Mesenchymal Stem Cell Preparation and Transfection‐free Ferumoxytol Labeling for MRI Cell Tracking

Li Liu1, Chien Ho2

1 Present address: National Institute of Neurological Disorders and Stroke, National Institute of Health, Bethesda, 2 Department of Biological Sciences, Carnegie Mellon University, Pittsburgh
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2B.7
DOI:  10.1002/cpsc.38
Online Posting Date:  November, 2017
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Mesenchymal stem cells (MSCs) are multipotent cells and are the most widely studied cell type for stem cell therapies. In vivo cell tracking of MSCs labeled with an FDA‐approved superparamagnetic iron‐oxide (SPIO) particle by magnetic resonance imaging (MRI) provides essential information, e.g., MSC engraftment, survival, and fate, thus improving cell therapy accuracy. However, current methodology for labeling MSCs with Ferumoxytol (Feraheme®), the only FDA‐approved SPIO particle, needs transfection agents. This unit describes a new “bio‐mimicry” protocol to prepare more native MSCs by using more “in vivo environment” of MSCs, so that the phagocytic activity of cultured MSCs is restored and expanded MSCs can be labeled with Ferumoxytol, without the need for transfection agents and/or electroporation. Moreover, MSCs re‐size to a more native size, reducing from 32.0 to 19.5 μm. The MSCs prepared from this protocol retain more native properties and would be useful for biomedical applications and MSC‐tracking studies by MRI. © 2017 by John Wiley & Sons, Inc.

Keywords: Ferumoxytol (Feraheme®); magnetic resonance imaging; stem cell culture and expansion; stem cell tracking; mesenchymal stem cells

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation and Culture of MSCs
  • Basic Protocol 2: Bio‐Mimicry Method to Prepare MSCs and to Label MSCs with Ferumoxytol
  • Support Protocol 1: Characterization of Prepared and Labeled MSCs
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


Basic Protocol 1: Isolation and Culture of MSCs

  • Dulbecco's modified Eagle's medium (DMEM; see recipe)
  • Ice
  • 70% isopropyl alcohol (First Priority, cat. no. MS073)
  • Brown Norway (BN) rat
  • Isoflurane (Bulter Schein, cat no. 11695‐6776‐1)
  • Trypan blue solution (Sigma‐Aldrich, cat. no. T8154)
  • Phosphate‐buffered saline (PBS; Mediatech, cat. no. 21‐040‐CV)
  • 15‐ml and 50‐ml sterile conical centrifuge tubes (Sigma‐Aldrich, cat. no. CLS430791 and CLS430829, respectively)
  • 2‐ml and 5‐ml pipettes (Sigma‐Aldrich, cat. no. CLS4021 and P7615, respectively)
  • 6‐well culture dishes (10‐cm; Sigma‐Aldrich, cat. no. CLS3516)
  • 27‐G needles (Becton, Dickinson and Company, cat. no. 301629)
  • 10‐ml syringes (Sigma‐Aldrich, cat. no. Z248029)
  • Steel surgical forceps (straight and curved), sterilized
  • Sponges, sterilized
  • Steel surgical scissors, sterilized
  • 70‐mm filter, optional
  • Hemacytometer with coverslips (Sigma‐Aldrich, cat. no. Z359629)
  • Incubator with both temperature and gas composition controls

Basic Protocol 2: Bio‐Mimicry Method to Prepare MSCs and to Label MSCs with Ferumoxytol

