The main objective of this research is to define metabolite alterations of embryonic stem cell lines in response to irradiation.

1D-1H NMR Data Summary for HEK293

Figure 1. HEK293 cells grown to 85% confluence and irradiated. For each radiation dosage; Control (0 Gy), Low (0.2 Gy) & High (2 Gy) dosage, three independent cell samples were used.

Control vs. High Dosage

PPM 0.009

p -value 0.023

Fold Change 1.4

Down-regulated Metabolites Glycogen Biuret

Embryonic Stem Cells (ESCs) are group of unspecialized cells derived from the inner cell mass

Low Dosage vs. High Dosage

of a blastocyst that are capable of self-renewal and differentiation into all cell lineages. In general Stem cells have remarkable ability to regenerate and replace themselves, with a capacity to study both normal and diseased state of a cell or a tissue. Functional studies in stem cell research involve genomics, proteomics and metabonomics. The field of Metabonomics has proved to be extremely helpful in bridging the gap between genomics and proteomics. Several high-throughput technologies like Nuclear Magnetic Resonance (NMR) can be used to detect the metabolites in tissues, cells and biological fluids. Radiation is both a carcinogen and therapeutic agent. Radiation or chemically induced changes in an organism, results in disturbance in the concentration of endogenous metabolites. Metabolic profiling of stem cells and their derivatives is still very limited, and often these cells have been defined by their functional properties. Mouse Embryonic Stem cells (mESCs) & Human Embryonic Kidney cells (HEK293) have been used as the model cell lines for evaluating the effect of radiation at variable doses. Studying these metabolites, would give a better understanding of the cells physiology.

0.969 1.009 1.049 1.089 2.168 2.208 2.447 3.247 3.566 3.606 4.006 4.166 4.206 7.084 7.124 7.203 8.482 8.562 9.362

0.01 0.02 0.04 0.03 0.00 0.00 0.03 0.03 0.01 0.02 0.01 0.04 0.03 0.02 0.05 0.04 0.00 0.02 0.02

1.3 1.3 1.4 1.3 1.3 1.2 1.3 1.2 1.6 1.4 2.3 1.3 1.3 1.5 1.6 1.3 1.2 1.4 1.7

D-Glucono-1,5-lactone D-Fructose-2,6-bisphosphate Phenyl pyruvic acid sodium salt 3-hydroxy cinnamic acid Phenylacetic acid 6-Phosphogluconic acid trisodium salt Sodium glycolate

High Dosage Spectrum

B
Low Dosage Spectrum

Figure 2. 1D- 1H NMR spectroscopy was performed on all the Magnification 400X samples NMR spectra for Control, Low dosage (0.2 Gy) treated and High dosage (2 Gy) treated HEK293 cells. NMR spectral data was processed with MestReNova software.

Control Spectrum

Mouse Embryonic Fibroblasts

Figure 4. Identification of Metabolites. Fold Change (F.C) & p-value are two important parameters for metabolite identification. Peaks with F.C>1 & p-value<0.05 were selected and metabolites were confirmed using the Madison Metabolomics Consortium Database.
P4.03 BxPc-3

Embryonic Stem Cell Culture

mESCs (grown over MEFs) & HEK293 cells cultured in 2D (in vitro) are
mESCs (grown over MEFs) & HEK293 cultured in vitro

Irradiation
(True Beam Radiation System) Cells are exposed to radiation at variable dosage (0.2 Gy & 2 Gy)
Irradiation mouse embryonic fibroblast
Figure 3. Plot of Fold Change vs. Chemical shifts. Statistical tools like Fold Change & Paired t-test were used to determine significant chemical shifts and associated intensity differences among the samples. Fold change was calculated by Group 1 (average):Group 2 (average) for each chemical shift.

mouse embryonic stem cells

Figure 5. Co-culture of mESCs on mitotically inactive MEF. mESCs will be used for future embryonic stem cell irradiation experiments

Nuclear Magnetic Resonance

NMR sample preparation is done following a defined protocol & 1H NMR (1D and 2D) spectroscopy is used to reveal metabolic profile
1H

NMR Spectroscopy

The HEK293 cell irradiation experiments revealed several metabolites that were down-regulated post-irradiation. These metabolites were more significant in High dosage (2 Gy) irradiated cells as compared to low dosage (0.2 gy) irradiated cells. Further 2D-1H NMR would provide better understanding of the metabolites that might be down-regulated (or up-regulated) as a result of irradiation in HEK293 cells. This experiment helped to establish Standard Operating Protocol (SOP) for identifying potential metabolites in irradiated cells. Identification of the potential metabolites as well as their down-regulation/up-regulation with respect to irradiation sets the pavement for future irradiation experiments using Mouse embryonic stem cells (mESCs) and then slowly transform to Human embryonic stem cells (hESCs).

Data Analysis
Specific NMR data processing (MestReNova) and statistical tools are used to report significant metabolites
This research is funded by the Undergraduate Research Grant 2012 and the ASI r-Grant 2011 This study has been possible with the collaboration of Dr. Richard Dunia at the Fresno Cancer Center.
Data Analysis