hESC-MSC were comparable to BMSC and AMSC and hence can be used as an alternative source of MSC for clinical applications.

Human embryonic stem cells (hESCs) are capable of extensive self-renewal and expansion and can differentiate into any somatic tissue, making them useful for regenerative medicine applications. Allogeneic transplantation of hESC-derived tissues from results in immunological rejection absent adjunctive immunosuppression. The goal of our study was to generate a universal pluripotent stem cell source by nucleofecting a mutated human leukocyte antigen G (mHLA-G) gene into hESCs using the PiggyBac transposon. We successfully generated stable mHLA-G(EF1α)-hESC lines using chEF1α promoter system that stably expressed mHLA-G protein during prolonged undifferentiated proliferation andin differentiated embryoid bodies as well as teratomas. Morphology, karyotype, and telomerase activity of mHLA-G expressing hESC were normal. Immunofluorescence staining and flow cytometry analysis revealed persistent expression of pluripotent markers, OCT-3/4 and SSEA-4, in undifferentiated mHLA-G(EF1α)-hESC. Nucleofected hESC formed teratomas and when directed to differentiate into epidermal precursors, expressed high levels of mHLA-G and keratinocyte markers K14 and CD29. Natural killer cell cytotoxicity assays demonstrated a significant decrease in lysis of mHLA-G(EF1a)-hESC targets relative to control cells. Similar results were obtained with mHLA-G(EF1α)-hESC-derived epidermal progenitors (hEEP). One way mixed T lymphocyte reactions unveiled that mHLA-G(EF1a)-hESC and -hEEP restrained the proliferative activity of mixed T lymphocytes. We conclude that heterologous expression of mHLA-G decreases immunogenicity of hESCs and their epidermal differentiated derivatives.

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Human leukocyte antigen (HLA)-G is a non-classical HLA class I molecule thought to play a key role in maternal-fetal tolerance. Although initial studies suggested that HLA-G expression is restricted to extravillous cytotrophoblasts, expression was subsequently reported in a wide variety of other human tissues and tumor cells. However, consensus as to the validity of these collective findings has proven difficult because the antibodies used to define the temporal and spatial expression patterns of HLA-G remain incompletely characterized. The aim of our study was to reassess two of the most widely used HLA-G antibodies (MEM-G/9 and 4H84) in HLA-G-positive (JEG-3 and HLA-G transduced) and -negative (dermal fibroblast, mesenchymal stem cell, K562, and Jurkat) lines using flow cytometry, immunofluorescence, and western blotting. We found that MEM-G/9 recognized HLA-G3 by flow cytometry, indicating that its epitope is present on the α1 domain of HLA-G. Although 4H84 preferably recognized unfolded HLA-G-free chains, it showed strong non-specificity under certain methodological conditions.

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Disintegrins and disintegrin-like peptides interact with integrins and interfere with cell-cell and cell-matrix interactions. A disintegrin-like snake venom gene, Acocostatin was cloned from the venom gland mRNA of Agkistrodon contortrix contortrix. Acocostatin belongs to the PIII-SVMP subfamily of disintegrin-like peptides. The recombinant acocostatin peptide was produced and purified as GST-fusion. The GST-acocostatin peptide, at 44 μg/mL, inhibited platelet aggregation by 30% in PRP and 18% in whole blood. In addition GST-acocostatin, at 220μg/mL, inhibited SK-Mel-28 cell migration by 48%, but did not inhibit T24 cell migration. The GST-acocostatin peptide ability to induce apoptosis on HUVEC, HeLa, and SK-Mel-28 cells was determined using Annexin-V-FITC and chromatin fragmentation assays after 24 h of treatment. At 5 μM GST-acocostatin peptide, 19.68% +/− 3.09 of treated HUVEC, and 35.86% +/− 2.05 of treated HeLa cells were in early apoptosis. The GST-acocostatin peptide also caused chromatin fragmentation of HUVEC and HeLa cells as determined by fluorescent microscopy and Hoechst staining. The GST-acocostatin peptide failed to induce apoptosis of SK-Mel-28 cells. We characterized the HUVEC, HeLa, and T24 integrin expression by flow cytometry, as the first step in determining GST-acocostatin binding specificity. Our results indicate that HUVEC express αv, αvβ3, αvβ5, α6, β1, and β3 integrin receptors. HeLa cells express α1, α2, α6, αv, αvβ5, and β1 integrin receptors. T24 cells express α1, α3, α6, αv, αvβ3, αvβ5, β1, β3, and β6 integrin receptors.

These findings provide insight regarding the relationship between MSC passage number and MSC immunosuppressive properties and suggest that AMSCs hold advantages over BMSCs for immunomodulatory therapeutic purposes.