第相同的MNC剂量在I / R后可显着改善收缩功能（图G和H）并减轻心脏肥大（图I）。综上所述，这些数据表明，全身性输注不能实现先前报道的MNC治疗对啮齿动物心脏的有益作用。相比之下，MNC的直接肌内递送侧翼在心脏梗塞区域的两侧，尽管有显着改善，但在2周内改善了心脏功能。

我们的数据不支持全身注入干细胞或祖细胞样细胞播种远端器官并产生旁分泌介导的心脏年轻化的机制（图G）。输注后保留的微量MNC被困在脉管系统中（图B到E），似乎低于定量水平，可能对心脏产生整体旁分泌作用，尽管通过分泌因子的局部信号传导可能发生在其中少数跨国公司在场。相反，在梗塞区域的两侧直接注射MNC对I / R小鼠有益。出于患者安全的原因，迄今为止进行的许多临床试验都使用了血管输注，但对这种途径的功效了解甚少，特别是因为大多数动物模型数据都使用了实质实质内直接注射。⁵基于心脏细胞的疗法在人类中的未来应用需要对为什么选择全身性血管输注进行仔细的重新检查，特别是因为我们现在了解到这些细胞不会通过毛细血管进入，也不是从远端区域持续存在。分泌的旁分泌因子的全球来源。

作者感谢辛辛那提儿童医院医学中心综合小鼠和癌症核心组织的Jeff Bailey和Victoria Summey协助静脉输液。所有流式细胞仪数据均使用辛辛那提儿童医院医学中心风湿病科研究流式细胞仪核心所维护的设备进行采集。

这项研究得到了美国国立卫生研究院（NIH）的资助以及霍华德·休斯医学研究所和美国心脏协会MERIT奖（授予JDM）的支持。RJV得到了美国国立卫生研究院（NIH）的国家研究服务奖（F32 HL128083）和美国心脏协会的职业发展奖（19CDA34670044）的支持。

没有。

本研究中使用的材料，数据集和方案的要求应直接提供给相应的作者，并应合理的要求提供给研究者。

https://www.ahajournals.org/journal/circ