Isolation of bone marrow mesenchymal stem cells in transgenic pigs and co-culture with porcine islets
-
摘要:
目的 探讨α-1, 3-半乳糖基转移酶（GGTA1）基因敲除（GTKO）、GTKO/人补体调节蛋白hCD46插入、单磷酸胞嘧啶-N-乙酰神经氨酸羟化酶（CMAH）/GGTA1双基因敲除（Neu5GC/Gal）猪骨髓间充质干细胞（BMSC）的分离培养，以及与猪胰岛共培养对胰岛的保护作用。 方法 从不同转基因修饰GTKO、GTKO/hCD46及Neu5GC/Gal猪中提取骨髓，采用全骨髓法分离猪BMSC后进行培养。对BMSC进行形态学观察，并使用流式细胞术鉴定BMSC表面标志物。同时，观察BMSC诱导的多向分化，通过绿色荧光蛋白（GFP）转染标记猪BMSC来实现对BMSC的标记和示踪。将GFP转染标记的猪BMSC与猪胰岛细胞共培养，观察猪胰岛形态变化，与单纯猪胰岛细胞培养组进行比较。 结果 猪来源的BMSC在体外培养时呈梭形，表达标志物CD29、CD44、CD73、CD90、CD105及CD166，不表达CD34、CD45，具有向脂肪细胞、成骨细胞、软骨细胞分化的能力；通过GFP转染标记的猪BMSC能够实现BMSC的标记和示踪，且在细胞分裂后的子代细胞中也能够稳定表达。猪BMSC对胰岛细胞有一定保护能力。 结论 成功建立了GFP标记的GTKO、GTKO/hCD46及Neu5GC/Gal猪来源的BMSC，其对胰岛细胞具有一定的保护能力。 Abstract:Objective To investigate the isolation and culture of porcine bone marrow mesenchymal stem cell (BMSC) with α-1, 3-galactosyltransferase (GGTA1) gene knockout (GTKO), GTKO/ human CD46 (hCD46) insertion and cytidine monopho-N-acetylneuraminic acid hydroxylase (CMAH)/GGTA1 gene knockout (Neu5GC/Gal), and the protective effect of co-culture with porcine islets on islet cells. Methods Bone marrow was extracted from different transgenic pigs modified with GTKO, GTKO/hCD46 and Neu5GC/Gal. Porcine BMSC were isolated by the whole bone marrow adherent method and then cultured. The morphology of BMSC was observed and the surface markers of BMSC were identified by flow cytometry. Meantime, the multi-directional differentiation induced by BMSC was observed, and the labeling and tracing of BMSC were realized by green fluorescent protein (GFP) transfection. The porcine BMSC transfected with GFP were co-cultured with porcine islet cells. Morphological changes of porcine islet cells were observed, and compared with those in the porcine islet cell alone culture group. Results BMSC derived from pigs were spindle-shaped in vitro, expressing biomarkers of CD29, CD44, CD73, CD90, CD105 and CD166 rather than CD34 and CD45. These cells were able to differentiate into adipocytes, osteoblasts and chondrocytes. Porcine BMSC with GFP transfection could be labeled and traced, which could be stably expressed in the daughter cells after cell division. Porcine BMSC exerted certain protective effect on islet cells. Conclusions GFP-labeled porcine BMSC modified with GTKO, GTKO/hCD46 and Neu5GC/Gal are successfully established, which exert certain protective effect upon islet cells. -
图 1 猪BMSC形态学表现（×100）
注：A图为野生型外三元猪BMSC；B图为野生型巴马猪BMSC；C图为野生型五指山猪BMSC；D图为GTKO/hCD46五指山猪BMSC；E图为GTKO巴马猪BMSC；F图为GTKO五指山猪BMSC；G图为野生型藏猪BMSC；H图为Neu5GC/Gal五指山猪BMSC。
Figure 1. Morphological findings of BMSC of pig
图 2 GTKO五指山猪BMSC的三系分化结果（×100）
注：A图为原位茜素红染色；B图为油红-O染色；C图为阿利新蓝染色。
Figure 2. The trilineage differentiation results of BMSC of GTKO Wuzhishan pig
图 3 GTKO五指山猪BMSC表面标志物表达情况
Figure 3. Expression of cell surface markers of BMSC of GTKO Wuzhishan pig
图 4 GTKO五指山猪BMSC标记示踪结果（×100）
注：A图为BMSC培养36 h后GFP表达情况；B图为BMSC培养48 h后GFP表达情况。
