This work was supported by a grant from the National Institute of Arthritis and Musculoskeletal and Skin Diseases: 3R01AR054458-02S2.

1. Day 1
1) Plate cells
<ol>
	<li>Plate hMSC in a T75 flask at a confluency of 80% at least 18-24 hours prior to labeling. Refer to Table 1 for instruction for alternate vessels.</li>
</ol>
2. Day 2
2) Prepare labeling solution. This preparation will label one (1) T75 flask at 80% confluency with a concentration of 400 &mu;g Fe/ml. Refer to Table 1 for instruction for alternate vessels.
<ol>
	<li>Create a solution (solution 1) by mixing 1 ml of serum-free media and 93.1 &mu;l of ferumoxytol (stock: 30 mg/ml) in a 15 ml conical tube.</li>
	<li>Create a second solution (solution 2) by mixing 1 ml of serum-free media and 7 &mu;l of protamine sulfate (stock: 10 mg/ml) in a second 15 ml conical tube.</li>
	<li>Allow each solution to rest for 5 minutes.</li>
	<li>Add solutions 1 and 2 together. Gently mix the new labeling solution. Allow the solution to rest for 5 minutes to permit USPIO-protamine sulfate complexes to form.</li>
	<li>Add 5 ml of serum-free media to the mixed 2 ml of SPIO/protamine sulfate solution for a final volume of 7 ml.</li>
</ol>
3) Prepare cells for labeling
<ol>
	<li>Aspirate the media from cells.</li>
	<li>Gently wash cells once with 2-3 ml of pre-warmed Mg/Ca-free D-PBS or serum-free media to rinse away residual serum proteins and or other media components that could impair contrast agent uptake and labeling efficiency. Aspirate out the rinse fluid.</li>
</ol>
4) Label cells
<ol>
	<li>Add the complete 7 ml of labeling solution to cells.</li>
	<li>Place cells into an incubator (37&deg;C/ 5% CO2) and allow the cells to incubate in the labeling solution for 4 hours.</li>
	<li>Add 700 &mu;l of FCS to the labeling solution of the cells to achieve a final serum concentration of 10%. Serum is added to re-establish the enriched environment of which the cells are accustomed, and to assist in minimizing cell death that could result from an abrupt shift to 24 hour exposure to a serum-free environment.</li>
	<li>Allow the cells to incubate with the labeling solution for an additional 20 hours.</li>
</ol>
3. Day 3
5) Prepare labeled cells
<ol>
	<li>Aspirate the labeling solution from cells.</li>
	<li>Gently rinse cells with 2-3 ml of pre-warmed Mg/Ca-free D-PBS. Aspirate out the PBS. It is important to use Mg/Ca-free PBS as the Mg and Ca will impair the efficiency of Trypsin in the next step.</li>
	<li>Add 2 ml of pre-warmed 0.05% Trypsin to the cells and tilt the flask back and forth to ensure the entire surface of the flask is covered with a thin layer of Trypsin. Place the cells in an incubator (37&deg;C/ 5% CO2) and allow the cells to rest for 5-7 minutes or until cells begin to detach from the plate. It may be necessary to confirm detachment under a microscope. Gently tap the sides of the flask if necessary to facilitate cell-surface detachment.</li>
	<li>Add 4 ml of pre-warmed complete media to the flask to neutralize the Trypsin. Gently rinse the flask by pipetting the Trypsin/media/cell solution up and down several times. Collect the entire solution in a 15 ml conical tube. Centrifuge the cell solution at 400 RCF for 5 minutes.</li>
