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.
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.
Manganese Labeling of Human Embryonic Stem Cells
Iron Labeling of Human Embryonic Stem Cells
Performing Cellular Magnetic Resonance Imaging
Analyzing the MRI Results
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.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Manganese chloride tetrahydrate | Reagent | Sigma | M8054 | |
(S)-(-)-Bay K8644 | Reagent | Sigma | B133 | |
(+)-Verapamil hydrochloride, minimum 99.0% titration | Reagent | Sigma | V4629 | |
Sodium Chloride 0.85-0.90% W/V | Reagent | VWR | 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 | 10828 | |
DMEM/F12 (1:1) | Reagent | Gibco | 11330 | |
2- mercaptoethanol 98+% | Reagent | Sigma | M 3148 | |
L-Glutamine 200mM 100x | Reagent | Gibco | 25030 | |
Penicillin-Streptomyocin 10,000 units/ml | Reagent | Gibco | 15140-122 | |
MEM Non-Essential Amino Acids 100x solution | Reagent | Gibco | 11140 | |
Signa HDx 3T MRI | Tool | GE Medical Systems | ||
clinical Knee coil | Tool | GE Medical Systems | ||
DPBS | Reagent | Gibco | 14190 |