Stem Cell Media Cultures

All cell culture procedures were performed under sterile conditions in laminar class II biohazard safety cabinet (ESCO). The cell cultures were incubated at 37oC in 5% CO2 humidified incubators (RSBiotech).
MSC were cultured in MSC complete medium made up of Dulbecco’s Modified Eagle’s medium with nutrient mixture F-12 (HAM)[1:1] (DMEM/F12) with GLUTAMAX -I (Gibco, Invitrogen, USA), supplemented with 10% pre-selected foetal bovine serum (Stem Cell Technology Inc.), 1% of Penicillin /Streptomycin (Gibco, Invitrogen), 0.5% Fungizone (Gibco, Invitrogen), 0.1% Gentamicin (Gibco, Invitrogen), with or without 40ng/ml basic fibroblast growth factor (bFGF) (Peprotech, USA) . The serum used was pre-selected by the manufacturer to support MSC growth optimally in vitro culture while preserving MSC multi-potency to differentiate into chondro-, adipo- and osteogenic pathways.
3.3 T cell media

PBMC were cultured in complete T cell medium made up of RPMI 1640 (Gibco BRL, Invitrogen) supplemented with 10% foetal bovine serum (Gibco BRL, Invitrogen) or Human AB Serum and 1% Penicillin/Streptomycin (Gibco BRL, Invitrogen), 0.5% Fungizone (Gibco BRL, Invitrogen), 0.1% Gentamicin (Gibco BRL, Invitrogen).

3.4 Flowcytometry Analysis

3.4.1 Immunophenotyping

The surface markers of cells were determined by direct immunofluorescence staining and analysed by flowcytometer. All antibodies were listed in Table 1. Cells were harvested and washed with 0.5% of BSA in 1X PBS (phosphate-buffered saline). Aliquots of cells from 105 to 106 were labelled with anti-human monoclonal antibodies, for 20 minutes at 2-8oC and washed with 0.5% of BSA in 1X PBS. All antibodies are raised in mice against human epitopes. The fluorochrome analysis was included with appropriate FITC-, PE-, PE-CY5-, PERCP-, APC-, conjugated isotype controls. The stained samples were assessed by using the FACSCalibur flowcytometer (Becton Dickinson). Gating at FACS acquisition was applied to select the target cells population and exclude any dead cells and debris. Ten to hundred thousands of events/cells were acquired and the data were analysed using CellQuestPro software by manufacturer.
Antibody Predominant reactivity Clone/source

Anti-huCD105-PE Endoglin 166707/R&D System
Anti-huCD73-PE T, B, DC & stem cells AD2/BD
Anti-huCD90-FITC Thy-1 cells 5E10/BD
Anti-huCD29-APC thrombocytes, monocytes, MAR4/BD
lymphocytes
Anti-huCD45-PerCP leucocytes 2D1/BD
Anti-huCD34-FITC HSC 8G12/BD
Anti-huCD80-FITC Activated B cells & DC L307.4/BD
Anti-huCD86-FITC Activated B cells & monocytes 2331(FUN-1)/BD
HLA-A,B,C-PE-Cy5 MHC-I expressing cells G46-2.6/BD
HLA-DR,DP,DQ-FITC MHC-II expressing cells TÜ39/BD
Anti-huCD4-PerCP CD4 T cells SK3/BD
Anti-huCD8-APC CD8 T cells SK1/BD
Anti-huCD25-FITC Activated T cells & B cells 2A3/BD
Anti-huCD69-PE Activated T, B & NK cells L78/BD
Anti-huLAP(TGF-β1) cells expressing LAP (TGF-β1) 27232

Abbreviations
hu Human
FITC Fluorescein isothiocynate
PE Phycoerythrin
PERCP Peridinin chlorphyll protein
APC Allophycoerythrin
DC Dendritic cells
NK Natural Killer

