Summary

将人 iPSC 稳健分化为纯脂肪细胞群以研究脂肪细胞相关疾病

Published: February 09, 2022
doi:

Summary

该方案允许从诱导多能干细胞(iPSC)产生纯脂肪细胞群。维甲酸用于将iPSCs分化为间充质干细胞(MSC),用于产生脂肪细胞。然后,采用基于尼罗河红染色的分选方法获得纯脂肪细胞。

Abstract

诱导多能干细胞(iPSC)技术的最新进展允许产生不同的细胞类型,包括脂肪细胞。然而,目前的分化方法效率低,不能产生均匀的脂肪细胞群。在这里,我们通过使用基于全反式视黄体的方法以高产量生产间充质干细胞(MSCs)来规避这个问题。通过调节控制细胞增殖、存活和粘附的途径,我们的分化策略允许有效生成分化成纯多能MSC群体的胚胎体(EB)。该方法产生的大量MSC为产生脂肪细胞提供了理想的来源。然而,脂肪细胞分化导致的样品异质性仍然是一个挑战。因此,我们使用基于尼罗河红的方法使用FACS纯化含脂成熟脂肪细胞。这种分选策略使我们能够建立一种可靠的方法来使用具有降低样品异质性和增强细胞功能的脂肪细胞池来模拟脂肪细胞相关的代谢紊乱。

Introduction

间充质干细胞(MSCs)是产生中胚层起源细胞(如脂肪细胞,骨细胞和软骨细胞)的有效短暂资源,可以进一步用于模拟其各自的遗传疾病。然而,以前的方法依赖于从成人组织1获得这些MSC,这带来了从供体获得大量MSC的挑战,并且限制了它们在次优培养条件下保持功能存活12。这些障碍产生了对体外产生MSCs的方案的巨大需求。人诱导多能干细胞(iPSCs)可用作MSC的宝贵来源,表现出MSC特征345。iPSCs衍生的间充质干细胞可用作多种疾病的治疗选择。此外,iPSCs衍生的MSCs产生脂肪细胞的能力使其成为研究人类脂肪生成,肥胖和脂肪细胞相关疾病的有价值的体外人类模型。

目前脂肪细胞的分化方案可分为两组,一组涉及使用化学或基于蛋白质的混合物分化脂肪细胞,结果产量为30%-60%678,9另一组涉及遗传操作以稳健地诱导控制脂肪细胞发育的关键转录因子,以产生80%-90%10的产量11.然而,基因操作并不能概括脂肪细胞分化的自然过程,并且经常掩盖脂肪生成过程中到达的微妙范式,使其对疾病建模目的无效1213。因此,我们提出了一种通过使用尼罗河红荧光标记含脂脂肪细胞来将化学衍生的成熟脂肪细胞与未成熟脂肪细胞进行分类的方法。

在这里,我们提出了一种协议,涉及将iPSC衍生的拟胚体(EB)与全反式 维甲酸瞬时孵育以产生大量快速增殖的MSC,这可以进一步用于产生脂肪细胞14。我们还提出了一种通过使用亲脂性染料荧光标记其脂滴来从异质分化池中分选化学衍生的成熟脂肪细胞的方法;尼罗河红。这将允许产生具有增强功能的成熟脂肪细胞的纯群体,以准确模拟脂肪细胞相关的代谢紊乱。

Protocol

该研究已获得适当的机构研究伦理委员会的批准,并按照1964年《赫尔辛基宣言》及其后来的修正案或类似的伦理标准中规定的伦理标准进行。该协议已获得HMC(编号16260/16)和QBRI(编号:2016-003)的机构审查委员会(IRB)的批准。这项工作还针对H1和H9等hESC进行了优化。血液样本是在完全知情同意的情况下从健康个体获得的。iPSCs由健康个体的外周血单核细胞(PBMC)产生。 <stro…

