人诱导多能干细胞分化为间充质基质细胞的两种代表性方法比较
Summary
该协议描述并比较了将hiPSC分化为间充质基质细胞（MSCs）的两种代表性方法。单层法的特点是成本更低，操作更简单，更容易成骨分化。胚状体（EBs）方法的特点是时间消耗更短。
Abstract
间充质基质细胞（MSCs）是成体多能干细胞，在再生医学中得到了广泛的应用。由于体细胞组织来源的 MSC 受到有限捐赠、质量差异和生物安全性的限制，过去 10 年中，从人类诱导多能干细胞 （hiPSC） 中产生 MSC 的努力大幅增加。过去和最近将hiPSCs分化为MSCs的努力都集中在两种培养方法上：（1）胚状体（EBs）的形成和（2）单层培养的使用。该协议描述了从hiPSCs中提取MSC的这两种代表性方法。每种方法都有其优点和缺点，包括时间、成本、细胞增殖能力、MSC标志物的表达及其 体外分化能力。该协议表明，这两种方法都可以从hiPSCs中获得成熟且功能性的MSC。单层法的特点是成本更低、操作更简单、更容易成骨分化，而EB法的特点是时间消耗更短。
Introduction
间充质基质细胞 （MSC） 是中胚层来源的成体多能干细胞1。间充质干细胞存在于几乎所有结缔组织中2.自 1970 年代首次发现 MSC 并于 1987 年由 Friedenstein 等人成功从骨髓中分离出来 3,4,5 以来，各种人类体细胞（包括胎儿和成人）组织已被用于分离 MSC，例如骨骼、软骨、肌腱、肌肉、脂肪组织和造血支持基质 1,2,6,7.间充质干细胞表现出高增殖能力和可塑性，可分化为许多体细胞谱系，并可迁移到受伤和发炎的组织 2,8,9。这些特性使间充质干细胞成为再生医学的潜在候选者10.然而，体细胞组织来源的 MSC （st-MSCs） 受到有限的捐赠、有限的细胞增殖能力、质量差异以及可能从捐赠者传播病原体（如果有的话）的生物安全问题的限制11,12。
人诱导多能干细胞 （hiPSC） 来源于具有转录因子（Oct4、Sox2、Klf4 和 c-Myc）的成体细胞重编程，其功能与胚胎干细胞相似13,14。与st-MSCs相比，iPSC-MSCs具有无限供应、成本更低、纯度更高、质量控制方便、易于规模化生产和基因修饰等优点15,16,17。
由于 iPSC-MSC 的这些优势，已经报道了多种从 iPSC 驱动 MSC 的方法。这些分化方法以两种培养方法为中心：（1）胚状体（EBs）的形成和（2）使用单层培养物11,18,19,20。在此，对两种方法中的每一种都具有代表性的方法进行了表征。此外，还访问了两种基于时间、成本、增殖能力、MSC生物标志物表达和体外分化能力的代表性方法之间的比较。
Protocol
1. hiPSCs维持 hiPSC的解冻从液氮中取出细胞，在37°C水浴中快速解冻细胞。将解冻细胞转移到用 3 mL iPSC 维持培养基制备的 15 mL 管中（材料表）。轻轻混合培养基。 以300 ×g 离心5分钟。除去上清液，轻轻将细胞重悬于含有 10 μM Y-27632 的 1 mL iPSC 维持培养基中（上下移液细胞 2-3 次）。 将细胞悬浮液转移到涂有生长因子降低 （GFR） -细胞?…
Representative Results
按照方案（图1A），通过EB形成和单层培养方法将hiPSC分化为MSC。在分化过程中，细胞表现出不同的代表性形态（图1B，C）。 如 图1B所示，hiPSCs菌落在分化前表现出典型的致密形态，具有由紧密堆积的细胞组成的清晰边界。hiPSCs在振荡器上解离和培养24小时后形成的均匀球形EB。在MSCs分化培养基中?…
Discussion
在该协议中，检查了将hiPSCs分化为MSCs的两种代表性方法20,21,22,23,24,25,26,27,28,30。两种方法都能够从hiPSCs中衍生MSCs。通过细胞形态（<strong class="…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们非常感谢毛和胡实验室的所有成员，无论是过去还是现在，都为这个项目进行了有趣的讨论和巨大的贡献。我们感谢国家儿童健康临床医学研究中心的大力支持。本研究得到了国家自然科学基金（U20A20351毛建华，82200784立丹胡）、浙江省自然科学基金（No.LQ22C070004 Lidan 胡）。
Materials
| Alizarin red staining kit | Beyotime Biotechnology | C0148S | |
| Anti-human-CD105 (PE) | Biolegend | 323206 | |
| Anti-human-CD34 (FITC) | Biolegend | 343503 | |
| Anti-human-CD45 (APC) | Biolegend | 304011 | |
| Anti-human-CD73( APC) | Biolegend | 344006 | |
| Anti-human-CD90 (FITC) | Biolegend | 328108 | |
| Ascorbic acid | Solarbio | A8100 | |
| BMP-6 | Novoprotein | C012 | |
| Carbon dioxide level shaker | Crystal | CO-06UC6 | |
| Compensation Beads | BioLegend | 424601 | |
| CryoStor CS10 | STEMCELL Technology | 07959 | |
| Dexamethasone | Beyotime Biotechnology | ST1254 | |
| DMEM/F12 medium | Servicebio | G4610 | |
| Fetal bovine serum | HAKATA | HS-FBS-500 | |
| FGF2 | Stemcell | 78003.1 | |
| Gelatin | Sigma-Aldrich | G2500-100G | |
