早产儿和足月婴儿脐带源性间充质干细胞的分离与鉴定
1Department of Pediatrics,Kobe University Graduate School of Medicine, 2Department of Pathology,Kobe Children’s Hospital, 3Department of Pediatrics,Hyogo College of Medicine, 4Department of Developmental Pediatrics,Shizuoka Children’s Hospital, 5Department of Pediatrics,Nihon University School of Medicine
Summary
人类脐带 (uc) 可以在围生期获得, 这是早产、足月和产后分娩的结果。在该协议中, 我们描述了在妊娠19-40周时从胎儿婴儿中分离和表征 uc 衍生的间充质干细胞 (uc-mscs) 的方法。
Abstract
间充质干细胞具有相当大的治疗潜力, 在生物医学领域引起越来越大的兴趣。骨髓间充质干细胞最初是从骨髓 (bm) 中分离出来并具有特征的, 然后从包括脂肪组织、滑膜、皮肤、牙髓和胎儿附属物 (如胎盘、脐带血) 和脐带 (uc)) 在内的组织中获得。骨髓间充质干细胞是一种异质细胞群, 有能力 (1) 在标准培养条件下坚持塑料, (2) cd73+/cd90+/cd105 +/cd45-/cd34-/cd14–/cd19 的表面标记表达–-hla-dr-表型, 和 (3) 三系分化为脂肪细胞、骨细胞和软骨细胞, 目前由国际细胞治疗学会 (isct) 定义.虽然 bm 是 msc 中应用最广泛的来源, 但 bm 吸入的侵入性在伦理上限制了其可访问性。bm 中获得的骨髓间充质干细胞的增殖和分化能力普遍随着供体年龄的扩大而下降。相反, 从 uc 中获得的胎儿骨髓间充质干细胞具有旺盛增殖和分化能力等优点。u c 抽样不存在伦理问题, 因为它通常被视为医疗废物。人类 uc 开始发展与羊膜腔的持续增长在4-8 的怀孕, 并不断增长, 直到达到50-60 厘米的长度, 它可以在整个新生儿分娩期间被隔离。为了深入了解难治性疾病的病理生理学, 我们使用了不同胎龄婴儿的 uc 衍生骨髓间充质干细胞 (uc-mscs)。在该协议中, 我们描述了在妊娠19-40周时从胎儿婴儿中分离和表征 uc-mscs 的方法。
Introduction
间充质干细胞 (mscs) 最初是从骨髓 (bm)1, 2 中分离和表征的, 但也可以从各种组织中获得, 包括脂肪组织、滑膜、皮肤、牙髓和胎儿附属物3. 骨髓间充质干细胞被认为是异质细胞群, 可以增殖并分化为脂肪细胞、骨细胞和软骨细胞。此外, 骨髓间充质干细胞具有迁移到损伤部位、抑制和调节免疫反应、重塑和修复损伤的能力。目前, 不同来源的骨髓间充质干细胞作为细胞治疗的来源, 包括移植物抗宿主病、心肌梗死和脑梗死4, 5,引起了越来越大的兴趣..
虽然 bm 是 msc 最有特色的来源, 但 bm 吸入的侵入性在伦理上限制了其可访问性。bm 中获得的骨髓间充质干细胞的增殖和分化能力普遍随着供体年龄的扩大而下降。相反, 从胎盘、脐带血 (ucb) 和脐带 (uc) 等胎儿附属物中获得的胎儿骨髓间充质干细胞具有优势, 包括对取样的伦理关注较少, 以及对增殖和分化能力的鲁棒性 6,7. 在通常作为医疗废物丢弃的胎儿附属物中, ucb 和 uc 被视为胎儿器官, 而胎盘被视为胎儿器官。此外, 需要在新生儿分娩的确切时刻对胎盘和 ucb 进行取样和收集, 而胎盘和 uc 则可以在新生儿分娩后收集和处理。因此, uc 是一个有前途的 msc 来源的细胞治疗 8,9。
人类 uc 开始发展与羊膜腔在4-8 的妊娠, 继续增长, 直到50-60 厘米的长度, 并可以在整个新生儿分娩期间隔离10。为了深入了解难治性疾病的病理生理学, 我们使用了不同妊娠年龄11岁、12岁的婴儿的 uc 衍生骨髓间充质干细胞 (uc-mscs)。在该协议中, 我们描述了如何在妊娠19-40周时从胎儿婴儿中分离和表征 uc-mscs。
Protocol
这项研究的人体样本使用得到了神户大学医学研究生院伦理委员会的批准 (第1370号和第1694号批准), 并按照批准的准则进行。 1. uc-mscs 的分离和培养 请注意:已成功地从200多个受该协议影响的 uc-mscs 中分离、培养和扩展 (超过第4段)。在200多家用品中, 100% 的 uc-msc 隔离成功, 不到5% 的人出现意外污染, 不到15% 的人表现出生长抑制, 80% 以上的人显示 u…
Representative Results
图 1总结了从 uc 集合到 msc 区域性的过程。大约5-10 厘米长的 uc 可以从所有新生儿剖宫产中收集。uc 开始发展在4-8 的妊娠, 并继续增长, 直到50-60 厘米的长度, 如图 2所示。uc 中有两条动脉 (a)、一条静脉 (v)、脐带衬里 (cl) 和沃顿果冻 (wj), 如图 3和图 4所示。uc-mscs 可以从所有电源线区?…
Discussion
骨髓间充质干细胞可以从各种组织中分离出来, 并且是异质性的细胞群, 并不都表达相同的表型标记。在这里, 我们概述了一个协议, 指导从早产儿和足月婴儿的收集和解剖 uc, 并实现 pc-mscs 的隔离和培养。根据该协议, 我们成功地从妊娠19-40周的胎儿婴儿中分离出符合 isct 标准的uc-msc, 并证明它们代表了难治性疾病病理生理学的某些方面在产前发展期间 11,…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了 jps kakenhi 的援助赠款 (c) (赠款编号: 25461644) 和青年科学家 (b) (赠款编号: 15k1914、2686045、17k16 298) 的支持。
Materials
| 50mL plastic tube | AS One Coporation, Osaka, Japan | Violamo 1-3500-22 | |
| 12-well plate | AGC Techno Glass, Tokyo, Japan | Iwaki 3815-012 | |
| 60mm dish | AGC Techno Glass, Tokyo, Japan | Iwaki 3010-060 | |
| Cell strainer (100 μm) | Thermo Fisher Scientific, Waltham, MA | Falcon 35-2360 | |
| Cell strainer (70 μm) | Thermo Fisher Scientific, Waltham, MA | Falcon 35-2350 | |
| Alpha MEM | Wako Pure Chemical, Osaka, Japan | 135-15175 | |