Representative Results

间充质分化过程中细胞的示意图和形态:iPSC 分化为 MSC 涉及跨越 EB 形成、MSC 分化和 MSC 扩增的不同发育阶段(图 1)。在这些发育阶段,细胞由于受到不同的刺激化学物质而获得各种形态。在开始分化时,细胞被铺在悬浮液中,并且预期是圆形的,具有明确的细胞边界,同时直径为小到中等尺寸(图2)。在分化的初始阶段选择悬浮培养细胞使其与自?…

Discussion

该协议至关重要,因为它能够提供高产量和高效率的MSC。这种大规模生产MSCs是通过将iPSCs衍生的EB与10μM的RA1415瞬时孵育来实现的。用 10 μM RA 瞬时处理将 MSC 产量提高了 11.2 至 1542 倍1415该方案适用于 iPSC 和 hPSC。在此剂量和治疗持续时间下,RA通过直接或间接调节参与细胞增殖,凋亡以及细胞间和ECM细?…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

这项工作由卡塔尔国家研究基金(QNRF)资助(资助号NPRP10-1221-160041)。Maryam Aghadi得到了卡塔尔国家研究基金(QNRF)的GSRA奖学金的支持。

Materials

Adiponectin Abcam ab22554 Adipocyte maturation marker
anti-CD105 BD Pharmingen 560839 MSC differentiation marker
anti-CD14 BD Pharmingen 561712 MSC differentiation marker
anti-CD19 BD Pharmingen 555415 MSC differentiation marker
anti-CD34 BD Pharmingen 555824 MSC differentiation marker
anti-CD44 abcam ab93758 MSC differentiation marker
anti-CD45 BD Pharmingen
560975
MSC differentiation marker
anti-CD73 BD Pharmingen 550256 MSC differentiation marker
anti-CD90 BD Pharmingen 555596 MSC differentiation marker
bFGF R&D 233-FP MSC culture media supplement
C/EBPA Abcam ab40761 Adipocyte maturation marker
Dexamethasone Torics 1126 Adipocyte differentiation media supplement
FABP4 Abcam ab93945 Adipocyte maturation marker
Fetal bovine serum ThermoFisher 10082147 MSC culture media supplement
Glutamax ThermoFisher 35050-061 MSC culture media supplement
IBMX Sigma Aldrich I5879 Adipocyte differentiation media supplement
Indomethacin Sigma Aldrich I7378 Adipocyte differentiation media supplement
Insulin Sigma Aldrich 91077C Adipocyte differentiation media supplement
Knockout DMEM ThermoFisher 12660012 Basal media for preparing matrigel
Low glucose DMEM ThermoFisher 11885084 MSC culturing media
Matrigel Corning 354230 Coating matrix
MEM-alpha ThermoFisher 12561056 Adipocyte differentiation media
Nilered Sigma Aldrich 19123 Sorting marker for adipocyte
Penicillin ThermoFisher 15140122 MSC/Adipocyte media supplement
Phosphate-buffered saline ThermoFisher 14190144 wash buffer
Pierce™ 20X TBS Buffer Thermo Fisher 28358 wash buffer
PPARG Cell Signaling Technology 2443 Adipocyte maturation marker
ReLeSR Stem Cell Technologies 5872 Dissociation reagent
Retinoic acid Sigma Aldrich R2625 MSC differentiation media supplement
Rock inhibitor Tocris 1254/10 hPSC culture media supplement
Roziglitazone Sigma Aldrich R2408 Adipocyte differentiation media supplement
StemFlex ThermoFisher A334901 hPSC culture media
Triton Thermo Fisher 28314 Permebealization reagent
Trypsin ThermoFisher 25200072 Dissociation reagent
Tween 20 Sigma Aldrich P7942 Wash buffer