Figure 4. Labeling and tracing results of BMSC of GTKO Wuzhishan pig
图 5 猪胰岛与GTKO五指山猪BMSC的共培养结果（×100）
注：A图为猪胰岛单独培养；B图为GFP转染标记的BMSC单独培养；C图为猪胰岛与GFP转染标记的BMSC共培养。
Figure 5. Co-culture results of porcine pancreatic islets and BMSC of GTKO Wuzhishan pig
-
[1] HELMAN A, MELTON DA. A stem cell approach to cure type 1 diabetes[J]. Cold Spring Harb Perspect Biol, 2021, 13(1): a035741. DOI: 10.1101/cshperspect.a035741. [2] NAQVI RA, NAQVI AR, SINGH A, et al. The future treatment for type 1 diabetes: pig islet- or stem cell-derived β cells?[J]. Front Endocrinol (Lausanne), 2023, 13: 1001041. DOI: 10.3389/fendo.2022.1001041. [3] 罗说明, 周智广. 1型糖尿病治疗新技术的现状与未来[J]. 中国医师杂志, 2023, 25(3): 321-324. DOI: 10.3760/cma.j.cn431274-20230215-00159.LUO SM, ZHOU ZG. Current status and future of new technologies in the treatment of type 1 diabetes[J]. J Chin Physician, 2023, 25(3): 321-324. DOI: 10.3760/cma.j.cn431274-20230215-00159. [4] 杨玉伟, 张婷, 李万里, 等. 胰岛移植即刻经血液介导的炎症反应应对策略[J]. 器官移植, 2023, 14(3): 352-357. DOI: 10.3969/j.issn.1674-7445.2023.03.005.YANG YW, ZHANG T, LI WL, et al. Therapeutic strategy for instant blood-mediated inflammatory reaction after islet transplantation[J]. Organ Transplant, 2023, 14(3): 352-357. DOI: 10.3969/j.issn.1674-7445.2023.03.005. [5] MARFIL-GARZA BA, IMES S, VERHOEFF K, et al. Pancreatic islet transplantation in type 1 diabetes: 20-year experience from a single-centre cohort in Canada[J]. Lancet Diabetes Endocrinol, 2022, 10(7): 519-532. DOI: 10.1016/S2213-8587(22)00114-0. [6] WALKER S, APPARI M, FORBES S. Considerations and challenges of islet transplantation and future therapies on the horizon[J]. Am J Physiol Endocrinol Metab, 2022, 322(2): E109-E117. DOI: 10.1152/ajpendo.00310.2021. [7] MARFIL-GARZA BA, SHAPIRO AMJ, KIN T. Clinical islet transplantation: current progress and new frontiers[J]. J Hepatobiliary Pancreat Sci, 2021, 28(3): 243-254. DOI: 10.1002/jhbp.891. [8] QU Z, LOU Q, COOPER DKC, et al. Potential roles of mesenchymal stromal cells in islet allo- and xenotransplantation for type 1 diabetes mellitus[J]. Xenotransplantation, 2021, 28(3): e12678. DOI: 10.1111/xen.12678. [9] NGUYEN TT, PHAM DV, PARK J, et al. Engineering of hybrid spheroids of mesenchymal stem cells and drug depots for immunomodulating effect in islet xenotransplantation[J]. Sci Adv, 2022, 8(34): eabn8614. DOI: 10.1126/sciadv.abn8614. [10] KIKUCHI T, NISHIMURA M, KOMORI N, et al. Development and characterization of islet-derived mesenchymal stem cells from clinical grade neonatal porcine cryopreserved islets[J]. Xenotransplantation, 2023,DOI: 10.1111/xen.12831[Epub ahead of print [11] 师越, 李燕, 金慧方, 等. 人间充质干细胞质量研究及评价进展[J]. 