Heterologous expression of mutated HLA-G1 reduces alloreactivity of human dermal fibroblasts.

AIM: To engineer a stable HLA-G molecule and evaluate its immunomodulatory properties in transgenic human dermal fibroblasts (HDFs). MATERIALS & METHODS: A mutated HLA-G1 (mHLA-G1) molecule was generated by modifying the endoplasmic reticulum retrieval motif and 3'-untranslated region miRNA-binding sites of HLA-G1. Immunomodulatory properties of transgenic HDF-mHLA-G1 were evaluated in vitro. RESULTS: Stable mHLA-G1 expressing HDF cells were successfully generated and flow cytometry analysis revealed that mHLA-G1 efficiently localized to the cell surface. Natural killer cell-mediated cytolysis of HDF-mHLA-G1/green fluorescent protein (GFP) was reduced by 73% compared with HDF-GDP. HDF-mHLA-G1/GFP decreased phytohemagglutinin-activated peripheral blood mononuclear cell proliferation by 30% versus HDF-GFP. CONCLUSION: We are the first to successfully create a human fibroblast source with reduced alloreactivity using a novel mHLA-G1 construct. This approach may be extended to other cell types including human embryonic stem cells for use in allogeneic transplantation for cell-based regenerative medicine applications.

Heterelogous expression of mutated HLA-G decreases immunogenicity of human embryonic stem cells and their epidermal derivatives.

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.

Inhibition of DNA methylation enhances HLA-G expression in human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are immunosuppressive multipotent cells under investigation for potential therapeutic applications in regenerative medicine and prevention of graft-versus-host disease. Human leukocyte antigen (HLA)-G contributes to the immunomodulatory properties of MSCs. HLA-G expression in MSCs is very low and diminishes during in vitro expansion. Epigenetic regulation activates HLA-G expression in some cancer cell lines but not in MSCs. In the present study, adipose- and bone marrow-derived MSCs were exposed to the DNA demethylating agent 5-aza-2-deoxycytidine (5-aza-dC) and histone deacetylase inhibitor valproic acid (VPA) and HLA-G mRNA levels assessed using semi-quantitative reverse-transcription PCR. Exposure to 5-aza-dC resulted in HLA-G1 and -G3 upregulation in both early and late passage MSCs. VPA treatment did not induce HLA-G expression in both bone marrow and adipose derived MSCs. Our results provide the first evidence that HLA-G3 could be expressed in MSCs and that methylation-mediated repression is partly responsible for the observed low levels of HLA-G expression in MSCs. Our findings provide insight that treatment of MSCs with specific epigenetic regulatory modulators may improve their immunoregulatory capability for therapeutic applications.

Human embryonic stem cell derived-mesenchymal stem cells: an alternative mesenchymal stem cell source for regenerative medicine therapy.

AIM: To enumerate and characterize mesenchymal stem cells (MSC) derived from human embryonic stem cells (hESC) for clinical application. MATERIALS & METHODS: hESC were differentiated into hESC-MSC and characterized by the expression of surface markers using flow cytometry. hESC-MSC were evaluated with respect to growth kinetics, colony-forming potential, as well as osteogenic and adipogenic differentiation capacity. Immunosuppressive effects were assessed using peripheral blood mononuclear cell (PBMC) proliferation and cytotoxicity assays. RESULTS: hESC-MSC showed similar morphology, and cell surface markers as adipose (AMSC) and bone marrow-derived MSC (BMSC). hESC-MSC exhibited a higher growth rate during early in vitro expansion and equivalent adipogenic and osteogenic differentiation and colony-forming potential as AMSC and BMSC. hESC-MSC demonstrated similar immunosuppressive effects as AMSC and BMSC. CONCLUSION: hESC-MSC were comparable to BMSC and AMSC and hence can be used as an alternative source of MSC for clinical applications.

Temporal HLA profiling and immunomodulatory effects of human adult bone marrow- and adipose-derived mesenchymal stem cells.