Referencias

  1. Hass, R., Kasper, C., Bohm, S., Jacobs, R. Different populations and sources of human mesenchymal stem cells (MSC): A comparison of adult and neonatal tissue-derived MSC. Cell Communication and Signaling: CCS. 9, 12 (2011).
  2. Wagner, W., et al. Aging and replicative senescence have related effects on human stem and progenitor cells. PLoS One. 4 (6), 5846 (2009).
  3. Brown, P. T., Squire, M. W., Li, W. J. Characterization and evaluation of mesenchymal stem cells derived from human embryonic stem cells and bone marrow. Cell and Tissue Research. 358 (1), 149-164 (2014).
  4. Trivedi, P., Hematti, P. Derivation and immunological characterization of mesenchymal stromal cells from human embryonic stem cells. Experimental Hematology. 36 (3), 350-359 (2008).
  5. Barberi, T., Willis, L. M., Socci, N. D., Studer, L. Derivation of multipotent mesenchymal precursors from human embryonic stem cells. PLoS Medicine. 2 (6), 161 (2005).
  6. Xiong, C., et al. Derivation of adipocytes from human embryonic stem cells. Stem Cells and Development. 14 (6), 671-675 (2005).
  7. Cuaranta-Monroy, I., et al. Highly efficient differentiation of embryonic stem cells into adipocytes by ascorbic acid. Stem Cell Research. 13 (1), 88-97 (2014).
  8. van Harmelen, V., et al. Differential lipolytic regulation in human embryonic stem cell-derived adipocytes. Obesity (Silver Spring). 15 (4), 846-852 (2007).
  9. Noguchi, M., et al. In vitro characterization and engraftment of adipocytes derived from human induced pluripotent stem cells and embryonic stem cells. Stem Cells and Development. 22 (21), 2895-2905 (2013).
  10. Ahfeldt, T., et al. Programming human pluripotent stem cells into white and brown adipocytes. Nature Cell Biology. 14 (2), 209-219 (2012).
  11. Lee, Y. K., Cowan, C. A. Differentiation of white and brown adipocytes from human pluripotent stem cells. Methods in Enzymology. 538, 35-47 (2014).
  12. Abdelalim, E. M. Modeling different types of diabetes using human pluripotent stem cells. Cellular and Molecular Life Sciences: CMLS. 78 (6), 2459-2483 (2021).
  13. Abdelalim, E. M., Bonnefond, A., Bennaceur-Griscelli, A., Froguel, P. Pluripotent stem cells as a potential tool for disease modelling and cell therapy in diabetes. Stem Cell Reviews and Reports. 10 (3), 327-337 (2014).
  14. Karam, M., Younis, I., Elareer, N. R., Nasser, S., Abdelalim, E. M. Scalable Generation of mesenchymal stem cells and adipocytes from human pluripotent stem cells. Cells. 9 (3), (2020).
  15. Karam, M., Abdelalim, E. M. Robust and highly efficient protocol for differentiation of human pluripotent stem cells into mesenchymal stem cells. Methods in Molecular Biology. , (2020).
  16. Li, L., Bennett, S. A., Wang, L. Role of E-cadherin and other cell adhesion molecules in survival and differentiation of human pluripotent stem cells. Cell Adhesion & Migration. 6 (1), 59-70 (2012).
  17. Lai, L., Bohnsack, B. L., Niederreither, K., Hirschi, K. K. Retinoic acid regulates endothelial cell proliferation during vasculogenesis. Development. 130 (26), 6465-6474 (2003).
  18. Chanchevalap, S., Nandan, M. O., Merlin, D., Yang, V. W. All-trans retinoic acid inhibits proliferation of intestinal epithelial cells by inhibiting expression of the gene encoding Kruppel-like factor 5. FEBS Letters. 578 (1-2), 99-105 (2004).
  19. di Masi, A., et al. Retinoic acid receptors: from molecular mechanisms to cancer therapy. Molecular Aspects of Medicine. 41, 1 (2015).
  20. Simandi, Z., Balint, B. L., Poliska, S., Ruhl, R., Nagy, L. Activation of retinoic acid receptor signaling coordinates lineage commitment of spontaneously differentiating mouse embryonic stem cells in embryoid bodies. FEBS Letters. 584 (14), 3123-3130 (2010).