| Fetal bovine serum | Sigma Aldrich, St. Louis, MO | 172012 | |
| Reduced serum medium | Thermo Fisher Scientific, waltham, MA | OPTI-MEM Gibco 31985-070 | |
| Antibiotic-antimycotic | Thermo Fisher Scientific, Waltham, MA | Gibco 15240-062 | |
| Trypsin-EDTA | Wako Pure Chemical, Osaka, Japan | 209-16941 | |
| PBS | Takara BIO, Shiga,Japan | T900 | |
| Purified enzyme blends | Roche, Mannheim, Germany | Liberase DH Research Grade 05401054001 | |
| PE-conjugated mouse primary antibody against CD14 | BD Bioscience, Franklin Lakes, NJ | 347497 | Lot: 3220644, RRID: AB_400312 |
| PE-conjugated mouse primary antibody against CD19 | BD Bioscience, Franklin Lakes, NJ | 340364 | Lot: 3198741, RRID: AB_400018 |
| PE-conjugated mouse primary antibody against CD34 | BD Bioscience, Franklin Lakes, NJ | 555822 | Lot: 3079912, RRID: AB_396151 |
| PE-conjugated mouse primary antibody against CD45 | BD Bioscience, Franklin Lakes, NJ | 555483 | Lot: 2300520, RRID: AB_395875 |
| PE-conjugated mouse primary antibody against CD73 | BD Bioscience, Franklin Lakes, NJ | 550257 | Lot: 3057778, RRID: AB_393561 |
| PE-conjugated mouse primary antibody against CD90 | BD Bioscience, Franklin Lakes, NJ | 555596 | Lot: 3128616, RRID: AB_395970 |
| PE-conjugated mouse primary antibody against CD105 | BD Bioscience, Franklin Lakes, NJ | 560839 | Lot: 4339624, RRID: AB_2033932 |
| PE-conjugated mouse primary antibody against HLA-DR | BD Bioscience, Franklin Lakes, NJ | 347367 | Lot: 3219843, RRID: AB_400293 |
| PE-conjugated mouse IgG1 k isotype | BD Bioscience, Franklin Lakes, NJ | 555749 | Lot: 3046675, RRID: AB_396091 |
| PE-conjugated mouse IgG2a k isotype | BD Bioscience, Franklin Lakes, NJ | 555574 | Lot: 3035934, RRID: AB_395953 |
| PE-conjugated mouse IgG2b k isotype | BD Bioscience, Franklin Lakes, NJ | 555743 | Lot: 3098896, RRID: AB_396086 |
| Viability dye | BD Bioscience, Franklin Lakes, NJ | Fixable Viability Stain 450 562247 | |
| Blocking reagent | Dainippon Pharmaceutical, Osaka, Japan | Block Ace UKB80 | |
| FCM | BD Bioscience, Franklin Lakes, NJ | BD FACSAria III Cell Sorter | |
| FCM software | BD Bioscience, Franklin Lakes, NJ | BD FACSDiva | |
| Adipogenic differentiation medium | Invitrogen, Carlsbad, CA | StemPro Adipogenesis Differentiation kit A10070-01 | |
| Osteogenic differentiation medium | Invitrogen, Carlsbad, CA | StemPro Osteogenesis Differentiation kit A10072-01 | |
| Chondrogenic differentiation medium | Invitrogen, Carlsbad, CA | StemPro Chondrogenesis Differentiation kit A10071-01 | |
| Formaldehyde | Polyscience, Warrigton, PA | 16% UltraPure Formaldehyde EM Grade #18814 | |
| Oil Red O | Sigma Aldrich, St. Louis, MO | O0625 | |
| Arizarin Red S | Sigma Aldrich, St. Louis, MO | A5533 | |
| Toluidine Blue | Sigma Aldrich, St. Louis, MO | 198161 | |
| Microscope | Keyence, Osaka, Japan | BZ-X700 |
References
- Friedenstein, A. J., Petrakova, K. V., Kurolesova, A. I., Frolova, G. P. Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation. 6 (2), 230-247 (1968).