国际生物医学工程杂志, 2023, 46(3): 275-280. DOI: 10.3760/cma.j.cn121382-20230411-00315.SHI Y, LI Y, JIN HF, et al. Research progress in quality research and evaluation of human mesenchymal stem cells[J]. Int J Biomed Eng, 2023, 46(3): 275-280. DOI: 10.3760/cma.j.cn121382-20230411-00315. [12] WANG Y, FANG J, LIU B, et al. Reciprocal regulation of mesenchymal stem cells and immune responses[J]. Cell Stem Cell, 2022, 29(11): 1515-1530. DOI: 10.1016/j.stem.2022.10.001. [13] WANG LT, LIU KJ, SYTWU HK, et al. Advances in mesenchymal stem cell therapy for immune and inflammatory diseases: use of cell-free products and human pluripotent stem cell-derived mesenchymal stem cells[J]. Stem Cells Transl Med, 2021, 10(9): 1288-1303. DOI: 10.1002/sctm.21-0021. [14] 潘兴华, 王颖翠, 张梦园, 等. 脐带间充质干细胞临床研究的伦理与安全问题[J]. 西南国防医药, 2018, 28(1): 4-6. DOI: 10.3969/j.issn.1004-0188.2018.01.002.PAN XH, WANG YC, ZHANG MY, et al. Ethical and safety issues in clinical research of umbilical cord mesenchymal stem cells[J]. Med J Natl Defend Forces Southwest China, 2018, 28(1): 4-6. DOI: 10.3969/j.issn.1004-0188.2018.01.002. [15] NISHIMURA M, NGUYEN L, WATANABE N, et al. Development and characterization of novel clinical grade neonatal porcine bone marrow-derived mesenchymal stem cells[J]. Xenotransplantation, 2019, 26(3): e12501. DOI: 10.1111/xen.12501. [16] GARCIA GA, OLIVEIRA RG, DARIOLLI R, et al. Isolation and characterization of farm pig adipose tissue-derived mesenchymal stromal/stem cells[J]. Braz J Med Biol Res, 2022, 55: e12343. DOI: 10.1590/1414-431X2022e12343. [17] TERATANI T, KASAHARA N, FUJIMOTO Y, et al. Mesenchymal stem cells secretions enhanced ATP generation on isolated islets during transplantation[J]. Islets, 2022, 14(1): 69-81. DOI: 10.1080/19382014.2021.2022423. [18] 淮国丽, 杜嘉祥, 潘登科. 基因编辑猪用于急性肝衰竭治疗的路径探讨[J]. 临床肝胆病杂志, 2022, 38(10): 2214-2218. DOI: 10.3969/j.issn.1001-5256.2022.10.004.HUAI GL, DU JX, PAN DK. The discussion on the genetically modified pigs for the treatment of acute liver failure[J]. J Clin Hepatol, 2022, 38(10): 2214-2218. DOI: 10.3969/j.issn.1001-5256.2022.10.004. [19] SYKES M, SACHS DH. Progress in xenotransplantation: overcoming immune barriers[J]. Nat Rev Nephrol, 2022, 18(12): 745-761. DOI: 10.1038/s41581-022-00624-6. [20] QI C, PANG D, YANG K, et al. Generation of PCBP1-deficient pigs using CRISPR/Cas9-mediated gene editing[J]. iScience, 2022, 25(10): 105268. DOI: 10.1016/j.isci.2022.105268. [21] PIERSON RN 3RD. Progress toward pig-to-human xenotransplantation[J]. N Engl J Med, 2022, 386(20): 1871-1873. DOI: 10.1056/NEJMp2118019. [22] DOS SANTOS RMN. Kidney xenotransplantation: are we ready for prime time?[J]. Curr Urol Rep, 2023, 24(6): 287-297. DOI: 10.1007/s11934-023-01156-7. [23] BURLAK C, TAYLOR RT, WANG ZY, et al. Human anti-α-fucose antibodies are xenoreactive toward GGTA1/CMAH knockout pigs[J]. Xenotransplantation, 2020, 27(6): e12629. DOI: 10.1111/xen.12629. [24] DING F, LIN Y, LIU G, et al. Immune disguise: the mechanisms of Neu5Gc inducing autoimmune and transplant rejection[J]. Genes Immun, 2022, 23(6): 175-182. DOI: 10.1038/s41435-022-00182-8. [25] YOON S, LEE S, PARK C, et al. An efficacious transgenic strategy for triple knockout of xeno-reactive antigen genes GGTA1, CMAH, and B4GALNT2 from Jeju native pigs[J]. Vaccines (Basel), 2022, 10(9): 1503. DOI: 10.3390/vaccines10091503. [26] JAGDALE A, NGUYEN H, LI J, et al. Does expression of a human complement-regulatory protein on xenograft cells protect them from systemic complement activation?