	<li>Carefully aspirate the supernatant without disturbing the cell pellet. Resuspend the cells in 5 ml of media (serum containing or serum-free). Centrifuge the cell solution at 400 RCF for 5 minutes.</li>
	<li>Repeat the cell wash described in step 5.5. In total, cells will be rinsed three times to remove residual, free contrast agent in media and on the surface of cells.</li>
	<li>Count cells at this point to attain the most accurate cell count, as cells will be lost during previous washing. Perform a viability test of cells. Cells are now ready for subsequent analysis and or experimental application. MR imaging can be performed with T1-w and T2-w parameters, because although ferumoxytol is a T2-w contrast agent, ferumoxytol also exhibits T1 effects. Representative T2-w MRI parameters that can be used to image ferumoxytol labeled cells include: FSE or SE_Multislice sequences with a TR of 2000-2500 ms and a TE of 60-80 ms. To acquire a T1-w image, decrease the TE value on a FSE or SE_Multislice sequence or use a GRE sequence with a high TR and low TE. Figure 4 demonstrates a potential in vivo application for labeled stem cell imaging.</li>
</ol>
4. Representative Results:
Labeled cells demonstrate a significant darkening or negative contrast effect on T2-weighted MR images and a brightening or positive contrast effect on T1-weighted MR images (Figure 2). Longitudinal MR imaging and viability tests performed 5 days post labeling demonstrated no significant MR signal and no significant impact on viability compared to unlabeled controls (data not shown).
Labeled stem cells can subsequently be differentiated into various cell types or intravenously injected for in vivo investigation.
<img alt="Figure 1" src="/files/ftp_upload/3482/3482fig1.jpg" /> 
Figure 1. Schematic time line for labeling stem cells with iron oxide nanoparticle, ferumoxytol.
<img alt="Figure 2" src="/files/ftp_upload/3482/3482fig2.jpg" /> 
Figure 2. MR images of sagittal cross sections thrrough eppendorf tubes with cells in a cell pellet. A) Unlabeled controls. B) Labeled cells demonstrating a significant T2 or negative contrast agent effect on T2-weighted FSE or SE_Multislice imaging and a T1 or positive contrast agent effect on the T1-weighted GRE sequence.
<img alt="Figure 3" src="/files/ftp_upload/3482/3482fig3.jpg" /> 
Figure 3. Sagittal cross sections of eppendorf tubes with cells in cell pellets. As little as 10,000 ferumoxytol labeled cells can be detected via T2-w MR imaging.
<img alt="Figure 4" src="/files/ftp_upload/3482/3482fig4.jpg" /> 
Figure 4. MR visualization of ferumoxytol-labeled stem cells injected into murine cerebral ventricles. A) Axial, MR image of murine barin with no injection of USPIO-labeled stem cells. B) Axial, C) coronal, and D) sagittal MR images depicting the T2, negative contrast effect of USPIO-labeled stem cells (white arrows) injected in a murine brain.
<img alt="Table 1" src="/files/ftp_upload/3482/3482table1.jpg" /> 
Table 1. Quantity adaptions for labeling cells in alternate vessels.