  Additional Materials (also see protocol 1)
  • MSC cells (see protocol 1), incubated 7 days
  • BN rat
  • 0.25% trypsin/1 mM ethylenediaminetetraacetic acid (EDTA) (Thermo Fisher Scientific, cat. no. 25200056)
  • Ferumoxytol (see recipe for stock solution)
  • Centrifuge (no temperature control is needed)
  • Inverted microscope equipped with a suitable camera and cell‐size measuring software
  • 5‐mm NMR tubes (Sigma‐Aldrich, cat. no. NORS55007)
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
  Al Battah, F., De Kock, J., Ramboer, E., Heymans, A., Vanhaecke, T., Rogiers, V., & Snykers, S. (2011). Evaluation of the multipotent character of human adipose tissue‐derived stem cells isolated by Ficoll gradient centrifugation and red blood cell lysis treatment. Toxicology In Vitro, 25, 1224–1230. doi: 10.1016/j.tiv.2011.05.024.
  Alper, J. (2009). Geron gets green light for human trial of ES cell‐derived product. Nature Biotechnology, 27, 213–214. doi: 10.1038/nbt0309‐213a.
  Bang, O. Y., Lee, J. S., Lee, P. H., & Lee, G. (2005). Autologous mesenchymal stem cell transplantation in stroke patients. Annals of Neurology, 57, 874–882. doi: 10.1002/ana.20501.
  Bashir, M. R., Bhatti, L., Marin, D., & Nelson, R. C. (2015). Emerging Applications for Ferumoxytol as a Contrast Agent in MRI. Journal of Magnetic Resonance Imaging, 41, 884–898. doi: 10.1002/jmri.24691.
  Bianco, P. (2014). "Mesenchymal" stem cells. Annual Review of Cell and Developmental Biology, 30, 677–704. doi: 10.1146/annurev‐cellbio‐100913‐013132.
  Bolli, R., Chugh, A. R., D'Amario, D., Loughran, J. H., Stoddard, M. F., Ikram, S., … Anversa, P. (2011). Cardiac stem cells in patients with ischaemic cardiomyopathy (SCIPIO): Initial results of a randomised phase 1 trial. Lancet, 378, 1847–1857. doi: 10.1016/S0140‐6736(11)61590‐0.
  Chen, C. L., Zhang, H., Ye, Q., Hsieh, W. Y., Hitchens, T. K., Shen, H. H., … Ho, C. (2011a). A new nano‐sized iron oxide particle with high sensitivity for cellular magnetic resonance imaging. Molecular Imaging and Biology, 13, 825–839. doi: 10.1007/s11307‐010‐0430‐x.
  Chen, P. M., Yen, M. L., Liu, K. J., Sytwu, H. K., & Yen, B. L. (2011b). Immunomodulatory properties of human adult and fetal multipotent mesenchymal stem cells. Journal of Biomedical Science, 18, 49. doi: 10.1186/1423‐0127‐18‐49.
  Christodoulou, I., Kolisis, F. N., Papaevangeliou, D., & Zoumpourlis, V. (2013). Comparative evaluation of human mesenchymal stem cells of fetal (Wharton's Jelly) and adult (adipose tissue) origin during prolonged in vitro expansion: Considerations for cytotherapy. Stem Cells International, 2013, 246134. doi: 10.1155/2013/246134.
  Colter, D. C., Class, R., DiGirolamo, C. M., & Prockop, D. J. (2000). Rapid expansion of recycling stem cells in cultures of plastic‐adherent cells from human bone marrow. Proceedings of the National Academy of Sciences of the United States of America, 97, 3213–3218. doi: 10.1073/pnas.070034097.
  Colter, D. C., Sekiya, I., & Prockop, D. J. (2001). Identification of a subpopulation of rapidly self‐renewing and multipotential adult stem cells in colonies of human marrow stromal cells. Proceedings of the National Academy of Sciences of the United States of America, 98, 7841–7845. doi: 10.1073/pnas.141221698.