[J]. Int J Surg, 2020, 83: 184-188. DOI: 10.1016/j.ijsu.2020.09.034. [27] CHABAN R, MCGRATH G, HABIBABADY Z, et al. Increased human complement pathway regulatory protein gene dose is associated with increased endothelial expression and prolonged survival during ex-vivo perfusion of GTKO pig lungs with human blood[J]. Xenotransplantation, 2023, 30(4): e12812. DOI: 10.1111/xen.12812. [28] BURDORF L, LAIRD CT, HARRIS DG, et al. Pig-to-baboon lung xenotransplantation: extended survival with targeted genetic modifications and pharmacologic treatments[J]. Am J Transplant, 2022, 22(1): 28-45. DOI: 10.1111/ajt.16809. [29] KIKUCHI T, NISHIMURA M, HIRATA M, et al. Development and characterization of Gal KO porcine bone marrow-derived mesenchymal stem cells[J]. Xenotransplantation, 2021, 28(6): e12717. DOI: 10.1111/xen.12717. [30] TECTOR AJ, MOSSER M, TECTOR M, et al. The possible role of anti-Neu5Gc as an obstacle in xenotransplantation[J]. Front Immunol, 2020, 11: 622. DOI: 10.3389/fimmu.2020.00622. [31] GALILI U. The alpha-gal epitope and the anti-Gal antibody in xenotransplantation and in cancer immunotherapy[J]. Immunol Cell Biol, 2005, 83(6): 674-686. DOI: 10.1111/j.1440-1711.2005.01366.x. [32] RAO JS, HOSNY N, KUMBHA R, et al. HLA-G1+ expression in GGTA1KO pigs suppresses human and monkey anti-pig T, B and NK cell responses[J]. Front Immunol, 2021, 12: 730545. DOI: 10.3389/fimmu.2021.730545. [33] CHABAN R, HABIBABADY Z, HASSANEIN W, et al. Knock-out of N-glycolylneuraminic acid attenuates antibody-mediated rejection in xenogenically perfused porcine lungs[J]. Xenotransplantation, 2022, 29(6): e12784. DOI: 10.1111/xen.12784. [34] LANDSTRA CP, NIJHOFF MF, ROELEN DL, et al. Diagnosis and treatment of allograft rejection in islet transplantation[J]. Am J Transplant, 2023, 23(9): 1425-1433. DOI: 10.1016/j.ajt.2023.05.035. [35] JEYAGARAN A, LU CE, ZBINDEN A, et al. Type 1 diabetes and engineering enhanced islet transplantation[J]. Adv Drug Deliv Rev, 2022, 189: 114481. DOI: 10.1016/j.addr.2022.114481. [36] KABAKCHIEVA P, ASSYOV Y, GERASOUDIS S, et al. Islet transplantation-immunological challenges and current perspectives[J]. World J Transplant, 2023, 13(4): 107-121. DOI: 10.5500/wjt.v13.i4.107. [37] SONG N, SCHOLTEMEIJER M, SHAH K. Mesenchymal stem cell immunomodulation: mechanisms and therapeutic potential[J]. Trends Pharmacol Sci, 2020, 41(9): 653-664. DOI: 10.1016/j.tips.2020.06.009. [38] SHEN Z, HUANG W, LIU J, et al. Effects of mesenchymal stem cell-derived exosomes on autoimmune diseases[J]. Front Immunol, 2021, 12: 749192. DOI: 10.3389/fimmu.2021.749192. -
下载:
点击查看大图
图(7)
计量
- 文章访问数: 235
- HTML全文浏览量: 153
- PDF下载量: 26
- 被引次数: 0