- Caplan, A. I. Mesenchymal stem cells. Journal of Orthopaedic Research. 9 (5), 641-650 (1991).
- Crisan, M., et al. A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell. 3 (3), 301-313 (2008).
- Bianco, P., Robey, P. G., Simmons, P. J. Mesenchymal Stem Cells: Revisiting History, Concepts, and Assays. Cell Stem Cell. 2 (4), 313-319 (2008).
- Bianco, P. 34;Mesenchymal" stem cells. Annual Review of Cell and Developmental Biology. 30 (1), 677-704 (2014).
- Baksh, D., Yao, R., Tuan, R. S. Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived from umbilical cord and bone marrow. Stem Cells. 25 (6), 1384-1392 (2007).
- Manochantr, S., et al. Immunosuppressive properties of mesenchymal stromal cells derived from amnion, placenta, Wharton’s jelly and umbilical cord. Internal Medicine Journal. 43 (4), 430-439 (2013).
- Arutyunyan, I., et al. Umbilical Cord as Prospective Source for Mesenchymal Stem Cell-Based Therapy. Stem Cells International. 6901286, (2016).
- Davies, J. E., Walker, J. T., Keating, A. Concise Review: Wharton’s Jelly: The Rich, but Enigmatic, Source of Mesenchymal Stromal Cells. Stem Cells Translational Medicine. 6 (7), 1620-1630 (2017).
- Zhu, D., Wallace, E. M., Lim, R. Cell-based therapies for the preterm infant. Cytotherapy. 16 (12), 1614-1628 (2014).
- Iwatani, S., et al. Gestational Age-Dependent Increase of Survival Motor Neuron Protein in Umbilical Cord-Derived Mesenchymal Stem Cells. Frontiers in Pediatrics. 5, 194 (2017).
- Iwatani, S., et al. Involvement of WNT Signaling in the Regulation of Gestational Age-Dependent Umbilical Cord-Derived Mesenchymal Stem Cell Proliferation. Stem Cells International. , 8749751 (2017).
- Mennan, C., et al. Isolation and characterisation of mesenchymal stem cells from different regions of the human umbilical cord. BioMed Research International. 916136, (2013).
- Capelli, C., et al. Minimally manipulated whole human umbilical cord is a rich source of clinical-grade human mesenchymal stromal cells expanded in human platelet lysate. Cytotherapy. 13 (7), 786-801 (2011).
- Lu, L. L., et al. Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials. Haematologica. 91 (8), 1017-1026 (2006).
- Tong, C. K., et al. Generation of mesenchymal stem cell from human umbilical cord tissue using a combination enzymatic and mechanical disassociation method. Cell Biology International. 35 (3), 221-226 (2011).
- Han, Y. F., et al. Optimization of human umbilical cord mesenchymal stem cell isolation and culture methods. Cytotechnology. 65 (5), 819-827 (2013).
- Paladino, F. V., Peixoto-Cruz, J. S., Santacruz-Perez, C., Goldberg, A. C. Comparison between isolation protocols highlights intrinsic variability of human umbilical cord mesenchymal cells. Cell Tissue Bank. 17 (1), 123-136 (2016).
- Dominici, M., et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 8 (4), 315-317 (2006).
- Mareschi, K., et al. Expansion of mesenchymal stem cells isolated from pediatric and adult donor bone marrow. Journal of Cellular Biochemistry. 97 (4), 744-754 (2006).
- Choumerianou, D. M., et al. Comparative study of stemness characteristics of mesenchymal cells from bone marrow of children and adults. Cytotherapy. 12 (7), 881-887 (2010).
- Hong, S. H., et al. Ontogeny of human umbilical cord perivascular cells: molecular and fate potential changes during gestation. Stem Cells and Development. 22 (17), 2425-2439 (2013).
Cite This Article
Iwatani, S., Yoshida, M., Yamana, K., Kurokawa, D., Kuroda, J., Thwin, K. K. M., Uemura, S., Takafuji, S., Nino, N., Koda, T., Mizobuchi, M., Nishiyama, M., Fujioka, K., Nagase, H., Morioka, I., Iijima, K., Nishimura, N. Isolation and Characterization of Human Umbilical Cord-derived Mesenchymal Stem Cells from Preterm and Term Infants. J. Vis. Exp. (143), e58806, doi:10.3791/58806 (2019).