Medical College of Yangzhou University, Yangzhou, 225001, Jiangsu, China.
Department of Cardiology, Northern Jiangsu People's Hospital, Yangzhou, 225001, Jiangsu, China.
Stem Cell Rev Rep. 2023 Jan;19(1):213-229. doi: 10.1007/s12015-022-10432-x. Epub 2022 Aug 4.
The identification in murine bone marrow (BM) of CD133 + /Lin-/CD45- cells, possessing several features of pluripotent stem cells, encouraged us to investigate if similar population of cells could be also isolated from the swine BM. Heart failure is the terminal stage of many cardiovascular diseases, and its key pathological basis is cardiac fibrosis (CF). Research showed that stem cell derived exosomes may play a critical role in cardiac fibrosis. The effect of exosomes (Exos) on CF has remained unclear.
To establish an isolation and amplification method of CD133 + /Lin-/CD45- cells from newbron swine BM in vitro, explore an highly efficient method to enrich swine bone marrow derived CD133 + /Lin-/CD45- cells and probe into their biological characteristics further. Furher more, to extract exosomes from it and explore its effect on CF.
The mononuclear cells isolated from swine bone marrow by red blood cell (RBC) lysing buffer were coated by adding FcR blocking solution and coupled with CD133 antibody immunomagnetic beads, obtaining CD133 + cell group via Magnetic Activated Cell Sorting (MACS). In steps, the CD133 + /Lin-/CD45- cells were collected by fluorescence-activated cell sorting (FACS) labeled with CD133, Lin and CD45 antibodies, which were cultured and amplified in vitro. The biological features of CD133 + /Lin-/CD45- cells were studied in different aspects, including morphological trait observed with inverted microscope, ultrastructural characteristics observed under transmission electron microscope, expression of pluripotent markersidentified by immunofluorescent staining and Alkaline phosphatase staining. The Exos were extracted using a sequential centrifugation approach and its effects on CF were analyzed in Angiotensin II (Ang-II) induced-cardiac fibrosis in vivo. Rats in each group were treated for 4 weeks, and 2D echocardiography was adopted to evaluate the heart function. The degree of cardiac fibrosis was assessed by Hematoxylin-Eosin (HE) and Masson's trichrome staining.
The CD133 + /Lin-/CD45- cells accounted for about 0.2%-0.5% of the total mononuclear cells isolated from swine bone marrow. The combination of MACS and FACS to extract CD133 + /Lin-/CD45- cells could improved efficiency and reduced cell apoptosis. The CD133 + /Lin-/CD45- cells featured typical traits of pluripotent stem cells, the nucleus is large, mainly composed of euchromatin, with less cytoplasm and larger nucleoplasmic ratio, which expressed pluripotent markers (SSEA-1, Oct-4, Nanog and Sox-2) and alkaline phosphatase staining was positive.Animal experiment indicated that the cardiac injury related indexes (BNP、cTnI、CK-MB and TNF-α), the expression of key gene Smad3 and the degree of cardiac fibrosis in Exo treatment group were significantly reduced compared with the control group. 4 weeks after the treatment, cardiac ejection fraction (EF) value in the model group showed a remarkable decrease, indicating the induction of HF model. While Exo elevated the EF values, demonstrating cardio-protective effects.
The CD133 + /Lin-/CD45- cells derived from swine bone marrow were successfully isolated and amplified, laying a good foundation for further research on this promising therapeutic cell. The Exos may be a promising potential treatment strategy for CF.
在鼠骨髓（BM）中鉴定出具有多能干细胞多种特征的 CD133+ / Lin- / CD45-细胞，这促使我们研究是否也可以从猪 BM 中分离出类似的细胞群。心力衰竭是许多心血管疾病的终末期，其关键病理基础是心脏纤维化（CF）。研究表明，干细胞衍生的外泌体可能在心脏纤维化中发挥关键作用。外泌体（Exos）对 CF 的影响仍不清楚。
建立一种从新生猪 BM 体外分离和扩增 CD133+ / Lin- / CD45-细胞的方法，探索一种高效富集猪骨髓来源 CD133+ / Lin- / CD45-细胞的方法，并进一步探讨其生物学特性。此外，从其中提取外泌体并探讨其对 CF 的影响。
采用红细胞（RBC）裂解缓冲液分离猪骨髓中的单个核细胞，加入 FcR 阻断溶液并偶联 CD133 抗体免疫磁珠，通过磁激活细胞分选（MACS）获得 CD133+细胞群。在步骤中，通过用 CD133、Lin 和 CD45 抗体标记，通过荧光激活细胞分选（FACS）收集 CD133+ / Lin- / CD45-细胞，在体外进行培养和扩增。从形态学特征观察倒置显微镜下的形态特征、透射电镜下的超微结构特征、免疫荧光染色和碱性磷酸酶染色鉴定多能标志物的表达等方面研究 CD133+ / Lin- / CD45-细胞的生物学特性。采用连续离心法提取外泌体，并分析其在血管紧张素 II（Ang-II）诱导的心脏纤维化中的作用。每组大鼠治疗 4 周，采用二维超声心动图评估心功能。苏木精-伊红（HE）和 Masson 三色染色评估心肌纤维化程度。
从猪骨髓中分离的单个核细胞中，CD133+ / Lin- / CD45-细胞约占 0.2%-0.5%。MACS 和 FACS 联合提取 CD133+ / Lin- / CD45-细胞可提高效率并减少细胞凋亡。CD133+ / Lin- / CD45-细胞具有多能干细胞的典型特征，细胞核较大，主要由常染色质组成，细胞质较少，核质比较大，表达多能标志物（SSEA-1、Oct-4、Nanog 和 Sox-2），碱性磷酸酶染色阳性。动物实验表明，与对照组相比，外泌体治疗组的心脏损伤相关指标（BNP、cTnI、CK-MB 和 TNF-α）、关键基因 Smad3 的表达和心肌纤维化程度均显著降低。治疗 4 周后，模型组的心脏射血分数（EF）值明显下降，提示 HF 模型的诱导。而外泌体升高了 EF 值，显示出心脏保护作用。
从猪骨髓中成功分离和扩增了 CD133+ / Lin- / CD45-细胞，为进一步研究这种有前途的治疗细胞奠定了良好的基础。外泌体可能是 CF 的一种有前途的潜在治疗策略。