<ol>
	<li>Narsinh, K. H., Plews, J., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+human+induced+pluripotent+and+embryonic+stem+cells:+fraternal+or+identical+twins?.">Comparison of human induced pluripotent and embryonic stem cells: fraternal or identical twins?.</a> Mol Ther. 19, 635-638 (2011).</li><li>Bulte, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+MRI+cell+tracking:+clinical+studies.">In vivo MRI cell tracking: clinical studies.</a> AJR. Am. J. Roentgenol. 193, 314-325 (2009).</li><li>Henning, T. D., Boddington, S., Daldrup-Link, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeling+hESCs+and+hMSCs+with+Iron+Oxide+Nanoparticles+for+Non-Invasive+in+vivo+Tracking+with+MR+Imaging.">Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging.</a> J. Vis. Exp. (13), e685-e685 (2008).</li><li>Tallheden, T., Nannmark, U., Lorentzon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+MR+imaging+of+magnetically+labeled+human+embryonic+stem+cells.">In vivo MR imaging of magnetically labeled human embryonic stem cells.</a> Life. Sci. 79, 999-1006 (2006).</li><li>Jung, C. W., Jacobs, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+and+chemical+properties+of+superparamagnetic+iron+oxide+MR+contrast+agents:+ferumoxides,+ferumoxtran,+ferumoxsil.">Physical and chemical properties of superparamagnetic iron oxide MR contrast agents: ferumoxides, ferumoxtran, ferumoxsil.</a> Magn. Reson. Imaging. 13, 661-674 (1995).</li><li>Coyne, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ferumoxytol+for+treatment+of+iron+deficiency+anemia+in+patients+with+chronic+kidney+disease.">Ferumoxytol for treatment of iron deficiency anemia in patients with chronic kidney disease.</a> Expert. Opin. Pharmacother. 10, 2563-2568 (2009).</li><li>Li, Z., Suzuki, Y., Huang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+reporter+gene+and+iron+particle+labeling+for+tracking+fate+of+human+embryonic+stem+cells+and+differentiated+endothelial+cells+in+living+subjects.">Comparison of reporter gene and iron particle labeling for tracking fate of human embryonic stem cells and differentiated endothelial cells in living subjects.</a> Stem Cells. 26, 864-873 (2008).</li><li>Metz, S., Bonaterra, G., Rudelius, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capacity+of+human+monocytes+to+phagocytose+approved+iron+oxide+MR+contrast+agents+in+vitro.">Capacity of human monocytes to phagocytose approved iron oxide MR contrast agents in vitro.</a> Eur. Radiol. 14, 1851-1858 (2004).</li><li>Nedopil, A., Klenk, C., Kim, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MR+signal+characteristics+of+viable+and+apoptotic+human+mesenchymal+stem+cells+in+matrix-associated+stem+cell+implants+for+treatment+of+osteoarthritis.">MR signal characteristics of viable and apoptotic human mesenchymal stem cells in matrix-associated stem cell implants for treatment of osteoarthritis.</a> Invest. Radiol. 45, 634-640 (2010).</li><li>Kraitchman, D. L., Heldman, A. W., Atalar, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+magnetic+resonance+imaging+of+mesenchymal+stem+cells+in+myocardial+infarction.">In vivo magnetic resonance imaging of mesenchymal stem cells in myocardial infarction.</a> Circulation. 107, 2290-2293 (2003).</li><li>Stuckey, D. J., Carr, C. A., Martin-Rendon, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron+particles+for+noninvasive+monitoring+of+bone+marrow+stromal+cell+engraftment+into,+and+isolation+of+viable+engrafted+donor+cells+from,+the+heart.">Iron particles for noninvasive monitoring of bone marrow stromal cell engraftment into, and isolation of viable engrafted donor cells from, the heart.</a> Stem Cells. 24, 1968-1975 (2006).</li><li>Henning, T. D., Sutton, E. J., Kim, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+ferucarbotran+on+the+chondrogenesis+of+human+mesenchymal+stem+cells.">The influence of ferucarbotran on the chondrogenesis of human mesenchymal stem cells.</a> Contrast. Media. Mol. Imaging. 4, 165-173 (2009).</li><li>Arbab, A. S., Yocum, G. T., Kalish, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+magnetic+cell+labeling+with+protamine+sulfate+complexed+to+ferumoxides+for+cellular+MRI.">Efficient magnetic cell labeling with protamine sulfate complexed to ferumoxides for cellular MRI.</a> Blood. 104, 1217-1223 (2004).</li><li>Nedopil, A. J., Mandrussow, L. G., Daldrup-Link, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implantation+of+Ferumoxides+Labeled+Human+Mesenchymal+Stem+Cells+in+Cartilage+Defects.">Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects.</a> J. Vis. Exp. (38), e1793-e1793 (2010).</li><li>Arbab, A. S., Yocum, G. T., Wilson, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+transfection+agents+in+forming+complexes+with+ferumoxides,+cell+labeling+efficiency,+and+cellular+viability.">Comparison of transfection agents in forming complexes with ferumoxides, cell labeling efficiency, and cellular viability.</a> Mol Imaging. 3, 24-32 (2004).</li><li>Babic, M., Horak, D., Trchova, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(L-lysine)-modified+iron+oxide+nanoparticles+for+stem+cell+labeling.">Poly(L-lysine)-modified iron oxide nanoparticles for stem cell labeling.</a> Bioconjug Chem. 19, 740-750 (2008).</li><li>Golovko, D. M., T, H. e. n. n. i. n. g., Bauer, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+stem+cell+labeling+with+ferucarbotran+and+protamine.">Accelerated stem cell labeling with ferucarbotran and protamine.</a> Eur. Radiol. 20, 640-648 (2010).</li><li>Lu, M., Cohen, M. H., Rieves, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FDA+report:+Ferumoxytol+for+intravenous+iron+therapy+in+adult+patients+with+chronic+kidney+disease.">FDA report: Ferumoxytol for intravenous iron therapy in adult patients with chronic kidney disease.</a> Am. J. Hematol. 85, 315-319 (2010).</li></ol>