  Estrada, J. C., Torres, Y., Benguria, A., Dopazo, A., Roche, E., Carrera‐Quintanar, L., … Bernad, A. (2013). Human mesenchymal stem cell‐replicative senescence and oxidative stress are closely linked to aneuploidy. Cell Death & Disease, 4, e691. doi: 10.1038/cddis.2013.211.
  Friedenstein, A. J., Petrakova, K. V., Kurolesova, A. I., & Frolova, G. P. (1968). Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation, 6, 230–247. doi: 10.1097/00007890‐196803000‐00009.
  Hare, J. M., Fishman, J. E., Gerstenblith, G., DiFede Velazquez, D. L., Zambrano, J. P., Suncion, V. Y., … Lardo, A. (2012). Comparison of allogeneic vs autologous bone marrow‐derived mesenchymal stem cells delivered by transendocardial injection in patients with ischemic cardiomyopathy: The POSEIDON randomized trial. Jama : The Journal of the American Medical Association, 308, 2369–2379. doi: 10.1001/jama.2012.25321.
  Heldman, A. W., DiFede, D. L., Fishman, J. E., Zambrano, J. P., Trachtenberg, B. H., Karantalis, V., … Hare, J. M. (2014). Transendocardial mesenchymal stem cells and mononuclear bone marrow cells for ischemic cardiomyopathy: The TAC‐HFT randomized trial. Jama : The Journal of the American Medical Association, 311, 62–73. doi: 10.1001/jama.2013.282909.
  Hoch, A. I., & Leach, J. K. (2015). Concise review: Optimizing expansion of bone marrow mesenchymal stem/stromal cells for clinical applications. Stem Cells Translational Medicine, 4, 412. doi: 10.5966/sctm.2013‐0196erratum.
  Horn, P., Bork, S., Diehlmann, A., Walenda, T., Eckstein, V., Ho, A. D., & Wagner, W. (2008). Isolation of human mesenchymal stromal cells is more efficient by red blood cell lysis. Cytotherapy, 10, 676–685. doi: 10.1080/14653240802398845.
  Karantalis, V., & Hare, J. M. (2015). Use of Mesenchymal Stem Cells for Therapy of Cardiac Disease. Circulation Research, 116, 1413–1430. doi: 10.1161/Circresaha.116.303614.
  Khurana, A., Chapelin, F., Beck, G., Lenkov, O. D., Donig, J., Nejadnik, H., … Daldrup‐Link, H. E. (2013). Iron administration before stem cell harvest enables MR imaging tracking after transplantation. Radiology, 269, 186–197. doi: 10.1148/radiol.13130858.
  Kumamoto, M., Nishiwaki, T., Matsuo, N., Kimura, H., & Matsushima, K. (2009). Minimally cultured bone marrow mesenchymal stem cells ameliorate fibrotic lung injury. European Respiratory Journal, 34, 740–748. doi: 10.1183/09031936.00128508.
  Li, X., Zhang, Y., & Qi, G. (2013). Evaluation of isolation methods and culture conditions for rat bone marrow mesenchymal stem cells. Cytotechnology, 65, 323–334. doi: 10.1007/s10616‐012‐9497‐3.
  Liu, L., Tseng, L., Ye, Q., Wu, Y. L., Bain, D. J., & Ho, C. (2016). A New Method for Preparing Mesenchymal Stem Cells and Labeling with Ferumoxytol for Cell Tracking by MRI. Sci. Rep, 6, 26271. doi: 10.1038/srep26271.
  Ma, S., Xie, N., Li, W., Yuan, B., Shi, Y., & Wang, Y. (2014). Immunobiology of mesenchymal stem cells. Cell Death and Differentiation, 21, 216–225. doi: 10.1038/cdd.2013.158.
  Nadri, S., Soleimani, M., Hosseni, R. H., Massumi, M., Atashi, A., & Izadpanah, R. (2007). An efficient method for isolation of murine bone marrow mesenchymal stem cells. International Journal of Developmental Biology, 51, 723–729. doi: 10.1387/ijdb.072352ns.