| GlutaMAX | Gibco | 35050061 | |
| human IgG1 isotype control APC | BioLegend | 403505 | |
| human IgG1 isotype control FITC | BioLegend | 403507 | |
| human IgG1 isotype control PE | BioLegend | 403503 | |
| Human TGF-β1 | Stemcell | 78067 | |
| Human TruStain FcX | BioLegend | 422301 | |
| IBMX | Beyotime Biotechnology | ST1398 | |
| Indomethacin | Solarbio | SI9020 | |
| Insulin | Beyotime Biotechnology | P3376 | |
| iPSC maintenance medium | STEMCELL Technology | 85850 | |
| ITS Media Supplement | Beyotime Biotechnology | C0341-10mL | |
| Matrigel, growth factor reduced | BD Corning | 354230 | |
| Oli Red O staining kit | Beyotime Biotechnology | C0158S | |
| Proline | Solarbio | P0011 | |
| Sodium pyruvate | ThermoFisher | 11360-070 | |
| TGFβ3 | Novoprotein | CJ44 | |
| Toluidine blue staining kit | Solarbio | G2543 | |
| TrypLE Express Enzyme(1x) | Gibco | 12604013 | |
| Ultra-Low Attachment 6 Well Plate | Costar | 3471 | |
| Versene | Gibco | 15040-66 | |
| Y-27632 | Stemcell | 72304 | |
| α-MEM | Hyclone | SH30265 | |
| β-glycerophosphate | Solarbio | G8100 |
References
- Weng, Z., et al. Mesenchymal stem/stromal cell senescence: Hallmarks, mechanisms, and combating strategies. Stem Cells Translational Medicine. 11 (4), 356-371 (2022).
- Soliman, H., et al. Multipotent stromal cells: One name, multiple identities. Cell Stem Cell. 28 (10), 1690-1707 (2021).
- Friedenstein, A. J., Chailakhyan, R. K., Gerasimov, U. V. Bone marrow osteogenic stem cells: in vitro cultivation and transplantation in diffusion chambers. Cell and Tissue Kinetics. 20 (3), 263-272 (1987).
- Friedenstein, A. J., et al. Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method. Experimental Hematology. 2 (2), 83-92 (1974).
- Friedenstein, A. J., Gorskaja, J. F., Kulagina, N. N. Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Experimental Hematology. 4 (5), 267-274 (1976).
- El Agha, E., et al. Mesenchymal stem cells in fibrotic disease. Cell Stem Cell. 21 (2), 166-177 (2017).
- Mushahary, D., Spittler, A., Kasper, C., Weber, V., Charwat, V. Isolation, cultivation, and characterization of human mesenchymal stem cells. Cytometry A. 93 (1), 19-31 (2018).
- Ullah, M., Liu, D. D., Thakor, A. S. Mesenchymal stromal cell homing: Mechanisms and strategies for improvement. iScience. 15, 421-438 (2019).
- Regmi, S., et al. Enhanced viability and function of mesenchymal stromal cell spheroids is mediated via autophagy induction. Autophagy. 17 (10), 2991-3010 (2021).
- Hoang, D. M., et al. Stem cell-based therapy for human diseases. Signal Transduction and Targeted Therapy. 7 (1), 272 (2022).