All contributors to this study offer no disclosures.

Improving the efficacy of stem cell engraftments is critical for the advancement of regenerative medicine. A non-invasive visualization technique for stem cells in vivo significantly enhances our ability to understand mechanisms that lead to successful engraftment outcomes. Magnetic labeling for MR visualization, such as the procedure we have demonstrated, allows in vivo tracking of stem cells with MR imaging. Magnetically labeled stem cells have previously been transplanted into target tissues and have...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>D-MEM High Glucose </td>
 <td>Sigma</td>
 <td>D5648</td>
 <td>Or other base medium for desired stem cell line to be used</td>
 </tr>
 <tr>
 <td>D-PBS (Ca++, Mg++ free)</td>
 <td>GIBCO</td>
 <td>14190-144</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Trypsin-EDTA 0.05%</td>
 <td>Invitrogen</td>
 <td>25300-120</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum (FBS)</td>
 <td>Hyclone</td>
 <td>SH30071.03</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ferumoxytol
 (Feraheme)</td>
 <td>AMAG</td>
 <td>59338-0775-01</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Protamine Sulfate</td>
 <td>APP Pharm.</td>
 <td>22930</td>
 <td>&nbsp;</td>
 </tr>
</tbody>
</table>

labeling stem cells with ferumoxytol, an fda-approved iron oxide nanoparticle

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo kinetics of transplanted cells. A non-invasive method to repetitively monitor transplanted stem cells in vivo would allow investigators to directly monitor stem cell transplants and identify successful or unsuccessful engraftment outcomes.
A wide range of stem cells continues to be investigated for countless applications. This protocol focuses on 3 different stem cell populations: human embryonic kidney 293 (HEK293) cells, human mesenchymal stem cells (hMSC) and induced pluripotent stem (iPS) cells. HEK 293 cells are derived from human embryonic kidney cells grown in culture with sheared adenovirus 5 DNA. These cells are widely used in research because they are easily cultured, grow quickly and are easily transfected. hMSCs are found in adult marrow. These cells can be replicated as undifferentiated cells while maintaining multipotency or the potential to differentiate into a limited number of cell fates. hMSCs can differentiate to lineages of mesenchymal tissues, including osteoblasts, adipocytes, chondrocytes, tendon, muscle, and marrow stroma. iPS cells are genetically reprogrammed adult cells that have been modified to express genes and factors similar to defining properties of embryonic stem cells. These cells are pluripotent meaning they have the capacity to differentiate into all cell lineages 1. Both hMSCs and iPS cells have demonstrated tissue regenerative capacity in-vivo.
Magnetic resonance (MR) imaging together with the use of superparamagnetic iron oxide (SPIO) nanoparticle cell labels have proven effective for in vivo tracking of stem cells due to the near microscopic anatomical resolution, a longer blood half-life that permits longitudinal imaging and the high sensitivity for cell detection provided by MR imaging of SPIO nanoparticles 2-4. In addition, MR imaging with the use of SPIOs is clinically translatable. SPIOs are composed of an iron oxide core with a dextran, carboxydextran or starch surface coat that serves to contain the bioreactive iron core from plasma components. These agents create local magnetic field inhomogeneities that lead to a decreased signal on T2-weighted MR images 5. Unfortunately, SPIOs are no longer being manufactured. Second generation, ultrasmall SPIOs (USPIO), however, offer a viable alternative. Ferumoxytol (FerahemeTM) is one USPIO composed of a non-stoichiometric magnetite core surrounded by a polyglucose sorbitol carboxymethylether coat. The colloidal, particle size of ferumoxytol is 17-30 nm as determined by light scattering. The molecular weight is 750 kDa, and the relaxivity constant at 2T MRI field is 58.609 mM-1 sec-1 strength4. Ferumoxytol was recently FDA-approved as an iron supplement for treatment of iron deficiency in patients with renal failure 6. Our group has applied this agent in an &ldquo;off label&rdquo; use for cell labeling applications. Our technique demonstrates efficient labeling of stem cells with ferumoxytol that leads to significant MR signal effects of labeled cells on MR images. This technique may be applied for non-invasive monitoring of stem cell therapies in pre-clinical and clinical settings.

