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 JoVE Biology

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

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1Division of Cardiovascular Medicine, Stanford University

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    Summary

    In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

    Date Published: 8/03/2008, Issue 18; doi: 10.3791/827

    Cite this Article

    Yamada, M., Yang, P. In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging. J. Vis. Exp. (18), e827, doi:10.3791/827 (2008).

    Abstract

    Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.

    In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

    Protocol

    Manganese Labeling of Human Embryonic Stem Cells

    1. Trypsinize the human embryonic stems which have been cultured in feeder-free conditions for 5 minutes. Neutralize the trypsin by adding culture media.
    2. Spin down the cells by centrifuging at 800 rpm for 5 minutes at 20° Celsius. After re-suspending the cell pellet, count the number of cells by trypan blue staining. Based on the cell count obtained, divide the cells into four aliquots of 3 million cells into conical tubes. Pellet the cells again by centrifugation.
    3. Just before starting the manganese labeling, dissolve fresh manganese chloride with 0.9% sodium chloride solution to make a 0.1mM manganese chloride solution.
    4. In order to observe calcium channel activity, four samples were prepared. The first sample is our control which contains cells incubated in 0.9% sodium chloride alone. The second sample contains cells incubated in 0.1 mM MnCl2. Sample 3 and 4 contain cells incubated in 0.1 mM MnCl2 with either 5 μM of the Ca2+-channel agonist, (s)-(-)-Bay K8644, or 250 μM of the Ca2+-channel antagonist, verapamil. Incubate the four samples at 37° Celsius with 5% CO2 for 30 minutes.
    5. After 30 minutes, spin down the cells at 800 rpm for 5 minutes at 20° Celsius. Then aspirate the supernatant and wash the labeled cells twice with PBS. After washing, re-suspend each pellet with 200 μL PBS and transfer into PCR tubes. These pellets are usually white.

    Iron Labeling of Human Embryonic Stem Cells

    1. Mix clinical-grade protamine sulfate with distilled water to get a 1 mg/mL stock solution.
    2. In a tube containing human embryonic stem cell culture media, add ferumoxides at 100 μg/mL followed by addition of 12 μg/mL protamine sulfate. Mix the solution vigorously for five minutes.
    3. Add mixed solution to the equal amount of the cell culture media to achieve a final concentration of 50 μg/mL of ferumoxides and 6 μg/mL of the protamine sulfate to label cells. Incubate cells in this solution for 12 hours.
    4. Wash the cells twice with PBS, with the last wash containing 10U/ml heparin to dissolve the extracellular ferumoxides-protamine sulfate complex. After washing, trypsinize cells to obtain a single cell suspension.
    5. Count the cells and aliquot into the required number of samples. After washing, re-suspend each pellet with 200 μL PBS and transfer into PCR tubes. These pellets should be dark orange brown in color.

    Performing Cellular Magnetic Resonance Imaging

    1. Make a phantom in order to stabilize the tubes containing the cell pellets and also to avoid artifacts from the surrounding air during the scanning. To do so, mix 0.8% of agar and 1% of copper sulfate in distilled water and bring it to a boil by microwaving for 5-7 minutes. Cool this mixture for at least 1 hour prior of scanning to attain a gel.
    2. The tubes containing the labeled cell pellets are placed in the phantom for scanning. In vitro cellular MRI is performed at Signa HDx 3T MRI (GE Medical Systems) by using clinical knee coil.
    3. Scan the manganese labeled cells for each of the three samples using a spin echo sequence: TR; Repetition time = 800 msec. TE; Echo time = minimum, FOV; field of view = 12 x 12 cm; matrix = 256x256, NEX 1.
    4. Scan the iron labeled cells by using a gradient-echo sequence. We use the following parameters for this imaging: TR = 100 msec; TE = 10 msec, FA; flip angle = 30°; FOV = 12 x 12 cm; matrix = 256 x 256, NEX 1.

    Analyzing the MRI Results

    1. Analyze images of MRI scanning from 4 different samples of manganese-labeled cells:
      • Sample #1 is our control which contains cells incubated in 0.9% sodium chloride alone.
      • Sample #2 contains cells incubated in 0.1 mM MnCl2.
      • Sample #3 contains cells incubated in 0.1 mM MnCl2 with 5 μM of the Ca2+-channel agonist, (s)-(-)-Bay K8644.
      • Sample #4 contains cells incubated in 0.1 mM MnCl2 with 250 μM of the Ca2+-channel antagonist, verapamil.
    2. As predicted, differences in signal intensities from manganese labeled cells are visible. Compared to the cells labeled purely with manganese, the signal intensity clearly increases in the presence of the calcium channel agonist and decreases in the presence of calcium channel antagonist. The area where signal intensity has increased for manganese labeled cells are measured by using Image J (NIH, Bethesda, MD, USA). The same software is used for analyzing the area of dark off-resonance signal from iron labeled cells. The size of the dark signal from the iron-labeled cells depends on the number of cells or the number of iron particles contained by cells. In this case, one million cells shows a smaller area of dark signal compared to the signal from the three million cells.

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    Discussion

    These results indicate that manganese does enter via the voltage-gated calcium channel, and manganese can be used as MRI contrast agent, which can also show the cell viability. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of human embryonic stem cells into the myocardium.

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    Disclosures

    Materials

    Name Type Company Catalog Number Comments
    Manganese chloride tetrahydrate Reagent Sigma-Aldrich M8054
    (S)-(-)-Bay K8644 Reagent Sigma-Aldrich B133
    (+)-Verapamil hydrochloride, minimum 99.0% titration Reagent Sigma-Aldrich V4629
    Sodium Chloride 0.85-0.90% W/V Reagent VWR international VW 3257-1
    Feridex I.V. Reagent Berlex Laboratories NDC 59338-7035-5 ferumoxides injectable solution 11.2mg iron/mL
    Protamine sulfate Reagent American Pharmaceutical Partners NDC 63323-229-05 50 mg (10 mg/mL)
    Knockout SR (Serum replacement for ES cells) Reagent GIBCO, by Life Technologies 10828
    DMEM/F12 (1:1) Reagent GIBCO, by Life Technologies 11330
    2- mercapt–thanol 98+% Reagent Sigma-Aldrich M 3148
    L-Glutamine 200mM 100x Reagent GIBCO, by Life Technologies 25030
    Penicillin-Streptomyocin 10,000 units/ml Reagent GIBCO, by Life Technologies 15140-122
    MEM Non-Essential Amino Acids 100x solution Reagent GIBCO, by Life Technologies 11140
    Signa HDx 3T MRI Tool GE Healthcare
    clinical Knee coil Tool GE Healthcare
    DPBS Reagent GIBCO, by Life Technologies 14190

    References

    Comments

    4 Comments

    I like this kind of publication very much. It showed me the experiment in details and most important, the author offered me many tips. I learned a lot. Thanks for your beautiful work!
    Reply

    Posted by: AnonymousOctober 21, 2008, 10:26 PM

    How to assess manganese labeling of human embryonic stem cells ,such as iron stained with Prussion blue?
    Reply

    Posted by: AnonymousFebruary 21, 2009, 7:14 AM

    there is no specific immunohistological stain for manganese. perhaps, an indirect assessment using calcium-channel by calcium analogues such as a fura will be the only method. thnx for a very pertinent question.  phil
    Reply

    Posted by: AnonymousApril 17, 2009, 8:28 PM

    How to assess manganese labeling of human embryonic stem cells ,such as iron stained with Prussion blue?
    Reply

    Posted by: AnonymousFebruary 21, 2009, 7:18 AM

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