- Jiang, B., et al. Concise review: Mesenchymal stem cells derived from human pluripotent cells, an unlimited and quality-controllable source for therapeutic applications. Stem Cells. 37 (5), 572-581 (2019).
- Soontararak, S., et al. Mesenchymal stem cells (MSC) derived from induced pluripotent stem cells (iPSC) equivalent to adipose-derived MSC in promoting intestinal healing and microbiome normalization in mouse inflammatory bowel disease model. Stem Cells Translational Medicine. 7 (6), 456-467 (2018).
- Di Baldassarre, A., Cimetta, E., Bollini, S., Gaggi, G., Ghinassi, B. Human-induced pluripotent stem cell technology and cardiomyocyte generation: Progress and clinical applications. Cells. 7 (6), 48 (2018).
- Shi, Y., Inoue, H., Wu, J. C., Yamanaka, S. Induced pluripotent stem cell technology: a decade of progress. Nature Reviews. Drug Discovery. 16 (2), 115-130 (2017).
- Levy, O., et al. Shattering barriers toward clinically meaningful MSC therapies. Science Advances. 6 (30), eaba6884 (2020).
- Zhao, C., Ikeya, M. Generation and applications of induced pluripotent stem cell-derived mesenchymal stem cells. Stem Cells International. 2018, 9601623 (2018).
- Path, G., Perakakis, N., Mantzoros, C. S., Seufert, J. Stem cells in the treatment of diabetes mellitus – Focus on mesenchymal stem cells. Metabolism. 90, 1-15 (2019).
- Zhou, Y., et al. One-step derivation of functional mesenchymal stem cells from human pluripotent stem cells. Bio-Protocol. 8 (22), e3080 (2018).
- Hua, Z., et al. Low-intensity pulsed ultrasound promotes osteogenic potential of iPSC-derived MSCs but fails to simplify the iPSC-EB-MSC differentiation process. Frontiers in Bioengineering and Biotechnology. 10, 841778 (2022).
- Dupuis, V., Oltra, E. Methods to produce induced pluripotent stem cell-derived mesenchymal stem cells: Mesenchymal stem cells from induced pluripotent stem cells. World Journal of Stem Cells. 13 (8), 1094-1111 (2021).
- Zhang, W., et al. Aging stem cells. A Werner syndrome stem cell model unveils heterochromatin alterations as a driver of human aging. Science. 348 (6239), 1160-1163 (2015).
- Liu, G. H., et al. Modelling Fanconi anemia pathogenesis and therapeutics using integration-free patient-derived iPSCs. Nature Communications. 5, 4330 (2014).
- Kubben, N., et al. Repression of the antioxidant NRF2 pathway in premature aging. Cell. 165 (6), 1361-1374 (2016).
- Duan, S., et al. PTEN deficiency reprogrammes human neural stem cells towards a glioblastoma stem cell-like phenotype. Nature Communications. 6, 10068 (2015).
- Zhang, J., et al. Exosomes released from human induced pluripotent stem cells-derived MSCs facilitate cutaneous wound healing by promoting collagen synthesis and angiogenesis. Journal of Translational Medicine. 13, 49 (2015).
- Hu, G. W., et al. Exosomes secreted by human-induced pluripotent stem cell-derived mesenchymal stem cells attenuate limb ischemia by promoting angiogenesis in mice. Stem Cell Research & Therapy. 6 (1), 10 (2015).
- Kang, R., et al. Mesenchymal stem cells derived from human induced pluripotent stem cells retain adequate osteogenicity and chondrogenicity but less adipogenicity. Stem Cell Research & Therapy. 6 (1), 144 (2015).
- Wang, L. T., et al. Differentiation of mesenchymal stem cells from human induced pluripotent stem cells results in downregulation of c-Myc and DNA replication pathways with immunomodulation toward CD4 and CD8 cells. Stem Cells. 36 (6), 903-914 (2018).
- 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).
- Kim, S., Kim, T. M. Generation of mesenchymal stem-like cells for producing extracellular vesicles. World Journal of Stem Cells. 11 (5), 270-280 (2019).
Cite This Article
Wang, F., Gao, L., Fu, X., Yan, Q., Hu, L., Mao, J. Comparison of Two Representative Methods for Differentiation of Human Induced Pluripotent Stem Cells into Mesenchymal Stromal Cells. J. Vis. Exp. (200), e65729, doi:10.3791/65729 (2023).