Watch this Scientific Journal Video about Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle at JoVE.com

Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

We describe a technique for labeling and tracking stem cells with FDA-approved, superparamagnetic iron oxide (SPIO), ferumoxytol (Feraheme). This cellular imaging technique that utilizes magnetic resonance (MR) imaging for visualization, is readily accessible for long-term monitoring and diagnosis of successful or unsuccessful stem cell engraftments in patients.

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo ...

labeling-stem-cells-with-ferumoxytol-an-fda-approved-iron-oxide

Research

JoVE Journal

Medicine

20.8K visualizações.  Molecular Imaging Program at Stanford (MIPS). Nós descrevemos uma técnica para rotulagem e rastreamento de células-tronco com FDA-approved, óxido de ferro superparamagnético (SPIO), ferumoxytol (Feraheme). Esta técnica de imagem celular que utiliza a ressonância magnética (RM) para visualização, é facilmente acessível a longo prazo de monitoramento e diagnóstico de engraftments tronco bem ou mal sucedidas de células em pacientes.

Vídeo: Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Continuous Manual Exchange Transfusion for Patients with Sickle Cell Disease: An Efficient Method to Avoid Iron Overload

Children with sickle cell anemia (SCA) may be at risk of cerebral vasculopathy and strokes, which can be prevented by chronic transfusion programs. Repeated transfusions of packed red blood cells (PRBCs) is currently the simplest and most used technique for chronic transfusion programs. However, iron overload is one of the major side effects of this therapy. More developed methods exist, notably the apheresis of RBC (erythrapheresis), which is currently the safest and most efficient method. However, it is costly, complicated, and cannot be implemented everywhere, nor is it suitable for all patients. Manual exchange transfusions combine one or more manual phlebotomies with a PRBC transfusion.
At the Reference Center of Sickle Cell Disease, we set up a continuous method of manual exchange transfusion that is feasible for all hospital settings, demands no specific equipment, and is widely applicable. In terms of HbS decrease, stroke prevention, and iron overload prevention, this method showed comparable efficiency to erythrapheresis. In cases where erythrapheresis is not available, this method can be a good alternative for patients and care centers.

We have outlined a method of continuous manual exchange transfusion for the treatment of sickle cell disease in patients. This safe protocol was designed to effectively limit iron overload in patients in need of chronic transfusions and can be used extensively without any special equipment.

Children with sickle cell anemia (SCA) may be at risk of cerebral vasculopathy and strokes, which can be prevented by chronic transfusion programs. ...

Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was synthesized to enhance imaging sensitivity for extrapulmonary tuberculosis (ETB). An SPIO nanoprobe was synthesized and conjugated with MtbsAb. The purified SPIO-MtbsAb nanoprobe was characterized using TEM and NMR. To determine the targeting ability of the probe, SPIO-MtbsAb nanoprobes were incubated with Mtb for in vitro imaging assays and injected into Mtb-inoculated mice for in vivo investigation with magnetic resonance (MR). The contrast enhancement reduction on magnetic resonance imaging (MRI) of Mtb and THP1 cells showed proportional to the SPIO-MtbsAb nanoprobe concentration. After 30 min of intravenous SPIO-MtbsAb nanoprobe injection into Mtb-infected mice, the signal intensity of the granulomatous site was enhanced by 14-fold in the T2-weighted MR images compared with that in mice receiving PBS injection. The MtbsAb nanoprobes can be used as a novel modality for ETB detection.

To improve serological diagnostic tests for Mycobacterium tuberculosis antigens, we developed superparamagnetic iron oxide nanoprobes to detect extrapulmonary tuberculosis.

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was ...