Table 1: Anti-human antibodies used for flowcytometric analysis. All antibodies were purchased from Becton Dickinson/Pharmingen except CD105 and LAP (TGF Beta I) from R&D System. All antibodies were used at volume 10µl/106 cells in 100µl of total staining volume.
3.4.2 Cell Cycle Analysis

DNA content was identified using propidium iodine (PI). Briefly, cells were harvested from experimental culture, washed and then fixed in 70% alcohol in -20oC for overnight. Then, the fixed cells were washed and incubated with 100µg/ml Propidium Iodide (PI) (Molecular Probe, Invitrogen) and 20ng/ml RNase (Sigma) in PBS for 30 minutes. In T cells cell cycle analysis (section 6.2.5), 100ng/ml of fluorescein isothiocyanate (FITC) was added into the staining buffer to identify the intracellular protein. The results were acquired by flowcytometry and analysis was done by FCS Express V3.

3.5 Tritium thymidine (3H-TdR) incorporation assays

Proliferation assays were measured by tritium thymidine (3H-TdR) incorporation which reveals the proportion of cells in S phase of the cell cycle. In brief, at final 18 hours of incubation, 3H-TdR (0.037 MBq/well [0.5 μCi/well]) (Pelkin Elmer) were added to pulse the cultures. At the end of incubation, the cells were harvested onto glass fibre filter mats A (Perkin Elmer) using a 96 well plate manual cell harvester (MACH IIIM-FM, Tomtec, Inc. Hamden, CT USA). Scintillation cocktail was added to amplify the signal. Liquid scintillation spectroscopy was counted by Microbeta Trilux beta counter (Pelkin Elmer).
3.6 Statistical Analysis

Values for all measurements are presented as mean ± SD or stated otherwise. Comparisons for all pairs were performed by Student’s t-test. Significance levels were set at p value of 0.05.

Glycation and Mesenchymal Stem Cell Function

New cells are often produced in the body during growth and development. In addition, new cells also develop as the body repairs and remodels its tissues after an injury. These new cells come from mesenchymal stem cells (MSCs), which are considered as multipotent cells. MSCs are found in various parts of the body during growth and development, but in adults, they are present in the bone marrow, where they later differentiate, mature and migrate to become more specialized cells with unique functions. These cells’ potential to develop into bone cells, cartilage cells, muscle cells and fat cells makes their role in regeneration, repair and remodelling important, especially when the body undergoes the normal process of aging or recovers from disease or injury.

Research shows, however, that the potential of stem cells to proliferate and mature into specialized cells may be hindered by compounds known as advanced glycation end-products (AGEs). These compounds are formed by a chemical reaction called glycation, which involves attaching sugar molecules to proteins without the use of enzymes. This process initiates a complex series of molecular rearrangements and dehydrations that produces cross-linked proteins, resulting in the disruption of normal metabolic processes.

Glycation and AGEs

The body normally metabolizes substances such as simple sugars and proteins to produce energy, build tissues and many more functions. In the molecular level, chemical reactions such as glycosylation occur, in which a carbohydrate molecule attaches to another protein molecule to form another substance. These chemical reactions are often catalyzed by enzymes resulting in the formation of various glycans, which are involved in many structural and functional roles in the cells. However, sometimes, nonenzymatic chemical reactions (like glycation) occur, resulting in the formation of unstable substances such as AGEs.
AGEs have been shown to crosslink with various proteins inside, as well as outside, the cells, resulting in alterions in the mechanical properties of various tissues in the body. They can also modulate many cellular processes that lead to aging, chronic inflammation, and disease. Although AGEs normally form at slow rates in the body even before birth, they constantly accumulate with time. They have been found in the tissues of aged individuals, and have also been associated in age-related diseases such as heart disease, diabetes, and renal disease.