  21. De Angelis, M. T., Parrotta, E. I., Santamaria, G., Cuda, G. Short-term retinoic acid treatment sustains pluripotency and suppresses differentiation of human induced pluripotent stem cells. Cell Death & Disease. 9 (1), 6 (2018).
  22. Li, L., Dong, L., Wang, Y., Zhang, X., Yan, J. Lats1/2-mediated alteration of hippo signaling pathway regulates the fate of bone marrow-derived mesenchymal stem cells. BioMed Research International. 2018, 4387932 (2018).
  23. Moldes, M., et al. Peroxisome-proliferator-activated receptor gamma suppresses Wnt/beta-catenin signalling during adipogenesis. The Biochemical Journal. 376, 607-613 (2003).
  24. Ross, S. E., et al. Inhibition of adipogenesis by Wnt signaling. Science. 289 (5481), 950-953 (2000).
  25. Wang, Y. K., Chen, C. S. Cell adhesion and mechanical stimulation in the regulation of mesenchymal stem cell differentiation. Journal of Cellular and Molecular Medicine. 17 (7), 823-832 (2013).
  26. Mohsen-Kanson, T., et al. Differentiation of human induced pluripotent stem cells into brown and white adipocytes: role of Pax3. Stem Cells. 32 (6), 1459-1467 (2014).
  27. Billon, N., et al. The generation of adipocytes by the neural crest. Development. 134 (12), 2283-2292 (2007).
  28. Li, N., Kelsh, R. N., Croucher, P., Roehl, H. H. Regulation of neural crest cell fate by the retinoic acid and Pparg signalling pathways. Development. 137 (3), 389-394 (2010).
  29. Ussar, S., et al. ASC-1, PAT2, and P2RX5 are cell surface markers for white, beige, and brown adipocytes. Science Translational Medicine. 6 (247), (2014).
  30. Festy, F., et al. Surface protein expression between human adipose tissue-derived stromal cells and mature adipocytes. Histochemistry and Cell Biology. 124 (2), 113-121 (2005).
  31. Cai, L., Wang, Z., Ji, A., Meyer, J. M., vander Westhuyzen, D. R. Scavenger receptor CD36 expression contributes to adipose tissue inflammation and cell death in diet-induced obesity. PLoS One. 7 (5), 36785 (2012).
  32. Mesuret, G., et al. A neuronal role of the Alanine-Serine-Cysteine-1 transporter (SLC7A10, Asc-1) for glycine inhibitory transmission and respiratory pattern. Scientific Reports. 8 (1), 8536 (2018).
  33. Silverstein, R. L., Febbraio, M. CD36, a scavenger receptor involved in immunity, metabolism, angiogenesis, and behavior. Science Signaling. 2 (72), (2009).
  34. Brooimans, R. A., van Wieringen, P. A., van Es, L. A., Daha, M. R. Relative roles of decay-accelerating factor, membrane cofactor protein, and CD59 in the protection of human endothelial cells against complement-mediated lysis. European Journal of Immunology. 22 (12), 3135-3140 (1992).
  35. Davies, A., et al. CD59, an LY-6-like protein expressed in human lymphoid cells, regulates the action of the complement membrane attack complex on homologous cells. The Journal of Experimental Medicine. 170 (3), 637-654 (1989).
  36. Lapid, K., Graff, J. M. Form(ul)ation of adipocytes by lipids. Adipocyte. 6 (3), 176-186 (2017).
  37. Aldridge, A., et al. Assay validation for the assessment of adipogenesis of multipotential stromal cells–a direct comparison of four different methods. Cytotherapy. 15 (1), 89-101 (2013).
  38. Schaedlich, K., Knelangen, J. M., Navarrete Santos, A., Fischer, B., Navarrete Santos, A. A simple method to sort ESC-derived adipocytes. Cytometry A. 77 (10), 990-995 (2010).
  39. Costa, L. A., et al. Functional heterogeneity of mesenchymal stem cells from natural niches to culture conditions: implications for further clinical uses. Cellular and Molecular Life Sciences: CMLS. 78 (2), 447-467 (2021).

Play Video

Citar este artículo
Aghadi, M., Karam, M., Abdelalim, E. M. Robust Differentiation of Human iPSCs into a Pure Population of Adipocytes to Study Adipocyte-Associated Disorders. J. Vis. Exp. (180), e63311, doi:10.3791/63311 (2022).

View Video