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical hurdles such as invasive cell delivery and tracking with low transplantation efficiency remain challenges in translational stem-based therapy. This article describes an emerging technique for stem cell labeling and tracking based on the labeling of the mesenchymal stem cells (MSCs) with superparamagnetic iron oxide (SPIO)&#160;nanoparticles, as well as intranasal delivery of the labeled MSCs. These nanoparticles are fluorescein isothiocyanate (FITC)-embedded and safe to label the MSCs, which are subsequently delivered to the brains of TBI-induced mice by the intranasal route. They are then tracked non-invasively in vivo by real-time magnetic resonance imaging (MRI). Important advantages of this technique that combines SPIO for cell labeling and intranasal delivery include (1) non-invasive, in vivo MSC tracking after delivery for long tracking periods, (2) the possibility of multiple dosing regimens due to the non-invasive route of MSC delivery, and (3) possible applications to humans, owing to the safety of SPIO, non-invasive nature of the cell-tracking method by MRI, and route of administration.

Presented here is a protocol for non-invasive mesenchymal stem cell (MSC) delivery and tracking in a mouse model of traumatic brain injury. Superparamagnetic iron oxide&#160;nanoparticles are employed as a magnetic resonance imaging (MRI) probe for MSC labeling and non-invasive in vivo tracking following intranasal delivery using real-time MRI.

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical ...

Estimation of Urinary Nanocrystals in Humans using Calcium Fluorophore Labeling and Nanoparticle Tracking Analysis

Kidney stones are becoming more prevalent worldwide in adults and children. The most common type of kidney stone is comprised of calcium oxalate (CaOx) crystals. Crystalluria occurs when urine becomes supersaturated with minerals (e.g., calcium, oxalate, phosphate) and precedes kidney stone formation. Standard methods to assess crystalluria in stone formers include microscopy, filtration, and centrifugation. However, these methods primarily detect microcrystals and not nanocrystals. Nanocrystals have been suggested to be more harmful to kidney epithelial cells than microcrystals in vitro. Here, we describe the ability of Nanoparticle Tracking analysis (NTA) to detect human urinary nanocrystals. Healthy adults were fed a controlled oxalate diet prior to drinking an oxalate load to stimulate urinary nanocrystals. Urine was collected for 24 hours before and after the oxalate load. Samples were processed and washed with ethanol to purify samples. Urinary nanocrystals were stained with the calcium binding fluorophore, Fluo-4 AM. After staining, the size and count of nanocrystals were determined using NTA. The findings from this study show NTA can efficiently detect nanocrystalluria in healthy adults. These findings suggest NTA could be a valuable early detection method of nanocrystalluria in patients with kidney stone disease.

The objective of this study was to determine whether nanoparticle tracking analysis (NTA) could detect and quantify urinary calcium containing nanocrystals from healthy adults. The findings from the current study suggest NTA could be a potential tool to estimate urinary nanocrystals during kidney stone disease.

Kidney stones are becoming more prevalent worldwide in adults and children. The most common type of kidney stone is comprised of calcium oxalate (CaOx) ...

Measurement of Tissue Non-Heme Iron Content using a Bathophenanthroline-Based Colorimetric Assay

Iron is an essential micronutrient. Both iron overload and deficiency are highly detrimental to humans, and tissue iron levels are finely regulated. The use of experimental animal models of iron overload or deficiency has been instrumental to advance knowledge of the mechanisms involved in the systemic and cellular regulation of iron homeostasis. The measurement of total iron levels in animal tissues is commonly performed with atomic absorption spectroscopy or with a colorimetric assay based on the reaction of non-heme iron with a bathophenanthroline reagent. For many years, the colorimetric assay has been used for the measurement of the non-heme iron content in a wide range of animal tissues. Unlike atomic absorption spectroscopy, it excludes the contribution of heme iron derived from hemoglobin contained in red blood cells. Moreover, it does not require sophisticated analytical skills or highly expensive equipment, and can thus be easily implemented in most laboratories. Finally, the colorimetric assay can be either cuvette-based or adapted to a microplate format, allowing higher sample throughput. The present work provides a well-established protocol that is suited for the detection of alterations in tissue iron levels in a variety of experimental animal models of iron overload or iron deficiency.

Here, a protocol for the measurement of the non-heme iron content in animal tissues is provided, using a simple, well-established colorimetric assay that can be easily implemented in most laboratories.

Iron is an essential micronutrient. Both iron overload and deficiency are highly detrimental to humans, and tissue iron levels are finely regulated. The ...