  Nejadnik, H., Lenkov, O., Gassert, F., Fretwell, D., Lam, I., & Daldrup‐Link, H. E. (2016). Macrophage phagocytosis alters the MRI signal of ferumoxytol‐labeled mesenchymal stromal cells in cartilage defects. Scientific Reports, 6, 25897. doi: 10.1038/srep25897.
  Ng, C. P., Sharif, A. R., Heath, D. E., Chow, J. W., Zhang, C. B., Chan‐Park, M. B., … Griffith, L. G. (2014). Enhanced ex vivo expansion of adult mesenchymal stem cells by fetal mesenchymal stem cell ECM. Biomaterials, 35, 4046–4057. doi: 10.1016/j.biomaterials.2014.01.081.
  Nguyen, P. K., Riegler, J., & Wu, J. C. (2014). Stem cell imaging: From bench to bedside. Cell Stem Cell, 14, 431–444. doi: 10.1016/j.stem.2014.03.009.
  Noad, J., Gonzalez‐Lara, L. E., Broughton, H. C., McFadden, C., Chen, Y., Hess, D. A., & Foster, P. J. (2013). MRI tracking of transplanted iron‐labeled mesenchymal stromal cells in an immune‐compromised mouse model of critical limb ischemia. NMR in Biomedicine, 26, 458–467. doi: 10.1002/nbm.2884.
  Perin, E. C., Willerson, J. T., Pepine, C. J., Henry, T. D., Ellis, S. G., & Zhao, D. X. (2012). Cardiovascular Cell Therapy Research NetworkEffect of transendocardial delivery of autologous bone marrow mononuclear cells on functional capacity, left ventricular function, and perfusion in chronic heart failure: The FOCUS‐CCTRN trial. Jama : The Journal of the American Medical Association, 307, 1717–1726. doi: 10.1001/jama.2012.418.
  Peterbauer‐Scherb, A., van Griensven, M., Meinl, A., Gabriel, C., Redl, H., & Wolbank, S. (2010). Isolation of pig bone marrow mesenchymal stem cells suitable for one‐step procedures in chondrogenic regeneration. Journal of Tissue Engineering and Regenerative Medicine, 4, 485–490. doi: 10.1002/term.262.
  Samsonraj, R. M., Rai, B., Sathiyanathan, P., Puan, K. J., Rotzschke, O., Hui, J. H., … Cool, S. M. (2015). Establishing criteria for human mesenchymal stem cell potency. Stem Cells, 33, 1878–1891. doi: 10.1002/stem.1982.
  Soleimani, M., & Nadri, S. (2009). A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow. Nature Protocols, 4, 102–106. doi: 10.1038/nprot.2008.221.
  Squillaro, T., Peluso, G., & Galderisi, U. (2016). Clinical Trials With Mesenchymal Stem Cells: An Update. Cell Transplantation, 25, 829–848. doi: 10.3727/096368915X689622.
  Thu, M. S., Bryant, L. H., Coppola, T., Jordan, E. K., Budde, M. D., Lewis, B. K., … Frank, J. A. (2012). Self‐assembling nanocomplexes by combining ferumoxytol, heparin and protamine for cell tracking by magnetic resonance imaging. Nature Medicine, 18, 463–467. doi: 10.1038/nm.2666.
  Wang, W. B., Yen, M. L., Liu, K. J., Hsu, P. J., Lin, M. H., Chen, P. M., … Yen, B. L. (2015). Interleukin‐25 Mediates Transcriptional Control of PD‐L1 via STAT3 in Multipotent Human Mesenchymal Stromal Cells (hMSCs) to Suppress Th17 Responses. Stem Cell Reports, 5, 392–404. doi: 10.1016/j.stemcr.2015.07.013.
  Zhang, W., Zhang, F., Shi, H., Tan, R., Han, S., Ye, G., … Liu, X. (2014). Comparisons of rabbit bone marrow mesenchymal stem cell isolation and culture methods in vitro. PLoS One, 9, e88794. doi: 10.1371/journal.pone.0088794.
PDF or HTML at Wiley Online Library