How AGEs Affect Stem Cell Function

Previous studies have suggested that AGEs may be involved in the development of age-related disorders including musculoskeletal diseases such as osteoarthritis, a chronic disabling disorder common among the elderly. Research shows that the accumulation of AGEs affects the collagen in bone, explaining the increase in bone fragility and risk for fracture in elderly individuals. Other studies have shown that a reduction in the number of MSCs and a decreased ability of MSCs to differentiate into osteoblasts and osteocytes (mature bone cells) are associated with increased AGE accumulation. Increased AGE levels have also been shown to affect proteoglycan synthesis in the cartilage, resulting in reduced chondrocyte (cartilage cell) formation. These processes lead to a decreased capacity for tissue repair, which may explain the loss of cartilage in osteoarthritis.
To investigate how these processes occur, scientists have done experiments that showed how AGEs inhibited the proliferation and differentiation of stem cells into specialized adipose (fat), chondrocyte (cartilage) and osteocyte (bone) cells. They found that AGEs inhibit the growth in different cell lines. In addition, they also found that AGEs induced cell death or apoptosis, which may explain some of the complications associated with diabetes, such as diabetic neuropathy. AGEs also interfered with bone mineralization, which may be linked to increased bone fragility in aging individuals.
Other scientific investigations have also shown that an increase in the expression of receptors for AGEs (RAGE) is associated with peripheral neuropathy, another complication of diabetes. Late complications of diabetes, such as numbing and tingling sensation in the feet may be due to morphological and functional changes in the peripheral nerve axons, epidermal nerve fibers, and dorsal root ganglion. One of the mechanisms that have been proposed in these changes is the accumulation of AGEs and the expression of RAGE in the peripheral nervous system.
The increased risk of development of different types of solid tumors in patients with diabetes has been a subject of investigation for years. Some authors propose that RAGE induction may participate in the origin of cancer stem cells in diabetic patients who have malignant tumors in their colon, although its mechanisms are yet unclear.
Other investigators who looked into the effects of AGEs on stem cell function also found that AGEs caused abnormal MSC growth and migration, and triggered the production of pro-inflammatory factors involved in the cardiovascular complications of diabetes. AGEs have been shown to increase the intracellular formation of reactive oxygen species (ROS) and the number of apoptotic cells. Although new studies suggest a therapeutic role for stem cells in various diseases such as ischemic cardiomyopathy or acute myocardial infarction, this is mitigated in patients with diabetes because of the inhibitory effects of AGEs on MSCs. Studies on stem cell transplantation to treat age-related diseases such as brain degeneration, heart disease, and diabetes are being done to explore the potential of stem cells to replace damaged cells and tissues. However, studies suggest that in patients with diabetes, AGEs induce the production of inflammatory substances called chemokines/cytokines, which inhibit the growth and migration of stem cells. Doctors treating diabetic patients who are undergoing stem cell therapy should therefore emphasize measures to control blood sugar levels and inflammation.
For stem cell therapy to work, the transplanted stem cells must be able to proliferate, differentiate into mature specific cells, survive in the body, integrate, and restore the function of the tissue. Scientists and physicians must determine the factors that will influence these processes for therapy to be successful, including the possible role AGEs play in it.

Works Cited

Kume , Kato S, Yamagishi S, Inagaki, Y, et al. Advanced Glycation End-Products Attenuate Human Mesenchymal Stem Cells and Prevent Cognate Differentiation Into Adipose Tissue, Cartilage, and Bone. J Bone Miner Res, 2005. 20: 1647–1658. doi: 10.1359/JBMR.050514

Yang K, Wang XQ, He YS, et al. Advanced glycation end products induce chemokine/cytokine production via activation of p38 pathway and inhibit proliferation and migration of bone marrow mesenchymal stem cells. Cardiovasc Diabetol, 2010, 9:66.

Toth C, Rong L, Yang C, et al. Receptor for Advanced Glycation End Products (RAGEs) and Experimental Diabetic Neuropathy
Diabetes. 2008;57(4):1002-1017.

Hu X, Cheng Y. Possible participation of receptor for advanced glycation end products (RAGE) in the origin of cancer stem cells in diabetic patients with colon cancer. Med Hypotheses. 2013 May;80(5):620-3.