AIM: To investigate the temporal HLA expression profile and immunomodulatory function of mesenchymal stem cells (MSCs) during in vitro expansion. MATERIALS & METHODS: Adult bone marrow-derived MSCs (BMSCs) and adipose-derived MSCs (AMSCs) were cultured and HLA class I and II mRNA expression were investigated during serial expansion using semiquantitative reverse-transcription PCR. The immunomodulatory properties of MSCs were monitored using peripheral blood mononuclear cell (PBMC) proliferation and cytotoxicity assays. RESULTS: Semiquantitative reverse-transcription PCR revealed that classical HLA class I molecules were highly expressed in MSCs and remained relatively stable during extended culture. Variable expression levels of HLA class II molecules were detected in both BMSCs and AMSCs across passages. AMSCs were more resistant to PBMC-mediated cytotoxicity and suppressed PBMC proliferation more than BMSCs, although the effect was diminished with increasing passage. CONCLUSION: 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.

Mutated HLA-G3 localizes to the cell surface but does not inhibit cytotoxicity of natural killer cells.

HLA-G plays an important role in the induction of immune tolerance. Various attempts to produce good manufacturing practice levels of HLA-G as a therapeutic molecule have failed to date partly due to the complicated structure of full-length HLA-G1. Truncated HLA-G3 is simpler and easier to produce than HLA-G1 and contains the expected functional epitope in its only α1 monomorphic domain. In this study, we engineered the ER retrieval and retention signal on HLA-G3's cytoplasmic tail by replacing its RKKSSD motif with RAASSD. We observed that mutated HLA-G3 was highly expressed on the cell surface of transduced K562 cells but did not inhibit cytotoxicity of natural killer cells.

Full-length HLA-G1 and truncated HLA-G3 differentially increase HLA-E surface localization.

Human leukocyte antigen (HLA)-E plays a role in immune tolerance induction and its transport to the cell surface is limited and dependent on the availability of HLA class I signal peptide. The role of HLA-G in regulating HLA-E surface localization remains controversial. The aim of our study was to clarify whether full-length and truncated HLA-G isoforms regulate HLA-E surface localization. Using a retroviral expression system and flow cytometric analysis, we found that surface HLA-E levels were significantly higher in HLA-G1 (34.1±4.4%, p<0.005) and -G3 (15.3±1.8%, p<0.04) versus empty vector (9.0±1.0%) transductants. Biotinylation and Western blot studies revealed HLA-E surface protein was increased by 4.5- and 1.3-fold in HLA-G1 and -G3 versus empty vector transductants. Although no significant differences in transcript and protein levels were detected between HLA-G1 and -G3 transductants, surface levels of HLA-G1 were 2.5-fold higher than HLA-G3 by flow cytometric analysis and Western blotting. Taken together, our data demonstrate that full-length HLA-G1 and truncated -G3 differentially increase HLA-E surface localization.

Functional analysis of a recombinant PIII-SVMP, GST-acocostatin; an apoptotic inducer of HUVEC and HeLa, but not SK-Mel-28 cells.

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 220 µg/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, αvß3, αvß5, α6, ß1, and ß3 integrin receptors. HeLa cells express α1, α2, α6, αv, αvß5, and ß1 integrin receptors. T24 cells express α1, α3, α6, αv, αvß3, αvß5, ß1, ß3, and ß6 integrin receptors.

Small cutaneous wounds induce telogen to anagen transition of murine hair follicle stem cells.

BACKGROUND: Recent studies suggest that large (>1 cm) diameter circular cutaneous wounds induce follicular neogenesis in mice. However, the effects of non-circular wounds or smaller circular wounds on the hair follicle cycle remain poorly understood. OBJECTIVE: We investigated whether non-circular wounds or smaller (≤1 cm) circular wounds could induce early entry of hair follicle stem cells into anagen. METHODS: We created different shaped and sized full-thickness incisional wounds on the dorsal skin of 6-wk old mice (BALB/c, C57BL/6, and CD1), and then assessed for hair growth, wound contraction rates, and growth factor and/or immunomodulatory cytokine involvement. RESULTS: By day 16 and through day 26 post-wounding, we observed hair growth in the skin around the wound, but not in distant unwounded skin of BALB/c and C57BL/6 mice. In contrast, CD1 mice showed hair growth in both the wounded and unwounded skin. In all mice, the hair growth pattern was independent of wound type. The area of hair growth induced by a 2 cm linear wound was roughly 2-fold that induced by a 1 cm linear wound and 4-fold that of a 0.5 cm linear wound. Increased γ-glutamyl transpeptidase activity was observed in hair follicles growing in the wounded area as well as an up to 8-fold upregulation of Wnt- and Shh-dependent signaling, consistent with anagen growth. CONCLUSION: Our data strongly support that small cutaneous wounds induce telogen to anagen transition of murine hair follicle stem cells.