Pre-clinical evaluation of Magnetically labeled Cells for Cellular MRI

以前，我们证明了通过将阿魏酸甘油酯(F)与鱼精蛋白(P)和肝素(H)相结合，可以产生一种可用于标记细胞的自组装纳米复合体。HPF的透射电子显微镜显示，这些纳米复合体呈球形，中间的HP被F包围。将干细胞和T细胞在HFP纳米复合体中在无血清介质中孵育2小时，然后在完全介质中进行细胞标记。HPF标记不影响细胞的活力、增殖能力、凋亡率、活化、表型表面标志的表达或分化能力。将HPF标记的骨髓间充质干细胞植入大鼠脑内，3T时的磁共振成像显示，与周围实质相比，大鼠脑内可检测到1000个细胞，T2*减少了50%。小鼠脑内注射HPF标记的神经干细胞也进行了临床前的安全性/毒性研究，结果显示没有明显的临床或行为变化，没有神经或全身毒性，也没有铁在肝脏或脾中的异常积聚。HPF标记技术已经扩大，NIH细胞处理部门的cGMP设施能够在生物工厂标记BMSCs，而标记的低温保存细胞产品的BMSC功能或活性不会发生变化。HPF方法还被用于标记表达胞嘧啶脱氨酶的基因工程神经干细胞(NSC)，作为正在进行的治疗复发胶质母细胞瘤患者的临床试验的一部分。HPF标记的神经干细胞将被直接植入手术切除视野周围的大脑深处，以便它们能够迁移到残留肿瘤或卫星转移的区域。MRI监测了30天内HPF标记的神经干细胞在脑内的迁移情况。在希望之城进行的初步研究报告称，标记细胞移植后基本上没有不良事件。我们最近优化了HPF标记方法，将三种药物加入细胞的顺序改为FHP，与HPF标记的细胞相比，BMSC和NSC的细胞内铁含量显着增加。我们还报告了优化H、P和F组分在不同比例和混合顺序下的物理化学特性，以产生流体力学大小不同的NCS。NC的大小取决于药物在介质中的混合顺序。HPF或FHP的电子显微镜显示，F位于球形HP络合物的表面。与FHP NCS孵育的人干细胞相比，与相同浓度的F孵育的HPF NCS相比，每个细胞的铁浓度显著增加。这些结果表明，FHP可以用于临床翻译研究中的干细胞标记。 由于皮质发育不成熟而导致的长期神经功能障碍正在成为先天性心脏病(CHD)的主要挑战。通过将超顺磁性氧化铁纳米颗粒注射到缺氧暴露的仔猪回脑回的侧脑室，我们能够标记体内的神经干细胞，并通过体外MRI追踪它们在皮质和嗅球中的分布模式。结果表明，低氧减少了脑室下区(SVZ)的增殖和神经发生，并伴随着皮质生长的减少。结果表明，SVZ神经干细胞有助于围产期皮质发育，提示恢复SVZ神经干细胞的神经发生潜能是改善CHD皮质生长的候选治疗靶点。

Previously, we demonstrated that by combining ferumoxytol (F) with protamine (P) and heparin (H) resulted in a self assembling nanocomplex that could be used to label cells. Transmission electron microscopy of HPF revealed that these nanocomplexes were spheroid shaped with the HP in center surrounded by F. Incubating stem cells and T-cells in HFP nanocomplexes in serum free media for 2 hours followed by complete media resulted in cell labeling. HPF labeling did not impair the cells viability, proliferative capacity, apoptotic rate, activation, phenotypic surface marker expression, or capacity to differentiate. MRI at 3T of HPF labeled BMSC implanted in the rat brain demonstrated the ability to detect 1000 cells with a 50% decrease in T2* in the rat brain compared to the surrounding parenchyma. Pre-clinical safety/toxicity studies of intracerebrally administrated HPF-labeled NSCs in mice were also performed, and demonstrated no significant clinical or behavioral changes, no neuronal or systemic toxicities, and no abnormal accumulation of iron in the liver or spleen. The HPF labeling technique has been scaled up, and the NIH Cell Processing Section cGMP facility was able to label BMSCs in biofactories with no changes in BMSC function or viability of the labeled cryopreserved cell product. The HPF method was also used to label genetically engineered neural stem cells (NSC) that express cytosine deaminase as part of an ongoing clinical trial to treat patients with recurrent glioblastoma. The HPF labeled NSC are to be directly implanted deep into brain around the periphery of the surgical resection sight in order that they can migrate to areas of residual tumors or satellite metastasis. MRI monitored the migration of the HPF labeled NSC in the brain over 30 days. Initial studies performed at City of Hope report that there was essential no adverse events following the implantation of labeled cells. We have recently optimized the HPF labeling approach by changing the order that the three drugs are added to cells to FHP resulted in significant increase in the intracellular iron content in BMSC and NSC as compared to HPF labeled cells. We also report on the physicochemical characteristics for optimizing the H, P, and F components in different ratios, and mixing sequences, producing NCs that varied in hydrodynamic size. NC size depended on the order in which drugs were mixed in media. Electron microscopy of HPF or FHP showed that F was located on the surface of spheroidal shaped HP complexes. Human stem cells incubated with FHP NCs resulted in a significantly greater iron concentration per cell compared to that found in HPF NCs with the same concentration of F. These results indicate that FHP could be useful for labeling stem cells in translational studies in the clinic. Long-term neurological deficits due to immature cortical development are emerging as a major challenge in congenital heart disease (CHD). By injection superparamagnetic iron oxide nanoparticles into the lateral ventricles of hypoxic exposed of the gyrencephalic piglet brain a model of CHD we were able to label neural stem cells in vivo and track their pattern of distribution into the cortex and olfactory bulbs by ex vivo MRI. The results showed that hypoxia reduces proliferation and neurogenesis in the subventricular zone (SVZ), which is accompanied by reduced cortical growth. The findings demonstrated that SVZ neuronal stem cells (NSC) contribute to perinatal corticogenesis and suggest that restoration of SVZ NSCs' neurogenic potential is a candidate therapeutic target for improving cortical growth in CHD.